Construction Of Key Enzymes Coupling System And Its Catalytic Properties In Preparation Of L-tert-leucine

Currently,the market capitalisation of chiral drugs increases rapidly,so the production of intermediates attracts great attention.Amino acids which have optical properties are used widely as prosthetic group,chiral materials and the inducer of asymmetry reactions.Besides,the optical amino acids are functional components of protein,peptide and other products,such as pesticide and medicine.L-tert-leucine(L-Tle),one of the crucial amino acids in this field,is extensively used as the basic module for pharmaceutically active compounds and as form boards in asymmetric synthesis.And L-Tle is the important intermediate of drugs aming to HIV or tumor fighting agents.Therefore,there exists a great market demand of chemicals and basic intermediate in agricultural and medical industry.This thesis mainly focuses on screening and exploring oxidordeuctase from marine source to make novel biological catalysts,constructing the cofactor regeneration system by coupling leucine dehydrogenase(LeuDH)and formate dehydrogenase(FDH),the use of synthetic biology technology to regulate and coordinate the related key enzymes of the biological preparation of L-Tle,improve and enhance the activity and catalytic performance of key enzymes,optimize the system and so on.This study aims to improve and optimize the multi-enzyme biosystem,which would enhanced the production efficiency of L-Tle by enzymatic method.The details of this thesis are summarized as follows:Firstly,a leudh,from the marine bacterium Alcanivorax dieselolei,was obtained through PCR and encoded a novel LeuDH which showed very low similarity to known proteins and displayed the highest similarity to the LeuDH(42.6%)from Thermoactinomyces intermedius.The LeuDH showed optimal activity at 30℃ and pH 6.5,and was identified to be extremely cold-adaptative retaining over 90%activity in the range temperature 0℃-37℃.The enzyme showed better stability in more weakly alkali environment(pH 6.0-8.5)than Thermoactinomyces intermedius’s LeuDH.The optimal substrate concentration was established and the e.e.value of the LeuDH catalyzing trimethylpyruvic acid(TMA)to L-Tle was more than>99%.Secondly,a strategy,constructing a regulatory enzymes(LeuDH and FDH)-coordinate expression system for biosynthesis chiral drug intermediates,was developed and evaluated by connecting protein-protein expressions using synthetic biology control components,regulating the strength of RBS and detecting the system production capacity of chiral amino acid.These results demonstrated that the multiple enzymes system had good enantioselectivity,low cost,high stability and approximately 100%substrate conversion.These methods pave a new method of exploring metabolic mechanism of functional genes and engineering whole cell-catalysts for synthesizing chiral amino acids or chiral a-Hydroxy acids.Thirdly,through comparing and analyzing its spatial structure with other FDHs,the catalysis,substrate and coenzyme binding sites,67P,98P,99F,93V,94G,119N,123V,256D,257A,260A,283V,287Q,311H,313S,314G and 321T of the CboFDH were identified.To improve its performance,amino acids concentrated on the enzyme active site and in the conserved NAD+ and substrate binding motif were mutated.Among these mutants,V120S had the highest catalytic efficiency(kcat/Km)with COONH4 as it enhanced the catalytic velocity(kcat)and kcat/Km 3.48-fold and 1.60-fold,respectively.And the double mutant V120S-N187D had the highest kcat/Km with NAD+ as it displayed an approximately 1.50-fold increase in kcat/Km.The single and double CboFDH mutants exhibited higher thermostability than the wide type.The structure-function relationship of single and double CboFDH mutants was analyzed by homology models and site parsing.Fourthly,an efficient optimization method for improving productivity of L-Tle synthesis was developed.And the mathematical model between different fermentation conditions and L-Tle yield was also determined in the form of the equation by using uniform design and regression analysis.The multivariate regression equation was conveniently implemented in water,with space-time yield of 505.92 g L-1d-1 and e.e.value of>99%,respectively.