| Streptococcus thermophilus is an important group with the status of "Generally Regarded as Safe"(GRAS)for the manufacture of dairy products including yogurts and cheese.It could intake lactose and hydrolyze it into glucose and galactose by ?-galactosidase.Glucose is converted to lactate through glycolysis pathway while galactose is secreted to the environment,leading to the large amount of galactose and lactose left in the products.The intracellular ?-galactosidase can not only hydrolyze lactose to produce lactate but also exerting transglycosylation function to form oligosaccharides,which could reduce the content of galactose.Therefore,regulating its decomposition activity and transglycosylation activity is conducive to the production of low-sugar and healthy dairy products.However,there is little report about the transglycosylation active site of ?-galactosidase from S.thermophilus.Recently,cell immobilized enzymes have drawn lots of attention because of its high activity and stability,leading to the rapid development of surface display system.But the surface display system of S.thermophilus was rarely reported.In this study,bioinformatic analysis and rational reconstruction was performed on the ?-galactosidase from S.thermophilus SDMCC050237 to improve its transglycosylation activity and the function of enolase from S.thermophilus for surface display was identified and studied.The specific experimental results are as follows:1.Screening of ?-galactosidase with high activity in S.thermophilus?-galactosidase plays an important role in the food industrial application of S.thermophilus.In this study,the ability of S.thermophilus strains to produce p-galactosidase was tested by LM17 plate containing X-Gal and the activity of the enzymes were detected using oNPG as substrate.It was found that there were differences in the activity of?-galactosidase among different strains.After mixing the intracellular crude enzyme showing higher hydrolytic activity with lactose solution,we preliminarily analyzed the sugar composition of the reaction solution by TLC.The results showed that the transglycosylation activity of different ?-galactosidase are weak and no significant difference were observed under natural conditions.2.Enzymatic characterization of ?-galactosidase BgaQ and the optimization of conditions for transglycosylationUsing S.thermophilus CGMCC7.179 as material,we cloned the gene bgaQ and overexpressed BgaQ protein in E.coli DE3 using expression plasmid pET28a(+).After purifying the enzyme by nickel column affinity chromatography and dialyzing,we obtained the 113 kDa target protein.The activity of BgaQ was 4725.3 ±155.4 U/mg for oNPG and 88.3±1.4 U/mg for lactose respectively.The results of enzymatic properties investigation showed that the optimum temperature of BgaQ was 50? and the temperature tolerance of BgaQ ranged from 37?-60?.The stability of the enzyme declined under high temperature.The optimum pH of BgaQ was 8.5 and this enzyme was stable under different pH value.Besides.Na4+,K+,Mg2+,Ba2+ and Co2+ could enhance the activity of the enzyme,while Fe2-,Ni2+ and Co2+ can inhibit the activity of the enzyme,which is intolerant of Zn2+ and Cu2+.The optimal lactose concentration,temperature and pH for GOS production were 25%,50? and 6.5 respectively.The optimum reaction conditions were as follows:lactose concentration of 15%,pH=6.5,and reaction time of 5 h at 50?.The GOS production accounted for 45.0±3.4%.3.Site-directed mutagenesis enhancing transglycosylation activity of p-galactosidase?-galactosidase of S.thermophilu,s has both the hydrolytic activity and transglycosylation activity,the industrial application of which has important significance and broad prospects.In this study,homologous modeling was performed on the screened(3-galactosidase BgaQ according to bioinformatics and the two glutamic acid active sites were identified by sequence overlapping.Using ?-3'-galactosyl-lactose as substrate.we analyzed the active sites by Autodocking and obtained potential transglycosylation related sites,which were aligned with those of other ?-galactosidases.Eventually we constructed the mutant protein mBgaQ with Y801H/P802G.The activity of mBgaQ for oNPG and lactose were 5957.7±209.1 U/mg and 81.9±3.6 U/mg respectively,which was basically the same as BgaQ.The enzyme properties showed that the optimum temperature of mBgaQ was 50?.It could maintain activity ranging from 37?-45? and its tolerance against high temperature decreased.The optimum pH of mBgaQ was 8.5.It was stable in acidic and neutral environments while the stability declined in alkaline environments.Besides,Na+,K+ and Mg2+ could improve the activity of the enzyme.Zn2+,Mn2+,Ni2+ and Co2+ repressed the activity of the enzyme,which was intolerant of Cu2+The results of transglycosylation optimization showed that the optimum lactose concentration,pH and temperature for GOS formation using mBgaQ were 25%,6.5 and 42?,respectively.The optimum reaction conditions were as follows:lactose concentration of 15%,pH=6.5,and reaction time of 5 h at 50?.The final GOS ratio was 58.3±1.4%.,which was 13%higher than that of wide type protein.What's more,the time curves of GOS production by mBgaQ and BgaQ at low lactose concentration was tested.Results showed that the highest GOS production converted by mBgaQ was accounted for 26.7±0.5%,which was 6%higher than that of BgaQ while left less galactose than BgaQ.4.Establishment of a surface display system for S.thermophilus based on enolaseEnolase is a key enzyme in glycolysis pathway.It could not only be active in the cytoplasm,but also be secreted to the cell surface,which is a so-called "moonlighting protein".An enolase gene-enoM was identified from the genome of S.thermophilus CGMCC 7.179 and the protein sequence of EnoM was aligned w ith those of pathogenic Streptococci.The results showed high homology,indicating that the enolase EnoM of.S.thermophilus has potential for surface display.Then we used pET28a(+)plasmid to overexpress and purify GFP protein and GFP-EnoM fusion protein in E.coli DE3 respectively.The binding reaction showed that fluorescence intensity of cells incubated with GFP-EnoM increased significantly,indicating that EnoM anchored GFP protein on the cell surface.To explore the binding mechanism of EnoM,we pretreated the cells with different chemical reagents and no fluorescence was detected in SDS-treated cells,suggesting that EnoM bound to some specific proteins on the surface.Subsequently,we investigated the feasibility of EnoM for surface display in lactic acid bacteria and found that it was function in other LAB strains.To further confirm the surface display function of EnoM,we displayed the high molecular weight ?-galactosidase mBgaQ on the cell surface of S.thermophilus and maintained its activity.The immobilized enzyme could retain 64%of the maximum activity after being used 8 times.In order to verify whether EnoM can achieve target protein expression in.S.thermophilus,we constructed the recombinant strain S.thermophilus CGMCC7.179/pLEISS-D2-gfp-enoM.The intracellular and surface proteins of the recombinant strain were extracted for Western Blot detection.It was found that GFP-EnoM fusion protein was successfully expressed but not anchored to the surface.which may be related to the insufficient secretion and the loss of pathogenicity in S.thermophilus. |
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