半乳糖o乙酰转移酶基因的克隆表达及功能研究粮食油脂及植物蛋白工程专业论文.docxVIP

ABSTRACTLactic ABSTRACT Lactic acid bacteria(LAB)are generally regarded aS safe food-grade microo唱anisms， and exopolysacch撕de(EPS)is an importam seconda巧metabolite of LAB．EPS is able to boost i删nuIli饥 against caJlcer aJld other 如nctions， moreoVer， EPS haVe a great Value in industrial印plications．HoweVer，the production of EPS is relatiVely 10w． How to improVe the yield of LAB expolysaccharide is a research hotspot，the key pan is researclling the mechallism of expolysaccharide biosynthesis．In the process of EPS fomation，acetyl group are c删ed on these carbohydrate chains．The de伊ee of the acetoxylation，eSpecially 0-acetylation，not only challges the physical stmctWe of the sugar chain，but also hinders the syne唱y between polysacch撕de chains． The whole genome sequencing of三口c幻6口c讲螂砘缈口门Q向cfP，2s ZW3 carries a 1 4．4 kb EPS gene cluster(Ⅵ验NGj 283 to WANGj 299)，which contains 1 7 EPS—related genes． WA NG—l 29 1 was picked out fIrom exopolysacch撕de genes to be mIrther studied． Bioinfo姗atics a11alysis showed that it belongs to LbetaH super雠1ily which oWns ConseⅣed Domain LbH MAT like that catalyzes t11e CoA—d印endent acetylation of t11e 6一hydroxyl group of their respective sugar substrates and is aIl in昀cellular protein． Tb identify t11e fhnction of WAN(Ll 29 1，it was expressed in E∞f，BL2 1(DE3)with plasndd pET3 0a as plasmid Vector．After inducing with IPTGl SDSanalysis of cell lysates showed that there waS a new baIld in site of 20kD as expected，t11en the protein waLs purified with Ni什一NTA a伍nity c11romatogr锄．By a11alyzing the reaction products after galactose， acetyl·CoA a11d protein mixed in 3 7℃，we fouIld that galactoside 0一ace够ltransferaSe waS pmduced by maSs spectmm detection．Characteristic enz)rme l(inetic data were also obtained and optimum actiVit)，was observed at pH 7．4 and 30℃．A Michaelise-Menten kinetic equation could be fit to the experimental data a11d a Km of O．6 m MⅥ，as calculated． For understanding仕le mnction ofⅥ‘州Gj29 1 in the bios)，11t11esis of EP