| Galactosides are a group of substances that are widely found in plants and animals and are involved in a variety of physiological activities;and are widely used in food,pharmaceuticals,health products,and cosmetics.As predominant galactolipids,galactoglycerides are a group of highly valuable compounds that can be used in a wide range of pharmaceutical and food applications.However,current synthetic methods are limited by high cost and low product purity.Galactooligosaccharide(GOS)is an officially acknowledged prebiotic with the physiological activity of promoting intestinal probiotics and inhibiting the growth of harmful bacteria.The main approaches to obtaining GOS are extraction and fermentation.The extraction method is complicated,with high costs and limited yields.The fermentation method has low yields,complex product composition,and is difficult to isolate and purify.Enzymatic synthesis can be carried out under mild conditions,with high yields and the ability to synthesize products with high regioselectivity and stereoselectivity.In this thesis,we explored the application of synthesizing structured galactoglyceride by Tn0602 cascaded lipase.In addition,we rationally designed the β-glucosidase Tn0602 derived from Thermotoga neapolitana RKU-10 to reveal the catalytic mechanism of GOS3 synthesized by Tn0602 sitedirected mutagenesis.Mutants with increased transglycosylation/hydrolysis ratios and improved GOS production yield were obtained.In this study,we constructed a novel in vitro synthetic pathway.Cheap substrates lactose and glycerol are involved and synthesize structured galactosylmono/diacylglycerol in coordination with Tn0602 and lipase.A t-butanolwater system was used to optimize the yield of the Tn0602-catalysed transglycosylation reaction of lactose and glycerol to synthesize the intermediate β-galactosylglycerol.In a system with an initial concentration of 1 M lactose,5 M glycerol,and 30% water content,Tn0602 achieved a yield of 416.9 mmol/L after 12 hours.The esterification reaction of intermediate β-galactosyl glycerol with lauric acid catalyzed by the lipase Novozym 435 resulted in the successful synthesis of monogalactosylmono/bilaurate esters.This work expands the application of Tn0602 and,more importantly,enables the large-scale synthesis of galactolipids,thus promoting their widespread application.Tn0602 catalyzes the exclusive synthesis of GOS3 from lactose.To investigate the specificity in synthesizing GOS3 and reaching a higher degree of polymerization of GOS,rational design and molecular modification were carried out around the +2aglycone site of the GH1 family thermophilic β-glucosidase Tn0602.Six single-site mutants were constructed around the +2 site based on the molecular docking results.At 75°C,the product synthesized by the single-site mutant F226 G was a mixture of GOS3 and GOS4 with yields of 32.0% and 5.1%,respectively,along with a 10%increase in transglycosylation activity and a 2-fold increase in the transglycosylation/hydrolytic ratio compared to the wild type.The molecular docking of the mutant with GOS3 indicates that the +2 region blocked the space of the active site and the repulsion with the glycosyl ligand affects the degree of polymerization of the Tn0602-catalyzed product.GOS with a higher degree of polymerization is more tolerant to fermentation by regulated intestinal bacteria,thus exerting a prebiotic effect at a more distal position in the gut.An increase in the yield of GOS synthesized by Tn0602 based on improved product polymerization could make it a more valuable candidate for industrial enzymes.According to previous studies,mutants F414 S and F414Y(-1 glycone site)have higher transglycosylation activity compared to the wild type.The two sites,F226 and F414,are far apart and have little potential to interact;therefore,F226 G was chosen to superimpose with F414 S and F414 Y,respectively,to obtain the most significant superimposition effect.Thus,superimposed mutants F226G/F414 S and F226G/F414 Y at the +2/-1 site were constructed,and their viability for GOS synthesis was explored.Total GOS yields for both F226G/F414 S and F226G/F414 Y synthesis exceeded those of F226 G and wild type at 75°C,p H 6.5.The yield of GOS3 synthesized by F226G/F414 S after 12 h of reaction was 240.80 m M,and the yield of GOS4 after 4 h was 72.75 m M,which is the highest yield reported so far.Analysis of the product structures by 1D and 2D NMR revealed that the GOS synthesized by Tn0602 is a mixture of both β(1 → 3)and β(1 → 4)bonds.Based on molecular dynamics simulations over a range of 300 ns,it was discovered that the single point mutation F226 G resulted in higher flexibility of the +2 site and that the flexibility of the +2 site had a significant impact on the degree of product polymerization.F226G/F414 S has steady stability of the enzyme-GOS3 intermediate,which enables the stable and highly productive synthesis of GOS3 and GOS4.However,due to the structural limitations of the catalytic pocket,the production of GOS5 was not observed,by reducing the space block at the +2 site made it possible to produce high purity GOS3 and GOS4 mixture with Tn0602. |
