Gene cloning,enzymatic properties and activity mechanism of β-glucosidase Bgls3 from rumen microbiota of cattle
Due to difficulties in the separation and extraction of β-glucosidase,as well as its low enzymatic activity,the industrial ap-plications of β-glucosidase have been restricted.β-glucosidase gene Bgls3 was cloned from the metagenome of rumen microorganisms domesticated from Miscanthus sinensis and subjected to bioinformatics analysis.The recombinant plasmid pET30a(+)-Bgls3 was con-structed and successfully expressed in Escherichia coli BL21(DE3).Subsequently,the enzymatic properties of Bgls3 were investigated,and the catalytic mechanism of Bgls3 was analyzed by homology modeling,molecular docking,and molecular dynamics.The results showed that the gene encoding β-glucosidase,Bgls3,consists of 2 340 base pairs,which encodes 779 amino acids.Bgls3 possesses structural domain characteristics of β-glucosidase.Through SDS-PAGE electrophoresis,the molecular weight of Bgls3 was determined to be 85.27 ku.Bgls3 enzyme showed good activity in a wide temperature and pH range,and the maximum enzyme activity can reach 306.2 U/mg.Homology modeling was used to obtain the structural model of Bgls3,which exhibited structural features consistent with those of glycoside hydrolase family 3.The molecular docking results revealed that the substrate pNPG binds to the pocket region be-tween protein domains Ⅰ and Ⅱ of Bgls3.Key amino acids involved in the hydrolysis of pNPG by Bgls3 were identified as D88,K193,E294,Y240,R160,W273,S395,E486 and H194.Molecular dynamics analysis showed that hydrogen bonding,hydrophobic in-teractions,electrostatic interactions,and water bridge interactions were the fundamental forces involved in the binding of Bgls3 with pNPG,and amino acid residues relevant to the activity of Bgls3 were determined.The results of this study expanded the enzyme re-sources for the study and application of β-glucosidase and provided a theoretical basis for rational modification of Bgls3.
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