Cell wall strength of Hansenula polymorpha in continuous cultures in relation to the recovery of methanol oxidase (MOX)

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.Abbreviations and symbols C_MOX MOX activity [MOX units·g X^–1] - D dilution rate [h^–1] - MOX methanol oxidase - k_c decay rate constant of A 610 nm [min^–1] - k_d decay constant of MOX activity [min^–1] - k_dis dissociation rate constant [min^–1] - k_MOX release rate constant of MOX activity [min^–1] - k_p release rate constant of protein [min^–1] - R recovery efficiency of enzyme [-] - St stability of enzyme [-]