Heterologous Expression And Molecular Modification Of Thermotoga Sp.?-Glucosidase,and Its Application In Preparation Of Gentiooligosaccharides

Gentiooligosaccharides are a kind of functional oligosaccharides with probiotic function.Its?-1,6 glucosidic bonds are not easy to be digested,which is beneficial to the growth of intestinal microorganisms such as Bifidobacterium and Lactobacillus.At present,only Japan can mass-produce gentiooligosaccharides,and China is still in its infancy.A good?-glucosidase is essential for the production of gentiooligosaccharides.This study successfully recombined the expression of?-glucosidase gene TsBgl1A in Bacillus subtilis.We obtained three mutants with improved conversion rate through site-directed mutation,and combining the 3D spatial position of the mutation site,the reasons for the increase in the conversion rate of the mutant were analyzed from the aspects of transglycosylation/hydrolysis ratio and kinetic parameters site.Finally,carried out shake flask optimization and preliminary exploration of 3-L fermentor fermentation among the mutant G224A with the highest conversion rate.The main findings are as follows:?1?The?-glucosidase gene TsBgl1A derived from Thermotoga sp.KOL6 was successfully expressed recombinantly in B.subtilis WS11.Afterwards,the enzymatic properties of the recombinant enzyme were explored,and the optimum temperature and optimum pH were measured to be 90?and 6.0.The recombinant enzyme has good pH stability and temperature stability.The recombinase has good hydrolytic activity to the four disaccharide substrates and pNPG.?2?We explored the effects of different conditions on the preparation of gentiooligosaccharides by recombinant enzymes to determine the optimal enzyme conversion conditions.The reaction temperature was 80?,pH 6.0,the amount of enzyme added was 500U·g^-1,the substrate concentration and the reaction time were 1200 g·L^-11 and 24 h.At this time,the conversion rate could reach 14.48%.The highest yield of gentiooligosaccharides are 175g·L^-1.Based on the industrial production of gentiooligosaccharides,when glucose with a substrate concentration of 800 g·L^-1 was selected as the optimal substrate concentration,the conversion rate of recombinant TsBgl1A to produce gentiooligosaccharides was 10.41%.?3?Through the analysis of the structure of the recombinant enzyme,it was found that the G224 site is likely to have an effect on the transglycosylation activity.Therefore,for the G224site design constructed 10 mutant and successfully expressed.Under the optimal enzyme conversion conditions of wild-type enzymes,explore the mutant's ability to produce gentiooligosaccharides,we found that the conversion rate of three mutants G224A,G224V and G224N was improved compared to the wild type.The rates were 17.01%,14.14%,and 15.45%,which were 1.7 times,1.35 times,and 1.48 times of the wild type enzymes.The reasons for the effects of the mutants G224A,G224N and G224V are analyzed.From the structural point of view,A and V are hydrophobic amino acids with small side chains.They can be used as a hydrophobic bridge through a glucose molecule to generate interactions with the+1 site.The force makes the enzyme more prone to transglycosylation reaction.N is a polar amino acid,and its selectivity to the?-1,6 bond type of gentiobiose has been improved,making the catalytic reaction of the enzyme more conducive to the production of gentiooligosaccharides.We further explored the enzymatic reaction kinetics parameters.It was found that the K_m value of gentiobiose of the three dominant mutants decreased significantly compared with the wild type,from 3.68 mM to 1.81 mM,2.39 mM and 1.15 mM in the wild type.This shows that the mutant's affinity for gentiobiose is significantly increased.In addition,the water/hexanol two-phase reaction system was used to determine the transglycosylation/hydrolysis ratio of the enzyme,and it was found that the transglycosylation/hydrolysis ratio of the three dominant mutants increased significantly compared with the wild type ratio.This further illustrates that the three dominant mutants of G224A,G224N,and G224V can increase the transglycosylation activity of the enzyme,and finally increase the conversion rate of gentiooligosaccharides.?4?The dominant mutant G224A was optimized for fermentation at the shake flask level and preliminary exploration of high-density fermentation at the 3-L fermentor level.The fermentation enzyme activity of mutant G224A in TB medium is 10.35 U·mL^-1.The optimized results of the shake flask fermentation are as follows:15 g·L^-1 industrial peptone,5 g·L^-1 angel corn syrup as combined nitrogen source,5 g·L^-1 glucose as carbon source,and the final concentration of 1 mM Ba²⁺,shake flask fermentation for 24 h,the enzyme activity can reach16.32 U·mL^-1.After that,a high-density fermentation test was carried out on the 3-L fermentor.Based on the shake flask results,the initial medium and the feed medium of the fermenter were determined.The enzyme activity after expanded culture can reach 297 U·mL^-1.30 times before optimization.The scale-up culture of mutant G224A was successfully achieved.