Abstract
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.
Highlights
Lipases (E.C. 3.1.1.3) (Enzyme Committee 3.1.1.3) are unique biocatalysts that catalyze hydrolysis reactions in aqueous conditions and esterification, interesterification, and transesterification reactions in microaqueous media [1]
The results showed in this table were averages calculated from at least three independent experiments by spectrophotometric method described in the text; b Specific activity of purified recombinant lipase to pNP caprylate under optimal temperature and pH of each enzymes; c The melting temperature of mutants were calculated from the Circular dichroism (CD) data using Global Analysis
The biochemical properties of SMG1 can be altered by mutagenesis of N277
Summary
Lipases (E.C. 3.1.1.3) (Enzyme Committee 3.1.1.3) are unique biocatalysts that catalyze hydrolysis reactions in aqueous conditions and esterification, interesterification, and transesterification reactions in microaqueous media [1] They have attracted much attention due to their remarkable characterization, such as specific regio- and stereoselectivity, high activity, and stability in non-aqueous environments [2]. Residues forming the catalytic cavity, such as those in the lid region or around active site, were considered as the “hot spots” in protein engineering for altering the enzyme properties, such as catalytic activity, substrate specificity, and thermostability [8,9,10]. The catalytic activity, substrate preference and thermostability of the purified mutants were investigated and the structural molecular basis of SMG1 mutants was discussed
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