Expression And Enzymatic Properties Of Thermobifida Fusca Cutinase In Saccharomyces Cerevisiae

Cutinase, one group of versatile enzymes, can catalyze a wide variety of estershydrolysis and transesterification. It has great potential applications for dairy and oil modifiedof food industry. And it also can be applied in the synthesis of pharmaceuticals and chemicals.The gene encoding cutinase from Thermobifida fusca and signal peptide were firstlysynthesized according to codon usage bias of Saccharomyces cerevisiae in this study. Toaccomplish the secretion of cutinase,-factor signal peptide and kozark sequence were addedat the N terminal of cutinase. By guidance of the α-factor signal peptide, the T. fusca cutinasewas successfully expressed and secreted extracellularlly with the GAL1expression system.After successfully constructed the recombinant yeast, the effect of concentration of galactoseand induction on cutinase production were deeply evaluated. Moreover, we investigated theeffect of different combination of mixture of carbon source on cutinase production by therecombinant yeast. As a result, raffinose was proved to be much more active than other sugarsapplied for expression of cutinase.To increase the level of cutinase, four different strong promoters (ADH1, HXT1, TEF1,TDH3) were compared and evaluated for cutinase production. Among the four constitutivepromoters, promoter TEF1exhibited an outstanding property and significantly increased thelevel of the recombinant cutinase. Then, altering the culture volume of recombinant S.cerevisiae in the shake-flask, the effect of the dissolved oxygen level on the cutinaseproduction was evaluated. The internal relationship of cutinase production, cell biomass anddissolved oxygen (DO) was deeply investigated in a3L fermenter. As a result, it would befavourable if dissolved oxygen came to a certain level which can keep the cells growingnormally. Furthermore, the cutinase activity increased with the accumulation of cell biomass.By fed-batch fermentation with a constant feeding approach, a high level cutinase of29.7U/mL was achieved.After purified by saturated ammonium sulphate, Phenyl HP column chromatography andDEAE Sepharose column chromatography respectively, the electrophoretic homogeneityrecombinant cutinase by yeast was obtained. The enzymatic properties of cutinase wereanalyzed. As a result, the optimum temperature was60°C and had very good thermal stability.The cutinase exhibited a optimum pH of8.0and had better stability in the range of6.0~9.0,which indicated that the cutinase had higher production and better stability in the lightalkaline reaction system. Furthermore, the cutinase expressed by recombinant yeast had arelatively good catalytic efficiency. Cr2+and Hg2+inhibited enzyme activity intensely. Cu2+,Zn2+, Fe2+and Pb2+inhibited recombinant cutinase in some extent while Ni2+,Ca2+, Mg2+hadno significant effect on the cutinase activity. Triton X-100, Tween20, SDS inhibited thecutinase activity in a certain extent while TDOC had no obvious influence on the cutinaseactivity.