【摘要】 目的:为提高β-葡萄糖苷酶的产量,用毕赤酵母取代理氏木霉用于生产,以弥补理氏木霉在大规模生产中的缺陷。方法:用套叠PCR法从理氏木霉基因组中扩增β-葡萄糖苷酶基因(bglⅠ)。用T4DNA连接酶和限制性DNA内酶将bglⅠ重组于P.pastoris表达载体pPIC9K的多克隆位点,获得含bglⅠ的重组表达载体pPIC9K-bglⅠ。通过电转法将其pPIC9K-bglⅠ载体转化于P.pastoris基因组,筛选高G418抗性以及高表达bglⅠ酶的重组子作为工程菌。结果:用BMGY-BMMY培养基体系,在摇瓶中发酵48 h,表达BglⅠ30 mg/L,在P.pastoris中表达的BglⅠ能水解对硝基苯-β-D-葡萄糖苷具有β-葡萄糖苷酶活性。其酶活力为56 U/L发酵液。结论:通过这种方法,可以成功地用毕赤酵母表达理氏木霉的β-葡萄糖苷酶基因。更多还原

【Abstract】 Objective:Construction of recombinant P.pastoris replaced of Trichoderma reesei to produce of β-glucosidase to increase the yield of the enzyme.Method:β-glucosidase gene(bgl) was amplified from Trichoderma reesei.After sequence,the DNA segment was inserted directly into pPIC9K to generate P.pastoris expression plasmid of pPIC9K-bglⅠ which was then transformed into the P.pastoris of GS115 by electroporation method.A strain on higher anti-G418 was used as engineering strain.Result:With the medium system of BMGY-BMMY to ferment the engineering strain for 48 h,30 mg/L of recombinant β-glucosidase is secreted into the fermentation liquid,which can hydrolyze 4’-Nitrophenyl-beta-D-glucopyranoside to show of β-glucosidase activity in the degree of 56 U/L fermentation liquid.Conclusion:The β-glucosidase gene from Trichoderma reesei was successfully expressed by recombinant P.pastoris.更多还原