南极假丝酵母脂肪酶A的表面展示及酶学性质研究

采用a凝集素作为载体蛋白,首次将南极假丝酵母脂肪酶A展示在酿酒酵母细胞表面,通过MD平板筛选获得表面展示型的CALA酵母工程菌株。免疫荧光检测显示CALA被成功展示在酵母细胞壁表面,重组子经诱导后能在三丁酸甘油酯板上形成透明圈,说明展示的CALA具有活性。重组酵母在液体培养基培养72 h,活性达到最高,为80.4 U/g干细胞。酿酒酵母展示的CALA最适温度及pH值为70°C和pH 8.0。经50°C保温2 h,仍含有60%水解酶活力。展示的CALA在pH 7.0和pH 8.0溶液中比较稳定。经DMSO处理2 h,展示的CALA仍保持70%的活性。以上结果表明酵母展示的CALA可作为一种有潜质商业用途的全细胞催化剂。

Cell surface display of Candida antarctica lipase A by saccharomyces cerevisiae and its practical properties

Lipase A gene of Candida Antarctica (cala) was displayed for the first time on the surface of Saccharomyces cerevisiae using a-agglutinin as an anchor protein, recombinant strains were screened on minimal dextrose plates. Results of immunofluorescence microscopy assay revealed that the CALA-a-agglutinin fusion proteins were successfully anchored on the S. cerevisiae cell surface. Furthermore, yeast displayed CALA produced halo on the MD plate containing tributyrin, suggesting that CALA was active on the cell wall. Yeast displayed CALA displayed maximal activity after 72 h cultivation in YNB-CAA medium, with an activity of 80.4 U/g dry cells. The optimal temperature and pH for yeast displayed CALA were 70 °C and pH 8.0, respectively. It remained 60% of activity after incubation at 50 °C for 2 h and the lipase still remained 70% activity after incubated in buffer containing 30% dimethyl sulfoxide for 2 h. Those properties make yeast displayed CALA great potential for industrial application.