%0 Journal Article
%T Codon optimization, expression and enzymatic comparison of Rhizopus oryzae lipases pro-ROL and m-ROL in Pichia pastoris<br>米根霉脂肪酶基因pro-ROL和m-ROL在毕赤酵母中的密码子优化、表达和酶学性质的比较分析
%A Jiangke Yang
%A Xiangxiang Yan
%A Xiangxiang Yan
%A Bo Zhang
%A <br>杨江科
%A 严翔翔
%A 黄日波
%A 张搏
%J 生物工程学报
%D 2011
%I 
%X 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.
%K Rhizopus oryzae lipases
%K m-ROL
%K pro-ROL
%K overlap extension PCR
%K Pichia pastoris<br>米根霉脂肪酶
%K m-ROL
%K pro-ROL
%K 重叠延伸PCR
%K 毕赤酵母
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=042A5D990BB41D6197B133C62A321570&yid=9377ED8094509821&vid=DB817633AA4F79B9&iid=59906B3B2830C2C5&sid=0420ACD7465FFFB2&eid=DC8E8C328836185A&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=25