ALBERT

Description: 〈p〉Publication date: November 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 130〈/p〉 〈p〉Author(s): Fernanda Valadares, Thiago A Gonçalves, André Damasio, Adriane MF Milagres, Fabio M Squina, Fernando Segato, André Ferraz〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Secretome evaluations of lignocellulose-decay basidiomycetes can reveal new enzymes in selected fungal species that degrade specific substrates. Proteins discovered in such studies can support biorefinery development. Brown-rot (〈em〉Gloeophyllum trabeum〈/em〉) and white-rot (〈em〉Pleurotus ostreatus〈/em〉) fungi growing in sugarcane bagasse solid-state cultures produced 119 and 63 different extracellular proteins, respectively. Several of the identified enzymes are suitable for 〈em〉in vitro〈/em〉 biomass conversion, including a range of cellulases (endoglucanases, cellobiohydrolases and β-glucosidases), hemicellulases (endoxylanases, α-arabinofuranosidases, α-glucuronidases and acetylxylan esterases) and carbohydrate-active auxiliary proteins, such as AA9 lytic polysaccharide monooxygenase, AA1 laccase and AA2 versatile peroxidase. Extracellular oxalate decarboxylase was also detected in both fungal species, exclusively in media containing sugarcane bagasse. Interestingly, intracellular AA6 quinone oxidoreductases were also exclusively produced under sugarcane bagasse induction in both fungi. These enzymes promote quinone redox cycling, which is used to produce Fenton’s reagents by lignocellulose-decay fungi. Hitherto undiscovered hypothetical proteins that are predicted in lignocellulose-decay fungi genomes appeared in high relative abundance in the cultures containing sugarcane bagasse, which suggests undisclosed, new biochemical mechanisms that are used by lignocellulose-decay fungi to degrade sugarcane biomass. In general, lignocellulose-decay fungi produce a number of canonical hydrolases, as well as some newly observed enzymes, that are suitable for 〈em〉in vitro〈/em〉 biomass digestion in a biorefinery context.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Gyumin Son, Thi Thanh Hanh Nguyen, Byeongsu Park, Sohyung Kwak, Juhui Jin, Young-Min Kim, Young-Hwan Moon, Sunghee Park, Seong-Bo Kim, Doman Kim〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Transglycosylation is one of enzymatic methods to improve the physical and biochemical properties of various functional compounds. In this study, stevioside glucosides were synthesized using sucrose as a substrate, stevioside (Ste) as an acceptor, and dextransucrase from 〈em〉Leuconostoc mesenteroides〈/em〉 B-512 F/KM. The highest Ste conversion yield of 98% was obtained with 50 mg/mL Ste, 800 mM sucrose, and dextransucrase 4 U/mL at 28 °C for 6 h. The concentration of Ste was unchanged while of Ste-G1 was increased from 7.7 mM to 9.1 mM as the Ste acceptor reaction digest was treated with dextranase from 〈em〉Lipomyces starkeyi〈/em〉. Ste-G1 (13-〈em〉O〈/em〉-β-sophorosyl-19-〈em〉O〈/em〉-β-isomaltosyl-steviol), Ste-G2 (13-〈em〉O〈/em〉-(β-(1→6) glucosyl)-β-glucosylsophorosyl-19-〈em〉O〈/em〉-β-isomaltosyl-steviol), and Ste-G2′ (13-〈em〉O〈/em〉-β-sophorosyl-19-〈em〉O〈/em〉-β-isomaltotriosyl-steviol) were determined by NMR. These glucosylated Ste showed increased stabilities at pH 2, 60 °C for 48 h as compared to Ste. Ste-G1, Ste-G2, and Ste-G2′ inhibited the insoluble glucan synthesis from sucrose by mutansucrase from 〈em〉Streptococcus muntans〈/em〉 by the transfer of the glucosyl group of sucrose to Ste-G1, Ste-G2, and Ste-G2′. The relative water solubility of curcumin, pterostilbene or idebenone was increased by Ste or Ste glucosides treatment. Ste and Ste-G1 restored cell viability in RAW264.7 cells at concentrations up to 8 mg/mL and inhibited nitric oxide production in LPS-induced RAW264.7 cells with IC〈sub〉50〈/sub〉 of 3.29 and 1.87 mg/mL.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301504-ga1.jpg" width="339" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Stefan Simić, Sanja Jeremic, Lidija Djokic, Nataša Božić, Zoran Vujčić, Nikola Lončar, Ramsankar Senthamaraikannan, Ramesh Babu, Igor M. Opsenica, Jasmina Nikodinovic-Runic〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Biocatalytic oxidations mediated by laccases are gaining importance due to their versatility and beneficial environmental effects. In this study, the oxidation of 1,4-dihydropyridines has been performed using three different types of bacterial laccase-based catalysts: purified laccase from 〈em〉Bacillus licheniformis〈/em〉 ATCC 9945a (〈em〉Bli〈/em〉Lacc), 〈em〉Escherichia coli〈/em〉 whole cells expressing this laccase, and bacterial nanocellulose (BNC) supported 〈em〉Bli〈/em〉Lacc catalysts. The catalysts based on bacterial laccase were compared to the commercially available 〈em〉Trametes versicolor〈/em〉 laccase (〈em〉Tv〈/em〉Lacc). The oxidation product of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate was obtained within 7–24 h with good yields (70–99%) with all three biocatalysts. The substrate scope was examined with five additional 1,4-dihydropyridines, one of which was oxidized in high yield. Whole-cell biocatalyst was stable when stored for up to 1-month at 4 °C. In addition, evidence has been provided that multicopper oxidase CueO from the 〈em〉E. coli〈/em〉 expression host contributed to the oxidation efficiency of the whole-cell biocatalyst. The immobilized whole-cell biocatalyst showed satisfactory activity and retained 37% of its original activity after three biotransformation cycles.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301498-ga1.jpg" width="500" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Abi L. Anello, Leonardo Aguilera, Marcela Kurina-Sanz, Maximiliano Juri Ayub, María Laura Mascotti〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Aldo-keto reductases (AKRs) are nicotinamide-dependent enzymes that catalyze the transformation of aldehydes and ketones into alcohols. They are spread across all phyla, and those from microbial origin have proved to be highly robust and versatile biocatalysts. In this work, we have discovered and characterized a microbial AKR from the yeast 〈em〉Rhodotorula mucilaginosa〈/em〉 by combining genome-mining and expression assays. The new enzyme, named AKR3B4, was expressed by a simple protocol in very good amounts. It displays a selective substrate profile exclusively transforming aldehydes into alcohols. Also, AKR3B4 shows very good stability at medium temperatures, in a broad range of pH values and in the presence of green organic solvents. Conversion assays demonstrate it is an excellent biocatalyst to be used in the synthesis of aromatic alcohols, and also to produce furan-3-ylmethanol and the valuable sweetener xylitol. These results show that AKR3B4 displays attractive features so as to be used in chemoenzymatic processes.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Giovanni Ausanio, Valeria Califano, Aniello Costantini, Giuseppe Perretta, Antonio Aronne, Giovanni Piero Pepe, Filomena Sannino, Luciano R.M. Vicari〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉β-glucosidase (BG) plays a key role in determining the efficiency of the enzymatic complex cellulase for the degradation of cellulose into sugars. It hydrolyses the cellobiose, an inhibitor of the enzymatic complex. Therefore, the immobilization of BG is a great challenge for the industrial application of cellulases. Cellulases usually contains a BG amount insufficient to avoid inhibition by cellobiose. Here the BG was immobilized by matrix assisted pulsed laser evaporation (MAPLE) technique. The frozen matrix was composed of water, water/m-DOPA and water/m-DOPA/quinone. The effect of the excipients on the final conformation of the enzyme after the MAPLE processing was determined. The enzyme secondary structure was studied by FTIR analysis. The catalytic performances of the deposited films were tested in the cellobiose hydrolysis reaction. The results demonstrate that the presence of the oxidized form of m-DOPA, the O-quinone form, can protect the protein native structure, with the laser inducing little or no damage. In fact, only the samples deposited from this target preserved the secondary structure of the polypeptide chain and allowed a complete hydrolysis of cellobiose for four consecutive runs, showing a high operational stability of the biocatalyst.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301528-ga1.jpg" width="390" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 20 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Wei Shen, Chen Yi, Shuai Qiu, Dan-Na Wang, Ya-Jun Wang, Yu-Guo Zheng〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉〈em〉t〈/em〉-Butyl 6-cyano-(3〈em〉R〈/em〉,5〈em〉R〈/em〉)-dihydroxyhexanoate ((3〈em〉R〈/em〉,5〈em〉R〈/em〉)-〈strong〉2〈/strong〉) is an important building-block of atorvastatin. In our previous work, a variant 〈em〉Kl〈/em〉AKR-Y295W-W296 L (designated as M1) of 〈em〉Kluyveromyces lactis〈/em〉 aldo-keto reductase (wild type (WT) 〈em〉Kl〈/em〉AKR, M0) was developed, which possessed strict diastereoselectivity but moderate activity towards 〈em〉t〈/em〉-butyl 6-cyano-(5〈em〉R〈/em〉)-hydroxy-3-oxohexanoate ((5〈em〉R〈/em〉)-〈strong〉1〈/strong〉). To further improve its catalytic performance, semi-rational engineering of M1 in present work was performed, and the “best” varaint 〈em〉Kl〈/em〉AKR-Y295W-W296L-I125V-S30P-Q212R-I63W (M8) was developed. M8’s 〈em〉K〈/em〉〈sub〉m〈/sub〉〈sup〉B〈/sup〉 towards (5〈em〉R〈/em〉)-〈strong〉1〈/strong〉 was 2.02 mM, and the catalytic efficiency (〈em〉k〈/em〉〈sub〉cat〈/sub〉/〈em〉K〈/em〉〈sub〉m〈/sub〉〈sup〉B〈/sup〉) value was 36.31 s〈sup〉-1〈/sup〉  mM〈sup〉-1〈/sup〉, which was 1.9-fold higher than that of the parent M1. Compared with M1, the half-life 〈em〉t〈/em〉〈sub〉1/2〈/sub〉, 〈math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si4.svg"〉〈msubsup〉〈mtext〉 T〈/mtext〉〈mtext〉S50〈/mtext〉〈mn〉50〈/mn〉〈/msubsup〉〈/math〉 and 〈math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si3.svg"〉〈msubsup〉〈mtext〉T〈/mtext〉〈mtext〉P50〈/mtext〉〈mn〉50〈/mn〉〈/msubsup〉〈/math〉 of M8 were improved. Under the optimized conditions, (5〈em〉R〈/em〉)-〈strong〉1〈/strong〉 at load of up to 80 g L〈sup〉-1〈/sup〉 was completely reduced in 1.5 h by M8 along with 〈em〉Exiguobacterium sibiricum〈/em〉 glucose dehydrogenase (〈em〉Es〈/em〉GDH) for cofactor regeneration, producing (3〈em〉R〈/em〉,5〈em〉R〈/em〉)-〈strong〉2〈/strong〉 in 〈em〉de〈/em〉〈sub〉p〈/sub〉 〉 99.5% and space-time yields (STY) of 660.0 g L〈sup〉-1〈/sup〉 d〈sup〉-1〈/sup〉.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301516-ga1.jpg" width="301" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 17 August 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Kamila Wlizło, Jolanta Polak, Anna Jarosz-Wilkołazka, Rebecca Pogni, Elena Petricci〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Transformation of 2-amino-3-methoxybenzoic acid into novel and eco-friendly orange dye (N15) was performed using native and immobilised laccase (LAC) from 〈em〉Pleurotus ostreatus〈/em〉 strain. A several parameters affecting laccase-mediated transformation efficiency included the selection of pH, buffer, reaction temperature, substrate, and laccase concentration as well as the type of carrier and LAC storage conditions were evaluated. The optimal conditions for N15 dye synthesis were 40 mM sodium-tartrate buffer pH 5.5 containing 3 mM of the substrate, efficiently transformed by 2 U of free laccase per 1 mmol of the substrate. Laccase was immobilised on porous Purolite® carriers, which had never been tested as a support for oxidoreductases. Immobilised laccase, characterised by a high immobilisation yield, was obtained by adsorption of laccase on a porous acrylic carrier with octadecyl groups (C〈sub〉18〈/sub〉) incubated in optimum conditions of 40 mM phosphate buffer pH 7.0 containing 1 mg of laccase per 1 g of the carrier (wet mass). The immobilised LAC showed the highest storage stability for 21 days and higher thermostability at 40℃ and 60℃ in comparison to its native form. The N15 dye showed good dyeing properties towards natural fibres, and the dyed fibre demonstrated resistance to different physicochemical factors during use, which was confirmed by commercial quality tests. The N15 dye is a phenazine, i.e. a heterogenic compound containing amino-, methoxy-, and three carboxyl functional groups with the molecular weight of approximately 449.37 U.