Lan Dongming, Wang Qian, Xu Jinxin, Zhou Pengfei, Yang Bo, Wang Yonghua
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China.
State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
Int J Mol Sci. 2015 Mar 31;16(4):7273-88. doi: 10.3390/ijms16047273.
Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273-278 and the 3104 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.
热稳定性和底物特异性是酶在工业应用中的重要特性，可通过蛋白质工程加以改善。来自球形马拉色菌的SMG1脂肪酶是一种单酰基甘油和二酰基甘油脂肪酶（MDL），对单酰基甘油和二酰基甘油具有活性，但对三酰基甘油无活性。SMG1脂肪酶被认为是一种潜在的生物催化剂，可应用于油脂改性，其晶体结构表明，一个有趣的残基——Asn277可能有助于稳定对酶特性至关重要的273 - 278环和3104螺旋。在本研究中，为了探索其在影响稳定性和催化活性方面的作用，用天冬氨酸（D）、缬氨酸（V）、亮氨酸（L）和苯丙氨酸（F）对N277进行了诱变。圆二色性（CD）光谱分析和半衰期测量表明，N277D突变体具有更好的热稳定性。N277D突变体的解链温度和半衰期分别为56.6℃和187分钟，而SMG1野生型（WT）分别为54.6℃和121分钟。对SMG1突变体进行了生化特性分析，以测试诱变是否影响催化特性。N277D具有与SMG1 WT相似的酶学性质，但与其他SMG1突变体相比，N277F表现出不同的底物选择性谱。对SMG1三维模型的分析表明，N277D通过其带负电荷的羧基与R227带正电荷的胍基形成盐桥，这可能有助于赋予N277D更高的温度稳定性。这些发现不仅为理解脂肪酶结构/功能关系的分子基础提供了一些线索，也为工程改造适合工业应用的MDL脂肪酶奠定了框架。
