Construction and High Expression of β-glucosidase I Gene from Aspergillus aculeatus NO.F 50 in Pichia pastoris
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Abstract:
β-Glucosidaseiswidely used in synthesis of alkyl glucosides and aryl glucosides,as well asdegradation of cellulose. The β-glucosidaseI gene from Aspergilus aculeatus No.F50 was cloned, andmultiple copies of expression cassette 5’AOX-ABGL-TT was inserted intosecreted expression vector pHKA, which could cut apart by the restriction enzymes Bgl II and BamH I. And thenthey were connected together by the use of DNA ligase. The constructed pHKA-(ABGL)3 recombinant plasmid was linearized and integrated into Pichia Pastoris GS115 strain by electroporation. Esculin high-throughput screening method was used to detect positive clones which were cultured in shake flask. In the methanol induction of 120 hours, the supernatant activity was detected using pNPG as substrate. The strain had the maximum supernatant activity of 83.15 U/mL, 3.35 fold higher than previous report. Then it was selectedto realize high-density fermentation using 50 L fed-batch fermentor. The supernatant hydrolytic activity of fermentation in 50 L fermentor wasup to 979 U/mL, which was 2.94 fold higher than control, and protein expression level reached 12.0 mg/mL.
