The Establishment Of A New Method For The Detection Of Bacillus Subtilis Acetolactate Synthase Activity And The Study Of The Substrate Preference Of This Enzyme

Acetohydroxyacid synthase(AHAS)is one of the rate-limiting enzymes that catalyze the synthesis of the three essential branched-chain amino acids including isoleucine,leucine,and valine in various organisms.Since branched-chain amino acids can only be biosynthesized in bacteria,fungi,algae,and higher plants,but not in animals and human bodies,people have to obtain these essential constituents from foods.Therefore,AHAS has been regarded as a potential target for developing novel herbicides and antimicrobial agents.Acetolactate synthase(ALS)is a key enzyme that catalyzes the synthesis of in isobutanol biosynthetic pathway of microorganisms,and it is of great significance for the regulation of isobutanol metabolic pathway.So in order to clarify the functions and catalytic mechanism of AHAS/ALS,it is of great importance to set up methods for measuring the activity of AHAS/ALS.Currently GC and UV methods,especially the UV protocols are frequently employed to determine AHAS/ALS activity.However,these methods can not fully meet the needs of various determinations due to their intrinsic drawbacks,so in order to understand more of the enzyme we still need to establish new analytical methods.In this paper,1,2-dia mino-4,5-methylenedioxybenzene(DMB)was used as a derivatization reagent to establish a simple and convenient pre-column derivatization-HPLC method to determine the activity of Bacillus subtilis ALS and its substrates preference.The derivatization of the ketoacids(pyruvate and 2-ketobutyrate)and the ?-dicarbonyl compounds(butanedione and 2,3-pentanedione)were carried out under experimental conditions listed below:excessive DMB(the molar ratio of ketoacids / ?-dicarbonyl?10:1),reaction at 80 ?for 1 h.The HPLC detection conditions for the derivatives were as follows:ODS column(250?4.6 mm,5 ?m),acetonitrile: water=35%: 65% isocratic elution,elution time was 25 min,and detection wavelength was 364 nm.When pyruvate,2-ketobutyrate,butanedione,and 2,3-pentanedione were detected using DMB as a derivatizing agent,sodium pyruvate was detectable in a linear range of 0.02-1.0 m M and the equation was y=15384x-345.4 with R2=0.993;2-ketobutyrate had good linearity in the range of 0.02-0.8m M,the linear equation was y=3423x-118.5 with R2=0.995;2,3-butanedione was detected in the linear range of 0.02-1.0 m M,the equation was y=3344x-6.886 with R2=0.997;2,3-pentanedione was detected in the linear range of 0.02-1.0 m M,and the equation is y=1259x-30.93 with R2=0.998.Based on the above experimental results,a simple and efficient pre-column derivatization-HPLC method was established to determine the activityof ALS.This method can be used for the determination of the ALS activity not only in the single substrate(pyruvate)reaction,but also in the dual substrates(pyruvate and 2-ketobutyrate)reaction.Furthermore,the activity of ALS can be evaluated from both the substrate consumption and the product generation.Using the newly established precolumn derivatization-HPLC method,we measured the steady-state kinetic parameters of ALS in both the single substrate and the dual substrates processes.More importantly,we emphatically investigated the substrates preference of the enzyme in the reaction employing pyruvate and 2-ketobutyrate as dual substrates.In summary,a new precolumn derivatization-HPLC method for determining the activity of ALS using DMB as a derivatization reagent has been established in the research.The method is simple,accurate,and reliable and can be extended to determine the substrates preference of ALS in the dual substrates process.This work lays a solid ground for the further elucidation of the catalytical mechanism of ALS.