ISSN:
0006-3592
Schlagwort(e):
protien stability
;
protein surface charge
;
6- amino-penicillanic acid
;
biocatalysis
;
Chemistry
;
Biochemistry and Biotechnology
Quelle:
Wiley InterScience Backfile Collection 1832-2000
Thema:
Biologie
,
Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
Notizen:
Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. © 1995 John Wiley & Sons, Inc.
Zusätzliches Material:
8 Ill.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1002/bit.260480208
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