棘孢曲霉β-葡萄糖苷酶I的毕赤酵母多拷贝体系构建及其在高密度发酵中的表达

Construction and High Expression of β-glucosidase I Gene from Aspergillus aculeatus NO.F 50 in Pichia pastoris

摘要:

β-葡萄糖苷酶目前广泛应用于合成烷基糖苷、芳基糖苷和辅助纤维素酶水解纤维素。本研究将来自棘孢曲霉（Aspergilus aculeatus）的β-葡萄糖苷酶I (ABGL)在毕赤酵母中通过构建多拷贝表达盒的方法实现高效表达，利用同尾酶Bgl II/BamH I反复酶切和用DNA连接酶连接的方法，成功构建了含多拷贝表达盒5’AOX-ABGL-TT的pHKA-(ABGL)3的分泌重组质粒，并线性化重组质粒转化毕赤酵母GS115，经过七叶苷显色筛选平板筛选获得高表达的GS115/pHKA-(ABGL)3菌株，且在50升发酵罐实现高密度发酵表达。研究表明，以pNPG为水解底物，在甲醇诱导120 h时摇瓶发酵液上清酶活可达83.15 U/mL，较之前报道的酶活提高了3.35倍；同时在50 L发酵罐实现高密度发酵，在甲醇诱导120 h时发酵罐上清酶活可达979 U/mL，较对照菌株的发酵罐上清液酶活提高2.94倍，且总蛋白表达量可以达到12 g/L。

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.