Candida boidinii kaynaklı NaD+ bağımlı format dehidrogenaz enziminin üretilmesi, enzim aktivitesinin ve termal stabilitesinin artırılması / Production of NAD + dependent formate dehydrogenase enzyme from candida boidinii and enhancement of enzyme activity and thermal stability

Abstract

Enzymes have a broad range of utility in different industries and they are important for biochemical kits used for diagnostics as well. Formate (formic acid) is an essential endogenous single-carbon metabolite under normal circumstances, it is found in human serum and urine at particular levels. Formate levels were shown to increase in human serum, urine and exhaled breathe condensate due to metabolism of different substances especially in cases of methanol intoxication, vitamins B deficiencies, asthma and various psychiatric disorders. Format Dehydrogenase (FDH) is an enzyme that shows absolute specificity for formate and removes electrons from the format ion in the environment and transfers them to NAD+. The amount of NADH + H at the end of the reaction is calculated as a direct proportion to the amount of the formate entering the reaction. With the thesis project, our aim is to produce NAD + dependent FDH enzyme by recombinant DNA technology and increase enzyme activity and thermal stability. In our study, 1095 base pair gene encoding the Candida boidinii (Cbo)FDH protein was amplified by PCR using the genomic DNA from C. boidinii as a template. The product was cloned into pET-23b(+) vector and transferred to Escherichia coli BL21 (DE3) cells for expression. The affinity His-Trap column was used for purification of the enzyme. Enzyme activity measurements were performed spectrophotometrically by monitoring the formation of NADH at 340 nm. To predict the influence of mutations done on intramolecular interactions, we introduced the available crystal structure (PDB:5DN9) to Gaussian 09 software and was changed the mutation cites. Subsequently, we modelled the structure of amino acid substituted enzyme, into almost real form in its environment by geometrical optimization. The results were visualized by pymol software and discussed. The determined mutations were performed by the directed mutation method for increase the enzyme activity and thermal stability. Kinetic and thermostability studies were performed for both the wild type and mutant FDHs. In this study different metal ions and organic solvents effect on enzyme activity of wild type FDH and mutant FDH strains were investigated as well. The thermal stability assays and secondary structures of the enzymes were performed by circular dichroism(CD) spectroscopy. According to the results of the study, the optimal pH for wild type CboFDH produced by recombinant DNA technology was 7.4 and optimal temperature was 40°C ; pH and temperatures optimal for mutant enzymes were as follows: Phe285ThrFDH pH 7, temperature 40 °C , Val120ThrFDH pH 7, temperature 40 °C , Gln287GluFDH pH 7, temperature 60 °C, His311GlnFDH pH 7, temperature 65 °C and for the Phe285Thr/His311GlnFDH, pH 8 and the optimal temperature was found to be 65 °C. Thermal denaturation (Tm) temperature values of wild type and mutant enzymes with α-helix secondary structures determined by CD device were determined as 64 °C for wild type CboFDH, 70 °C for Gln287GluFDH, 77 °C for His311GlnFDH and 73 °C for Phe285Thr/His311GlnFDH. Enzyme kinetics studies for wild type CboFDH for formate revealed that the KM value was 2 mM and Kcat value was 9.2 x 102 S-1, for NAD the KM value was 1.5 mM and Kcat was 2.62 x 104 S-1; for Phe285ThrFDH the KM for formate was 0.97 mM and the Kcat value was 6.23 x 1063 S-1, the KM value for NAD was 2.3 mM and the Kcat value was 4.59 x 103 S-1; for Val120ThrFDH the KM value for formate was 1.3 mM and Kcat was 6.23 x 103 S-1 , KM value for for the NAD 7.9 mM and Kcat of 1.04 x 104 S-1; for Gln287GluFDH, the KM value for formate was 1.65 mM and the Kcat was 4.42 x 103 S-1, the KM value for NAD was 3.3 mM and the Kcat value was 9.51 x 103 S-1; for His311GlnFDH, the KM value for the format was 1.5 mM and the Kcat was 3.28 x 103 S-1, the KM value for NAD was 1.6 mM, the Kcat value was 8.2 x 103 S-1; for Phe285Thr / His311GlnFDH, the KM value for the formate was 1.4 mM and the Kcat was 6.95 x 103 S-1, the KM value for NAD was 7 mM, and the Kcat value was 1.21 x 104 S-1. The effect of 11 different metals in different concentrations on the enzyme activity was found that all metals except CuCl2 found to enhance the activity for wild type CboFDH and mutant FDHs by up to 80%; CuCl2 was found to reduce the activity up to 77% in higher concentrations. The effect of 5 different organic solvents in different concentrations on the enzyme activity was found that acetone, ethanol, metanol and propanol increase the activity for wild type CboFDH and mutant FDHs in range between 20%-31% but chloroform was found to decrease the activity between 37%- 97% at increasing concentrations in all enzymes. This study is important in terms of providing data on the production of a wide range of NAD+-dependent FDH enzymes, which are absolutely specific to the formate, which is an important indicator of the levels in some pathological conditions, and the production of large-scale NAD+-dependent FDH enzyme. Keywords: Candida boidinii, Formate Dehydrogenase, Formate, Protein Engineering, Recombinant DNA Technology, Thermal Stability{{abstract}}