Yeast-spore Encapsulation Of Glucose Dehydrogenase Variant And (S)-carbonyl Reductase ? Efficiently Catalyzes Chiral Synthesis

Saccharomyces cerevisiae spore is hypopus with good resistance.It has good application prospect using yeast-spore in enzyme encapsulation to perform catalytic reaction.Candida parapsilosis(S)-specific carbonyl reductase(SCR?)with Bacillus sp.glucose dehydrogenase(GDH)were coupled in S.cerevisiae AN120 spores.The yeast-spore encapulation catalyzed the synthesis of(S)-1-phenyl-1,2-ethanediol with glucose as cosubstrate to strengthen the cofactor-regeneration.The existence of glucose is very easy to make spore generation.In addition to natural substrate glucose,GDH can catalyze D-xylose.And the presence of xylose does not make spore germination.To improve microencapsulation stability and inhibit spore germination,we designed the site-directed mutagenesis in the substrate binding pocket of GDH to improve GDH activity towards xylose.SCR? and GDH mutant were coupled in S.cerevisiae AN120 spores to obtain the yeast-spore microencapsulation.The spore microencapsulation realized efficient multi-batch asymmetric chiral transformation without the addition of cycloheximide to inhibit spore germination.The main results are as follows:(1)Seven recombinant Escherichia coli strains harboring glucose dehydrogenase mutants: E.coli BL21/pET-gdh/K207 A,E.coli BL21/pET-gdh/E220 K,E.coli BL21/pET-gdh/Q252 K,E.coli BL21/pET-gdh/A258 F,E.coli BL21/pET-gdh/A258 H,E.coli BL21/ pET-gdh/A258 W and E.coli BL21/pET-gdh/A258 Y were constructed.All the mutants were highly expressed in E.coli BL21.The purified enzyme with a single band was obtained through two procedures including HisTrap HP affinity chromatography and Superdex 200 gel chromatography.(2)The enzyme activity results showed that the mutant enzymes A258 F,A258H,A258 W,and A258 Y improved activity of 1–4 folds towards xylose.Among all the mutants,A258 F exhibited the highest activity.Under the optimal conditions: 55? and pH 7.0,A258 F showed a specific activity of 7.59 U?mg-1 towards xylose,which was about 4 times higher than that of the wild-type.Similar to the wild type,A258 F had good organic solvent tolerance.It retained more than 90% of the whole activity after incubation in 50%(v/v)octane and nonane,over 85% in 50% cyclohexane and decane,and over 50% in ethyl caprylate and ethylene glycol for 24 hour.(3)The kinetic results showed that A258 F had 2.6 folds increase in the kcat value and 3 folds decrease in the Km value.The variations of A258 F in kcat and Km values towards xylose led to 7.8 folds increase in the kcat/Km value with respect to the wild-type.The results indicated that the catalytic efficiency of A258 F towards xylose was obviously improved.The kcat/Km ratio of A258 F was 1.3 folds higher towards glucose than that of the wild type,suggesting that the catalytic efficiency of A258 F towards glucose was slightly improved.(4)The recombinant S.cerevisiae AN120/TEF-scr?-gdh/A258 F was constructed.When cultured with potassium acetate as the sole energy source,yeast spores were induced to produce and the recombinant proteins were embedded in spores,and yeast-spore microencapsulation of enzymes was obtained.The target proteins SCR? and GDH(A258F)were proved to be expressed in S.cerevisiae AN120 spores by western blotting analysis.(5)Using spore microencapsulation of enzymes as biocatalyst,xylose as cosubstrate for cofactor regeneration,the asymmetric biotransformation was carried out and the optimal reaction conditions and multi-batch performance of spore microencapsulation of enzymes were investigated.When 2-hydroxyacetophenone concentration was 20 g?L-1,the ratio between 2-hydroxyacetophenone and xylose was 1:1,the product(S)-1-phenyl-1,2-ethanediol was produced with an optical purity of 99.7% and a yield of 95.8% at optimal temperature 35? and optimal pH 6.0 for 4 h.After being reused for 10 times,the microencapsulation catalyzed the biotransformation of(S)-1-phenyl-1,2-ethanediol with a yield of more than 60% without the addition of cycloheximide.