Format dehidrogenaz enziminin candida boidinii'den moleküler olarak klonlanması, ekspresyonu ve karakterizasyonu

Abstract

Enzyme production has an important place in modern biotechnological industry. Factors as expensiveness of enzyme extraction from plantal and animal sources, difficulty of getting standardization in terms of quality and quantity have increased the tendency toward microbial sources. With the development of recombinant DNA technology and its manipulability on microorganisms, the global industrial market for recombinant enzymes was indicated at 4.61 billion dollars in 2016. This market is expected to reach 6.3 billion dollars by 2022 in terms of value, at a compound annual growth rate of 5.8% from 2017. NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is an enzyme that catalyzes the oxidation of formate ions to CO2 concurrently reduction of NAD+ molecule to NADH. Expensive cofactors like NAD(H) and NADP(H) are needed in reactions where enzymatic reduction is important. Cofactor regeneration methods have been developed on the purpose of offering affordable alternatives. In these methods, FDH is one of the most preferred enzymes thanks to its advantageous properties. No by-product accumulation, inertness to the reaction substrate, suitable thermodynamic equilibrium, wide pH range features raise FDH enzyme into a privileged position. Synthetic production of this enzyme has gained importance in recent years since it is frequently used for the production of optically active compounds in pharmaceutical industry and diagnostic tests in clinical biochemistry laboratories. Studies showed that FDH enzyme can be produced recombinantly from various methylotrophic bacteria and yeasts. In this study, cloning of the gene encoding FDH enzyme from Candida boidinii (C. boidinii) ATCC 18810 yeast strain into Escherichia coli (E. coli) bacterium, expression and characterization of the gene product, FDH enzyme, was aimed. In line with this purpose, first, genomic DNA of C. boidinii was isolated, target gene was amplified with polymerase chain reaction (PCR) and the expected 1104 bp band was seen. Amplified gene was cloned into pTZR57R/T vector via TA cloning method and E. coli One Shot® Mach1™-T1R cells were transformed with the recombinant vector pTZR57R/T+FDH. The vector within the transformed cells was digested with NdeI and BamHI restriction enzymes and the target FDH gene transferred to pET-14b expression vector that was treated with the same restriction nucleases. E. coli One Shot® Mach1™-T1R cells were transformed with the new recombinant vector, pET-14b/FDH. Transformation success was verified with colony PCR, restriction enzyme digestion and sequence analysis. Afterwards, pET-14b/FDH vector was transferred to E. coli One Shot® BL21 (DE3) expression cells. FDH enzyme production was induced with IPTG and produced protein was purified with Ni-NTA affinity column chromatography. SDS-PAGE and Western blot analyzes were carried out to see the presence of FDH protein and both of them displayed expected 41 kDa bands. Finally, enzyme assays were done to determine ideal activity conditions. Results were evaluated by 340 nm wavelength absorbances of enzymatic reaction product, NADH. In consequence of characterization studies, it was determined that recombinant FDH enzyme works efficiently at pH 8.0 Tris buffer solution with 40 mM formate substrate concentration until the temperature reaches 60°C. This enzyme which is frequently used in pharmaceutical industry and clinical biochemistry laboratories is provided from foreign sources in our country thereby it becomes costly. This study is important in the way of providing data for national production of FDH enzyme and starting large scale production in industrial area.