扩展青霉碱性脂肪酶基因在毕赤酵母中的高效表达  

Overexpression of Penicillium expansum Lipase Gene in Pichia pastoris

作　　者：袁彩[1] 林琳[1] 施巧琴[1] 吴松刚[1] 

机构地区：[1]福建师范大学生物工程学院,福州350007

出　　处：《生物工程学报》2003年第2期231-235,共5页Chinese Journal of Biotechnology

基　　金：国家自然基金项目 (No.30 2 70 0 33);福建省自然科学基金重点项目 (No .B0 12 0 0 0 1)资助。~~

摘　　要：将编码扩展青霉碱性脂肪酶 (PEL)的cDNA克隆到酵母整合型质粒pPIC3.5K ,电转化His4缺陷型巴斯德毕赤酵母 (Pichiapastoris)GS115 ,通过橄榄油 MM平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDS PAGE分析、橄榄油检验板鉴定 ,表明扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中获得了高效表达。表达蛋白分泌至培养基中 ,分子量约 2 8kD ,与扩展青霉碱性脂肪酶大小一致 ,占分泌蛋白的 95 %。橄榄油检验板检验表明该表达蛋白可分解橄榄油 ,通过优化该表达菌的发酵条件 ,以橄榄油为底物进行酶活测定 ,其发酵液酶活可达 2 6 0u mL。The alkaline lipase gene of Penicillium expansum (PEL) was coloned into the yeast integrative plasmid pPIC3.5K, which was then transformed into His4 mutant yeast GS115. Recombinant Pichia strains were obtained by minimal olive oil-me- thanolplates screening and confirmed by PCR. The expression producus of PEL gene was analysis by SDS-PAGE and olive oil plate, the result indicated that PEL gene was functionally overexpressed in Pichia pastoris and up to 95% of the secreted protein. Recombinant lipase had a molecular mass of 28kD, showing a range similar to that of PEL, could hydrolyze olive oil and formed clear halos in the olive oil plates. Four different strategies (different media, pH, glycerol and methanol concentration) were applied to optimize the cultivation conditions, the activity of lipase was up to 260 u/mL under the optimal cultivation conditions. It is pointed out that the absence of the expensive biotin and yeast nitrogen base in the medium increased the lipase production. The possible reason of this result is absence of yeast nitrogen base increased the medium pH during cultivation, and PEL shows a higher stability at this condition. The lipase activity of the supernatant from the culture grown at pH 7 was higher than the one from the culture in the same medium at pH 6.0 is due to the pH stability of PEL too. The results also showed that the methanol and glycerol concentration had a marked effect on the production of lipase.

关 键 词：扩展青霉碱性脂肪酶 巴斯德毕赤酵母 基因表达 质粒 

分 类 号：Q78[生物学—分子生物学]