Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites
Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites
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DOI：
10.1021/jf0702949
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发表时间：
2007-06-27
影响因子：
6.1
通讯作者：
Shaw, Jei-Fu
中科院分区：
文献类型：
--
作者：
Lee, Li-Chiun;Chen, Yu-Ting;Shaw, Jei-Fu
Candida rugosa（以前称为Candida cylindracea）脂肪酶（CRL）是一种重要的工业酶，其广泛用于生物技术应用，例如脂肪酸的生产和各种酯的合成。CRL包含至少七种同工酶（LIP 1-LIP 7），它们共享相似的氨基酸序列但对底物具有不同的特异性。以前，据报道，LIP 4对长酰基链酯具有较高的酯酶活性，对甘油三酯具有较低的脂肪酶活性。LIP 4的A296和V344在其底物特异性中起决定性作用。在这项研究中，位点特异性饱和诱变已被用来研究的底物特异性的LIP 4。使用含有32个密码子的简并引物组将点突变分别引入A296和V344位置以产生两个变体文库。LIP 4变体在酵母毕赤酵母中异源表达。使用特异性平板测定法来鉴定在含有三丁酸甘油酯的培养基中产生脂肪酶的巴斯德毕赤酵母克隆。筛选对短脂肪酰基链甘油三酯（三丁酸甘油酯）具有高活性的LIP 4变体。特异性分析和生化特性表明，重组变体A296 I，V344 Q和V344 H具有与野生型LIP 4显著不同的性质。所有三种变体酶对三丁酸甘油酯的比活性均显著高于LIP 4。除了短链甘油三酯外，A296 I和V344 Q还改善了甘油三酯对测试的中链和长链甘油三酯的水解活性。结果表明，A296对脂肪酶活性和LIP 4的高温依赖性起重要作用，但对脂肪分解反应的链长特异性没有影响。V344残基对LIP 4的底物链长特异性有显著影响。
Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.