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S014102291930136X-ga1.jpg" width="301" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Hong Liu, Lingyan Dai, Fanyu Wang, Xin Li, Wei Liu, Bailing Pan, Chengtao Wang, Dongjie Zhang, Jingzhi Deng, Zhijiang Li〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉〈em〉Zygosaccharomyces rouxii〈/em〉 is a well-known salt-tolerant yeast. In our previous study, it was interesting that 〈em〉Z. rouxii〈/em〉 could produce higher levels of 4-hydroxy-2, 5-dimethyl-3(2 H)-furanone in 120 g/L D-fructose and 180 g/L NaCl involved YPD medium at 5 d. In order to explore the resistance and furanone production mechanisms of 〈em〉Z. rouxii〈/em〉 under D-fructose regulation, a comparative transcriptomics method in 〈em〉Z. rouxii〈/em〉 was to set to find differentially expressed genes, the physiological and biochemical indexes (growth and cell morphology, lipid peroxidation and relative electrical conductivity, the antioxidant enzymes activity), and the expression of oxidoreductase activity genes. The results indicated that a larger number of different expressed genes at transcriptome analysis, such as the series antioxidant enzymes were related to the resistance characteristics. Research had confirmed that the living cell numbers and cell areas of D-fructose regulation group were significantly lower than the controls at the initial stage, while those higher than of the controls at the late stage. During the fermentation period, the lipid peroxidation and the relative electrical conductivity of the yeast cell membrane were increased. And also the D-fructose regulation group present lower inhibition superoxide anion ability. The activity of CAT in the D-fructose regulation group was always higher than that of the control group. Only the activity of GSH-Px was found to be significantly increased at 1 d except for other enzymes activities. Most of the oxidoreductase activity genes, such as especially the GSH-Px gene under D-fructose regulation conditions were expressed at higher levels than those of control groups. Combining the levels of transcription and enzymes activity data, those could understand that exogenous D-fructose had a stress effect on 〈em〉Z. rouxii〈/em〉 at the early stage of culture. With the fermentation time progress, it was no longer a stressor substance for the 〈em〉Z. rouxii〈/em〉, and changed the nutrient to promote growth of 〈em〉Z. rouxii〈/em〉 in the later stages. During the whole process, GSH-Px was the main defense enzyme and CAT was the sustained defense enzyme. Therefore, the experimental results might provide effective mechanisms in 〈em〉Z. rouxii〈/em〉 for practical application of furanone production in the industry under exogenous D-fructose regulation.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Can Cui, Chao Guo, Hui Lin, Zhao-Yun Ding, Yan Liu, Zhong-Liu Wu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Styrene monooxygenases (SMOs) are two-component enzymes known to catalyze the epoxidation of styrene to (〈em〉S〈/em〉)-styrene oxide. In this work, we identified a new oxygenase component, named 〈em〉St〈/em〉StyA, from the genome of 〈em〉Streptomyces〈/em〉 sp. NRRL S-31. 〈em〉St〈/em〉StyA displayed complementary stereoselectivity to all of the known SMOs when coupled with a known reductase component (〈em〉Ps〈/em〉StyB), which made it the first natural SMO that produces (〈em〉R〈/em〉)-styrene oxide. Accordingly, a plasmid co-expressing 〈em〉St〈/em〉StyA and 〈em〉Ps〈/em〉StyB was constructed, which led to an artificial two-component SMO, named 〈em〉St〈/em〉StyA/B. When applied in the bio-epoxidation of nine aromatic alkenes, the enzyme showed activity toward five alkenes, and consistently displayed (〈em〉R〈/em〉)-selectivity. Excellent stereoselectivity was achieved for all five substrates with enantiomeric excesses ranging from 91% to 〉99%ee.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Tugce Akkas, Anastasia Zakharyuta, Alpay Taralp, Cleva W. Ow-Yang〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉In this study, we presented a new approach for immobilizing JBU (Jack bean urease), by producing urease cross-linked enzyme lyophilisates (CLELs). Through the use of bovine serum albumin (BSA), lyophilisation, cross-linking with dextran polyaldehyde (DPA), and optimizing cross-linker pH, the urease-CLELs produced show an increase in relative catalytic activity that is 1.47 times higher than that of free urease, while remaining stable up to temperatures of 85 °C. Urease-CLEL activity increases in direct proportion with the increasing BSA content due to the offered additional lysine (Lys) groups which are potential cross-linking points providing better immobilization and retention of JBU, while lyophilisation also enables stabilization by eliminating solvating water molecules and intra-molecular reactions that may block the cross-linking residues. Two most commonly used cross-linkers that are reacting with the available Lys groups, 〈em〉i.e.〈/em〉glutaraldehyde (GA) and bulkier alternative DPA, have been selected for the immobilization of urease. The catalytic activity increase with DPA suggests an improved access to the active site through hindering blockage, while the increase with alkaline pH of the cross-linkers indicates decreased buffer inhibition. The long lifetime (113% residual activity after 4 weeks), recyclability (132% residual activity after 10 cycles) and thermal stability (276% relative activity at 85 °C) of these urease-CLELs demonstrate that they are technologically attractive as green biocatalysts, while our immobilization approach offers an alternative to conventional methods for proteins that are difficult to immobilise.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301280-ga1.jpg" width="500" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Gomathi Manoharan, Thiagarajan Sairam, Rajesh Thangamani, Dhivya Ramakrishnan, Manish K.Tiwari, Jung-Kul Lee, Jeya Marimuthu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including 〈em〉Arbus precatorius〈/em〉, 〈em〉Bacopa monnieri,Citrus aurantifolia〈/em〉 and 〈em〉Datura metel〈/em〉 to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60–99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from 〈em〉Fusarium incarnatum〈/em〉 BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in 〈em〉E. coli〈/em〉 Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 10〈sup〉4〈/sup〉 s〈sup〉−1〈/sup〉 M〈sup〉-1〈/sup〉 with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.〈/p〉〈/div〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Mohammad Perwez, Jahirul Ahmed Mazumder, Meryam Sardar〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Cross-linked enzyme aggregate (CLEA) is a technology to overcome the limitation of enzymes for its application in chemical industries. The inability of repeated use of enzymes, stability and ease of separation from reaction mixture limits its applications. Here, magnetic combi-CLEA has been synthesised by adding amino-functionalized magnetic nanoparticles into pectinase ultra-clear (containing pectinases, xylanases and cellulases). Enzymes were precipitated on the surface of amino-functionalized magnetic nanoparticles with ethanol and cross-linked using glutaraldehyde. The structural characterization of magnetic combi-CLEA was studied by Scanning Electron Microscopy. Thermal stability was performed at 70 °C for pectinase and 80 °C for xylanase and cellulase respectively. Half-life (t〈sub〉1/2〈/sub〉) of the xylanase, cellulase and pectinase in free form remarkably enhance from 84.51, 29.36, and 25.29 min respectively to 533.07, 187.29 and 147.44 min in magnetic-combi CLEA respectively. Magnetic combi-CLEA can be efficiently reused till 12th cycle after which pectinase, xylanase and cellulase retain 86.45%, 90.3% and 88.62% activity respectively. Using this CLEA preparation bioethanol concentration increases to 1.82-fold as compared to free enzyme, when simultaneous saccharification and fermentation was performed using wheat straw as the substrate. Magnetic combi-CLEA can be used for a variety of industrial applications like food processing, textile industry and bioethanol production.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301279-ga1.jpg" width="240" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Yi Yan, Pu Jia, Yajun Bai, Tai-Ping Fan, Xiaohui Zheng, Yujie Cai〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Rosmarinic acid (RA), as a hydroxycinnamic acid ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid (3,4-DHPL), is a phenylpropanoid-derived plant natural product and has diverse biological activities. This work acts as a modular platform for microbial production using a two-cofactor (ATP and CoA) regeneration system to product RA based on a cell-free biosynthetic approach. Optimal activity of the reaction system was pH 8 and 30 °C. Total turnover number for ATP and CoA was 820.60 ± 28.60 and 444.50 ± 9.65, respectively. Based on the first hour data, the RA productivity reached 320.04 mg L〈sup〉−1〈/sup〉 h〈sup〉−1〈/sup〉 (0.889 mM L〈sup〉−1〈/sup〉 h〈sup〉−1〈/sup〉).〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Yosuke Masakari, Chiaki Hara, Yasuko Araki, Keiko Gomi, Kotaro Ito〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉FAD-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is an enzyme utilized industrially in glucose sensors. Previously, FAD-GDH isolated from 〈em〉Mucor prainii〈/em〉 (MpGDH) was demonstrated to have high substrate specificity for glucose. However, MpGDH displays poor thermostability and is inactivated after incubation at 45 °C for only 15 min, which prevents its use in industrial applications, especially in continuous glucose monitoring (CGM) systems. Therefore, in this study, a chimeric MpGDH (Mr144–297) was engineered from the glucose-specific MpGDH and the highly thermostable FAD-GDH obtained from 〈em〉Mucor〈/em〉 sp. RD056860 (MrdGDH). Mr144–297 demonstrated significantly higher heat resistance, with stability at even 55 °C. In addition, Mr144–297 maintained both high affinity and accurate substrate specificity for D-glucose. Furthermore, eight mutation sites that contributed to improved thermal stability and increased productivity in 〈em〉Escherichia coli〈/em〉 were identified. Collectively, chimerization of FAD-GDHs can be an effective method for the construction of an FAD-GDH with greater stability, and the chimeric FAD-GDH described herein could be adapted for use in continuous glucose monitoring sensors.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Topwe Milongwe Mwene-Mbeja, Amélie Dufour, Joanna Lecka, Brar Satinder Kaur, Céline Vaneeckhaute〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Enzymatic reactions refer to organic reactions catalyzed by enzymes. This review aims to enrich the documentation relative to enzymatic reactions occurring during the anaerobic degradation of residual organic substances with emphasis on the structures of organic compounds and reaction mechanisms. This allows to understand the displacement of electrons between electron-rich and electron-poor entities to form new bonds in products. The detailed mechanisms of enzymatic reactions relative to the production of biomethane have not yet been reviewed in the scientific literature. Hence, this review is novel and timely since it discusses the chemical behavior or reactivity of different functional groups, thereby allowing to better understand the enzymatic catalysis in the transformations of residual proteins, carbohydrates, and lipids into biomethane and fertilizers. Such understanding allows to improve the overall biomethanation efficiency in industrial applications.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Seung Soo Lee, Jaehyun Park, Yu Been Heo, Han Min Woo〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance the production of value-added chemicals in 〈em〉Corynebacterium glutamicum〈/em〉 as an industrial host. Here, we described the biotransformation of xylose to glycolate using engineered 〈em〉Corynebacterium glutamicum〈/em〉, a well-known industrial amino acid producer. A synthetic pathway involving heterologous D-tagatose 3-epimerase and L-fuculose kinase/aldolase reactions was introduced in 〈em〉C. glutamicum〈/em〉, resulting in 9.9 ± 0.01 g/L glycolate from 20 g/L xylose at a yield of 0.51 g/g (equal to 1.0 mol/mol). Additional glyoxylate reduction pathway developed by CRISPR-Cas12a recombineering has been introduced and attempted to increase the maximum theoretical molar yield of 2.0 (mol/mol). Due to the limitation of the CRISPR-Cas12a recombineering with TTTV PAM sites, advanced CRISPR-Cas systems were suggested for the next-round metabolic engineering for improving the glycolate yield to overcome the current genome-editing tool for metabolic engineering in 〈em〉C. glutamicum〈/em〉.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Carlos M. Verdasco-Martín, Carlos Corchado-Lopo, Roberto Fernández-Lafuente, Cristina Otero〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉 〈p〉Conjugated linolenic acid (CLA) has important health effects, and their phospholipids forms (PL) are advantageous vehicles of this bioactive agent. Acidolysis of soybean phosphatidylcholine (PC) with conjugated linolenic acid (CLA) catalyzed by Lecitase® Ultra immobilized on Duolite A658 was studied. This reaction is typically limited by hydrolysis, producing 60–90 % of lyso- and glycero-PC and yielding low the process efficiency. Drying the amphiphilic PC material was found the key factor for maximal diacylglycerol phosphatidylcholines (PC) production and different drying approaches were studied to maximize the formation of PC rich in CLA in a solvent free process.〈/p〉 〈p〉PC lyophilization for 24 h getting dry solid appearance (PC; 783 ± 11 mg water/Kg PC) or other standard protocols to reduce water content/activity of reaction medium, did not improve the reaction performance. However, adding 4 extra days to the second step of lyophilization at high vacuum (1 Pa) and moderate temperature (20 °C), followed by further PC dehydration with molecular sieves, drastically reduced the hydrolysis process by achieving a extensive PC dehydration (279 ± 4 mg water/Kg PC), obtaining for the first time 〉99% molar yield of diacyl-PC product. After 24 h of reaction, a diacyl-PC product with 72.3% CLA content was obtained. PC molecules containing two CLA were the major species formed.〈/p〉 〈/div〉

