Screening, Identification Of Fungi Producing β-glucosidase, And Purification, Characteristics, Cloning Of β-glucosidase

P-glucosidase is one important consist of cellulose hydrolases, and its activity impacts the level of hydrocellulose by cellulose hydrolases. For the past few years, with the extensive applications in food processing, forage producing, medicine, chemical industry,β-glucosidase is catching more and more attentions.One Aspergillus oryzae strain possessing higher level of P-glucosidase named giF-10 was isolated from the oil of Zhougong Mountain and fermented to make a great deal ofβ-glucosidase. The P-glucosidase producing by Aspergillus oryzae strain giF-10 was purified, analyzed in its kinds of characters,and its gene bgl was cloned in this paper.1.Three fungus which have cellulase activities were screened from the oil of Zhougong Mountain,Yaan. A strain named giF-10 was detectde to have the highest activity ofβ-glucosidase which can be 2.522U U/mL. Accordingly, giF-10 was choosed for futher study. Based on morphological characteristics, the texture of colony is rarefacting, distal end is radiated producing green and oval spores. Sequence alignment of ITS DNA amplified from chromosomal DNA through PCR, the length was 594bp.Utilizing the Neighbor-Joining of MEGA4.0 building the cladogram of phyletic evolution, proving both the ITS homology of giF-10 with Aspergillus oryzae and Aspergillus flavus are 99%, and the ITS homology of giF-10 with Aspergillus oryzae strain UPM A22 is 100%.So giF-10 was identified as Aspergillus oryzae.2. Aspergillus oryzae giF-10 was fermented by shake flask to get a great deal of P-glucosidase. Crude liquid enzyme was purified by using ammonium sulfate precipitation, Sephadex G-100 gel chromatography, DEAE Cellulose 52 ion exchange chromatography. Multiples of purification are 6.39,8.08 and 11.31, respectively. The specific activity of purified P-glucosidase is 40.84U/mg finally.3. Molecular weight ofβ-glucosidase was about 90kDa as identified by SDS-PAGE. Its optimum temperature and pH were 55℃and 4.5, respectively. It was stable uande 30-50℃and in pH4.0-6.0. Different metal ions showde different effects onβ-glucosidase activity. Mn2+enhanced its aactivited while Fe3+and Cu2+caused obvious inhibition. The enzyme showde stronger substrate specificity to salicin and cellobiose. Its Km for salicin and cellobiose are 0.676 mmol/L and 2.906 mmol/L, respectively.4.Primers were designde based on theβ-glucosidase gene of Aspergillus oryzae RIB40 published in genebank. With the primers, the nucleotide sequence of the P-glucosidase gene was amplified from chromosomal DNA through PCR. The length of the bgl gene was 2903 bp which shared a 99% sequence identity with the bgl gene from Aspergillus oryzae RIB40 submitted in GenBank, and wad severed by five introns. Exon was 2581bp, encoding 865 amine acids, containing a signal peptide length of 30 amine acids. Molecular weight of P-glucosidase was predictde to be 93.69 kDa through PROTEIN CALCULATOR v3.3.