Enzymatic Synthesis Of Galactooligosaccharide And Its Application In The Treatment Of Acute Ulcerative Colitis

Objective:Ulcerative colitis（UC）is a non-specific colonic and proctitis disease,which belongs to one of the inflammatory bowel disease（IBD）,and is the main cause of inflammatory related colorectal cancer.Galactooligosaccharides（GOS）is a kind of functional oligosaccharides,which can improve the intestinal flora and has a variety of biological activities.GOS is mainly synthesized byβ-galactosidase through transglycosylation.However,there are few reportedβ-galactosidases with transglycosylation activity.Therefore,it is of great significance to search forβ-galactosidase with high transglycosylation activity to prepare GOS for the treatment of acute ulcerative colitis.Research methods:The selective medium containing X-gal was used to screen the marine strains producingβ-galactosidase.The samples were obtained from the sediment surface of the Yellow Sea.The 16S r DNA of the high activity strain was amplified for sequence analysis and recombinant expression.The obtainedβ-galactosidase was purified and characterized.GOS produced byβ-galactosidase was analyzed by TLC.The mice with acute ulcerative colitis induced by dextran sodium sulfate（DSS）were treated with GOS synthesized by enzymatic method.During the feeding period,the basic information of each mouse was recorded every day,and the disease activity index（DAI）was evaluated.After 3 weeks,the colonic tissue and spleen of mice were collected,and the colonic tissue was taken for histological analysis to study the effect of GOS on the acute colitis model of mice.Results:The strain was identified as Alteromonas sp.QD01 by 16S r DNA gene sequence analysis.A newβ-galactosidase gene（gal2a）was cloned from the strain.The gene belongs to the second family of glycoside hydrolases（GH2）.The molecular weight of Gal2A was 117.07 k Da,and the optimum p H and temperature were 8.0 and 40℃,respectively.At the same time,Gal2A showed a wide range of p H stability in the p H range of 6.0-9.5.Most metal ions promoted the activity of Gal2A,especially Mn²⁺and Mg²⁺.TLC showed that Gal2A exhibited high transglycosylation activity and could catalyze the formation of GOS in milk and lactose.Compared with DSS induced acute ulcerative colitis mice,high-dose GOS and low-dose GOS pretreatment significantly reduced spleen weight,reversed weight loss and DAI index increase induced by DSS,and improved colon length shortening and histological damage.The effect of high dose GOS was more significant（P<0.05）.GOS has a good therapeutic effect on acute ulcerative colitis in mice.Conclusion:In this study,aβ-galactosidase-producing marine bacterium was screened,and a newβ-galactosidase was obtained by culturing the recombinant engineering bacterium and purifying the recombinant protein.Gal2A has high transglycosylation activity and can produce GOS.GOS has potential applications in medicine,food processing,and other fields in the ulcerative colitis.