Description: 〈p〉Publication date: February 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 121〈/p〉 〈p〉Author(s): Yuka Sasaki, Ryosuke Mitsui, Ryosuke Yamada, Hiroyasu Ogino〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Various recombinant proteins can be produced by the yeast 〈em〉Saccharomyces cerevisiae〈/em〉 cell factories; therefore, efficient recombinant protein production techniques are desirable. In this study, to establish an efficient recombinant protein production technique in 〈em〉S. cerevisiae〈/em〉, the secretory production of recombinant protein endoglucanase II (TrEG) was tested. We developed 2 novel methods for TrEG production via clustered regularly interspaced short palindromic repeat (CRISPR) -δ-integration as well as multiple promoter shuffling, which involved the pre-breakdown of the δ-sequence by the CRISPR system and subsequent δ-integration as well as the conjugation of 〈em〉TrEG〈/em〉 with various promoters and subsequent δ-integration, respectively. Moreover, simultaneous use of the CRISPR-δ-integration and multiple promoter shuffling methods was also examined. The CRISPR-δ-integration method was effective for improvement of the integrated 〈em〉TrEG〈/em〉 copy number and its activity, and the multiple promoter shuffling method was also beneficial for enhancing the transcriptional level of 〈em〉TrEG〈/em〉 and its activity. Furthermore, simultaneous use of CRISPR-δ-integration and multiple promoter shuffling methods was the most useful. The carboxymethyl cellulase activity of the 〈em〉TrEG〈/em〉 expressing transformant YPH499/24CP constructed by the method reached 559 U/L, and it was 17.3-fold higher than that of the transformant constructed by the conventional YEp type vector. Overall, the simultaneous use of CRISPR-δ-integration and multiple promoter shuffling can be useful and easily applied for recombinant protein production.〈/p〉〈/div〉

Description: 〈p〉Publication date: February 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 121〈/p〉 〈p〉Author(s): Yao Chen, Hao Chu, Wei Liu, Wei Feng〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉3,4 Dihydroxy phenyl 〈span〉l〈/span〉-alanine (L-DOPA) is the gold standard Parkinson's disease therapy. A heme-dependent peroxidase (HDP) catalyzes the 〈em〉ortho-〈/em〉hydroxylation of 〈span〉l〈/span〉-tyrosine to 〈span〉l〈/span〉-DOPA using H〈sub〉2〈/sub〉O〈sub〉2〈/sub〉 as the co-substrate. 〈span〉d〈/span〉-amino acid oxidase (DAAO) catalyzes the oxidative deamination of 〈span〉d〈/span〉-amino acids (e.g. 〈span〉d〈/span〉-alanine), and H〈sub〉2〈/sub〉O〈sub〉2〈/sub〉 is evolved. However, both the enzymes DAAO and HDP can be inactivated by H〈sub〉2〈/sub〉O〈sub〉2〈/sub〉 during the catalysis. In situ generation and utilization of H〈sub〉2〈/sub〉O〈sub〉2〈/sub〉 can siginificatly reduce the inactivation by H〈sub〉2〈/sub〉O〈sub〉2〈/sub〉. HDP exists as a monomer and DAAO is a dimeric enzyme. Herein, the C-terminus of HDP was specifically ligated to the N-terminus of the DAAO subunit with native peptide through the 〈em〉in vivo〈/em〉 monomer-subunit splicing. In the splicing product HDP&DAAO, HDP is close to the DAAO subunit at a molecular distance, and the transfer of H〈sub〉2〈/sub〉O〈sub〉2〈/sub〉 from DAAO to HDP is facilitated. In addition, HDP&DAAO exhibited a higher stability than HDP. Kinetics analysis showed that both the substrates 〈span〉l〈/span〉-tyrosine and 〈span〉d〈/span〉-alanine obey the Michaelis–Menten kinetics. For the deamination of 〈span〉d〈/span〉-alanine, the catalytic efficiency of HDP&DAAO is 3.05 times that of DAAO. For the sybthesis of 〈span〉l〈/span〉-DOPA from 〈span〉l〈/span〉-tyrosine, the catalytic efficiency of HDP&DAAO is 1.58 times that of HDP. Furthermore, HDP&DAAO was encapsulated within a Znic-based coordination polymer (Zn-CP). The morphorogy of HDP&DAAO/Zn-CP can be regulated by the enzyme concentration, the catalytic efficiency of the conjugates was found to be dependent on the morphorogy. The conjugates HDP&DAAO/Zn-CP exhibited a higher catalytic efficiency than free HDP&DAAO.〈/p〉〈/div〉

Description: 〈p〉Publication date: February 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 121〈/p〉 〈p〉Author(s): Zohreh Nowroozi-Nejad, Bahram Bahramian, Saman Hosseinkhani〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉We investigated the mechanism of luciferase immobilization on a solid surface through therphtalaldehyde in order to understand the role of this linker on stability and activity of luciferase. Metal organic frameworks (MOFs) are capable supports for the stabilization of some proteins and biomolecules, therefore, in this context for the first time, we report a light emmiting enzyme immobillization on one of these supports and then outline chemical developments in this process. To this end, Fe-MIL-88(NH2) was used to immobilize native luciferase and therphtalaldehyde linker was attached to the framework as an activated group. Interestingly, high loading capacity was observed in both bonding mechanisms. Furthermore, thermal stability and kinetic properties were improved very much but, thermodynamic/kinetic results and bonding efficiency is much better in the modified-MOF than native MOF. It was found that Schiff base mechanism between aldehyde as an active functional group in MOF and amine groups of enzyme led to the formation of imine bond. The surface chemical structure and morphologies of Fe-MIL-88(NH2) were characterized by FTIR, X-ray diffraction, Brunauer–Emmett–Teller (BET), FE-SEM, and TEM. In conclusion, using Fe-MIL-88(NH2) as a support to immobilize luciferase established simple immobilization process of luciferase with and without linker and it improved the remaining activity significantly.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022918304009-ga1.jpg" width="500" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 25 March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Zhiwei Deng, Zichao Wang, Pan Zhang, Pinghui Xia, Kedong Ma, Degang Zhang, Lin Wang, Yusuo Yang, Yuejing Wang, Shipeng Chen, Shilong Deng〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The effects of divalent copper (Cu(II)) on microbial community, enzymatic activity and functional genes in a sequencing batch reactor (SBR) at tetracycline (TC) stress were investigated. The enzymatic activity and functional genes abundance associated with nitrification and denitrification at a 20 mg L〈sup〉-1〈/sup〉 TC stress were higher than those at a mixtures stress of 20 mg L〈sup〉-1〈/sup〉 TC and 10 mg L〈sup〉-1〈/sup〉 Cu(II), while they were lower than those at a mixtures stress of 20 mg L〈sup〉-1〈/sup〉 TC and 40 mg L〈sup〉-1〈/sup〉 Cu(II). Compared to lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) production at a 20 mg L〈sup〉-1〈/sup〉 TC stress, they were lower at the TC stress with 10 mg L〈sup〉-1〈/sup〉 Cu(II), while they were higher at the TC stress with 40 mg L〈sup〉-1〈/sup〉 Cu(II). The incremental Cu(II) concentration at a 20 mg L〈sup〉-1〈/sup〉 TC stress could not change the result that the sensitivity of denitrifying enzymatic activity to TC was higher than nitrifying enzymatic activity. Compared to the relative abundance of nitrifers and denitrifers at a 20 mg L〈sup〉-1〈/sup〉 TC stress, the 10 mg L〈sup〉-1〈/sup〉 Cu(II) addition resulted in their increase, while they decreased as the 40 mg L〈sup〉-1〈/sup〉 Cu(II) addition. The relative abundance of genera 〈em〉Pseudomonas, Rivibacter〈/em〉 and 〈em〉Nitrobacter〈/em〉 at the stress of Cu(II) and TC were higher than those at TC stress, suggested they had an ability to resist the stress of Cu(II) and TC.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 28 March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Mingyue Piao, Donglei Zou, Xianghao Ren, Song Gao, Chuanyu Qin, Yunxian Piao〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉We developed a high efficiency bisphenol A (BPA) treating system in a fluidized bed reactor (FBR) using stabilized laccase (Lac) in mesoporous silica (SER/Lac). The SER/Lac, prepared by cross linking of each enzymes inside the porous silica using glutaraldehyde, presented improved stability than the free Lac over various pH and temperatures with shaking. When the SER/Lac was used for BPA biotransformation in a batch reaction, higher efficiency was achieved than the free or adsorbed Lac (ADS/Lac). Also, the SER/Lac presented better reusability compared to ADS/Lac, and it could be reused for three times without decrease of biotransformation efficiency, and half of it was remained after ten times use. Due to great stability and robustness property, SER/Lac was successfully applied for high efficiency and continuous BPA treatment in FBR with better performance than the batch reaction.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉 〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022918302345-ga1.jpg" width="301" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉 〈p〉Biotransformation of BPA in FBR〈/p〉 〈/div〉

