bsLDH enziminde Q102R ve Q203L çift mutantının substrat özgüllüğü ve aktivatör gereksinimine etkileri

Enzymes are biological catalysts that play role in chemical transformations, thus they are important for several branches of industries. The L-lactate dehydrogenase (LDH) from Bacillus stearothermophillus(bs) is substantial for the synthesis of pharmaceuticals and agrochemicals as it provides the production of chiral building blocks. It enables the reaction that generates hydroxy acids from their corresponding oxoacids. Moreover, because Bacillus stearothermophillus is a thermophilic bacterium, the enzyme is highly stable to heat. However, the enzyme has a limited substrate specificity. On the other hand, the enzyme is activated by an expensive activator, fructose 1,6 bisphosphate (FBP). FBP also shows undesirable co-factor complications for industrial processes. Besides, bsLDH activity is inhibited by excess substrate (pyruvate), which is once more a deleterious feature owing to its industrial use. All these characteristics make bsLDH an applicable target for protein engineering. Previous studies have provided significant developments over these unwanted properties by using rational and randomized mutagenic methods. For instance, by Wilks et al., a Q102R substitution (found in the naturally occurring malate dehydrogenases) on the bsLDH sequence has been shown to convert the substrate specificity from lactate to malate by 3 orders of magnitude. In another research, Allen and Holbrook succeeded in producing a mutant (6A) which is almost fully activated in the absence of FBP. This 6A mutant had three amino acid replacements: R118C, Q203L and N307S, resulting in a 70-fold activation. Normally FBP activates the enzyme by enabling structural rearrangements which turns the protein into tetrameric form from dimeric form. Because the Q203L is located at the dimer-dimer interface, this substitution is thought to have realized the substrate switch by altering the dimer-tetramer equilibrium. By our laboratory, similar protein engineering applications have yielded significant effects on bsLDH as well. For example, by D38R replacement, using PCR based overlap extension mutagenesis, the substrate inhibition was decreased threefold by Binay and Karaguler. In another study, recombinant colonies, formed by random mutagenesis were screened and a more efficient malate dehydrogenase, compared to Q102R variant, was produced. Our aim in this study is forming a double mutant form of bsLDH enzyme and investigate its effect both on the activator need and on substrate specificity. In order to achieve this, Q102R and Q203L mutants were constituted by site-directed mutagenesis in bsLDH sequence which was found in pQE2 vector. To be used in the PCR reaction, appropriate oligonucleotides were designed to generate Q102R and Q203L mutations. Following the PCR with these oligos, mutant dsDNAs were selected by Dpn I digestion. After that, mutated DNA was transformed into E.coli strain BL21 E. coli genotype fhuA2 [lon] ompT gal [dcm] ΔhsdS competent cells. Plasmid DNA was purified following the transformation and samples were sequenced to ensure to have achieved the correct mutations. According to the data obtained, Q102R and Q203L mutations are formed in bsLDH sequence. Mutated DNA was amplified in large-scale E.coli culture and the culture was precipitated in order to obtain protein from cells. Desired protein with 6xHis-tag was sorted from the protein pool by Ni-NTA resin system which selectively binds His-tagged proteins. Thereafter, by Sodium Dodecyl Sulphate Gel Electrophoresis (SDS-PAGE), the protein was visualized at the region of its molecular mass (35kDa) and have ensured to obtain the enzyme. By ultrafiltration, smaller proteins and other contaminants were removed and a purer enzyme was attained. With different concentrations of pyruvate as substrate, wild type and mutant enzyme kinetic measurements were realized in the absence and presence of FBP. Wild type data were found quite consistant with the previous studies. But we could not get detectable results in mutants with pyruvate. Actually, this phenomenon agrees with the Q102R mutant feature, which gives rise to a malate dehydrogenase upon mutation. Further experiments will be made with malate and oxaloacetate substrates to more precisely elucidate the double mutant effect.