The Study Of Cloning And Expression And Its Enzymatic Properties Of β-glucosidase Of GH1 Family From Lactobacillus Plantarum WU14 And Thermophilic Archaea

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,β-glucosidase.Among them,β-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.β-glucosidase is mainly derived from natural plants and microorganisms.Andβ-glucosidases from different environments have different enzymatic properties,such as extreme high/low temperature and extreme acid/alkali.Therefore,exploring the novelβ-glucosidase with good enzymatic properties and application potential has important significance.In this study,Glycoside Hydrolase（GH）family 1β-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 GH1familyβ-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-β-glucosidase.Their sequence identity was between 32-74%and they had two typical conserved glutamate catalytic sites of GH 1β-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-β-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 familyβ-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 identifiedβ-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 showedβ-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 K_mand V_maxwere 0.56 m M and 2199μmol/（mg·min）,and the catalytic efficiency(K_cat/K_m)was 3900（1/（m M s））,which was higher than most of GH1β-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 thatβ-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 GH1β-glucosidase from archaea.β-glucosidase occupies a very important role in many applications,so the study ofβ-glucosidase had high application value.β-glucosidase included phosphorylatedβ-glucosidase andβ-glucosidase.In this study,the 6-phospho-β-glucosidase derived from Lactobacillus plantarum WU14 andβ-glucosidase derived from thermoarchaea both had laid a theoretical foundation for the research ofβ-glucosidase produced by different microorganisms derived from the GH1 family and profound significance for the research ofβ-glucosidase.