Description: 〈p〉Publication date: February 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 121〈/p〉 〈p〉Author(s): Weixin Zhao, Guocheng Du, Song Liu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Self-assembling amphipathic peptides (SAPs) have been used as stabilization tags to improve enzyme stability but do not function uniformly well with all target enzymes. Here, the key factors involved in SAPs stabilization were identified as the SAP length and linker length and flexibility, using S1 (AEAEAKAK)〈sub〉2〈/sub〉 as an originated SAP and polygalacturonate lyase (PGL) as model protein. Biochemical analysis demonstrated that SAPs could induce loose protein oligomerization via intermolecular hydrophobic interactions. Based on this mechanism, a comprehensive protein stabilization strategy was proposed, in which a library of stabilizing tags through random combination of different SAPs and linker peptides was developed to design the fusion composition while the sodium chloride (NaCl) was used to enhance the intermolecular hydrophobic interactions. By using the strategy, the PGL, lipoxygenase (LOX) and L-asparaginase exhibited 33.25-, 17.55- and 15.6-fold increases, respectively, in the 〈em〉t〈/em〉〈sub〉1/2〈/sub〉 value relative to that of the corresponding wild-type enzyme. The SAP library therefore shows great application potential in stability enhancement of enzymes/proteins.〈/p〉〈/div〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): Shujin Pu, Xuan Zhang, Chengli Yang, Sidra Naseer, Xutong Zhang, Jie Ouyang, Dali Li, Junfang Yang〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Metal-organic framework (MOF) has generated a lot of research interests for it can be employed as immobilization material for enzyme. There are many small molecules in enzyme solution during the extraction process, such as NaCl. It is important to study the effects of the small molecules on MOFs. Here we report a facile and efficient method to encapsulate (〈em〉R〈/em〉)-1-phenylethanol dehydrogenase ((〈em〉R〈/em〉)-PEDH) into zeolitic imidazolate framework-8 (ZIF-8). In this work, the effects of NaCl on shape of ZIF-8 and enzyme encapsulation have been investigated. The scanning electron microscope (SEM) results showed that 0.1 M NaCl affect the morphology of ZIF-8 while the crystal structure was not changed analyzed by X-ray diffraction (XRD), and 0.1 M NaCl consisted in the encapsulation system of (〈em〉R〈/em〉)-PEDH@ZIF-8 enhanced the activity up to 2.5 folds than no NaCl consisted during prepared (〈em〉R〈/em〉)-PEDH@ZIF-8. The (〈em〉R〈/em〉)-PEDH@ZIF-8 (prepared with 0.1 M NaCl) enhanced the storage stability and resist ability against trypsin, and the (〈em〉R〈/em〉)-PEDH@ZIF-8 has been successfully reused.〈/p〉〈/div〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): Samkelo Malgas, Brett I. Pletschke〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Xylan, the most abundant hemicellulose in lignocellulosic biomass, requires a consortium of xylanolytic enzymes to achieve its complete de-polymerisation. As global interest in using xylan-containing lignocellulosic feedstocks for biofuel production increases, an accompanying knowledge on how to efficiently depolymerise these feedstocks into fermentable sugars is required. Since it has been observed that the same enzyme [i.e. an enzyme with the same EC (Enzyme Commission) classification] from different GH families can display different substrate specificities and properties, we evaluated GH10 (XT6) and 11 (Xyn2A) xylanase performance alone, and in combination, during xylan depolymerisation. Synergistic enhancement with respect to reducing sugar release was observed when Xyn2A at 75% loading was supplemented with 25% loading of XT6 for both beechwood glucuronoxylan (1.14-fold improvement) and wheat arabinoxylan (1.1-fold improvement) degradation. Following this, the optimised xylanase mixture was dosed with an oligosaccharide reducing-end xylanase (Rex8A) from either 〈em〉Bifidobacterium adolescentis〈/em〉 or 〈em〉Bacillus halodurans〈/em〉 for further synergistic enhancement. Dosing 75% of the xylanase mixture (Xyn2A:XT6 at 75:25%) with 25% loading of Rex8A led to an enhancement of reducing sugar (up to an 1.1-fold improvement) and xylose release (up to an 1.5-fold improvement); however, this effect was both xylan and Rex8A specific. Using thin layer chromatography, synergism appeared to be a result of the GH10 and 11 xylanases liberating xylo-oligomers that are preferred substrates of the processive Rex8As. Rex8As then hydrolysed xylo-oligomers to xylose - and xylobiose which was the preferred substrate for xylosidase, SXA. This likely explains why there was a significant improvement in xylose release in the presence of Rex8As. Here, it was shown that Rex8As are key enzymes in the efficient saccharification of hetero-xylan into xylose, a major component of lignocellulosic substrates.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022918304502-ga1.jpg" width="368" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: February 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 121〈/p〉 〈p〉Author(s): 〈/p〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): Xueqiao Xie, Peng Luo, Juan Han, Tong Chen, Yun Wang, Yunfeng Cai, Qian Liu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉In the present study, a novel and efficient immobilization for horseradish peroxidase (HRP) had been developed by using 6-arm magnetic composite microsphere (Fe〈sub〉3〈/sub〉O〈sub〉4〈/sub〉@PAA-6-arm-PEG-NH〈sub〉2〈/sub〉) containing 6-arm polyethylene glycol (6-arm-PEG-NH〈sub〉2〈/sub〉) and Fe〈sub〉3〈/sub〉O〈sub〉4〈/sub〉. The morphology and chemical properties of Fe〈sub〉3〈/sub〉O〈sub〉4〈/sub〉@PAA-6-arm-PEG-NH〈sub〉2〈/sub〉 were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectra (FTIR), X-ray powder diffraction (XRD), vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA). The immobilization conditions and loading capacity of the carrier toward HRP were also investigated. According to the results, the optimum immobilization conditions were as follows: glutaraldehyde concentration of 0.10 mol L〈sup〉−1〈/sup〉, the carrier concentration of 7 mg L〈sup〉−1〈/sup〉, the temperature of 35 °C and immobilization time of 180 min. The loading capacity of the immobilized HRP arrived to 139.82 mg g〈sup〉−1〈/sup〉 (RSD = 0.87%). Compared with free HRP, the stability of the immobilized HRP had been enhanced in a wide range of temperature and pH. The activity of the immobilized HRP retained 71.05% of its initial activity after 60 days storage. It was also found that the activity of the immobilized HRP retained 61.06% of its initial activity after eight times of successive reuse. The immobilized HRP could hydrolyze phenol to 94.4% within 10 min. It proved that the immobilized HRP could improve the performance of HRP, which had a good prospect in biological application.〈/p〉〈/div〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): Seyyed Soheil Rahmatabadi, Issa Sadeghian, Younes Ghasemi, Amirhossein Sakhteman, Shiva Hemmati〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The enzyme phenylalanine ammonia lyase (PAL) is of special importance for the treatment of phenylketonuria patients. The aim of this study was to find a stable recombinant PAL with suitable kinetic properties among all natural PAL producing species using 〈em〉in silico〈/em〉 and experimental approaches. To find such a stable PAL among 481 natural isoforms, 48,000 of 3-D models were predicted using the Modeller 9.10 program and evaluated by Ramachandran plot. Correlation analysis between Ramachandran plot and the energy of different thermodynamic components indicated that this plot could be an appropriate tool to predict protein stability. Hence, PAL6 from 〈em〉Lotus japonicus〈/em〉 (LjPAL6) was selected as a stable isoform. Molecular dynamic (MD) simulation for 50 ns and docking has been conducted for LjPAL6-phenylalanine complex. The best PAL-phenylalanine frame was selected by re-docking with 〈span〉l〈/span〉-phenylalanine (L-Phe) and root-mean-square deviation (RMSD) value. MD simulation showed that the complex has a good stability, depicted by the low RMSD value, binding free energy and hydrogen bindings. Docking results showed that LjPAL6 has a high affinity toward 〈span〉l〈/span〉-Phe according to the low level of binding free energy. By overexpressing 〈em〉Ljpal6〈/em〉 in 〈em〉E. coli〈/em〉 BL21, a total of 33.5 mg/l of protein was obtained, which has been increased to 83.7 mg/l via the optimization of LjPAL6 production using response surface methodology. The optimal pH and temperature were 8.5 and 50 °C, respectively. LjPAL6 showed a specific activity of 42 nkat/mg protein, with 〈em〉K〈/em〉〈sub〉m〈/sub〉, 〈em〉K〈/em〉〈sub〉cat〈/sub〉 and 〈em〉K〈/em〉〈sub〉cat〈/sub〉/〈em〉K〈/em〉〈sub〉m〈/sub〉 values of 0.483 mM, 7 S〈sup〉−1〈/sup〉 and 14.5 S〈sup〉−1〈/sup〉 mM〈sup〉−1〈/sup〉 for 〈span〉l〈/span〉-phe, respectively. In conclusion, finding models with the most reasonable stereo-chemical quality and lowest numbers of steric clashes would result in easier folding. Hence, 〈em〉in silico〈/em〉 analyses of bulk data from natural origin will lead one to find an optimal model for 〈em〉in vitro〈/em〉 studies and drug design.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022918303508-ga1.jpg" width="384" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): 〈/p〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): Valentina Perna, Andreas Baum, Heidi A. Ernst, Jane W. Agger, Anne S. Meyer〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyze the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Laccase activity is commonly determined by monitoring spectrophotometric changes (absorbance) of the product or substrate during the enzymatic reaction. Fourier Transform Infrared Spectroscopy (FTIR) is a fast and versatile technique where spectral evolution profiling, i.e. assessment of the spectral changes of both substrate and products during enzymatic conversion in real time, can be used to assess enzymatic activity when combined with multivariate data analysis. We employed FTIR to monitor enzymatic oxidation of monolignols (sinapyl, coniferyl and 〈em〉p〈/em〉-coumaryl alcohol), sinapic acid, and sinapic aldehyde by four different laccases: three fungal laccases from 〈em〉Trametes versicolor〈/em〉, 〈em〉Trametes villosa〈/em〉 and 〈em〉Ganoderma lucidum〈/em〉, respectively, and one bacterial laccase from 〈em〉Meiothermus ruber〈/em〉. By coupling the FTIR measurements with Parallel Factor Analysis (PARAFAC) we established a quantitative assay for assessing laccase activity. By combining PARAFAC modelling with Principal Component Analysis we show the usefulness of this technology as a multivariate tool able to compare and distinguish different laccase reaction patterns. We also demonstrate how the FTIR approach can be used to create a reference system for laccase activity comparison based on a relatively low number of measurements. Such a reference system has potential to function as a high-throughput method for comparing reaction pattern similarities and differences between laccases and hereby identify new and interesting enzyme candidates in large sampling pools.〈/p〉〈/div〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): Huahua He, Chao Zhai, Meng Mei, Yi Rao, Yao Liu, Fei Wang, Lixin Ma, Zhengbing Jiang, Guimin Zhang, Li Yi〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Porcine interferon-α (pIFN-α) could be used as the vaccine adjuvant to enhance the antiviral ability of porcine in swine industry. In here, a combinational strategy integrating codon optimization, multiple gene insertion, strong AOX1 promoter, and efficient secretion signal sequence was developed to obtain high-level secreted pIFN-α in 〈em〉Pichia pastoris〈/em〉 GS115. The codon optimized pIFN-α shared 76% sequence identity with the original pIFN-α, which was inserted into the 〈em〉P. pastoris〈/em〉 genome under AOX1〈sub〉d1-2x201〈/sub〉 promoter and MF4I secretion sequence. Our results showed positive correlation between the mRNA and secreted protein levels with the copy numbers of genome-integrated pIFN-α gene in the recombinant 〈em〉P. pastoris〈/em〉 strains. The recombinant opt-pIFN-α-6C strain bearing six copies of pIFN-α expression cassette produced the highest extracellular secretion of pIFN-α of 3.2 ± 0.1 mg/mL in shake flask experiment, and 17.0 ± 0.8 mg/mL in a 5 L high-cell-density cultivation after methanol induction of 84 h. The antiviral activity of secreted pIFN-α from the high-cell-density cultivation was determined to be approximately 2.8 ± 0.9 × 10〈sup〉9〈/sup〉 IU/mL against the vesicular stomatitis virus (VSV) infected Madin-Darby bovine kidney (MDBK) cells. This strategy provided an efficient way to generate recombinant 〈em〉P. pastoris〈/em〉 strains in a non-antibiotics-selection manner, which might also give general guidance for the heterologous expression of other proteins in 〈em〉P. pastoris〈/em〉.〈/p〉〈/div〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): Harshini Pakalapati, Mohammad Asad Tariq, Senthil Kumar Arumugasamy〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Recently enzymatic catalysts have replaced organic and organometallic catalysts in the synthesis of bio-resorbable polymers. Enzymatic polymerization is considered as an alternative to conventional polymerization as they are less toxic, environmental friendly and can operate under mild conditions. In this research, the enzymatic ring-opening polymerization (e-ROP) of e-caprolactone (e-CL) using Candida Antartica Lipase B (CALB) as catalyst to produce the Polycaprolactone. Two modelling techniques namely response surface methodology (RSM) and artificial neural network (ANN) have been used in this work. RSM is used to optimize the parameters and to develop a model of the process. ANN is used to develop the model to predict the results obtained from the experiment. The parameters involved are time, reaction temperature, mixing speed and enzyme-solvent ratio. The experimental result is Polydispersity index (PDI) of the polymer. The experimental data obtained was adequately fitted into second-order polynomial models. Simulation was done using artificial neural network model developed with Mean absolute error (MAD) value of 1.65 in comparison with MAD value of 7.4 for RSM. The Regression value (R〈sup〉2〈/sup〉) values of RSM and ANN were found to be 0.96 and 0.93 respectively. The predictive models were validated experimentally and were found to be in agreement with the experimental values.〈/p〉〈/div〉

