Production of D-malate by maleate hydratase from Pseudomonas pseudoalcaligenes

The biological activity of a chiral compound with respect to its pharmaceutical and agrochemical activity, flavour and taste can vary dramatically for the different enantiomers. Especially when using chiral compounds for pharmaceutical or agrochemical applications, the presence of the "wrong" stereoisomer can have severe effects on patients or may cause an additional environmental load. The availability of (cheap) optically pure compounds is, therefore, of prime importance, especially for the pharmaceuticals industry.There are several ways in which optically active compounds can be produced. One way is by using biocatalysts. During the last decade, the importance of biocatalysis for the production of optically pure fine-chemicaIs has increased, and it is expected to increase even further in the near future. Advantages of enzymes as catalysts are the fact that they work under mild reaction conditions and their reaction-, regio-, and stereospecificity. These last three aspects also imply that fewer side- products are produced which is positive in view of the increasing environmental concern. Enzymes that can transform a substrate for 100% into a 100% optically pure product, and which do not require cofactor recycling are, both from an economical and an environmental point of view, most suitable as biocatalysts.D-Malate is an optically active compound which can be used as a synthon in organic synthesis, as a resolving agent and as a ligand in asymmetric catalysis (Chapter 1).D-Malate can be produced in several ways, but the approach using the lyase maleate hydratase (malease; EC 4.2.1.31), which catalyzes the hydration of maleate toD-malate, seems to be the most promising approach for commercialization (Chapter 1).Lyases are enzymes that catalyze the cleavage of C-C, C-N, C-O and other bonds by elimination to produce double bonds or, conversely, catalyze the addition of groups to double bonds. These enzymes do not require cofactor recycling, show an absolute stereospecificity (100% e.e.) and can give a theoretical yield of 100%. Lyases are attracting increasing interest as biocatalysts for the production of optically active compounds, and have already found application in several large commercial processes (Chapter 2).We have screened more than 300 microorganisms for the presence of malease activity (Chapter 3). Many strains (n = 128) could convert maleate toD-malate with an enantiomeric purity of more than 97%. Accumulation of fumarate during incubation of permeabilized cells with maleate was shown to be indicative for the presence of the unwanted maleate cis-trans -isomerase activity, which ultimately results in the formation of the unwantedLenantiomer of malate. The ratio in which fumarate and malate accumulated could be used to estimate the enantiomeric composition of the malate formed (Chapter 3).Pseudomonas pseudoalcaligenes NCIMB 9867 was selected for more detailed studies, because it contained one of the highest malease activities, theD-malate formed had an enantiomeric purity of more than 99.97%, it did not degradeD-malate and because it did not show any side-product formation (Chapter 3).The highest malease activity in P.pseudoalcaligenes was observed when it was grown on 3-hydroxybenzoate (Chapter 4). Growth on gentisate also resulted in an enhanced malease activity. Both compounds are degraded via maleate in this microorganism. The specific malease activity of cells grown on 3-hydroxybenzoate was constant during the logarithmic phase, but dropped rapidly as soon as growth ceased.Malease from P. pseudoalcaligenes was purified (Chapter 5). The purified enzyme (89 kDa) consisted of two subunits (57 and 24 kDa). No cofactor was required for full activity of this colorless enzyme. The stability of the enzyme was dependent on the protein concentration and the presence of dicarboxylic acids. Maximum enzyme activity was measured at pH 8 and 45°C. The purified enzyme also catalyzes the hydration of citraconate (2-methylmaleate) formingD-(+)-citramalate and of 2,3- dimethylmaleate at, respectively, 54 and 0.8% of the rate of maleate hydration (30°C). The KM of malease for maleate was 0.35 mM, and for citraconate 0.20 mM. The productsD-malate andD-citramalate and the substrate analog 2,2-dimethylsuccinate were strong competitive inhibitors of malease (Chapter 5).Hydratases catalyze equilibrium reactions. To optimize the reaction conditions, data concerning the equilibrium constant are necessary to