The Study On Catalytic Performances And Assembly Characteristics Of Glucose Dehydrogenase-Leucine Dehydrogenase Fusion Enzyme Mediated By Rigid Peptide

L-tert-leucine(L-tle)is an important chiral amino acid belong to non-proteinogenic amino.It is widely used in pharmaceutical and food industries,especially as a pharmaceutical intermediate using in the synthesis of HIV protein inhibitors,anti-cancer and antiviral drugs.Leucine dehydrogenase(LeuDH,EC 1.4.1.9)can reduce trimethylpyruvic acid(TMA)to obtain pure L-tle under NADH which is regenerated by glucose dehydrogenase(GDH,EC 1.1.1.47).Inspired by the efficient and rapid catalytic reaction and structural stability of multi-enzyme complexes in nature,the multienzyme fusion coupling system may be an ideal method to improve the production efficiency of L-tle.A system of glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the synthesis of L-tle was constructed in this work,then the catalytic performances and assembly properties were studied.The following experiments were completed:The construction of recombinant E.coli.The pUC18-leudh and pUC18-gdh were used as templates to amplify gene fragments encoding LDH and GDH,respectively.The PCR products of leudh and gdh were used as templates to fuse gene fragments with rigid peptide R3 by Overlap extension PCR to obtain gdh-r3-leudh.The fusion gene and pET-28a plasmid were digested,then ligated together to structure recombinant plasmid pET-28a-leudh-r3-gdh.In addition,the recombinant plasmids pET-28a-leudh and pET-28a-gdh were also constructed.The above three kinds of recombinant plasmids were transformed into E.coli respectively to obtain recombinant E.coli.The induced expression and enzymatic properties of fusion enzyme.expression and purification recombinant protein.The three strains of recombinant E.coli were cultured to induce recombinant protein expression,and the fusion enzyme LeuDH-R3-GDH,free LeuDH and GDH were obtained respectively.Then the proteins were purified by His-Trap HP affinity column and analyzed by SDS-PAGE,the specific enzyme activity was determined finally.The kinetic parameters,optimal pH and temperature and thermalstability of the fusion enzyme and free enzymes were investigated respectively.(1)The Km value of fusion GDH and LeuDH was higher than that of free GDH and LeuDH,while the kcat and kcat/Km values of fusion GDH and LeuDH were lower than those of free GDH and LeuDH,the results showed that the affinity and catalytic efficiency of the substrate were decreased after double enzyme fusion.(2)The optimal pH of free GDH was 8.0,while the fusion GDH maintained more than 80%of reducing activity in the range of pH 6.0 to 9.0,the optimal temperature of free GDH and LeuDH were 70?,while the optimal temperature of fusion GDH and LeuDH were 80?,indicating that fusion enzyme had better adaptability of pH and temperature.(3)As the temperature increased,the activity of fusion enzyme decreased gradually,but the fusion GDH maintained 40%of reducing activity after incubation at 50? for 3 h,while free GDH almost deactivated after 0.5 h,indicating that the fusion enzyme had stronger thermalstability for the rigid peptide and fusion structure may enhance the stability of complex structure.The catalytic performances of fusion enzyme.The effects of the status of fusion enzyme,the concentration of cofactor,the concentration of substrate and other reaction conditions on L-tle synthesized by fusion enzyme were investigated separately;then the catalytic system was optimized and amplified.The optimum conditions for the catalytic reaction were:the whole cell of fusion enzyme,the concentration of NAD+was 0.4 mM,the concentration of substrate was 500 mM,30? and pH 9.0.Scale-up the catalytic system to 200 mL under the above optimized conditions,40 g/L of whole cell was used to catalyze 500 mM of substrate,and the yield of L-tle was 67.9%and the productivity achieved 2135.9 g/L/d after half an hour of reaction.The e.e.values were all greater than 99%.The catalytic performance of active inclusion bodies of fusion enzyme.It was found that a large number of inclusion bodies formed during the Intracellular overexpression of fusion enzyme.The analysis showed that 70%of the recombinant protein and more than 85%of activities of GDH and LeuDH were distributed in inclusion bodies,and it showed better catalytic performance than soluble fusion enzyme.The active inclusion bodies had a good reusability for the yeild of synthetized L-tle remained more than 90%compared to the first time after 6 consecutive recoveries of active inclusion bodies(a round of catalytic reaction lasted for 2 h).Using the substrate feeding mode,the concentration of L-tle reached 37.1 g/L in a 20 mL catalytic system after 6 hours,while concentration of L-tle could acheive about 48.4 g/L in a 200 mL catalytic system after 24 hours.The assembly characteristics of fusion enzyme.From the analysis of fusion enzyme assembly process,as the time of inducing fusion enzyme expression by IPTG advanced,the proportion of active inclusion gradually increased;extracellular fusion enzyme concentration increased protein concentration would produce precipitation,its supernatant and precipitated protein and enzyme activity distribution similar to the expression results of intracellular active inclusion bodies;dynamic light scattering analysis found that the fusion enzyme particle size and protein concentration were positively correlated,the results showed that the increase of protein concentration could promote the fusion enzyme self-assembly to form aggregates.From the morphology analysis of fusion enzyme assembly,after SEM observation,it was found that the fusion enzyme activity inclusion body assembled to form a regular structure,and the tructure of the aggregates after pure enzyme concentration was similar to that of the active inclusion bodies,and even assembled to form a more regular lamellar structure.AFM analysis of aggregates found that it formed the assembled multilayer lamellar structure.Combined with the assembly process and assembly morphology analysis of intracellular and extracellular fusion enzymes,it is speculated that the active inclusion bodies and insoluble aggregates may be supramolecular multi-enzyme complexes formed by the self-assembly behavior mediated by oligomeric forces at high concentrations.