Description: 〈p〉Publication date: March 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 122〈/p〉 〈p〉Author(s): Gema Núñez-López, Azucena Herrera-González, Lázaro Hernández, Lorena Amaya-Delgado, Georgina Sandoval, Anne Gschaedler, Javier Arrizon, Magali Remaud-Simeon, Sandrine Morel〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Fructosylation can significantly improve the solubility, stability and bioactivity of phenolic compounds, increasing their health benefits. Levansucrase from 〈em〉Gluconacetobacter diazotrophicus〈/em〉 (LsdA, EC 2.4.1.10) was found to transfer the fructosyl unit of sucrose to different classes of phenolic compounds. Among the various acceptors tested, the isoflavone puerarin and the phenol coniferyl alcohol were the most efficiently fructosylated compounds, with conversion rates of 93% and 25.1%, respectively. In both cases, mono-, di-, and trifructosides were synthesized at a ratio of 37:14:1 and 32:8:1, respectively. Structural characterization of the puerarin mono-fructoside revealed that the enzyme transferred the fructosyl moiety of sucrose to the O6-position of the glucosyl unit of puerarin. The water solubility of fructosyl-β-(2→6)-puerarin was increased 23-fold, up to 16.2 g L〈sup〉−1〈/sup〉, while its antioxidant capacity was only decreased 1.25-fold compared with that of puerarin.〈/p〉〈/div〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Flor Sánchez-Alejandro, Maria Camilla Baratto, Riccardo Basosi, Olivia Graeve, Rafael Vazquez-Duhalt〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Versatile peroxidase (VP) from 〈em〉Bjerkandera adusta〈/em〉 is an enzyme able to oxidize bulky and high-redox substrates trough a Long-Range Electron Transfer (LRET) pathway. In this study, the introduction of radical-forming aromatic amino acids by chemical modification of the protein surface was performed, and the catalytic implications of these additional surface active-sites on the oxidation of 2,6-dimethylphenol, Mn〈sup〉2+〈/sup〉 and Remazol Brilliant Blue R (RBBR) were determined. These three different substrates are oxidized in different active-sites of enzyme molecule, of which the high redox RBBR the only one that is transformed by an external radical formed on the protein surface. Both catalytic constants k〈sub〉cat〈/sub〉 and K〈sub〉M〈/sub〉 were significantly affected by the chemical modifications. Tryptophan- and tyrosine-modified VP showed higher catalytic transformation than the unmodified enzyme for RBBR, while the Mn〈sup〉2+〈/sup〉 oxidation was significantly reduced by all chemical modifications. Electron Paramagnetic Resonance studies demonstrated the formation of additional protein-based radicals after the chemical modification with radical-forming amino acids. In addition, the catalytic rate of the LRET-mediated transformation showed a good correlation with the ionization energy of the additional amino acid on the protein surface.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 12 September 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Longbao Zhu, Jing Yang, Guoqiang Feng, Fei Ge, Wanzhen Li, Ping Song, Yugui Tao, Zhemin Zhou〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Phenylalanine aminomutase (〈em〉Pa〈/em〉PAM) from 〈em〉Pantoea agglomerans〈/em〉 is a member of the MIO (4-methylene-imidazol-5-one) family of enzymes, which isomerizes 〈em〉α〈/em〉-phenylalanine to 〈em〉β〈/em〉-phenylalanine, and could be used to synthesize unnatural 〈em〉β〈/em〉-arylalanine. However, the mechanism of isomerization reaction is not clear. To investigate the mechanism, the gene (〈em〉pam〈/em〉), which encodes 〈em〉Pa〈/em〉PAM, was first expressed in 〈em〉E.coli〈/em〉, and recombinant 〈em〉Pa〈/em〉PAM was prepared using affinity chromatography. Then, 〈sup〉15〈/sup〉N-(2〈em〉S〈/em〉)-〈em〉α〈/em〉-phenylalanine, (2〈em〉S〈/em〉)-(3-〈sup〉2〈/sup〉H〈sub〉2〈/sub〉)-〈em〉α〈/em〉-phenylalanine and (2〈em〉S〈/em〉,3〈em〉S〈/em〉)-[2,3-〈sup〉2〈/sup〉H〈sub〉2〈/sub〉]-〈em〉α〈/em〉-phenylalanine were used as substrates to analyze the mechanism of isomerization reaction. The results of MS and NMR showed that the isomerization reaction was performed through the intramolecular exchange of NH〈sub〉2〈/sub〉 with 〈em〉pro〈/em〉-3〈em〉R〈/em〉 hydrogen of 〈em〉α〈/em〉-phenylalanine. The 〈em〉Pa〈/em〉PAM shuttles the 〈em〉α〈/em〉-NH〈sub〉2〈/sub〉 of 〈em〉α〈/em〉-phenylalanine to 〈em〉β〈/em〉 site to replace the 〈em〉pro〈/em〉-3〈em〉R〈/em〉 hydrogen. Simultaneously, the 〈em〉pro〈/em〉-3〈em〉R〈/em〉 hydrogen is shifted to 〈em〉α〈/em〉 site to produce 〈em〉β〈/em〉-phenylalanine. Furthermore, a key residue, Phe at position 455 in the active site, was determined to control the exchange way using molecular docking and sequence alignment of MIO family enzymes. The results indicated that the key 455 Phe residue is involved in changing the binding orientation of the carboxyl group of the intermediate 〈em〉trans〈/em〉-cinnamic acid to control the NH〈sub〉2〈/sub〉-H pair exchange.〈/p〉〈/div〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Fei Xue, Qin Chen, Yulong Li, Eryan Liu, Dengxin Li〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Novel carboxyl-functionalized core-shell magnetic cellulose microspheres (MCM〈sub〉S〈/sub〉) were prepared by surface modification with 1,2,3,4-butanetetracarboxylic acid (BTCA) and then applied in the immobilization of lysozyme via covalent bonding. The successful preparation of particles has been verified by transmission electron microscopy (TEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), and Fourier-transform infrared spectroscopy (FTIR) techniques. The optimal temperature and pH of the immobilized lysozyme were shown to be respectively 40 °C and 7. The immobilized lysozyme exhibited excellent performances within wide pH and temperature ranges as well as the high storage and thermal stabilities compared to free lysozyme. The apparent kinetic characterization of immobilized lysozyme revealed that its 〈em〉K〈sub〉m〈/sub〉〈/em〉 value was 1.37 times higher than that of free lysozyme and that its 〈em〉Vmax〈/em〉 was slightly lower. The immobilized lysozyme demonstrated an acceptable reusability and showed 51.9±2.2% of activity after six cycles. This study demonstrated the application potential of BTCA-modified MCM〈sub〉S〈/sub〉 as an immobilized carrier for lysozyme.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉 〈p〉Immobilized lysozyme onto BTCA-modified core-shell MCM to enhance bio-catalytic stability and activities.〈/p〉 〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301632-ga1.jpg" width="262" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉 〈/div〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Muhammad Waheed Iqbal, Tahreem Riaz, Hinawi A.M. Hassanin, Dawei Ni, Imran Mahmood Khan, Abdur Rehman, Shahid Mahmood, Muhammad Adnan, Wanmeng Mu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉〈span〉d〈/span〉-Ribulose and 〈span〉l〈/span〉-fuculose are potentially valuable rare sugars useful for anticancer and antiviral drugs in the agriculture and medicine industries. These rare sugars are usually produced by chemical methods, which are generally expensive, complicated and do not meet the increasing demands. Furthermore, the isomerization of 〈span〉d〈/span〉-arabinose and 〈span〉l〈/span〉-fucose by〈span〉D〈/span〉〈span〉d〈/span〉-arabinose and 〈span〉l〈/span〉-fucose by 〈span〉d〈/span〉-arabinose isomerase from bacterial sources for the production of 〈span〉d〈/span〉-ribulose and 〈span〉l〈/span〉-fuculose have not yet become industrial due to the shortage of biocatalysts, resulting in poor yield and high cost of production. In this study, a thermostable 〈span〉d〈/span〉-ribulose- and 〈span〉l〈/span〉-fuculose producing 〈span〉d〈/span〉-arabinose isomerase from the bacterium 〈em〉Thermanaeromonas toyohensis〈/em〉 was characterized. The recombinant 〈span〉d〈/span〉-arabinose isomerase from 〈em〉T. toyohensis〈/em〉 (Thto-DaIase) was purified with a single band at 66 kDa using His-trap affinity chromatography. The native enzyme existed as a homotetramer with a molecular weight of 310 kDa, and the specific activities for both 〈span〉d〈/span〉-arabinose and 〈span〉l〈/span〉-fucose were observed to be 98.08 and 85.52 U mg〈sup〉−1〈/sup〉, respectively. The thermostable recombinant Thto-DaIase was activated when 1 mM Mn〈sup〉2+〈/sup〉 was added to the reactions at an optimum pH of 9.0 at 75 °C and showed approximately 50% activity for both 〈span〉d〈/span〉-arabinose and 〈span〉l〈/span〉-fucose at 75 °C after 10 h. The Michaelis-Menten constant (〈em〉K〈sub〉m〈/sub〉〈/em〉), the turnover number (〈em〉k〈sub〉cat〈/sub〉〈/em〉) and catalytic efficiency (〈em〉k〈sub〉cat〈/sub〉〈/em〉/〈em〉K〈sub〉m〈/sub〉〈/em〉) for 〈span〉d〈/span〉-arabinose/〈span〉l〈/span〉-fucose were 111/81.24 mM, 18,466/10,688 min〈sup〉−1〈/sup〉, and 166/132 mM〈sup〉−1〈/sup〉  min〈sup〉−1〈/sup〉, respectively. When the reaction reached to equilibrium, the conversion rates of 〈span〉d〈/span〉-ribulose from 〈span〉d〈/span〉-arabinose and 〈span〉l〈/span〉-fuculose from 〈span〉l〈/span〉-fucose were almost 27% (21 g L〈sup〉−1〈/sup〉) and 24.88% (19.92 g L〈sup〉−1〈/sup〉) from 80 g L〈sup〉−1〈/sup〉 of 〈span〉d〈/span〉-arabinose and 〈span〉l〈/span〉-fucose, respectively.〈/p〉〈/div〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Sha Xu, Yu Zhang, Youran Li, Xiaole Xia, Jingwen Zhou, Guiyang Shi〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉L-tyrosine is an amino acid that has been widely used in the food, agriculture and pharmaceutical industries. In order to screen a tyrosine phenol-lyase (TPL) with excellent catalytic performance for L-tyrosine production, TPL genes from 〈em〉Citrobacter freundii〈/em〉 (CfTPL)〈em〉, Erwinia herbicola〈/em〉 (EhTPL) and 〈em〉Rhodobacter capsulatus〈/em〉 (TutA) were codon-optimized and overexpressed in 〈em〉Escherichia coli.〈/em〉 The results showed that EhTPL had the highest whole cell catalysis activity and tyrosine yield (3-fold that of CfTPL). The results of RT-qPCR and a stability analysis also revealed that EhTPL had a higher transcriptional level in whole cell catalysis, while CfTPL possessed greater stability. Conditions for the production by whole cell transformation were optimized in terms of reaction conditions and fed-batch strategy. Finally, the maximum production was obtained with a titer of 48.5 g·L〈sup〉−1〈/sup〉 by intermittent feeding with a conversion ratio of 75%.〈/p〉〈/div〉

Description: 〈p〉Publication date: February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 133〈/p〉 〈p〉Author(s): Tong-Yi Dou, Jing Chen, Chenglin Liu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉〈em〉Cellulosimicrobium cellulans〈/em〉, which is type species of the genus 〈em〉Cellulosimicrobium〈/em〉, produces xylanase predominant nanoscale multienzyme complexes, i.e., xylanosomes, when grown on water-insoluble polysaccharides. Here, we report on the isolation of similar multienzyme complexes (MECs) produced by two other species in genus 〈em〉Cellulosimicrobium〈/em〉 (〈em〉Cellulosimicrobium funkei〈/em〉 and 〈em〉Cellulosimicrobium terreum〈/em〉). Functional studies and subunit structure identifications using genomic sequencing and proteomic techniques were also performed. When compared with the xylanosomes produced by 〈em〉C. cellulans〈/em〉 F16, the isolated MECs showed a larger particle size and shared at least three conserved multidomain proteins. In addition, they also exhibited different enzymatic activities and subunit compositions, which indicates diverse capability and strategies in degrading hemicelluloses.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 15 October 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Sunirmal Sheet, Yesupatham Sathishkumar, Kuntal Ghosh, Mi-Sook Choi, Kwan Seob Shim, Yang Soo Lee〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The present study investigated the influence of low-shear modeled microgravity (LSMMG) conditions on Listeria monocytogenes stress response (heat, cold, and acid), membrane fatty acid composition, and virulence potential as well as stress-/virulence-associated gene expression. The results showed that LSMMG-cultivated cells had lower survival rate and lower D-values under heat and acid stress conditions compared to cells grown under normal gravity (NG). Interestingly, the cold resistance was elevated in cells cultivated under LSMMG conditions when compared to NG conditions. A higher amount of anteiso-branched chain fatty acids and lower ratio of iso/anteiso were observed in LSMMG cultured cells, which would contribute to increased membrane fluidity. Under LSMMG conditions, upregulated expression of cold stress-related genes (cspA, cspB, and cspD) was noticed but no change in expression was observed for heat (dnaK, groES, clpC, clpP, and clpE) and acid stress-related genes (sigB). The LSMMG-grown cells showed inferior virulence capacity in terms of infection, cell cycle arrest, and apoptosis induction in Caco-2 cells compared to those grown under NG conditions. Approximately 3.65, 2.13, 4.02, and 2.65-fold downregulation of prfA, hly, inlA, and bsh genes, respectively, in LSMMG-cultured cells might be the reason for reduced virulence. In conclusion, these findings suggest that growth under LSMMG conditions stimulates alterations in L. monocytogenes stress/virulence response, perhaps due to changes in lipid composition and related genes expression.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Shuhong Mao, Xiaotao Cheng, Zhangliang Zhu, Ying Chen, Chao Li, Menglu Zhu, Xin Liu, Fuping Lu, Hui-Min Qin〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉D-allulose has received increasing attention due to its excellent physiological properties and commercial potential. The D-allulose 3-epimerase from 〈em〉Rhodopirellula baltica〈/em〉 (RbDAEase) catalyzes the conversion of D-fructose to D-allulose. However, its poor thermostability has hampered its industrial application. Site-directed mutagenesis based on homologous structures in which the residuals on high flexible regions were substituted according to B-factors analysis, is an effective way to improve the thermostability and robustness of an enzyme. RbDAEase showed substrate specificity toward D-allulose with a 〈em〉K〈/em〉〈sub〉m〈/sub〉 of 58.57 mM and 〈em〉k〈/em〉〈sub〉cat〈/sub〉 of 1849.43 min〈sup〉−1〈/sup〉. It showed a melting temperature (〈em〉T〈/em〉〈sub〉m〈/sub〉) of 45.7 °C and half-life (〈em〉t〈/em〉〈sub〉1/2〈/sub〉) of 52.3 min at pH 8.0, 60 °C with 1 mM Mn〈sup〉2+〈/sup〉. The Site-directed mutation L144 F strengthened the thermostability to a Δ〈em〉t〈/em〉〈sub〉1/2〈/sub〉 of 50.4 min, Δ〈em〉T〈/em〉m of 12.6 °C, and Δ〈em〉T〈/em〉〈sub〉50〈/sub〉〈sup〉60〈/sup〉 of 22 °C. It also improved the conversion rate to 28.6%. Structural analysis reveals that a new hydrophobic interaction was formed by the mutation. Thus, site-directed mutagenesis based on B-factors analysis would be an efficient strategy to enhance the thermostability of designed ketose 3-epimerases.〈/p〉〈/div〉

