Preliminary Research On Characterization、Optimization And Application Of GH1 Β-glucosidase Bgl1356

β-Glucosidase can hydrolyze non-reducing β-D-glucoside bonds to produce β-D-glucoside and corresponding ligands,which plays an important role in the extraction process of plant functional substances.However,natural β-glucosidase has problems such as high acquisition cost and low enzyme activity,and it is difficult to apply to industrial production.Therefore,natural β-glucosidase with excellent properties needs to be developed urgently.In this study,a β-glucosidase gene bgl1356 with a length of 1356 bp and encoding 451 amino acids was screened from the soil metagenomic library established in the laboratory,and analyzed by bioinformatics technology.β-glucosidases all share the same conserved sequence TENG.The recombinant β-glucosidase Bgl1356 was obtained by prokaryotic expression of the gene bgl1356.The conditions of its inducible expression were optimized and the enzymatic properties of the recombinant enzyme were studied.The results showed that the best time for induction: 8 h;the best temperature: 25℃;the best IPTG concentration:0.8 mM.In terms of enzymatic properties,the optimum reaction temperature of Bgl1356 is 40℃,and the relative enzymatic activity is greater than 40% between30℃-70℃;the optimum reaction pH is 5.0,and the relative enzymatic activity retains 50%,at pH 5.0-8.0 Above,it has strong adaptability to temperature and pH.In addition,Bgl1356 has good tolerance to most metal ions,organic solvents,and salt and glucose solutions.The enzymatic kinetics calculation showed that the Vmax=3.786 mM/min,Km=0.2472 mM of Bgl1356.The five medium components of recombinant Bgl1356 strain were optimized by single factor method,and then the optimal fermentation conditions were obtained by response surface methodology design.The results showed that when the carbon source was 16 g/L starch,the nitrogen source was a 1:1 compound nitrogen source of peptone + yeast powder,and the inorganic salt was 8 g/L dipotassium hydrogen phosphate,the inoculum volume and the liquid volume were respectively at 12% and 50 mL,the maximum enzyme activity was 2596.3 U/L,which was 1.56 times higher than that before optimization.Bgl1356 was immobilized with chitosan as carrier to improve its enzymatic properties.The single factor method was used to obtain the optimal immobilization conditions of carrier dosage of 0.6 g,adsorption temperature of 40℃,adsorption time of 8 h,and optimal amount of adsorbed enzyme of 0.8 mL.The immobilized enzymatic properties were investigated,and it was found that the optimal reaction temperature of immobilized Bgl1356 increased by 10℃,and the thermal stability was significantly increased under the high temperature of 50℃-70℃;The activity was as high as more than 60%,indicating that the acid-base stability was also optimized;after 9 days of storage at low temperature and room temperature,the relative residual enzyme activity remained above 80%.The resveratrol glycosides in Polygonum cuspidatum were hydrolyzed by β-glucosidase Bgl1356,thereby increasing the content of resveratrol.First,the optimal enzymatic hydrolysis system was explored.The results showed that the content of resveratrol was the highest when the amount of enzyme added was 8 mL,the pH was 6.0,the enzymatic hydrolysis time was 4 h,and the enzymatic hydrolysis temperature was 45℃.Under the optimal enzymatic hydrolysis state,the hydrolysis rate of resveratrol glycosides reached 90%,and the content of resveratrol in Polygonum cuspidatum after enzymatic hydrolysis was increased by62.3% compared with that before enzymatic hydrolysis,which indicated that Bgl1356 had a higher concentration of resveratrol.The enzymatic hydrolysis process has high feasibility.In this study,Bgl1356 was explored from the aspects of bioinformatics,prokaryotic expression and enzymatic properties,optimization of fermentation conditions,immobilization and application potential,found that Bgl1356 has high application value in the process of hydrolyzing resveratrol glycosides and then extracting resveratrol.It also provides a good demonstration material for the properties and structures of GH1 family β-glucosidases,broadens the research field and types of β-glucosidases,and provides reference and data for the further development of new β-glucosidases.support.