Increasing The Activity Of Hyperthermophilic Esterase APE1547 By Protein Semi-rational Design

Esterases catalyze a diverse array of esterolytic transformations on a largenumber of natural and unnatural ester substrates. They are the most used enzymesin synthetic organic chemistry, catalyzing the hydrolysis of carboxylic acid estersin aqueous medium or the reverse reaction in organic solvents. Esterases oftenexhibit high stability and activity, especially in organic solvents. However, theproperties of enzymes do not always meet the requirements for a given applicationand thus they have to be further optimized for certain processes. For example,there remains a need for more diverse esterolytic and ester synthesizing enzymes,particularly those with thermostability can work under special condition.Recently, the directed evolution was emerged as a very elegant approach togenerate enzyme variants with improved properties. Directed evolution combinesrandom 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 detailed understanding of the structure informationand the catalytic mechanism. In addition, analysis of the improved properties andthe mutation sites could extend our knowledge on the structure-functionrelationship of the enzyme.In our earlier studies, hyperthermophilic esterase APE1547 from thethermophilic archaeon Aeropyrum pernix K1 has been cloned and over-expressedin Escherichia coli and the recombinant protein (apAPH) has been characterized.The enzyme shows extremely high thermostability (half life at 90℃ is 168 hours)and shows a wide range of substrate specificity: It can hydrolyze both long alkylchain ester and short acetyl peptide.In this study, the hyperthermophilic esterase APE1547 was subjected todirected evolution to generate mutants with increased esterase activity. Based onits extreme thermostability, a sensitive high throughput method was set up forscreening esterase activity at high temperature. By two successive rounds ofrandom mutagenesis, we got two mutants M010 and M020. The first mutantM010 has a single mutation site R526S and exhibits 1.5-fold improvement ofrelative activity than wild-type enzyme. Based on the mutant M010, the secondgeneration mutant M020 ( R526S/E88G/A200T/I519 ) exhibits 5.8-foldimprovement of total activity than wild-type enzyme and close relative activitywith M010. We found that the increased activity of M020 is due to the nearly4-fold higher expressed level than wild-type enzyme. We found that the increasedactivity of M020 is due to the nearly 4-fold higher expressed level than wild-typeenzyme. We found that thermostability of M020 is higher than wild-type enzymeand half life of M020 at 90℃ is 3 folds than wild-type enzyme. Elimination ofArg526 and E88 residues with electric charges possibly leads to enhancinghydrolytic effects of the two domains.The mutation site R526S increase the catalytic efficiency of the enzyme.Multiple sequence alignment showed that Arg526 is completely conserved amongall APHs. In addition, crystal structure analysis indicated that the Arg526 is about4? from the active site, which is the closest among the four mutation sites ofM020. The special location and the highly conservation suggests that the residue526 may playes an important role in catalytic function of this enzyme. Therefore,we chose Arg526 as a hotspot and saturation mutagenesis on this site wasperformed for future research.R526X saturation mutagenesis library was constructed by whole-plamidPCR and screened to get a serial of mutants with further improved activity.Sequencing of the improved mutants showed that hydrophobic mutant increasecatalytic efficiency most effectively: The large hydrophobic mutants (Ile, Leu, andVal) increased the catalytic efficiency by 3.4-to 5.7-fold. Determination of thekinetic parameters indicated that the increased activities are due to the significantincreases in kcat rather than Km. Interestingly, The R526X mutants showeddecreased peptidase activity. Therefore, their specificity toward ester substrate andpeptidase has dramatically changed. In the case of R526V, the esterase activitybecomes ~150 times higher than the peptidase activity, which is only 6.8 times ofwild type. These results unambiguously confirmed the importance of position 526in substrate discrimination and illustrate that enzymes can be evolved todiscriminate their substrates by a single mutation.We ultimately introduced R526V into M020 by site-directed mutagenesis andreplace Ser with optimized Val to get the mutant M030 (E88G/A200T/I519/R526V). Relative activity, protein expression and total activity of M030 exhibit5.8-fold, 4-fold and 22.8-fold higher than WT, so it is more suited for applicationof industrial production.