Protein engineering applications of novel esterase enzyme using rational design and directed evolution approaches

Abstract

Enzymes mostly are protein-structured biomolecules that act as catalysts in living organisms. They regulate the rate at which chemical reactions proceed without altering themselves. Enzymes should have properties that are suitable for the desired reaction conditions to be used as biocatalysts in industry (high activity, substrate specificity, resistance to organic solvents, etc.). However, enzymes do not always have the properties required for use in a variety of reaction conditions. So, it is necessary to develop the properties of enzymes for the desired reaction conditions. Protein engineering strategies can be used to create enzymes with unique properties for this purpose. Esterase enzyme has great industrial importance due to its use in many different areas such as detergent, food, cosmetics and pharmaceuticals. Since esterases, which partially dissolve in water and hydrolyze ester bonds, are still able to adapt to harsh industrial conditions and do not have the required level of activity, studies are ongoing to improve the existing properties of the esterase enzyme and to bring new features. Protein engineering methods are one of the most efficient ways to obtain enzymes that can work under harsh conditions. If these methods are applied to enzymes isolated from microorganisms living in extreme conditions it is possible to produce enzymes with superior abilities. Microorganisms living in extreme conditions can adapt to extreme conditions by the construction of certain mutations spontaneously. When protein engineering methods are applied to the adaptation tendencies of these microorganisms and their enzymes with superior properties, enzymes that can meet the needs of the industry can be obtained. Recently, our group isolated and identified microbial diversity of the samples by a sequence-based metagenomic approach using environmental samples collected from Acıgöl, which has a high salt concentration. The esterase(hAGEst) enzyme was produced recombinantly and its biochemical characterization was completed successfully. The amino acid sequence of the esterase enzyme is 91% similar to the esterase of the Halomonas gudaonensis organism, and the gene sequence is 75% similar to a part of the Halolamina sediminis genome. This thesis aimed to determine the three-dimensional(3-D) structure and improve the substrate specificity of the newly defined esterase enzyme using two basic approaches of protein engineering, which are widely used in the literature. Firstly, Site-Directed Mutagenesis was used, which is one of the basic methods of the rational design approach. To predict the 3-D structure of the new esterase protein, when the Protein Data Bank (PDB) was examined with the SWISS-MODEL software, it was seen that the new esterase(hAGEst) enzyme had the highest similarity (34%) with the esterase ybfF protein. Thanks to this method, the importance of these amino acids in the structure were investigated by replacing the 209th serin amino acid residue, which is estimated to be in the active region, and the 88th methionine, which is estimated to be in the oxyanion hole, with alanine amino acid. Primers were designed to obtain the Ser209Ala and Met88Ala mutations. The gene was amplified using the Q5 Site-Directed Mutagenesis Kit and was transformed into E. Coli C43 cells. Colonies were randomly selected from the resulting transformants sent for sanger sequencing. The DNA sequencing results indicated that the desired mutations were obtained successfully. Protein expression was initially stimulated by the addition of 1 mM IPTG, followed by a 6-hour incubation at 30 °C. The proteins from the pET-28a(+) vector containing the Ser209Ala and Met88Ala mutant esterase genes were then purified utilizing the 6xHis tag technique, and SDS-PAGE analysis was used as a control. Bands in the range of 25-35 kDa were observed after inducing gene expression with IPTG for Ser209Ala and Met88Ala. The results verified the molecular weight of esterases ranges between 27 and 54 kDa. In the next step, the protein purified by His-tag purification was removed from the unwanted proteins according to the SDS-PAGE images by ultrafiltration. The concentration of the pure protein was measured by using Bradford reagent. It was measured at 20 mg/mL for Ser209Ala mutant esterase, 20 mg/mL for Met88Ala mutant esterase and 30 mg/mL for wild-type esterase according to Bradford analysis. 