Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Appl Biochem Biotechnol. 2012 Mar;166(5):1355-67. doi: 10.1007/s12010-011-9524-4. Epub 2012 Jan 14.
A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for 48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99% after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.
脂肪酶基因 Yarrowia lipolytica lipase LIP2 (YlLIP2) 被克隆到组成型表达载体 pGAPZαA 中，并通过电转化进入毕赤酵母 X-33 菌株。通过高拷贝数和酶活性筛选获得的高产克隆被选为摇瓶和发酵罐培养的宿主菌株。结果表明，葡萄糖是 YlLIP2 生产的最佳碳源，在 28°C、pH7.0 下摇瓶培养 48 小时，重组 YlLIP2 的最大水解活性达到 1315U/ml。在 3L 和 10L 生物反应器中进行分批补料发酵，通过向生长培养基中连续添加葡萄糖来实现高细胞密度和 YlLIP2 产量。在 3L 生物反应器中获得的 YlLIP2 的最大水解活性和细胞密度分别为 10300U/ml 和 116g 干细胞重量（DCW）/l。在 10L 发酵罐中进一步提高了 YlLIP2 的峰值水解活性和细胞密度，分别达到 13500U/ml 和 120g DCW/l。上清液中的总蛋白浓度达到 3.3g/l，培养 80 小时后细胞存活率仍保持在 99%左右。此外，毕赤酵母 pGAP 和 pAOX1 系统中表达的重组 YlLIP2 具有相似的糖含量（约 12%）和生化特性。上述结果表明，毕赤酵母的 GAP 启动子表达系统通过高密度细胞培养有效地表达了 YlLIP2，可能是工业脂肪酶大规模生产中替代传统 AOX1 启动子表达系统的一种选择。
