Heterologous Expression And Biological Characteristics Of UGPase From Lactobacillus Acidophilus

UDP-glucose pyrophosphorylase(UGPase)can catalyze the synthesis and decomposition of UDP-glucose(UDPG),an important nucleotide sugar substrate,is a key rate-limiting enzyme in the process of glucose metabolism.They are widely distributed in nature.The biological characteristic of UGPase,active sites,polysaccharide yield,and freeze-drying stress are not clear in lactic acid bacteria.In this study,the UGPase gene of Lactobacillus acidophilus ATCC4356 was over-expressed in E.coli,and the biological characteristics were analyzed.The effects on polysaccharides yield and lyophilized survival rate of E.coli were investigated.The main results are as follows:1.Two genes(LBA1719,LBA0625)encoding Lactobacillus acidophilus UGPase(UGPase-1719,UGPase-0625)were cloned and connected with p ET-30a(+)vector,then transformed into E.coli Transetta(DE3)to construct recombined E-1719 and E-0625.The enzyme production conditions of the recombinant strain were optimized.When E-1719 was induced at 37?,induced by 0.2 mMIPTG for 4 h,the enzyme activity was highest(376.41U/L)which was 1.58 times that of the control group(238.94 U/L).When E-0625 was induced at 25?,induced by 0.4 mMIPTG for 6 h,the enzyme activity was the highest(366.66 U/L),which was 1.51 times that of the control group(242.61 U/L).The target protein was further purified by affinity chromatography.2.The bioinformatics software was used to predict and analyze the proteins UGPase-1719 and UGPase-0625,which was encoded by LBA1719 and LBA065,respectively.The results showed that UGPase-1719 and UGPase-0625 were belong to the glycosyltransferase family of stable hydrophilic proteins,containing 294 and 300 amino acids.Prediction analysis of the conserved domains revealed that UGPase-1719 and UGPase-0625 each had 20 conserved active sites.The homology modeling analysis of its secondary and tertiary structure showed that UGPase was mainly composed of three parts:?-helix,?-sheet and random coil.Among them,?-helix and random coil were the main secondary structures,which constituted the main skeleton of UGPase.3.The enzymatic properties of L.acidophilus UGPase were determined to determine the optimal reaction conditions.The optimal temperature of UGPase was 37?,the optimal p H was 10.0,and the optimal substrate binding concentration was 0.5 mM.High concentration substrate would inhibit enzyme activity.Mg²⁺was necessary for UGPase to exert catalytic activity.4.The different sites P9,A10,A11,L130,L263,respectively on recombinant plasmids E.coli E-1719 and E-0625 were mutated.According to the changes of the enzyme activity of UGPase to determine the key active sites of UGPase-1719 and UGPase-0625.The result showed that either site was mutated that had an effect on the enzyme activity of UGPase.Among them,when mutated the sites A11,L130,L263 of E-1719 and E-0625,the enzyme activity had significantly difference between the control group(P<0.01).Then,it indicated that these sites may be the key active sites of UGPase.Mutation of these sites prevented them from binding to the corresponding substrates,which to a certain extent inhibited the active center from functioning,resulting in diminished UGPase dimer or multimer activity.5.The yield of polysaccharide and lyophilized survival rate of the E.coli,which overexpressing UGPase were measured.The results showed that the polysaccharide production of strains E-1719 and E-0625 increased by 66.34%,48.79%respectively,compared with the control group,which were 1.66 times and 1.49 times of the production of the control group.The survival rate of recombinant bacteria E-1719 and E-0625 after freeze-drying were 12.59% and 2.48% higher than those of the control group,respectively.In this study,the UGPase of L.acidophilus was heterologously overexpressed and purified.For the first time,the enzymatic properties,active sites,and structural simulation of L.acidophilus UGPase were studied,which provided a theoretical basis for in-depth understanding of the biological characteristics of lactic acid bacteria UGPase.Overexpression of UGPase could increase polysaccharide yield and lyophilized survival rate.The method could be applied to improve the lyophilization survival rate of lactic acid bacteria and increase the activity of the starter.It also could promote the production of extracellular polysaccharides and improve the texture of fermented yogurt in-depth.Finally,it be used to industrial production of extracellular polysaccharides,which has good application prospects.