Improve Substrate Specificity And Activity Of Hyper-thermophilic Esterase By Protein And Solvent Engineering

An acylaminoacyl-peptidase (apAAP) from hyperthermophilic archaea Aeropyrum pernix K1 is a promiscuous acyl peptidase and esterase. Our previous studies showed that saturation mutation of conservative residues R526 near the active site can effectively improve the esterase activity, but does not change the selectivity to pNP-ester. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, the substrate preference of apAAP was further improved from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering.We start from the best mutant P01 (R526V), directed evolute its esterase activity toward long-chain acyl ester under the multiple selection pressure (high temperature and different carbon chain length of the substrate), applied of optimized high-throughput screening methods, found a key site: T560 among about 10000 mutants, which is 18.2 ? away from the active center. A mutant E01 (R526V/T560W) showed high catalytic efficiency (k_cat/K_m) for pNP-C12, which is about 14-fold higher than P01.The study found that was mainly due to improve the affinity for pNP-C12. We use SLITHER, a web server to generate contiguous conformations of a molecule along a curved tunnel inside E01 and P01, and the binding free energy profile along the predicted channel pathway. The results showed T560W improved SUBSTRATE TRAFFIC of pNP-C12 in E01.After that, we adopt semi-rational design methods combining the crystal structure analysis, retain conservative catalytic and oxygen hole residues, chose for saturation mutagenesis at 11 residues (P370, E419, Y444, Y446, Y449, W474, M477, F485, F488, I489 and T527) near the substrate-binding pocket and the catalytic residue Ser. Then screen for mutant of high activity to pNP-C8 and/or pNP-C12 at 60℃. Three mutants F488G/R526V/T560W (named S01), F488P/R526V/T560W (named S02) and W474V/R526V/T560W (named S03) were selected from variants, with catalytic efficiency (k_cat/K_m) for pNP-C12 increased of 3-, 1.5- and 4-fold compared with the mutant P01.To investigate the effects of the mutations on the catalytic mechanism, additional mutants with diverse side chains at positions 474 and 488 were constructed and subjected to kinetic analysis alongside the previously selected mutants. The combined mutant W474V/F488G/R526V/T560W (named D01) had the highest esterase activity of all the mutants investigated in this study. The catalytic efficiency (k_cat/K_m) of D01 for pNP-C12 was 24-fold higher than P01. Analysis of the structure revealed that Trp474 and Phe488 are respectively located at the bottom and the wall of the substrate-binding pocket. S01 and S03 had a deeper (11.4 (?) vs. 6.1 (?)) and larger (577(?)³ vs. 227 (?)³) substrate-binding pocket compared to P01 and E01, which facilitates the binding of substrates with long acyl chains. The long-chain substrate selectivity and the Volume of substrate binding pocket are positively correlated well. Conjecture that the increasing volumes of pocket reduce the steric hindrance of the long-chain substrate entering the active site, so the long-chain ester substrate selectivity of the mutants increases.Solvent engineering is an important technology that can regulate activity and selectivity of enzymes by changing media environment. Because most of the lipase family are interface enzyme whose hydrophobicity are pretty high, we add several water-miscible polar organic solvents to the reaction system, found that the catalytic efficiency of the mutants was significantly affected by polar solvents. In the presence of organic solvents, the wild-type apAAP, P01 and E01 mutants were strongly inhibited, whereas the activities of the mutants S01, S02, and D01 were simulated under the same conditions. In particular, the activity of the mutant D01 increased 240-fold compared with the wild type in the presence of 10% acetonitrile, 99-fold in 20% DMF and more than 280-fold in 30% DMSO. The ratio of the activities for pNP-C12 and pNP-C4 increased 12-, 18- and 19-fold for the mutant D01 in the presence of 10% acetonitrile, 30% DMSO and 20% DMF, respectively.MD simulations were run for the mutants S01, S02 and their parental enzyme P01, mimicking the condition when 10% acetonitrile was present. Detailed analysis revealed that several acetonitrile molecules penetrated the enzymes. The acetonitrile penetration for mutants S01 and S02 was more pronounced than for P01. Interestingly, the penetration of one acetonitrile molecule into mutant S01 was just occurred at the region where residue 488 was located. It means that the increase of substrate binding pocket is more favorable for polar solvent to permeate into the active site, change the catalytic site microenvironment, thus changing the enzyme catalysis efficiency.