Characterization Of Glycosyltransferase In Trehalose Preparation,Its Molecular Modification And Fermentation Optimization

Trehalose is a safe and non-toxic non-reducing disaccharide.It has special properties of stabilizing and protecting organisms or biological macromolecules,and can be applied to food,cosmetics,biological agents,medicine and agriculture.Trehalose can be obtained by using starch as a substrate and catalyzing by maltooligosyl trehalose synthase?MTSase?and maltooligosyl trehalose trehalohydrolase?MTHase?,which has low cost and great production potential.However,small molecular maltooligosaccharide by-products such as maltose,maltotriose and maltotetraose are often left in the reaction system,which restricts the increase in trehalose production.Glycosyltransferase is added to extend the sugar chain of these small molecular maltooligosaccharides by their glycosylation reaction,so that they can be further utilized by MTSase and MTHase,thereby increasing the yield of trehalose.In this study,seven different glycosyltransferases were selected for the preparation of trehalose,and the cyclodextrin glucosyltransferase derived from Bacillus circulans?BcCGT?was obtained with the best effect.The components of the system after enzyme conversion were analysis,and it was found that many small molecular maltooligosaccharides,which were mainly composed of maltose,were still left in system.In order to continue to increase the substrate utilization rate,a mutant M234I with enhanced affinity for the maltose as receptor was further obtained by directed evolution,which improved the trehalose yield.On this basis,the mutant M234I was recombinantly expressed in Bacillus subtilis,and the efficient preparation of the mutant M234I was achieved at the 3 L fermentor level.The main results were listed as follows:?1?Three 4-?-glycosyltransferase?4?GTase?genes derived from Arabidopsis thaliana,Archaeoglobus fulgidus and Corynebacterium glutamicum were synthesized and heterologously expressed using E.coli BL21?DE3?as a host.These 4?GTase,together with the three cyclodextrin glucosyltransferases?CGTase?derived from Paenibacillus macerans,Bacillus stearothermophilus and Bacillus circulans and the 4?GTase derived from Thermus aquaticus which were recombinantly expressed in the laboratory before,were used in the multi-enzyme complex system to produce trehalose with 150 g·L^-11 maltodextrin?DE 16?as substrate,and addition amount of each enzyme was optimized.The results showed that when 1.8U·mL^-1 of CGTase derived from Bacillus circulans?BcCGT?was added,the yield of trehalose was the highest,reaching 74.1%,which was 24.0%higher than the control.Analysis of the components of the trehalase conversion solution revealed that when the glycosyltransferase was added to the reaction system,the higher the trehalose content,the more the glucose content,and the lower the small molecular maltooligosaccharide such as maltose and maltotriose,maltose was found to be the highest proportion of these small molecular maltooligosaccharides.?2?In order to further increase the yield of trehalose,a high-throughput screening system for improving the affinity of BcCGT for maltose receptor was constructed using E.coli BL21?DE3?as the expression host.After screening the mutant library for Multiple batches,a mutant M234I with increased affinity for maltose as receptor was obtained,and the wild-type and mutant enzymes were isolated and purified,and the results showed that the maltose K_m vaule of the mutant M234I was was only 54.4%of the wild type.The mutant M234I was used in the multi-enzyme compounding system to produce trehalose,and the conversion rate of trehalose was further increased to 77.2%.The components after the enzyme reaction were analyzed,and the contents of glucose and trehalose in the system were increased,while the contents of small molecular maltooligosaccharides such as maltose,maltotriose and maltotetraose are reduced.?3?The BcCGT mutant M234I was recombinantly expressed in B.subtilis WS11 and the fermentation conditions of the recombinant strains were optimized at the shake flask level.The results showed that the best carbon source was glucose,and the best nitrogen source was Corn syrup?Long ke te?and Soy peptone with a ratio of 7:1.Based on the above results,the effect of the carbon-to-nitrogen ratio of the feed medium on the enzyme production of the recombinant bacteria in the 3 L fermentor was investigated.The fermentation results showed that the highest extracellular activity of BcCGT mutant M234I was up to 1423.2 U·mL^-1 when C/N=3:1.