Description: 〈p〉Publication date: February 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 133〈/p〉 〈p〉Author(s): Way-Rong Lin, I-Son Ng〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Microalgae biorefinery is an alternative, sustainable and promising trend to solve the problem of fossil oil depletion and carbon dioxide emission. However, considering the innate limitation of cell growth and oil content in microalgae, to accelerate metabolic balance by CRISPR/Cas9 system is attractive. At first, plasmid based from 〈em〉Agrobacterium tumefaciens〈/em〉 and a fragment of mGFP was transformed into 〈em〉Chlorella sorokiniana〈/em〉 and 〈em〉Chlorella vulgaris〈/em〉 FSP-E by electroporation, respectively. Selected colonies were tested by spectrophotometer and inverted fluorescence microscopy (IFM), and an increase of fluorescent was observed by 67% compared with that in wild type, which proved the 〈em〉Agrobacterium〈/em〉-mediated plasmid is suitable for gene insertion in 〈em〉Chlorella〈/em〉 species. Consequently, plasmid with similar structure as mentioned previously containing fragment of Cas9 with sgRNA designed on omega-3 fatty acid desaturase (〈em〉fad〈/em〉3) gene was constructed and showed a higher accumulation of lipid content by 46% (〈em〉w/w〈/em〉) in 〈em〉C. vulgaris〈/em〉 FSP-E. This is first-time to use CRISPR/Cas9 based technology for gene manipulation in 〈em〉Chlorella〈/em〉.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301966-ga1.jpg" width="305" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 27 December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Young-Wook Chin, Se-Won Jang, Hee-Soon Shin, Tae-Wan Kim, Sun-Ki Kim, Cheon-Seok Park, Dong-Ho Seo〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Amylosucrase (ASase) has great industrial potential owing to its multifunctional activities, including transglucosylation, polymerization, and isomerization. In the present study, the properties of 〈em〉Deinococcus geothermalis〈/em〉 ASase (DGAS) expressed in 〈em〉Corynebacterium glutamicum〈/em〉 (cDGAS) and purified via Ni-NTA affinity chromatography were compared to those of DGAS expressed in 〈em〉Escherichia coli〈/em〉 (eDGAS). The pH profile of cDGAS was similar to that of eDGAS, whereas the temperature profile of cDGAS was lower than that of eDGAS. The melting temperature of both enzymes did not differ significantly. Interestingly, polymerization activity was slightly lower in cDGAS than in eDGAS, whereas luteolin (an acceptor molecule) transglucosylation activity in cDGAS was 10% higher than that in eDGAS. Analysis of protein secondary structure via circular dichroism spectroscopy revealed that cDGAS had a lower strand/helix ratio than eDGAS. The present results indicate that cDGAS is of greater industrial significance than eDGAS.〈/p〉〈/div〉

Description: 〈p〉Publication date: November 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 130〈/p〉 〈p〉Author(s): Hyunjun Ko, Jung-Hoon Bae, Bong Hyun Sung, Mi-Jin Kim, Hyun-Ju Park, Jung-Hoon Sohn〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉A high yielding and straightforward production system of fructooligosaccharide (FOS) was developed for industrial production of prebiotics. To increase conversion yield of FOS from sucrose, recombinant yeast secreting inulosucrase from 〈em〉Lactobacillus reuteri〈/em〉 (LrInu) were constructed. Efficient secretion of LrInu was achieved by truncation of both amino- and carboxy-termini (LrInuΔNC) and by introducing an optimal secretion signal. The recombinant yeast produced 220 U/mL of recombinant LrInuΔNC into culture medium during fed-batch fermentation. By direct fermentation of recombinant yeast in medium containing sucrose, 128.4 g/L of FOS was produced with 85.6% conversion yield from 300 g/L sucrose, and the highest titer was 152.6 g/L from 400 g/L sucrose. The degree of polymerization of generated FOS was 2–20 indicating medium chain (mcFOS) range. This is the first report of industrially applicable production of mcFOS by recombinant yeast secreting bacterial inulosucrase.〈/p〉〈/div〉

Description: 〈p〉Publication date: November 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 130〈/p〉 〈p〉Author(s): Baolong Wang, Darren Nesbeth, Eli Keshavarz-Moore〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉This study investigates how sorbitol/methanol mixed induction affects fermentation performance, dewatering characteristics of cells during harvesting and the profile of host cell proteins (HCP) in the process fluid when producing the target recombinant protein aprotinin. Compared to standard methanol induction, sorbitol/methanol (1:1, C-mol/C-mol) mixed induction improved cellular viability from 92.8 ± 0.3% to 97.7 ± 0.1% although resulted in a reduced product yield from 1.65 ± 0.03 g L〈sup〉−1〈/sup〉 to 1.12 ± 0.07 g L〈sup〉−1〈/sup〉. On the other hand, average oxygen consumption rate (OUR) dropped from 241.4 ± 21.3 mmol L〈sup〉−1〈/sup〉 h〈sup〉−1〈/sup〉 to 145.5 ± 6.7 mmol L〈sup〉−1〈/sup〉 h〈sup〉−1〈/sup〉. Cell diameter decreased over time in the mixed induction, resulting in a D〈sub〉50〈/sub〉 value of 3.14 μm at harvest compared to 3.85 μm with methanol. The reduction in cell size enhanced the maximum dewatering efficiency from 78.1 ± 3.9% to 84.5 ± 3.3% as evaluated by using an established ultra scale-down methodology that models pilot and industrial scale disc stack centrifugation. Seventy host cell proteins (HCPs) were identified in clarified supernatant when using sorbitol/methanol mixed induction regimen. The total number of HCPs identified with standard methanol induction was nearly one hundred. The downstream process advantage of the mixed induction lies in improved product purity by reducing both cell mortality and level of released whole cell proteins. This needs to be balanced and optimised against the observed reduction in product yield during fermentation.〈/p〉〈/div〉

Description: 〈p〉Publication date: November 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 130〈/p〉 〈p〉Author(s): Xiao-Jian Zhang, Hao-Hao Fan, Nan Liu, Xin-Xin Wang, Feng Cheng, Zhi-Qiang Liu, Yu-Guo Zheng〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Transaminases (TAs, EC 2.6.1.X) are a class of promising catalysts for the synthesis of chiral amines. The addition of exogenous expensive cofactor, pyridoxal 5′-phosphate (PLP) leads to the high cost and complicated reaction composition and thus limits the industrial implementation of TAs. In this study, a novel self-sufficient biocatalyst was developed based on transaminase and PLP covalent co-immobilization with the activity recovery of 83.6% and the specific activity of 343.0 U/g catalyst. The self-sufficient BgTA biocatalyst was employed in the continuous biosynthesis of (〈em〉R〈/em〉)-sitagliptin in a recirculating packed bed reactor (RPBR) for 700 batches reaction without extra addition of PLP. The yield and 〈em〉e.e.〈/em〉 of (〈em〉R〈/em〉)-sitagliptin for each batch were above 90% and 99% respectively. The space-time yield was 40.0 g/(L·h) which was higher than ever reported. This is the first report about the covalent co-immobilization of TA and PLP with high activity and stability, and our work also contributed to the economic flexibility of chiral amine pharmaceutical intermediates biosynthesis.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919300997-ga1.jpg" width="500" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: October 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 129〈/p〉 〈p〉Author(s): Chan-Su Rha, Young Sung Jung, Dong-Ho Seo, Dae-Ok Kim, Cheon-Seok Park〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Amylosucrase (ASase) is a unique multifunctional enzyme exhibiting transglycosylation activity. In this study, the specificity of the transglycosylation activity of ASase was evaluated using several hydroxyflavones (HFVOs) and hydroxyflavanones (HFVAs). Our results reveal that the 3–OH and 7–OH positions of the mono-HFVOs and -HFVAs are resistant to transglycosylation by 〈em〉Deinococcus geothermalis〈/em〉 ASase (DGAS), whereas the 6–OH and 4ʹ–OH positions of the mono-HFVOs and -HFVAs exhibit relatively strong transglycosylation reactivities with the glucose donors released from sucrose by DGAS. Particularly, the 6–OH position is considerably more reactive (54-fold higher k〈sub〉cat〈/sub〉) than the 4ʹ–OH position in both HFVOs and HFVAs. Further, the transglycosylation reactions with di- and tri-HFVOs and HFVAs were also investigated and observed to exhibit similar results to those observed for the mono-HFVO and -HFVA molecules. The pH of the reaction influences the reactivity of certain hydroxyl residues, indicating that the pKa values of the hydroxyl groups may be crucial factors in the transglycosylation reactions. These observations help us understand the specificity of the transglycosylation activity of ASase and to predict the transglycosylation products of flavonoids.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919300985-ga1.jpg" width="365" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 6 April 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Bing-Mei Su, Xin-Qi Xu, Ren-Xiang Yan, Yong Xie, Juan Lin〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The recombinant rAgaZC-1 was a family GH50 β-agarase from 〈em〉Vibrio〈/em〉 sp. ZC-1 (CICC 24670). In this paper, the mutant D622G (i.e., mutate the aspartic acid at position 622 to glycine) had better thermo-stability than rAgaZC-1, showing 1.5℃ higher 〈em〉T〈/em〉〈sub〉50〈/sub〉〈sup〉10〈/sup〉 (the temperature at which the half-time is 10 min) and 4-folds of half-time at 41℃, while they had almost same optimum temperature (38.5℃), optimum pH (pH6.0) and catalytic efficiency. Thermal deactivation kinetical analysis showed that D622G had higher activation energy for deactivation, enthalpy and Gibbs free energy than rAgaZC-1, indicating that more energy is required by D622G for deactivation. Substrate can protect agarase against thermal inactivation, especially D622G. Hence the yield of agarose hydrolysis catalyzed by D622G was higher than that by rAgaZC-1. The models of D622G and rAgaZC-1 predicted by homology modeling were compared to find that it is the improved distribution of surface electrostatic potential, great symmetric positive potential and more hydrophobic interactions of D622G that enhance the thermo-stability.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 4 April 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Nimra Shakeel, Anees Ahmad, Mohd Imran Ahamed, Inamuddin, Abdullah M. Asiri〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The objective of this work is to introduce a nanocomposite based bioanode for biofuel cell application. The as-prepared Kraton/MWCNTs nanocomposite was used as a current enhancer and ferritin (Frt) as a mediator between glucose oxidase (GOx) and the electrode surface. The hybrid anode achieved enzymatic oxidation of glucose by the nanocomposite reflecting an efficient energy conversion from glucose. The resulting Kraton/MWCNTs/Frt/GOx bioande exhibited good catalytic activity towards glucose oxidation along with excellent stability. The maximum current density attained by the bioanode is 1.14 mA cm〈sup〉−2〈/sup〉 at the optimum glucose concentration of 60 mM. This enzymatic strategy can be employed to develop other polysaccharide or oligosaccharide fuel cells in which glucose oxidation is involved.〈/p〉〈/div〉

Description: 〈p〉Publication date: July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 126〈/p〉 〈p〉Author(s): Won Je Jang, Jong Min Lee, Md Tawheed Hasan, In-Soo Kong〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The beta-propeller phytase (BPP) is an enzyme that hydrolyzes phyate to release inorganic phosphorus. The BPP produced by 〈em〉Pseudomonas〈/em〉 sp. FB15 (PSphy) possesses an additional N-terminal domain that is not present in BPP produced by other 〈em〉Bacillus〈/em〉 species. In this study, BPP produced by 〈em〉Bacillus〈/em〉 sp. SJ-10 (SJ-10phy) was fused with the N-terminal domain of PSphy and the enzymatic properties of the resulting fusion protein (FUSJ-10phy) were investigated. FUSJ-10phy exhibited an optimal temperature that was 10 °C lower than that of the wild-type enzyme. A comparison of kinetic parameters showed that the turnover rate of FUSJ-10phy was 2.39-fold higher than that of SJ-10phy, representing a 1.79-fold increase in catalytic efficiency. In addition, BPP produced by 〈em〉Bacillus〈/em〉 sp. SJ-48 has relatively low sequence similarity with SJ-10phy, was fused with N-terminal domain (FUSJ-48phy). FUSJ-48phy also increased catalytic efficiency and changed the optimal temperature. These results indicate that, when fused to other BPPs, the N-terminal domain of PSphy increases catalytic efficiency and enzyme activity at lower temperatures.〈/p〉〈/div〉

