Yang Jiangke, Yan Xiangxiang, Huang Ribo, Zhang Bo
School of Biology andPharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China.
Sheng Wu Gong Cheng Xue Bao. 2011 Dec;27(12):1780-8.
Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.
米根霉脂肪酶（ROL）不仅是一种用于广泛生物技术领域的生物催化剂，也是研究分子内伴侣在脂肪酶翻译后加工过程中功能的模型。在本研究中，我们在毕赤酵母GS115中克隆并表达了含有米根霉HU3005前序列（pro-ROL）的成熟脂肪酶基因（m-ROL），并对其酶活性进行了表征。m-ROL在pH 9.0时对中链底物（C10和C12）表现出更高的水解活性，而pro-ROL更喜欢短链底物（C4），并在pH 8.0时表现出最大活性。此外，pro-ROL比m-ROL具有更好的热稳定性。m-ROL和p-ROL之间的这种酶活性差异可能是由于前序列影响了成熟脂肪酶结构域的折叠和构象。为了提高m-ROL在毕赤酵母中的表达水平，进行了重叠延伸PCR，用常用密码子替代m-ROL的八个较少使用的密码子。甲醇诱导表达72小时后，密码子优化的m-ROL的活性和蛋白质含量分别达到132.7 U/mL和50.4 mg/L，而亲本m-ROL和pro-ROL的活性分别为28.7 U/mL和14.4 mg/L、29.6 U/mL和14.1 mg/L。
