Summary
The changes in cell wall strength of Hansenula polymorpha have been investigated in continuous cultures with respect to the recovery of methanol oxidase (MOX). Cultures grown on several substrate mixtures that enable induction of MOX have been compared with cultures grown on methanol as the sole inducer. The effects of dilution rate (D) on lysis properties have been studied. The cell wall strength was consistently influenced by growth media and D. Media containing glycerol/methanol showed the slowest lysis kinetics, with a large fraction of non-degradable cell wall material. In continuous cultures grown on a mixture of glucose and methanol both the resistance to zymolyase and the mean cell wall thickness increased at D<0.1 h−1. The yield of MOX by zymolyase lysis is reproducible and up to 100% higher than that of the standard ultrasonic treatment. The lysis kinetics indicated that zymolyase punctures the cell wall; since the release rate of MOX is lower than that of protein, the cell contents will leak through. At D-values>0.2 h−1, both protein and MOX release rates increase, reflecting a change in lysis mechanism due to the increased fraction of thin daughter cells. Kinetic analysis of zymolyase lysis using both physical and enzymatic methods provides information for achieving optimal recovery of MOX.
Similar content being viewed by others
Abbreviations
- C MOX :
-
MOX activity [MOX units·g X−1]
- D :
-
dilution rate [h−1]
- MOX:
-
methanol oxidase
- kc :
-
decay rate constant of A 610 nm [min−1]
- kd :
-
decay constant of MOX activity [min−1]
- kdis :
-
dissociation rate constant [min−1]
- kMOX :
-
release rate constant of MOX activity [min−1]
- kp :
-
release rate constant of protein [min−1]
- R:
-
recovery efficiency of enzyme [-]
- St :
-
stability of enzyme [-]
References
Baratti J, Couderi R, Cooney CL, Wang DIC (1978) Preparation and properties of immobilised methanol oxidase. Biotech Bioeng 20:333–348
Bruinenberg PM (1985) The NADP(H) redox couple in yeast metabolism. Thesis TUD, Delft
Dijken van JP, Otto R, Harder W (1976) Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. Arch Microbiol 111:137–144
Eggeling L, Sahm S (1978) Derepression and partial insensitivity to carbon catabolite repression of the methanol dissimilating enzymes in Hansenula polymorpha. Eur J Appl Microbiol Biotechnol 5:197–202
Egli Th (1980) Wachstum von Methanol assimilierende Hefen. Thesis ETH no 6538, Zürich
Giuseppin MLF, van Eijk HJM, Bes BCM Molecular regulation of methanol oxidase by Hansenula polymorpha in continuous cultures. Biotechnol Bioeng (in press)
James CJ, Coakley WT, Hughes DE (1972) Kinetics of protein release from yeast sonicated in batch and flow systems at 20 kHz. Biotechnol Bioeng 14:33–43
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin reagent. J Biol Chem 193:265–271
Petersen GR (1985) Determining a carbohydrate profile for Hansenula polymorpha. Enzyme Microbiol Technol 7:339–345
Phillips Petroleum Company USP 4414329 (1983) Biochemical conversions by yeast fermentation at high cell densities
Veenhuis M, Dijken van JP, Harder W (1976) Cytochemical studies on the localisation of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha. Arch Microbiol 111:123–135
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Giuseppin, M.L.F., van Eijk, H.M.J., Hellendoorn, M. et al. Cell wall strength of Hansenula polymorpha in continuous cultures in relation to the recovery of methanol oxidase (MOX). Appl Microbiol Biotechnol 27, 31–36 (1987). https://doi.org/10.1007/BF00257250
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00257250