Purification And Characterization Of Maleate Hydratase From Arthrobacter Pascens DMDC12

Chiral compound are generally used in many fields including medicine and fine-chemicals industry. One of the major application areas in biotechnology is the production of optically active compounds for the fine-chemicals industry.D-malate is becoming an important chiral compound which is a widely used as a versatile exploitation foreground in chiral pharmaceutical synthesis, chiral additive reagent and chiral accessory reagent. For example, It is used as a starting material for the synthesis of a wide variety of pharmaceutical and chemical compounds, such as antibiotics, antivirals, antitumors, and HMG-CoA reductase inhibitor.Maleate hydratase (D-malate hydro-lyase EC4.2.1.31) catalyzes the hydration of maleate to D-malate by trans addition to the double bond. Sachs and Jensen first reported the existence of maleate hydratase in corn kernels, although the optical purity of the malate formed was not indicated.Subsequently, several workers reported the presence of the enzyme in mammalian kidneys and microorganisms. The methods for the microbial productions of D-malate from maleate were reported by Nakayama, Asano, Van der werf, and He.Dreyer purified the enzyme from rabbit kidney, it contains an iron-sulfur cluster and is dependent on a divalent mental ion and a sulfur compound for activity. Asano and Van der werf had purified and characterized maleate hydratase respectively from Arthrobacter sp MCI2612 and Pseudomonas pseudoalcaligenes NCIMB9867.This paper describes the purification and characterization of maleate hydratase from Arthrobacter pascens DMDC12. The properties of maleate hydratase have been studied.Maleate hydratase was purified from a crude extract of Arthrobacter pascens DMDC12 strain by DEAE Sepharose^TM Fast Flow and Phenyl-Sepharose HP column chromatographies. Three distinct maleate hydratase (MHaseA, MHaseB and MHaseC) was partially purified from stain Arthrobacter pascens DMDC12.Each of them catalyzed the stereospecific addition of water to maleate andcitraconate, forming D-malate and D-citramalate, respectively. But these three hydratase showed different activity ratio for malease to citraconase. Three kinds of maleate hydratases have different Km for maleate and citraconate. A interesting phenomenon is that three distinct maleate hydratase can catalyzed fumaric acid, but the hydration product did not completely characterize.Maximum enzyme activities were measured at different pH and temperature.Arthrobacter pascens DMDC12 is a halophile, that can possible explain the high NaCl and (NH^SC^ concentrations(0.5M¹.5M) did not appear to have unfavorable affect on activity of any enzyme.As part of development for industrial production, the stability of the hydratase was most important. The maleate hydratase from reported D-malate-producing strains were unstable.The lower temperature resulted in a better stabilization of maleate hydratase activity. So in purification, Addition of (NH^SC^ to the phosphate buffer stabilized the maleate hydratase from stain Arthrobacter pascens DMDC12. D, L-malate also stabilized maleate hydratase, therefore, phosphate buffer containing D, L-malate was used during the different purification steps.Malease from Arthrobacter sp. required activation by ferrous ion and sulfhydryl compounds such as glutathione, L-cysteine, 2-mercaptoethanol, dithiothreitol, but malease from P. pseudoalcaligenes does not required the addition of cofactors for full enzyme activity. In this study, three kinds of maleate hydratases were not activated by external supply of DTT, oppositely, DTT inhibited MhaseA and MhaseB activity. With the addition of ferrous ion, each of maleate hydratases from DMDC12 was stimulated. Hg2+ strongly inhibited MHaseA, MHaseB and MHaseC activity.These results suggested that the three maleate hydratase possessed different characteristics, and there are at least two new type of maleate hydratase in strain DMDC12.