Structure And Function Of β-glucosidase BGL Ⅲ From Rhizopus Stolonifer

Cellulase is a multi-enzyme consists of three different enzymes: exoglucanase, endoglucanase and β-glucosidase (BGL). It is the three enzymes that collaborative hydrolyze cellulose. P-glucosidase as a rate-limiting enzyme in the hydrolysis of cellulose, become the bottleneck of enzymatic hydrolysis of lignocellulose and bioethanol generation process. Currently, how to improve the β-glucosidase activity and optimized cellulase composition has become a major focus of research.Rhizopus stolonifer TP-02 is a strain of Ascomycetes, which has the advantages of high efficiency of lignocellulose degradation ability of zygomycotina Rhizopus stolonifer subspecies nod Rhizopus.On the basis of previous research, We cloned a new β-glucosidase gene and the key sites of the structure and function of the enzyme have been studied.Genetically engineered bacteria were successfully constructed and the enzyme activity is increased. Main research results are as follows:(1) The study successfully extracted Rhizopus stolonifer genomic DNA and single-stranded cDNA, cloned a new β-glucosidase gene named bgl3.bgl3 was expressed in a prokaryoticis.coli BL21, using esculin screening medium to screen positive clones. The recombinant bacteria fermentation experiments showed with IPTG induction, the activity reached a maximum value of 0.788 IU/mL after 12 h of fermentation.(2)Bioinformatics software was used to predict and analyze the structure and function of bgl3. Sequence analysis showed that the full-length of bgl3 was 1452bp, encoding 484 amino acids. BGIII which bgl3 encoded contained two typical GH1 characteristics sequences, belonging to Glycoside Hydrolase family 1. BGIII existed no typical transmembrane region which was in theory for soluble good globulin.(3)The amino acid sequence of BGIII and GH1 was compared and analyzed, DS 2.5 was used to build homology modeling. Cellobiose as the substrate molecule was docked, to simulate the interaction of enzyme and substrate in the active site. The result showed that the tertiary structure of BGLIII is a classical (α/β)8-TIM barrel which contained two glutamic acid residues as key active sites. Cellobiose closed to the binding pocket wall, deeping into the binding pocket and hydrolyzing to glucose under the action of the key residues. According to bioinformatics and molecular dynamics analysis, Trp 127 was determined and mutated to Ala, Phe and Leu with different side chains.Trp was located in the center of the active side and played an important role in the active site-substrate binding process. When Trp 127 mutated to Ala without side chain, the spatial distance with Glu173 increased and the steric hindrance was changed, therefore the conformation of glycoside binding site can not be maintained, resulting in a significant reduction in enzyme activity. When mutated to Phe with the same benzene ring, hydrogen bonds and hydrophobic force changed little Which further evidented the importance of spatial conformation of amino acids. When mutated to Leu with a non-benzene long chain, enzyme acivity was greatly improved. Probably because Leu could not olny maintain the original conformation but also shorened the spatial distance of Tyr315 and Glu384 with the substrate, which increased hydrogen bond energy. Thus the hydrolysis force of β-1,4 glycosidic bond enhanced,so did the hydrophobic interaction of glucose and glucose tolerance.(4) Site-directed mutagenesis was compeleted by overlap PCR. Three recombinant strains of E. coli was successfully screened. Fermentation results showed the activity of pET-22b(+)-W127L reached a maximum value of 1.090 IU/mL, compared with pET-22b(+)-W127 improved 34.07%. The SDS-PAGE results showed BGIII molecular weight was 54 kDa and pET-22b(+)-W127L had higher expression levels.(5) Purification and Characterization of the analysis was done. The optimum temperature and thermal stability studies showed the optimum temperature of BGLⅢ was 55℃, maintaining more than 90% of the enzyme activity between 30℃ to 60℃, which meaned a wide temperature adaptability; The enzyme activity decreased rapidly over 70℃. The optimum pH and pH stability studies showed the optimum pH was 5.0, the enzyme could keep more than 80% of the activity at pH 4 to pH 7.0. Effect of metal ions on BGLⅢ suggested that Mg2+、Mn2+ and Fe2+ had promotion on BGLⅢ activity, among them, Mg2+ was the most obvious. Hg2+、Ca2+、K+、Cu2+ and Zn2+ had inhibition on BGLⅢ activity, among them, Hg2+ and Cu2+ were the most obvious.(6) Glucoamylase promoter PGlaA gene fragment was cloned with Aspergillus niger genomic DNA as template. By overlap PCR method, the glaA was connected with bgl3 to construct hybrid gene glaA-bgl3. Homology arms and hygromycin B selection marker were built to glaA-bgl3. The heterozygous gene was successfully transformed creeping branches Rhizopus spores.The fermentation experiments of transformed strain and Rhizopus stolonifer TP-02 showed that the β-glucosidase activity was increased by 23.9%.On the basis of experiments and bioinformatics analysis, structure and function of Rhizopus stolonifer β-glucosidase bgl3 was analysed. Prokaryotic expression of recombinant bacteria and Rhizopus stolonifer transformants were constructed.The study might provide a basis for future study of Rhizopus stolonifer β-glucosidase.