1 µM of wild type enzyme, Ser209Ala and Met88Ala mutant enzymes were incubated in 1 mM para-nitrophenol (pNP) hexanoate substrate prepared in 1 mM Tris-HCl pH:8 buffer solution at 30 ºC for 20 minutes. For kinetic activity, three independent experiments were repeated and absorbance was measured at a wavelength of 410 nm. According to these results, the activity of wild type was found to be decreased by 87.1% and 17.7%, when compared to the activity of Ser209Ala and Met88Ala mutant esterases, respectively. These results show that the 88th methionine amino acid is located in the active site of the esterase enzyme due to the serious decrease in the activity and has a direct effect on the activity of the esterase enzyme. However, it was thought that the 88th methionine residue is not of great importance in the oxyanion hole, since a small decrease in the activity is observed by 17.7%. The second part of the study aimed to create a large library with the directed evolution approach. To improve the substrate specificity of the enzyme, the library was created by using the Error-Prone PCR . It would help to develop esterase enzyme for the needs of the industry. So, it may contribute to the national economy and literature by obtaining esterases with superior properties. Firstly, the enzymes studied in the literature, especially esterases, and the Error-Prone PCR conditions applied to these enzymes were examined in detail, the most suitable conditions for the hAGEst enzyme were determined and suitable primers were designed. A suitable Error-Prone PCR reaction to generate mutant enzyme variants was achieved by decreasing adenine and guanine nucleotide concentrations and increasing thymine and cytosine concentrations. Also, commercially available Taq DNA polymerase, which does not have proofreading, was used. In addition, by increasing the concentration of 5 mM MgCl2 and adding 0.25 mM MnCl2, optimum mutation formation was targeted. After cleavage with EP-PCR product and pET-28a(+) EcoRI and HindIII endonuclease enzymes, the determination of purity was calculated by measuring the ratio of absorbance with 260 nm and 280 nm NanoDrop equipment. Also, the qualities of samples were assessed by calculating the ratio of A260/A280. They were transformed into E. Coli cells by ligation according to the ratios of 5:1 and 7:1 (insert:vector). After establishing various genetic differences as a result of mutations, a library of 159 transformants was obtained. 58 mutants showing lipolytic activity were obtained with tributyrin agar plate screening. Protein expression was ensured by IPTG induction (incubation at 30 °C for 6 hours) of the obtained 58 mutants. Expressed proteins were extracted by using the BugBuster protein extraction reagent. The amount of protein was quantified for each of the 58 mutants by Bradford analysis and diluted to keep the equal amount of reacted protein. The activity of both wild-type and mutants was measured in triplicate with the substrates 4-nitrophenyl hexanoate (6C) and 4-nitrophenyl dodecanoate (12C). The absorbance value was measured three times for each of the 58 mutants, and these values were averaged. When the all kinetic activity comparison of mutants were examined, no mutant with significantly greater activity was found compared to wild type. After control cleavage, Mutant48, Mutant52 and Mutant53 were selected as they showed greater activity against the 12C dodecanoate substrate than the wild-type esterase enzyme. Mutant11 and Mutant36 were selected because they showed greater activity than the wild-type esterase enzyme against both 6C hexanoate and 12C dodecanoate substrates. Finally, Mutant6 and Mutant23 showed lower activity than the wild-type esterase enzyme for the two substrates; Mutant 11 was chosen randomly. DNA sequences of 8 selected mutants were analyzed. When sequencing data for these mutants using CLUSTAL OMEGA were aligned with wild type, it was seen that all the potential mutants had the same gene sequence as the wildtype esterase enzyme. As a result of the study, mutant esterase enzymes that could be used as a substrate for 6 and 12C fatty acids could not be obtained or due to the high mutation rate, mutated colonies lost their activity and were eliminated during activity screening. The library will continue to be scanned using different screening strategies. In further studies, fatty acids with a different number of C can be used as alternative substrates to observe the activity of mutants. Error-Prone PCR experiments might be set up under different conditions and the properties of the esterase enzyme might be improved by using protein engineering approaches consisting of combinations of different methods. In this way, it will be possible to contribute to the national economy and literature by obtaining esterases that can meet the needs of the industry.