determine the maximal obtainable yield. In literature no data were available concerning the equilibrium of the malease catalyzed reactions. Therefore, we determined the equilibrium constants ( Kapp ) for the malease catalyzed hydration reactions. The Kapp for the maleate, citraconate, and 2,3-dimethylmaleate hydration reactions were 2050, 104 and 11.2, respectively, under standard biochemical conditions (25°C, pH 7.0, I =0.1) (Chapter 5 and 6). The equilibrium constants for the maleate and citraconate hydration reactions make yields of more than 99% possible.As especially maleate has a high p K a 2 , the influence of the pH (6.0-8.5) on the Kapp was determined to describe the influence of the presence of the different forms of maleate (i.e. dianionic, monoanionic and diprotonated) on the equilibrium constant (Chapter 6). Also the influence of the temperature (10°C - 40°C) on Kapp was determined. From these experiments the Gibbs-free-energy change (ΔG°'), the enthalpy change (ΔH°'), and entropy change (ΔS°') under standard biochemical conditions for the maleate 2-and citraconate 2-hydration reactions were calculated (Chapter 6). Also the effect of the temperature on the maleate and citraconate hydration rates of the purified enzyme was determined to calculate the activation energy. At low temperatures the hydration rate of citraconate was higher, while at temperatures above 18°C the maleate hydration reaction was faster (Chapter 6).P. pseudoalcaligenes is not able to grow on maleate, probably because it lacks an uptake system for maleate (Chapter 3). Intact cells of P.pseudoalcaligenes do not show any accumulation ofD-malate from maleate but Triton X-100 treated cells showed accumulation ofD-malate from maleate, indicating that these cells were permeabilized or lysed (Chapter 7). Incubation of cells with Triton X-100 also resulted in an increase in the protein concentration of the supernatant, indicating the occurrence of lysis. Permeabilization and lysis were time-dependent: longer incubations resulted in higher malease activities and more protein in the supernatant. The permeabilization and lysis rates were also dependent on the Triton X-100 and biomass concentration (Chapter 7).Malease activity of permeabilized cells of P.pseudoalcaligenes decreased strongly when Na 2 - maleate concentrations higher than 0.6 M were used as the substrate (Chapter 8). When other counter- ions than Na +were used, in some instances the malease activity was found to be affected much less by high substrate concentrations. When for instance Mg 2+was used as the counter-ion, a much larger percentage of maleate is present as metal-substrate complex, thereby reducing the "real" substrate concentration resulting in less substrate inhibition. When Ca 2+and Ba 2+were used as the counter- ion, the malease activity was not at all affected by increasing substrate concentrations. The use of these metal-ions resulted in the formation of a crystal-liquid two-phase system, due to the low solubility of the metalsubstrate complex. In this situation, the "real" substrate concentration was independent on the total amount of substrate present. Ca 2+was the best counter-ion for the conversion of maleate intoD-malate. The use of this metal-ion resulted in the highest malease activities and the absence of substrate inhibition at high substrate concentrations (Chapter 8). In this way, high concentrations (up to 160 g/l) of either maleate or citraconate were converted by malease of P.pseudoalcaligenes intoD-malate andD-citramalate, respectively, with yields of more than 99%.To determine the potential of the crystal-liquid two-phase system for the large scale conversion of maleate intoD-malate, the conversion of one kilogram of maleate was studied (Chapter 9). At a substrate concentration of 200 g/l, permeabilized cells of P.pseudoalcaligenes (1 g/l protein) converted maleate within two days into 1. 15 kg ofD-malate with a yield of 99.4%.During our studies on theD-malate production from maleate with malease from P.pseudoalcaligenes, also three Japanese groups published about their studies on this conversion, using other biocatalysts. P.pseudbalcaligenes was the best malease containing biocatalyst (Chapter 10). It producedD-malate at the highest product concentrations, with the highest yield and the highest specific activity. Comparing these data with the data for the already commercialized process forL-malate production catalyzed by fumarase, suggests that commercialization of the malease process is very promising (Chapter 10).