Screening The Aspergillus Niger Strain Of Over-expression Thermostable β-glucosidase And Its Gene Cloning

Aspergillus niger is not only safe but also can produce more β-glucosidas-e than the other microorganism. As a foreign gene expression system, Aspergil-lus niger has more specific advatanges such as high secrete performance,high strength promoter, outstanding after-translate processing ability and maturefer-mentation technology than other foreign gene expression systems(bacterial,yeast). β-glucosidase(EC.3.2.1.21)plays a key role during cellulose degradation,while its low content and degradation activity is the bottleneck of the cellulose degra-dation process. It is significant to produce a new β-glucosidase thatcould deg-rade cellulose effectively by gene recombination technique.In this study Aspergillus niger3.316which could produce thermostableβ-glucosidase and Asperqillus niger3.2130which could produce high activityβ-glucosidase were screened out from five Asperqillus niger strains, and as researchobjects, some research were done to provide a basis for expressing Aspergillus nigerβ-glucosidase gene, such as optimization of fermentation contions, protoplast DESmutagenesis, isolation and purification of β-glucosidase and some research werestudied, gene cloning and sequence analysis. The main results were shown asfollows:(1) The best growth conditions for Aspergillus niger3.2130which could produce thehighest activity β-glucosidase by response surface analysis method were as below:bran17g/L, sulfate+peptone5g/L(1:1, W/W), salicin+CMC3g/L(1:2, W/W), initialpH5.0, the content of fermented bottle was63mL, the inoculation was7mL, therotation speed was154r/min. The activity of β-glucosidase could reached1.352U/mL under the optimized culture medium.(2) Two mutants which could produce low activity acid prolealytic enzyme werescreened from Aspergillus niger3.2130protoplast by DES mutagenesis, and wereexpected as the cloning host strains.(3) Pure β-glucosidase was obtained by the separation and purification, the molecular mass of the subunits was estimated to be127KDa by sodium dodecylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE), the optimal temperatureand pH value were60℃and4.4, respectively. The enzyme activity could keep above72.6%in65℃, and demonstrated it belonged to heat-resisting enzyme.(4) With genomic DNA of Aspergillus niger3.316as a template, β-glucosidase(bgl)gene was amplified by polymerase chain reaction(PCR), and designated as bgl3.316(GenBank accession number: JN969085). cDNA sequence was obtained by splicingexons of bgl gene, the analysis of amino acid gene indicated that the gene sequenceencoded a protein-bgl containing860amino acid. This work laid the foundation forthe latter part of the bgl gene expression in Aspergillus niger3.2130.