Production of d-malate and d-citramalate by Arthrobacter pascens DMDC12 having stable citraconase

doi:10.1016/S0032-9592(00)00219-3

Process Biochemistry

Volume 36, Issue 5, December 2000, Pages 407-414

https://doi.org/10.1016/S0032-9592(00)00219-3 Get rights and content

Abstract

Citraconate-utilizing bacteria were found to produce d-malate and d-citramalate from maleate and citraconate, respectively. A newly isolated strain DMDC12 showed very stable and high hydratase activity on maleate and citraconate. The strain DMDC12 was identified taxonomically as Arthrobacter pascens. The optical purities of the malate and citramalate produced by this strain were 97.2% and 99.9% d type, respectively. Under optimized conditions, 204.1 g/l of malate and 187.2 g/l of citramalate were produced from maleate and citraconate in 48 h using permeabilized resting cells, with the molar yields of 94.9% and 95.6%, respectively. The hydratase in A. pascens was very stable when 0.6 M of maleate or citraconate was added as the substrate. It was also very stable in the presence of 1 M NaCl. The hydratase was considered to be citraconase since it was strongly induced by citraconate and d-citramalate, but neither by maleate and d-malate nor by 3-hydroxybenzoate and gensitate. These suggested different characteristics between the citraconase of A. pascens and the reported malate-producing enzyme.

Introduction

Biological formation of optical materials for fine chemical industry has become a process of potential commercial interest, due to the growing industrial need for optically active synthons. d-Citramalate and especially, d-malate are optically active α-hydroxyacid, and are valuable sources of chirality for various applications. d-Malate is used as a starting material for the synthesis of a wide variety of pharmaceutical and chemical compounds, such as antibiotics [1], [2], [3], antivirals [4], antitumors [5], [6], [7], HMG-CoA reductase inhibitor [8], carnitine [9], antihistamines [10], novel biodegradable optically active network polyesters [11], bioresorbable composite polyester which is preferred for nerve growth channels [12], and many other compounds [13], [14]. d-Malic acid is also used as a resolving agent [15]. d-Citramalate is applied as a chiral synthon in the synthesis of chiral aliphatic sulfones [16], but the high cost of d-citramalate has reduced its application. Thus reduction in the cost of production of these optical materials will increase the scope of their commercial applications.

The methods for the production of d-malate from maleate by malease of various microorganisms were reported by Nakayama et al. [17], Nakajima et al. [18], Asano et al. [19], and Van der Werf et al. [20], [21]. The malease from Pseudomonas pseudoalcaligenes showed the highest activity [20]. d-Citramalate was produced enzymatically [21], [22]. Van der Werf et al. also reported on production of d-citramalate from citraconate by the malease of P. pseudoalcaligenes [21]. However, it is difficult to obtain large amounts of P. pseudoalcaligenes cells since 3-hydroxybenzoate was toxic to the cells [23] and the malease from this strain was unstable [24].

This study was aimed at isolating microorganisms which possess efficient and stable hydratase of maleate and citraconate. The induction of the hydratase of Arthrobacter pascens showed unique characteristics.

Section snippets

Isolation of citraconate-assimilating microorganisms

Citraconate-utilizing microorganisms were isolated from soil samples, with a medium containing 50.0 g of citraconic acid, 10.0 g of KH₂PO₄, 2.0 g of NH₄NO₃, 500 mg of MgSO₄ · 7H₂O, 15 mg of FeSO₄ · 7H₂O and 100 mg of yeast extract (YE) in 1000 ml distilled water. The pH was adjusted to 7.0 with NaOH. FeSO₄ · 7H₂O solution was sterilized separately and was then added to the rest of the medium under aseptic conditions. The agar (1.5%) plate medium had the same compositions except that 5.0 g/l of

Screening and identification of d-malate- and d-citramalate-producing strains

Using citraconate as a sole carbon source, 65 strains of citraconate-assimilating bacteria were isolated from soil with the screening medium described above. Out of these strains, 21 strains produced d-malate and d-citramalate from maleate and citraconate, and 8 strains produced more than 60 g/l of malate or citramalate in 20 h. The best producer, strain DMDC12, was selected and used in further experiments.

Taxonomic studies of strain DMDC12 were performed by the National Collection of

Screening for efficient-stable and distinct d-malate and d-citramalate-producing strains

Although some studies have been reported on d-malate production from maleate [17], [18], [19], [20], [21], the activity of malease was unstable and was considered unsuitable for long period operation for the production of d-malate and d-citramalate [24]. In order to isolate microorganisms which not only possess effective and stable hydratase, but also have different characteristics from the reported hydratase, citraconate was selected as a sole carbon source at a concentration of 5% (w/v). With

Acknowledgements

We thank Dr. James C. Ogbonna of this Institute for helpful discussion in the preparation of the manuscript.

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Production of d-malate and d-citramalate by Arthrobacter pascens DMDC12 having stable citraconase

Abstract

Introduction

Section snippets

Isolation of citraconate-assimilating microorganisms

Screening and identification of d-malate- and d-citramalate-producing strains

Screening for efficient-stable and distinct d-malate and d-citramalate-producing strains

Acknowledgements

Tetrahedron Lett

Tetrahedron

Tetrahedron Lett

Enzyme Microb Technol

J Biol Chem

J Biol Chem

Enantioselective syntheses of 3-substituted 4-(alkoxycarbonyl)-2-azetidinones from malic acid

J Org Chem

Stereocontrol in the addition of allyl metal reagents to an optically active imine derived from malic acid, leading to a formal synthesis of (+)-negamycin

Chem Lett

Synthesis and antiviral activity of 9-alkoxypurines. 2. 9-(2,3-dihydroxypropoxy)-, 9-(3,4-dihydroxybutoxy)-, and 9-(1,4-dihydroxybut-2-oxy) purines

J Med Chem

Synthetic studies of halichondrin B, an antitumor polyether macrolide isolated from a marine sponge. 2. Efficient synthesis of C16–C26 fragments via construction of the D ring by a highly stereocontrolled iodoetherification

Syn Lett

New chiral route to (-)-swainsonine via an aqueous acylnitroso cycloaddition approach

J Org Chem

Optical isomers of rocastine and close analogues: synthesis and H1 antihistaminic activity of its enantiomers and their structural relationship to the classical antihistamines

J Med Chem

Preparation and characterization of novel biodegradable optically active network polyesters from malic acid

Macromolecules