Alteration Of Substrate Specificity Of Thermophilic Esterase By Protein Engineering

Esterase is a hydrolase enzyme that splits esters into an organic acid and an alcohol with water. It has broad substrate specificity, also catalyze thioester, amide and phospholipid, and therefore apply in the field of food industry. Most natural esterases prefer for esters whose acyl-chain length is shorter than eight carbon atoms, while have poor substrate selectivity toward long-chain acyl esters. It is necessary to engineer the natural esterases to alter their substrate specificity toward the long-chain acyl ester.Recently, directed evolution and rational design were emerged as very elegant approaches to generate enzyme variants with improved properties. Directed evolution combines random mutagenesis with high-throughput screening for the improved properties. It has accomplished such impressive feats in optimizing the properties of enzyme, which could carry out without understanding the details of the structure information and the catalytic mechanism;Rational design is to engineer the protein on the basis of understanding structure-function relationship of the enzyme. However, it is very difficult for rational design that lack understanding of the structure-function relationship of the enzyme. Semi-rational design takes the advantages of rational design and directed evolution. Random mutations were limited to one or a few residues. So the mutant library for screening was smaller and the screening was more effective. In addition, activity and substrate specificity analysis of the mutation libraries could extend our knowledge on the structure-function relationship of the enzyme. We adopt semi-rational design targeting a thermophilic esterase to get one who has thermostability and substrate selectivity toward long-chain acyl ester. It is the double significance of the ideal theory and practice.In our prior studies, hyperthermophilic esterase APE1547 from the thermophilic archaeon Aeropyrum pernix K1 has been cloned and over-expressed in Escherichia coli. The recombinant protein (APE1547) also has been characterized and crystallized. The mutant R526V can effectively increase the esterase activity, but did not change the chain-length specificity of acyl ester. To further enhance substrate selectivity toward the long-chain acyl ester, we began with the best mutant R526V, applied of directed evolution and semi-rational design to improve the property of the enzyme. Firstly, we performed error-prone PCR on the catalytic domain and then constructed mutant library containing about ten thousand clones for high-throughput screening. We obtained the mutant APE1547/VW with 1.4 times catalytic efficiency higher than that of R526V for pNPC12. Secondly, according to crystal structure, we used semi-rational design approach to analyze binding site residues of substrate pocket and retained the key catalytic active sites and the 'oxygen hole' residues. By whole plasmid PCR we established saturated mutant library from which screen for variant of high activity to the long-chain ester substrate. We undertook the substrate specificity investigation by activity assay to two substrates of different length acyl chain, revealed the structure-function relationships in detail. Data analysis showed that the amino acid sites P370, E419, Y444, Y449, I489 and T527 had little influence to chain-length specificity. Site Y446 is relatively conservatism. Sites W474, M477, F485 and F488 had significantly influence to chain-length specificity, especially W474 and F488.We obtained optimal mutants F488G/VW, W474V/VW and W474V/F488G/VW which were favorable for long-chain ester substrate. They increased the catalytic efficiency by 3.14-,4.30- and 4.80-fold compared with R526V respectively. Computer modeling and docking experiments confirmed that site 488 was at the side wall of substrate binding pocket. Mutant F488G expanded the volume of the substrate pocket which would accommodate long-chain acyl ester better. Meanwhile mutant F488Y/VW and F488W/VW lost the activity to long-chain acyl ester; site 474 was at the bottom of substrate binding pocket. The mutant W474V deepened the substrate pocket from 6.07 ? to 11.04 ?, which eliminated the obstacle for the long-chain acyl ester. At the same time, the distance between the active site S445 and carbonyl oxygen of substrate were shortened from 7.6 ? to 5.0 ?, which led to increase substrate selectivity to the long-chain acyl ester.Interestingly, in the reaction system that contained 12% (v/v) concentration of acetonitrile, F488G/VW, W474V/VW and W474V/F488G/VW significantly increased activity to long-chain acyl ester substrate. The esterase activity was 10-fold higher than that of R526V. However, wild type displayed 60% reducing. The hydrophobic parameter of residue 488 and C50 displayed positive relevance. It was proposed that the smaller polarity of the side chain can make the desalvation easier, which decreased the free energy and enhanced the catalytic efficiency.In conclusion, random mutagenesis combined with saturation mutagenesis for all the amino acid residues at the substrate-binding site successfully identified the mutational site 474 and site 488 as hot spots for substrate selectivity toward the long-chain acyl ester. The kinetic analysis of the mutants W474V/F488G/VW revealed that the catalytic efficiency was 22.5-fold higher than that of R526V, which could lay a foundation for application of industrial production. The results obtained are intriguing from both an academic and biotechnological point of view.