| Gentiooligosaccharides are novel functional oligosaccharides composed of glucose units linked through ?-1,6-glycosidic bonds,examples of which include gentiobiose,gentiotriose and gentiotetraose.Gentiooligosaccharides have many excellent physiological functions,low-heat,low-sweet,low-water activity and can not broken down by digestive enzymes in the human body.At present,gentiooligosaccharides are widely used in chocolate,ice cream,coffee,spices,baked goods and beverages.In the present study,the P-glucosidase gene bgll derived from Trichoderma viride was ligated into vector pPIC9K and co-expressed in Pichia pastoris KM71 with disulfide isomerase pdi.A recombinant P.pastoris strain was successfully obtained to effectively secrete the expression of ?-glucosidase.And the fermentation conditions of recombinant P.pastoris KM71/pPIC9K-bgl1/pPICZ A-pdi were optimized in a 3.6 L bioreactor.The enzymatic properties of the recombinant p-glucosidase were studied,and the process conditions for the synthesis of gentiooligosaccharides via reverse hydrolysis and transglycoside activity of the recombinant P-glucosidase were optimized.The main findings of this paper are as follows:(1)The recombinant plasmid pPIC9K-bgl1 containing ?-glucosidase bgll from Trichoderma viride was constructed by overlapping PCR and integrated into Pichia pastoris KM71.In order to assist the formation of disulfide bonds and thus improve protein folding efficiency,protein disulfide isomerase pdi was co-expressed in the P.pastoris KM71/pPIC9K-bgl1/pPICZ-A-pdi strain,and fermentation in flasks resulted in enzyme activity of 143 U ·mL-1,which was 1.7-fold higher than that of the strain lacking pdi.(2)The recombinant P-glucosidase was purified by ammonium sulfate precipitation and size exclusion chromatography to investigate the enzymatic properties.The recombinant P-glucosidase had a specific activity of 610 U·mg-1 using pNPG as the substrate.Its optimal temperature and pH were 60 ? and 5.0,respectively,and its half-life was about 6 h at 60 ?and pH 5.0.Using pNPG as a substrate,Km,kcat,and kcat/Km of recombinant P-glucosidase were 0.19 mM,516.0 s-1,and 2.70×106M-1·s-1,respectively.Using cellobiose as a substrate,Km,kcat,and kcat/Km of recombinant ?-glucosidase were 0.38 mM,23.0 s-1,and 6.1×104 M-1·s-1,respectively.Using gentiobiose as a substrate,Km,kcat,and kcat/Km of recombinant?-glucosidase were 0.61 mM,15.0 s-1,and 2.5×104M-1·s-1,respectively.The results showed that the affinity of the recombinant P-glucosidase to the above three substrates was pNPG,cellobiose,and gentiobiose in descending order.(3)To optimize the production of ?-glucosidase,on the production of ?-glucosidase using P.pastoris KM71/pPIC9K-bgl1/pPICZ-A-pdi in a 3.6 L bioreactor were studied.The optimal conditions are as follows:when the induction temperature was kept.at 28 ?,the initial cell density OD600 was 150,methanol concentration was 1.0%,the production of P-glucosidase activity in the culture supernatant reached 1452 U· mL-1,which is 10.2 times higher than that of shake flask and 23.4-fold higher than the highest level of Trichoderma viride ?-glucosidase expression reported previously.(4)The process of preparation of gentiooligosaccharides by recombinant ?-glucosidase was optimized.Production of gentiooligosaccharides from glucose via ?-glucosidase-based reverse hydrolysis.The optimum conditions were as follows:pH 5.0,60 ?,the amount of?-glucosidase was 900 U·g-1 of glucose,glucose concentration of 800 g·L-1 achieved the maximum value of gentiooligosaccharides reached 130 g·L-1 and a conversion rate of 16.25%.Synthesis of gentiooligosaccharides from a mixture of glucose and cellobiose via?-glucosidase-based transglycosylation.The optimum conditions were as follows:pH 5.0,60 ?,the amount of ?-glucosidase was 400 U·L-1 of cellobiose,the molar ratio of glucose to cellobiose was 1:1,and the substrate concentration was 200 g·L-1 glucose and 400 g·L-1 cellobiose,the yield of gentiooligosaccharides reached 116 g·L-1 with a conversion rate of 19.4%. |
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