Description: 〈p〉Publication date: July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 126〈/p〉 〈p〉Author(s): Pengfei Cheng, Jiapao Wang, Yifeng Wu, Xinpeng Jiang, Xiaolin Pei, Weike Su〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉(〈em〉R〈/em〉)-pantolactone is a key chiral intermediate for synthesizing calcium (〈em〉R〈/em〉)-pantothenate. The commercial synthesis of (〈em〉R〈/em〉)-pantolactone is performed through the resolution of racemic pantolactone using lactonase-catalyzed enantioselective hydrolysis. The process needs highly toxic hydrogen cyanide and a tedious dynamic kinetic resolution. In this study, we investigated an alternative method to prepare (〈em〉R〈/em〉)-pantolactone through asymmetric reduction of ketopantolactone (KPL). An NADPH-dependent conjugated polyketone reductase gene from 〈em〉Candida dubliniensis〈/em〉 CD36 (〈em〉Cdu〈/em〉CPR) was functionally overexpressed in 〈em〉Escherichia coli〈/em〉 BL21 (DE3). Recombinant 〈em〉Cdu〈/em〉CPR belonged to the aldo-keto reductase superfamily, and showed high catalytic activity and stereoselectivity using KPL as the substrate. In a continuous feeding reaction, 200 mM ketopantolactone was reduced to (〈em〉R〈/em〉)-pantolactone with 98% conversion and 99% enantiomeric excess (〈em〉e.e.〈/em〉) within 2.0 h. The catalytic mechanism was further investigated. Tyr66 functions as a proton donor following hydrogen transfer from NADPH. Thr30 and His128 are critical residues to bind and orient KPL. Therefore, the recombinant 〈em〉Cdu〈/em〉CPR from 〈em〉C. dubliniensis〈/em〉 exhibited potential application in the asymmetric synthesis of (〈em〉R〈/em〉)-pantolactone.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 25 June 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Ramkrishna D. Singh, Sachin Talekar, Jane Muir, Amit Arora〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉In this work, the effect of particle size on alkali pretreatment of the almond shell was evaluated for recovery of hemicellulose. Further, endoxylanase from 〈em〉Thermomyces lanuginosus〈/em〉 was immobilized on Fe-based magnetic nanoparticles to enable reuse of enzyme. Reduction in particle size significantly influences the recovery of hemicellulose as particle size below 120 µm enable recovery of 97% available hemicellulose in 1 h at 121 °C with 2 M alkali. The enzyme could retain 93.3% of enzymatic activity upon immobilization onto magnetic support using glutaraldehyde (25 mM) and was at par with the free enzyme in terms of pH and temperature profile. The measurement of reaction kinetics (Km and Vmax) indicates similar values for free and immobilized enzyme. The structural and morphological analysis indicates presence near spherical magnetic core and successful cross-linking of the enzyme without alteration of the magnetic core. The immobilized enzyme was able to hydrolyze hemicellulose to produce XOS, the yield equivalent to 67.4% of that obtained using free enzyme at 50 °C. The comparison of XOS production ability at 50 and 60 °C, suggests that the immobilized enzyme retains activity as similar yield was obtained at both temperatures, whereas, the yield for free enzyme decreases significantly. The XOS yield on recycling of immobilized enzyme for three successive cycles was found to reduce to 41% of the initial cycle. However, in all cycles of enzymatic hydrolysis, the percentage of xylobiose was found to be above 90%.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S014102291930105X-ga1.jpg" width="301" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: November 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 130〈/p〉 〈p〉Author(s): Teisuke Takita, Kota Nakatani, Yuta Katano, Manami Suzuki, Kenji Kojima, Naoki Saka, Bunzo Mikami, Rie Yatsunami, Satoshi Nakamura, Kiyoshi Yasukawa〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉GH11 xylanase XynJ from 〈em〉Bacillus〈/em〉 sp. strain 41M-1 has a β-jellyroll fold composed of eight β strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the β strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13–Arg104 and Gly169–Tyr194, both of which are located in the β-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the 〈em〉k〈/em〉〈sub〉cat〈/sub〉 and 〈em〉K〈/em〉〈sub〉m〈/sub〉 values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 22 April 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Moon Won Min, Chae-Eun Kim, Sushma Chauhan, Hyeon Ji Park, Chang-Seo Park, Tae Hyeon Yoo, Taek Jin Kang〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Matrix metalloproteinases (MMPs) are zinc-dependent proteases involved in the degradation of extracellular matrix proteins. As one of the isoforms, MMP-1 breaks down collagen, and its activity is known to be important in wound healing. Its timely and adequate level of expression is pivotal because MMP-1 is also involved in the damage or aging of skins as well as in certain types of cancers. Thus, both assaying the MMP-1 activity and developing its inhibitors are of great importance. We here developed an in-house assay system that gave us the high degree of freedom in screening peptide inhibitors of MMP-1. The assay system utilized a circularly permutated fusion of β-lactamase and its inhibitory protein through an MMP-1-sensitive linker so that the activity of MMP-1 could be translated into that of β-lactamase. As a proof of concept, we applied the developed assay system to initial screens of MMP-1 inhibitors and successfully identified one lead peptide that inhibited the collagenase activity of the enzyme.〈/p〉〈/div〉

Description: 〈p〉Publication date: June 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 125〈/p〉 〈p〉Author(s): Baobei Wang, Xueshan Pan, Jing Jia, Weide Xiong, Emmanuel Manirafasha, Xueping Ling, Lu Yinghua〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉〈em〉Xanthophyllomyces dendrorhous〈/em〉 is an excellent industrial source for production of natural astaxanthin, but the yield of astaxanthin is relative low due to the contradiction between biomass weight and astaxanthin accumulation. Glutamate, a metabolite connecting nitrogen and carbon metabolisms, is probably a promising entry point to interfere cellular metabolisms. Thus, the effect of glutamate on cell growth and astaxanthin accumulation in 〈em〉X. dendrorhous〈/em〉 was investigated. Results showed that glutamate feeding facilitated glucose consumption and further led to the increment of astaxanthin content with little influence of cell growth. A comparative proteomics study was applied to decipher the regulatory mechanisms of enhanced astaxanthin biosynthesis in 〈em〉X. dendrorhous〈/em〉 as a response to the glutamate feeding. The expressions of proteins with the highest degree of fold change were involved in carbohydrate, amino acids, and carotenogenesis metabolisms as well as redox and stress-associated metabolisms. In addition, a possible regulatory model of enhanced astaxanthin accumulation in response to glutamate feeding in 〈em〉X. dendrorhous〈/em〉 is also proposed.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 24 April 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Fei Teng, Ran You, Meirong Hu, Weifeng Liu, Lei Wang, Yong Tao〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉D-glucuronic acid (GlcUA) is an important intermediate with numerous applications in the food, cosmetics, and pharmaceutical industries. Its biological production routes which employ myo-inositol oxygenase (MIOX) as the key enzyme are attractive. In this study, five diverse MIOX-encoding genes, from 〈em〉Cryptococcus neoformans〈/em〉, 〈em〉Chaetomium thermophilum〈/em〉, 〈em〉Arabidopsis thaliana〈/em〉, 〈em〉Thermothelomyces thermophila〈/em〉, and 〈em〉Mus musculus〈/em〉 were overexpressed in 〈em〉Escherichia coli〈/em〉, respectively. A novel MIOX from 〈em〉Thermothelomyces thermophila〈/em〉 (TtMIOX) exhibited high specific activity, and efficiently converted myo-inositol to GlcUA. Meanwhile, the degradation of GlcUA was inhibited by inactivation of uxaC from the 〈em〉Escherichia coli〈/em〉 genome. Finally, the BWΔuxaC whole-cell biocatalyst harboring TtMIOX resulted in the production of 106 g/L GlcUA within 12 h in a 1-L bioreactor, corresponding to a conversion of 91% and productivity of 8.83 g/L/h. This study provides a feasible method for the industrial production of GlcUA.〈/p〉〈/div〉

Description: 〈p〉Publication date: Available online 17 April 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): Cai-Xia Yao, Tian-Yang Lin, Yi-Long Su, Hui Zou, Zheng-Yu Yan, Sheng-Mei Wu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Macrophages eliminate and destroy invading bacteria and contaminants by engulfing them or secreting cytokines that trigger downstream immune responses. Consequently, impairment of the phagocytic functions of macrophages and/or suppressing their cytokine secretion are dangerous to organisms that rely on immune protection. Accordingly, exposure to environmental nanoparticles (NPs) that display immunomodulatory properties are serious. In this work, two types of NPs, i.e., mild-toxicity CuInS〈sub〉2〈/sub〉 NPs and high-toxicity CdTe NPs, were used to evaluate the effects of NP exposure for macrophages. Following incubation for 24 h, THP-1-derived macrophage viability was assessed using an MTT method after exposing the THP-1 cells to different concentrations of CuInS〈sub〉2〈/sub〉 or CdTe NPs. Phagocytosis assays demonstrated that both CuInS〈sub〉2〈/sub〉 and CdTe NPs impair phagocytic activity toward 〈em〉Staphylococcus aureus〈/em〉 (〈em〉S. aureus〈/em〉). After pretreatment with CuInS〈sub〉2〈/sub〉 and CdTe NPs at 4 μmol/L, THP-1 macrophages exhibited decreases in phagocytic ratio from ca. 32.9% to ca. 18.5% and 18.7%, respectively. Since the zeta potentials of intact and weathered CuInS〈sub〉2〈/sub〉 NPs were distributed over a wide range from positive to negative, large quantities of intact and weathered CuInS〈sub〉2〈/sub〉 NPs bore sufficient positive charge on their surfaces to induce membrane depolarization, thus theoretically providing electrostatic forces between 〈em〉S. aureus〈/em〉 and THP-1, which could induce downstream intracellular events that increase phagocytosis. However, real time polymerase chain reaction arrays revealed that transcription of the pro-inflammatory factors IL-1β, IL-6, and TNF-α decreased while that of the anti-inflammatory factor IL-10 increased after treatment with CuInS〈sub〉2〈/sub〉 NPs. Furthermore, transcription of TNF-α decreased while IL-10 increased after treatment with CdTe NPs. Thus, both kinds of NPs inhibited phagocytosis of 〈em〉S. aureus〈/em〉 by THP-1 to some extent, confirming that immunosuppression can occur when macrophages are exposed to environmental NPs.〈/p〉〈/div〉

Description: 〈p〉Publication date: June 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 125〈/p〉 〈p〉Author(s): 〈/p〉

Description: 〈p〉Publication date: Available online 16 July 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology〈/p〉 〈p〉Author(s): 〈/p〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Pornpimol Phuengmaung, Yoichi Sunagawa, Yosuke Makino, Takafumi Kusumoto, Satoshi Handa, Wasana Sukhumsirichart, Tatsuji Sakamoto〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉We previously described the fungus 〈em〉Penicillium chrysogenum〈/em〉 31B, which has high performance to produce the ferulic acid esterase (FAE) for de-esterifying ferulic acids (FAs) from sugar beet pulp. However, the characteristics of this fungus have not yet been determined. Therefore, in this study, we evaluated the molecular characteristics and natural substrate specificity of the 〈em〉Pcfae1〈/em〉 gene from 〈em〉Penicillium chrysogenum〈/em〉 and examined its synergistic effects on sugar beet pectin. The 〈em〉Pcfae1〈/em〉 gene was cloned and overexpressed in 〈em〉Pichia pastoris〈/em〉 KM71H, and the recombinant enzyme, named PcFAE1, was characterized. The 505 amino acids of PcFAE1 possessed a GCSTG motif (Gly164 to Gly168), characteristic of the serine esterase family. By comparing the amino acid sequence of PcFAE1 with that of the FAE (AoFaeB) of 〈em〉Aspergillus oryzae〈/em〉, Ser166, Asp379, and His419 were identified as the catalytic triad. PcFAE1 was purified through two steps using anion-exchange column chromatography. Its molecular mass without the signal peptide was 75 kDa. Maximum PcFAE1 activity was achieved at pH 6.0–7.0 and 50 °C. The enzyme was stable up to 37 °C and at a pH range of 3–8. PcFAE1 activity was only inhibited by Hg〈sup〉2+〈/sup〉, and the enzyme had activity toward methyl FA, methyl caffeic acid, and methyl 〈em〉p〈/em〉-coumaric acid, with specific activities of 6.97, 4.65, and 9.32 U/mg, respectively, but not on methyl sinapinic acid. These results indicated that PcFAE1 acted similar to FaeB type according the Crepin classification. PcFAE1 de-esterified 〈em〉O〈/em〉-[6-〈em〉O〈/em〉-feruloyl-β-〈span〉d〈/span〉-galactopyranosyl-(1→4)]-〈span〉d〈/span〉-galactopyranose, 〈em〉O〈/em〉-[2-〈em〉O〈/em〉-feruloyl-α-〈span〉l〈/span〉-arabinofuranosyl-(1→5)]-〈span〉l〈/span〉-arabinofuranose, and 〈em〉O〈/em〉-[5-〈em〉O〈/em〉-feruloyl-α-〈span〉l〈/span〉-arabinofuranosyl-(1→3)]-〈em〉O〈/em〉-β-〈span〉d〈/span〉-xylopyranosyl-(1→4)-〈span〉d〈/span〉-xylopyranose, indicating that the enzyme could de-esterify FAs decorated with both β-〈span〉d〈/span〉-galactopyranosidic and α-〈span〉l〈/span〉-arabinofuranosidic residues in pectin and xylan. PcFAE1 acted in synergy with endo-α-1,5-arabinanase and α-〈span〉l〈/span〉-arabinofuranosidase, which releases FA linked to arabinan, to digest the sugar beet pectin. Moreover, when PcFAE1 was allowed to act on sugar beet pectin together with Driselase, approximately 90% of total FA in the substrate was released. Therefore, PcFAE1 may be an interesting candidate for hydrolysis of lignocellulosic materials and could have applications as a tool for production of FA from natural substrates.〈/p〉〈/div〉

