Alanine scanning on the active site of a novel esterase

Enzymes are widely used in industrial applications and most of them are in the protein structure. Modifications in the protein sequence and protein structure by protein engineering strategies are often used to improve the properties of enzymes. Through all studies such as the finding of new enzymes, characterization and optimization of conditions, efforts are being made to reduce the production cost of the enzyme and to produce it at a higher scale. Esterase enzyme is a hydrolase enzyme used in many fields such as food, industry, health, medicine, detergent industry. It has an α/β hydrolase fold. Due to the high demand of industrial esterase enzymes, attempts are made to characterize novel esterases and increase their properties by using protein engineering approaches. In this thesis, the predicted information about the 3-D structure of a novel esterase enzyme is sought to be elucidated by one of the protein engineering methods which is named as site directed mutagenesis. In previous studies the esterase-encoding gene, isolated from microorganism cultures collected from Acıgöl, was introduced into pET28-a vector and introduced into competent C43 cells for the production of high amount in the laboratory conditions. In this study, pET28-a plasmid containing esterase gene was used as a template DNA. Protein sequence translation of the esterase gene was performed with the ExPASY tool followed by sequence comparison using the SWISS Model software. The closest predicted 3-D structure and protein in the Protein Data Bank database were investigated, and the closest protein was found to be the esterase ybfF protein from Escherichia coli (Ec_ybfF) with 34% similarity. It was noticed that the amino acids (H234 and S206) in the active region of the esterase ybfF protein were similar to the amino acids (H237 and S87) in a particular region of the novel protein obtained in this study. Alanine scanning of these specific regionhas been used to confirm the hypothesis that the amino acids H237 and S87 are members of the catalytic triad of our novel esterase enzyme. The reason for replacing the target amino acids with alanine by PCR reaction with the primers designed by QuikChange® is that alanine does not enforce the electrostatic or H bond effects because of the uncharged methyl groups in its R group and not being heavy. In addition, protein folding and stability do not undergo severe change when compared to the wild-type protein in three-dimensional structure. The mutant amplicons were disrupted by DpnI, the methylation-sensitive restriction enzyme, to remove the unmutated DNA. Subsequently, transformation of DpnI-applied PCR products into C43 cells was performed. Cultures obtained were plated on LB agar plates containing 100 μg/ml kanamycin and incubated overnight at 37 ° C. After isolating the plasmids, agarose gel electrophoresis was performed. The samples were sequenced and then the protein expression steps were followed to check the mutants were constructed. After addition of 0.01 mM IPTG, expression was induced by incubation at 30 ° C for 6 hours. Purification of the proteins of the pET28 vector carrying the H237A and S87A mutant esterase gene was then performed by the 6xHis tag procedure and the control was performed by SDS PAGE analysis. After gene expression induction with IPTG, bands between 25-35 kDa for H237A and S87A were detected. The result was compatible with previous studies while the molecular weight of the esterases in the literature changes between 27-54 kDa. Centrifugal ultrafiltration was performed to purify the obtained protein. Concentration by Bradford protein analysis was measured as 1.3 mg/ml for S87A mutant esterase, 4.5 mg/ml for H237A mutant esterase and 5.5 mg/ml for wild type. Kinetic measurement analysis of wild-type and mutant enzymes was performed at pH 8 with wavelength at 410 nm in the presence of 1 mM Para-nitrophenol (pNP) as substrate. Triplicate enzymatic activity measurements of wild-type and mutant enzymes, each incubated for 20 minutes at 30 ° C, were performed in three independent experiment sets. The kinetic measurements showed that H237A mutant and S87A mutant esterase enzymes lost their activities. These results prove that Serine and Histidine at 87 and 237 positions respectively are members of the catalytic triad of novel esterase enzyme and they are responsible for the catalytic activity of the enzyme.