The Establishment Of A New Method For The Detection Of The Acetohydroxy Acid Synthase Activity Of Thermotoga Maritima And The Study Of Its Catalytic Reaction

Acetohydroxy acid synthase(AHAS,EC 4.1.3.18)is a typical thiamine pyrophosphate(ThPP)-dependent enzyme.It is the rate-limiting enzyme in the first biosynthetic step of the three branched-chain amino acids(BCAAs),namely isoleucine,leucine,and valine which are essential for all living organisms.These important compounds can be biosynthesized by bacteria,fungi,and plants themselves,but must be obtained through various foods by animals and human because this kind of enzyme is not exsisted in them.Therefore,AHAS can be used as an effective screening target for antibiotics,herbicides and the like.At present,there are a few studies on the AHASs in extreme microorganisms.The exogenous AHAS gene in a recombinant E.coli strain preserved in our laboratory is derived from Thermotoga maritima,a model organism of the extreme thermophilic bacteria,which has an optimum growth temperature of 80?.The enzymes isolated from extreme thermophilic bacteria derived from marine extreme microorganisms have got extraordinary biological stability and can be used in extreme temperatures,pH,and pressure and exhibit biological activity at high ion intensity.Such an extreme thermophilic enzyme would offer more possibilities for biocatalysis and biotransformation.This thesis mainly includes two aspects of research.Firstly,we have established and evaluated a new pre-column derivatization-HPLC method to determine the activity of AHAS.We used in this protocol two well-studied derivatization reagents,namely 4-nitro-o-phenylenediamine(NPDA)and 2,4-dinitrophenylhydrazine(DNPH)to respectively derivatize and detec t the substrates of the AHAS reaction(?-keto acids)and the products(such as ?-aceto-?-hydroxylcarboxylic acids,?-diketones,acyloins,and aldehydes).Moreover,we calculated the specific activity of the enzyme via quantifications of the comsumptions of the substrates or the formations of the products.Comparing with the reported colorimetric(UV)method and gas chromatography(GC)method,the established method can not only simultaneously determine the residual amounts of the substrates and the productions of various products,clarifying the presence and concentrations of all the components in the reaction mixture,but also overcomes the drawbacks present in the UV method(incompetence in the measurement of the dual substrates reaction of AHAS)and the GC method(tedious operations and unavoidable interference in GC analysis).Secondly,by utilizing the established method and the combination with LC-MS determination,we found that besides the well-known catalytic mode of AHAS,namely the substrate pyruvate is employed as the donor substrate and another pyruvate or ?-ketobutyric acid is used as the acceptor substrate,there exists a new catalytic mode in the reaction mediated by AHAS of T.maritima(TmAHAS).This enzyme can also take ?-ketobutyric acid as a donor substrate and another ?-ketobutyric acid or pyruvate as the acceptor substrate.The reaction mode of dual donor substrates and dual acceptor substrates leads to very complicated products,including the main compounds acetolactate(also called ?-aceto-?-hydroxylpropionic acid,AL)and ?-aceto-?-hydroxyl-butyric acid(AHB)and the various byproducts.Based on further analysis of the reaction catalylized by AHAS and literature review,we successfully identified nine byproducts via combination of the pre-column derivatization-HPLC method with LC-MS determination.These byproducts include two aldehyde compounds acetaldehyde and propionaldehyde;three ?-diketones,namely 2,3-butanedione,2,3-pentanedione,and 3,4-hexanedione;and four acyloin compounds,acetoin(3-hydroxy-2-butanone),3-hydroxy-2-pentanone,2-hydroxy-3-pentanone,and 4-hydroxy-3-hexanone.Through these two experiments,we established a method for detecting AHAS activity in a single substrate(pyruvate)reaction,and explained the process of catalytic reaction under high temperature conditions and the constitution of the compound in the system after catalysis by TmAHAS.In this thesis we have built up and evaluated a novel pre-column derivatization-HPLC procedure to determine the activity of AHAS.Based on the method and under the assistance of LC-MS measurement,we systematically analyzed the consumption of the substrates and the formation and distribution of the products in the reaction catalyzed by TmAHAS.Furthermore,we discussed the insights of TmAHAS catalysis and the possible routes through which the products produce.These data lay solid ground for the further exploration of the catalytic mechanism of TmAHAS and the widening of its application in asymmetric organic synthesis.