Isolation, Purification And Structure Characterization Of Thermal Resistant β-glucosidase Of Aspergillus Niger

β-glucosidase is also known as cellobiose(EC3.2.1.21).It is the primary component of cellulases which can hydrolyze the glycosidic bond between glycosyl groups and hydroxyl or aryl groups to release glucose.Obtaining the β-glucosidase that possesses good thermal stability,high specific activity,and heavy output has been focused on cellulase study.β-glucosidase secreted by Aspergillus niger 3.316 possesses the good thermal resistance.In this study,the β-glucosidase was research object,the rough extraction conditions of fermentation broth were optimized,the β-glucosidase was isolated and purified,the tolerance of β-glucosidase for ethanol and PEG solution was characterized.The active site and thermal resistant mechanism of β-glucosidase were also characterized using biological information method.The main results were as follows:1.The rough extraction conditions of β-glucosidase fermentation broth were optimized using fractional precipitation of ammonium sulphate,ethanol,PEG,and ultrafiltration,the optimized conditions were 50% of ethanol precipitation combined with ultrafiltration.2.After the rough extraction of fermentation crude enzyme solution of Aspergillus niger,the β-glucosidase was isolated and purified by ion exchange and gel filtration using AKTA P-urifier100,the subunit molecular weight ofβ-glucosidase of Aspergillus niger 3.316 was 60 kDa using SDS-PAGE.3.The tolerance of β-glucosidase for ethanol and PEG was investigated by determining the change of activity of β-glucosidase,which β-glucosidase was pretreated from 10~60 minutes in the 10%~80% of ethanol and 10%~90%of PEG,respectively.The results showed that there were significantly difference among different pretreatment time,different concentration of ethanol or PEG as well as different pretreatment time and different concentration of ethanol or PEG.β-glucosidase was relatively well tolerated in 50% ethanol for 40 minutes pretreatment,and alsotolerated in 20% PEG for 30 minutes.The activity was 0.511U/mg,and 0.852U/mg,respectively.4.The extracted genomic DNA of Aspergillus niger 3.316 was used as template to ampilify the bgl gene by PCR,which the amplicon was 2?080 bp.The nucleotide sequence,amino acid sequence,and structure of encoded protein were characterized.The active site and thermal resistant mechanism were investigated based on some correlative literature and structure of encoded protein.The results showed that the molecular including 860 amino acid residues and 4 major hydrophobic regions with a maximum score of 1.343.The protein secondary structure might contain 63.26%random coils,20.58% α-helix,and 16.16% β-fold.Prediction of protein domain through NCBI CDD showed that there were three domains,(β/α)8 TIM barrel domain,(β/α)6 sandwich sandwiches domain,and fibronectin III-like domain,in which the former two domains could be classified to glucoside hydrolase family3.The function of the third domain was unknown but it was connected to the former two domains.The active site and themal resistant mechanism were predicted using the basic local alignment sequence tool(BLAST)and the SWISS MODEL,the results indicated that Asp280,Trp281,Gly294,Asn405,Pro410,and Glu509 were conservative amino acid which were located in the active center of protein.The active sites of the glucosidase might be Asp280 of(β/α)8 TIM barrel domain and Glu509 of(β/α)6 sandwich sandwiches domain.The stability of the glucosidase might be related to 211 amino acid residues in the hydrophobic region,48 prolines,and 20.58% α-helix.