The Study Of Cloning And Expression And Its Enzymatic Properties Of ?-glucosidase Of GH1 Family From Lactobacillus Plantarum WU14 And Thermophilic Archaea | Posted on:2022-10-23 | Degree:Master | Type:Thesis | Country:China | Candidate:T T Miao | Full Text:PDF | GTID:2480306731465064 | Subject:Bio-engineering | Abstract/Summary: | PDF Full Text Request | The biodegradation of cellulose is through synergistic effect of cellulases from microorganisms.It is widely used because of its simple process,high conversion rate and no pollution.Cellulases are multi-component enzymes,including Cellobiohydrolase,endoglucanase,?-glucosidase.Among them,?-glucosidase plays a vital role in the efficiency enhancement.It can hydrolyze cellobiose into glucose and effectively enhance the inhibitory effect.It has been widely used in industry,agriculture,food,medicine,bioenergy and other fields.?-glucosidase is mainly derived from natural plants and microorganisms.And?-glucosidases from different environments have different enzymatic properties,such as extreme high/low temperature and extreme acid/alkali.Therefore,exploring the novel?-glucosidase with good enzymatic properties and application potential has important significance.In this study,Glycoside Hydrolase(GH)family 1?-glucosidases derived from Lactobacillus plantarum WU14 and Thermofilum adornatum were heterologously expressed in Escherichia coli.The main results were as follow:(1)Through analyzed the genome of Lactobacillus plantarum WU14,8 GH1family?-glucosidase genes were found.Then the genes were successfully cloned by used polymerase chain reaction(PCR)technology.Based on sequence alignment and bioinformation,all of them were 6-phospho-?-glucosidase.Their sequence identity was between 32-74%and they had two typical conserved glutamate catalytic sites of GH 1?-glucosidases.SDS-PAGE results showed that all eight proteins were successfully expressed in E.coli.Except for Bgl AW14,Bgl CW14,and Bgl FW14which were partially soluble expression,others were all inclusion bodies.Bioinformatics analysis showed that 8 proteins had no signal peptide and transmembrane structure,stronger hydrophobicity.The prediction of subcellular location showed most genes were present in the cytoplasm.The cloning expression and biological sequence analysis of eight 6-phosphate-?-glucosidase genes would lay a theoretical foundation for further research on molecular mechanism of those derived from Lactobacillus plantarum.(2)Gene synthesis of the GH1 family?-glucosidase(Ta BG1)were carried out which derived from Thermofilum adornatum,and the gene encoded a protein with522 amino acids.The result of amino acid sequence alignment revealed that it was only 41%consistent with the identified?-glucosidase(Vul?Bgl1A)from uncultured archaeon.Ta BG1 was successfully expressed in E.coli by SDS-PAGE analysis,and its molecular weight was 59.0 k Da.Ta BG1 showed?-glucosidase activity,the optimum temperature and p H were 95°C and 5.0,respectively.Under optimal conditions,when the substrate was p NPG,the Kmand Vmaxwere 0.56 m M and 2199?mol/(mg·min),and the catalytic efficiency(Kcat/Km)was 3900(1/(m M s)),which was higher than most of GH1?-glucosidases reported in archaeal origin.Ta BG1 had substrate specificity.When the substrates were p NPX,p NPMan,o NPG and Sophorise,the corresponding enzyme activities(U/mg)were 96,32,70,and 73,indicated that?-glucosidase Ta BG1 had a positive effect on the p NPG substrate and relatively weak hydrolysis ability for other substrates.The enzyme activity of Ta BG1could be enhanced by Cd2+,but would be completed by Ag+or SDS,significantly affected by most mental ions.In addition,the enzyme had good glucose tolerance,which had a certain activation effect when in low concentration of glucose(<0.5 M),and still kept65%of the enzyme activity under 2 M glucose.The enzyme had tolerance to methanol and ethanol,when it in low-concentration methanol and ethanol buffer had a certain activation.Ta BG1 had many excellent enzymatic properties and application value.Further,using bioinformatics and molecular technology to study Ta BG1 would provide a theoretical and application basis for the molecular mechanism of GH1?-glucosidase from archaea.?-glucosidase occupies a very important role in many applications,so the study of?-glucosidase had high application value.?-glucosidase included phosphorylated?-glucosidase and?-glucosidase.In this study,the 6-phospho-?-glucosidase derived from Lactobacillus plantarum WU14 and?-glucosidase derived from thermoarchaea both had laid a theoretical foundation for the research of?-glucosidase produced by different microorganisms derived from the GH1 family and profound significance for the research of?-glucosidase. | Keywords/Search Tags: | GH1 family, ?-glucosidase, Gene cloning, Expression, Properties | PDF Full Text Request | Related items |
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