Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 11529, Taiwan.
J Agric Food Chem. 2011 Oct 12;59(19):10693-8. doi: 10.1021/jf202161a. Epub 2011 Sep 14.
Recombinant Candida rugosa lipase 5 (LIP5) has been functionally expressed along with other isoforms in our laboratory. However, the characterization and codon optimization of LIP5 have not been done. In this work, we characterized, codon-optimized and compared LIP5 with commercial lipase. LIP5 activity on hydrolysis of p-nitrophenyl (p-NP) butyrate was optimal at 55 °C as compared with 37 °C of the commercial lipase. Several assays were also performed to determine the substrate specificity of LIP5. p-NP butyrate (C(4)), butyryl-CoA (C(4)), cholesteryl laurate (C(12)), and N-carbobenzoxy-l-tyrosine-p-nitrophenyl ester (l-NBTNPE) were found as preferred substrates of LIP5. Interestingly, LIP5 specificity on hydrolysis of amino acid-derivative substrates was shown to be the highest among any lipase isoforms, but it had very weak preference on hydrolyzing triacylglycerol substrates. LIP5 also displays a pH-dependent maximum activity of a lipase but an esterase substrate preference in general. The characterization of LIP5 along with that of LIP1-LIP4 previously identified shows that each lipase isoform has a distinct substrate preference and catalytic activity.
我们实验室已成功表达了重组假丝酵母脂肪酶 5（LIP5）及其它同工酶。然而，LIP5 的特性分析和密码子优化尚未进行。在这项工作中，我们对 LIP5 进行了特性分析、密码子优化，并与商业脂肪酶进行了比较。与商业脂肪酶的最适温度 37°C 相比，LIP5 水解对硝基苯丁酸酯（p-NP 丁酸酯）的活性在 55°C 时最佳。还进行了几项测定来确定 LIP5 的底物特异性。发现 p-NP 丁酸酯（C(4)）、丁酰辅酶 A（C(4)）、月桂酰胆固醇酯（C(12)）和 N-碳苄氧羰基-L-酪氨酸对硝基苯酯（l-NBTNPE）是 LIP5 的首选底物。有趣的是，与任何其它同工酶相比，LIP5 对水解氨基酸衍生底物的特异性最高，但对三酰基甘油底物的水解偏好性很弱。LIP5 还显示出依赖 pH 的脂肪酶最大活性，但总体上显示出酯酶底物偏好性。LIP5 的特性分析与先前鉴定的 LIP1-LIP4 的特性分析表明，每种脂肪酶同工酶都具有独特的底物偏好和催化活性。