Description: 〈p〉Publication date: December 2019〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 131〈/p〉 〈p〉Author(s): Neera Agarwal, Lokesh Kumar Narnoliya, Sudhir P. Singh〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Turanose is a natural isomer of sucrose. It is an emerging functional sweetener of the next generation. Turanose is catalytically synthesized from the sucrose biomass by employing amylosucrase enzyme. In this study, a novel gene encoding amylosucrase (〈em〉As〈sub〉met〈/sub〉〈/em〉) has been identified from the metagenome of a thermal aquatic habitat. 〈em〉As〈sub〉met〈/sub〉〈/em〉 exhibits 37–55% identity at the protein level with the known amylosucrases characterized till date. 〈em〉As〈sub〉met〈/sub〉〈/em〉 was cloned and expressed in 〈em〉Escherichia coli〈/em〉, followed by protein purification, and characterization. As〈sub〉met〈/sub〉 protein exhibited the maximum total activity at 9.0 pH and 60 °C temperature, whereas, 8.0 pH and 50 °C temperature were found optimum for transglycosylation activity. As〈sub〉met〈/sub〉 showed fairly high thermal tolerance at 50 °C. The conjugation of As〈sub〉met〈/sub〉 protein with functionalized iron nanoparticles significantly improved its thermal tolerance, showing hardly any loss in the enzyme’s activity even after 72 h of heat (50 °C) exposure. The turanose yield of about 47% was achieved from 1.5 M sucrose, containing 0.5 M fructose in the reaction. Turanose was purified (˜95%) via a bio-physical process, and characterized by TLC, HPLC, and NMR. The novel amylosucrase gene was demonstrated to be a potential candidate for turanose production, utilizing various sucrose containing feedstocks.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Yue Zhang, Wenhui Sun, Nora Mohamed Elfeky, Yuepeng Wang, Dongying Zhao, Hao Zhou, Jingyun Wang, Yongming Bao〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Lipase ZC12, a cold-adapted lipase derived from 〈em〉Psychrobacter〈/em〉 sp. ZY124, can be effectively activated by Ca〈sup〉2+〈/sup〉. Inspired by this significant property, we developed a novel immobilized lipase ZC12/Ca〈sub〉3〈/sub〉(PO〈sub〉4〈/sub〉)〈sub〉2〈/sub〉 hybrid nanoflowers (LHNs). The LHNs have been characterized as a regular hierarchical flowerlike structure nanoflowers by scanning electron microscopy (SEM). Compared with free lipase ZC12, the LHNs exerted enhanced enzymatic activity of 206% and 2.31-fold in 〈em〉k〈/em〉〈sub〉cat〈/sub〉/〈em〉K〈/em〉〈sub〉m〈/sub〉 value, especially high specific activity at low temperature. After 7 successive cycles, the LHNs could still maintain its initial activity, demonstrating superior durability than the free lipase ZC12. Meanwhile, its stability basically kept unchanged in a wide range of temperature and pH. Finally, fructose laurate was transformed by the LHNs with 57.39% conversion rate which is twice as much as the free lipase. To sum up, these results validated that LHNs could emerge as an efficient immobilized lipase and possess the promising potential for practical applications.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301462-ga1.jpg" width="429" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): M. Serrano-Arnaldos, J.J. García-Martínez, S. Ortega-Requena, J. Bastida, F. Máximo, M.C. Montiel〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉This work describes for the first time the green synthesis of neopentyl glycol diheptanoate in a solvent-free medium via an enzymatic pathway. The process has been carried out in an open-air reactor in order to ease water removal through evaporation and shift the chemical equilibrium towards product formation. The inhibiting effect of high concentrations of heptanoic acid has been put into evidence by a reduction of initial reaction rate when esterification was performed with stoichiometric amounts of substrates. Therefore, in this work different strategies for the stepwise addition of heptanoic acid are proposed, and best results were obtained when stoichiometric quantities of acid were divided in four equal amounts and added when previous batch was consumed. Biocatalyst Novozym® 435 concentration and temperature were optimised, giving yields of 90% in neopentyl glycol diheptanoate when 7.5% (w/w) and 70 °C were used. With a remaining 7% of heptanoic acid (probably caused by the alcohol evaporation) the addition of neopentyl glycol led to a conversion of 95%. Thus, product can be used in cosmetics without further purification and can be labelled as environmentally-friendly synthesized because of its enzymatic origin.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301383-ga1.jpg" width="500" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Svetlana Dekina, Irina Romanovska, Oleg Sevastyanov, Yevgeniia Shesterenko, Alexandra Ryjak, Ludmila Varbanets, Dzubluk Natalia, Eugene Muratov〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉The biologically active polymeric material with entrapped peptidase 〈em〉Bacillus thuringiensis〈/em〉 var. 〈em〉israelensis〈/em〉 with caseinolytic, collagenase and elastase activities was developed as a promising product for medical use. We have evaluated and reported here the following physical-chemical and biochemical characteristics of entrapped enzyme: peptidase/polymer interaction and morphology analyses, film thickness, water content, time of dissolution in water and physiological saline, kinetic of casein hydrolysis and pH- and thermoprofiles of proteolytic activity. Scanning electron microscopy images shows the relative uniformity of the film surface with entrapped peptidase. The released peptidase was characterized by increased proteolytic activity in the acidic (14%–35%) and alkaline (7%–32%) regions. After nine months of storage, peptidase in chitosan/polyvinyl alcohol films retains more than 95% of its initial proteolytic activity. We consider this film as a perspective biotechnological agent in medicine.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): You Jin Lim, Bora Lim, Hae Yeong Kim, Soon-Jae Kwon, Seok Hyun Eom〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Microorganism selection is critical to deglycosylation in soybean fermentation for producing beneficial phytochemicals. This study investigated isoflavone bioconversion in soybean extract inoculated with 〈em〉Lactobacillus plantarum〈/em〉 K2-12 and 〈em〉Lactobacillus curvatus〈/em〉 JD0-31 exhibiting different enzyme activities. 〈em〉L. plantarum〈/em〉 showed higher esterase (C4), esterase (C8), β-galactosidase, α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminase activities. We found that isoflavone bioconversion was distinguished into isoflavone backbone structure types. Malonyl- and acetyl- types of isoflavones except for malonyl daidzin were not significantly differed their contents between 〈em〉lactobacilli〈/em〉. Deglycosylating severity was observed in malonyl genistin in both 〈em〉lactobacilli〈/em〉, resulting mass production of genistein. On the other hand, daidzein glycosides were dependable to 〈em〉lactobacilli〈/em〉, in which 〈em〉L. plantarum〈/em〉 efficiently degraded malonyl daidzin and daidzin in fast time. Glycitein was most degradable among the three aglycones by fermentation. These results suggest that efficient control of isoflavone deglycosylation by 〈em〉Lactobacillus〈/em〉 species should be controlled to the inoculation period and select target isoflavones.〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Mariana de Melo Brites, Annie A. Cerón, Sirlene M. Costa, Rodrigo C. Oliveira, Humberto G. Ferraz, Luiz Henrique Catalani, Silgia A. Costa〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Cellulose triacetate (CTA〈sub〉B〈/sub〉) synthesized by cellulose extracted from sugarcane bagasse, and commercial cellulose acetate (CA) were used to produce nanofiber membranes contained bromelain by electrospinning technique. About 1.3 g of cellulose acetate per gram of bagasse were obtained, and both CTA〈sub〉B〈/sub〉 and CA was characterized by analysis of Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC). The nanofiber membranes were produced by electrospinning process testing the following conditions: voltage 25 kV, flow rate 4 mL/h and distance 10 cm, using acetone/ dimethylformamide (DMF) (85:15 m/ m) to 15% cellulose triacetate (70% CA + 30% CTA〈sub〉B〈/sub〉) or CA solutions. Scanning Electron Microscopy (SEM) was used to nanofiber membranes characterization. Bromelain was immobilized on the nanofiber membranes by crosslinking with glutaraldehyde and directly in the electrospinning step, the highest activity recovery was about 675% and in vitro controlled release tests were performed to semi-quantitatively evaluate the release of the enzyme bromelain thus demonstrating complete release process in 3 days.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S014102291930122X-ga1.jpg" width="332" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Lucas Dal Magro, Jakub F. Kornecki, Manuela P. Klein, Rafael C. Rodrigues, Roberto Fernandez-Lafuente〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉Pectin lyase (from Rohapect 10 L) was immobilized on glutaraldehyde supports at low ionic strength at pH 5, 6.5 or 8 and later incubated at pH 8 for 48 h. The activity recovery of the biocatalysts versus pectin was quite low, under 10% for all of the immobilized biocatalyst at 20 °C. However, a high stabilization was found when the enzyme was immobilized at pH 5, (e.g., the immobilized enzyme kept 83% of the activity when the free enzyme was fully inactivated (pH 4.8 and 55 °C in 5 h)). This biocatalyst increased the activity versus pectin in an almost exponential way when temperature increased until reach the maximum temperature used in the study (90 °C), conditions where the free enzyme was almost inactive. The immobilized biocatalyst was also active even at pH 9, where the free enzyme was fully inactive. This biocatalyst could be reused for pectin hydrolysis 5 times for 72 h reaction cycles at 40 °C maintaining more than 90% of the initial activity.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301358-ga1.jpg" width="353" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉

Description: 〈p〉Publication date: January 2020〈/p〉 〈p〉〈b〉Source:〈/b〉 Enzyme and Microbial Technology, Volume 132〈/p〉 〈p〉Author(s): Licheng Wu, Xiaolei Guo, Gaobing Wu, Pengfu Liu, Ziduo Liu〈/p〉 〈h5〉Abstract〈/h5〉 〈div〉〈p〉In our previous study, we produced α-keto acids by using an L-amino acid deaminase 〈em〉Pmi〈/em〉LAAD (wide-type) from 〈em〉Proteus mirabilis,〈/em〉 however, the catalytic efficiency was low due to its low substrate affinity. In this study, protein engineering of 〈em〉Pmi〈/em〉LAAD was performed to improve the α-keto acid production. 〈em〉Pmi〈/em〉LAAD was engineered by iterative CASTing to improve its catalytic performance. The four mutant 〈em〉Pmi〈/em〉LAAD-SAVS (〈em〉Pmi〈/em〉LAAD-Phe93Ser-Pro186Ala- Met394Val-Phe184Ser) with 6.6 -fold higher specific activity compared with that of wild-type 〈em〉Pmi〈/em〉LAAD has been obtained by high-throughput screening. Comparative kinetics analysis showed that the four mutant 〈em〉Pmi〈/em〉LAAD-SAVS had a higher substrate-binding affinity and catalytic efficiency than that of 〈em〉Pmi〈/em〉LAAD wild-type. The 〈em〉K〈/em〉〈sub〉m〈/sub〉, 〈em〉k〈/em〉〈sub〉cat〈/sub〉, and 〈em〉k〈/em〉〈sub〉cat〈/sub〉/〈em〉K〈/em〉〈sub〉m〈/sub〉 values of the 〈em〉Pmi〈/em〉LAAD(SAVS) variant was better (-42.7%, 75.11%, and 85.79%, respectively) than the corresponding values of 〈em〉Pmi〈/em〉LAAD wild type. Finally, the whole cell biocatalyst 〈em〉E. coli〈/em〉-pETDuet-1-〈em〉Pmi〈/em〉LAAD(SAVS) has been applied to α-keto acids production. The conversion rate of L-phenylalanine reached 99% by whole-cell biocatalyst 〈em〉E. coli〈/em〉-pETDuet-1-〈em〉Pmi〈/em〉LAAD(SAVS). The conversion of (D/L)-4-phenylalanine was reached 49.5% after 7 h by whole-cell biocatalyst 〈em〉E. coli〈/em〉-pETDuet-1-〈em〉Pmi〈/em〉LAAD(SAVS), while the conversion of 〈em〉E. coli〈/em〉-pETDuet-1-〈em〉Pmi〈/em〉LAAD (wild type) was only 18% after an extension of the reaction time (24 h). This study has developed a robust whole-cell 〈em〉E. coli〈/em〉 biocatalyst for α-keto acids production by protein engineering, and this strategy may be useful for the construction of other biotransformation biocatalysts.〈/p〉〈/div〉 〈h5〉Graphical abstract〈/h5〉 〈div〉〈p〉〈figure〉〈img src="https://ars.els-cdn.com/content/image/1-s2.0-S0141022919301310-ga1.jpg" width="254" alt="Graphical abstract for this article" title=""〉〈/figure〉〈/p〉〈/div〉