A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase

作者：Chen Hui; Wu Jianping^*; Yang Lirong; Xu Gang

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2013, 1834(12): 2494-2501.

DOI：10.1016/j.bbapap.2013.08.011

• Lipase
• Diastereopreference
• Site-directed mutagenesis
• Chemical modification
• Steric exclusion
• Structural rigidity

A combination of site-directed mutagenesis and chemical modification was employed to alter protein structure with the objective of improving diastereopreference over that achieved by simple site-directed mutagenesis. Conformational analysis using molecular dynamic (MD) simulation of Pseudomonas alcaligenes lipase (PAL) indicated that stronger steric exclusion and structural rigidity facilitated diastereopreference. A cysteine (Cys) residue was introduced using site-directed mutagenesis to construct variant A272C. The modifier 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was then reacted with the introduced Cys residue to provide stronger steric exclusion and structural rigidity. The modification was verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Diastereopreference was improved significantly. The diastereomeric excess (de(p)) of L-menthol increased from 35% with wild type PAL to 90% with A272C-DTNB modified PAL when the conversion ratio of L-menthyl propionate was nearly 100%. Conformation and kinetic parameter analysis showed that A272C-DTNB modified PAL exhibited stronger steric exclusion and increased structural rigidity around the modification site that inhibited the hydrolysis of non-targeted substrates. The combination of site-directed mutagenesis and chemical modification could be an effective method to alter protein properties and enhance diastereopreference through the combined effect of steric exclusion and structural rigidity.

• 出版日期2013-12
• 单位浙江大学