Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Abstract

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K m ‫؍‬ 7.9 mM; k cat /K m ‫؍‬ 0.73 mM ؊1 s ؊1 ). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k cat /K m ‫؍‬ 450 mM ؊1 s ؊1 ) than with NADPH (k cat /K m ‫؍‬ 5.5 mM ؊1 s ؊1 ), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu 2؉ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.Erythritol, a four-carbon polyol, is widely distributed in nature (16). Like most other polyols, erythritol is a metabolite or storage compound and is found in seaweed, mushrooms, and fruits. It is a noncaloric, noncariogenic sweetener that is safe for diabetics (35). Erythritol has about 70% of the sweetness of sucrose in a 10% (wt/vol) solution and a very high negative heat capacity, providing a strong cooling effect when dissolved (16). Erythritol can be synthesized from dialdehyde starch by a high-temperature chemical reaction in the presence of a nickel catalyst (39). This process has not been industrialized because of its low efficiency. Erythritol also can be produced by microbial methods that utilize osmophilic yeasts and some bacteria (42, 48), and it has been produced commercially by using a mutant of Aureobasidium that produces erythritol in high yield (44% [wt/wt] glucose) (20). Recently, a high-erythritol-producing yeast strain was isolated from honeycombs and the new isolate was identified as Candida magnoliae KFCC 11023 (44). This strain can produce erythritol in high yield (43% [wt/wt] glucose) when cultured appropriately (42, 44).There are many repor...

Purification and characterization of carbonyl reductase from Geotrichum candidum

“…strain S749 [10], while alkaline pH optimum (7.5-8.5) was observed in C. parapsilosis DSM 70125 [5]. Carbonyl reductases from G. capitatum JCM 3908 [14] and C. magnolia KFCC 11023 [23] had pH optima at neutral pH (7.0). The pH stability of carbonyl reductase from G. candidum was determined at various pH ranging from 4.4 to 6.4 and the enzyme showed higher stability at pH 5.4 with a half-life (t 1/2 ) of 7.13 h (data not shown).…”

“…magnolia KFCC 11023[23] and C. parapsilosis DSM 70125[5] are made up of two identical subunits (holoenzyme of 79 and 135 kDa, respectively). Heterodimeric carbonyl reductase has been reported in G. capitatum JCM 3908 with two subunits of 39 and 41 kDa…”

Primary Structure Analysis and Functional Expression of Erythrose Reductases from Erythritol-Producing Fungi (Trichosporonoides megachiliensisSNG-42)

“…We characterized three isozymes of erythrose reductases (ER-I, ER-II, and ER-III) biochemically from Trichosporonoides megachiliensis SNG-42, one of the strains used for commercial production of erythritol. 1,2) In Candida magnoliae, partial amino acid sequences of the enzyme, which had erythrose reductase activity, have been reported, 3) but the entire primary structures and the corresponding cDNAs for ERs remain unknown. In this paper, we report the primary structure, cDNA isolation, and functional expression of erythrose reductases in Escherichia coli.…”

Proteomics and physiology of erythritol-producing strains

“…Fumarate and 1.8-dihydroxynaphthalene-melanin inhibited the activity of erythrose reductase in an uncompetitive and non-competitive type, respectively [37,60]. Characterization of erythrose reductase in C. magnoliae indicated that it was one of the aldose reductase with a high preference for NADH in contrast to the typical aldose reductase acting with NADPH [36]. Recently, our laboratory identified the nucleotide sequence of erythrose reductase in C. magnoliae with N-terminal homology to the typical aldose reductase (data not shown).…”

“…Inside the cooling chamber with carbon dioxide, an MSK Cell Homogenizer (B. Braun Japan Co. Ltd.) disrupted the acetone-treated cells repeatedly for a short time. To obtain key enzymes in erythritol biosynthesis, T. corallina and C. magnoliae cells were ruptured by grinding with glass beads of 0.5 mm in diameter [35,36]. The cells were resuspended in disruption buffer containing 10 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride (PMSF) and 20 mM Tris-HCl (pH 7.8) for T. coralline or 50 mM potassium phosphate buffer (pH 7.0) for C. magnoliae.…”