Purification and characterization of a novel mannitol dehydrogenase from a newly isolated strain of Candida magnoliae

Abstract

Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37 degrees C and 10.0 and 40 degrees C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k(cat) = 823 s(-1), K(m) = 28.0 mM, and k(cat)/K(m) = 29.4 mM(-1) s(-1)) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

FIG. 1.

(A) Separation of MDH from C. magnoliae on DEAE-cellulose. The ammonium sulfate precipitate of C. magnoliae cell extracts was loaded onto a DEAE-cellulose column. (B) Separation of MDH from C. magnoliae on Cibacron Blue 3GA affinity resin. Active fractions from hydrophobic interaction chromatography were loaded onto a Cibacron Blue 3GA column. The proteins were eluted with a linear NaCl gradient, and each fraction (2 ml) was collected. Fractions were assayed for MDH activity by using fructose as a substrate. Bars I and II indicate the fractions used for native PAGE (inset). ○, protein; ▴, MDH activity; −, NaCl gradient.

FIG. 2.

PAGE and determination of molecular mass of MDH purified from the C. magnoliae. (A) Native PAGE; (A-a) activity staining after native PAGE; (B) SDS-PAGE. The enzyme solution was run on a 10% (wt/vol) polyacrylamide slab gel as described in Materials and Methods. The arrow indicates the protein band containing MDH. (C) Determination of the M_r of native C. magnoliae MDH, purified according to the present method, by gel filtration chromatography. The chromatography runs were performed as described in Materials and Methods. The arrow indicates the position for the MDH M_r from C. magnoliae. K_av = (V_e − V_o)/(V_t − V_o); V_e, elution volume of protein; V_o, elution volume of Blue Dextran 2000; V_t, total bed volume.

FIG. 3.

Comparison of internal amino acid sequences of C. magnoliae MDH with those of other SDRs, including MDHs (A. bisporus [26], Drosophila ADH [38], Bactrocera oleae ADH [7], E. coli gluconate DH [5], and Klebsiella aerogenes ribitol DH [18]). The domain names αB, βD, and βF were assigned by Jörnvall et al. (28). Residues given against a black background are those conserved in more than 90% of the sequences aligned, and boxed residues are conserved in more than half of the aligned sequences. The percentages are given according to the results of complete alignment with the 57 enzymes in reference .

FIG. 4.

Effects of substrate concentration on the activities of MDH. MDH activity of the enzyme (1 U) was measured in the presence of the indicated concentrations of d-fructose and 0.25 mM NADPH, at pH 7.5. The inset shows a Lineweaver-Burk plot of initial velocity versus various fixed d-fructose concentrations. Each value represents the mean of triplicate measurements and varied from the mean by not more than 10%.

FIG. 5.

Graphical analysis of the inhibition of C. magnoliae MDH by mannitol. The effects of increasing mannitol (product) concentration on the apparent K_m and V_max values for fructose and NADPH were examined. Analysis of these data by double-reciprocal plots indicated that mannitol inhibited MDH noncompetitively with respect to fructose (A) and NADPH (B). In panel C, the secondary plots for noncompetitive inhibition with fructose and NADPH are shown. The mannitol product binds to MDH with a K_i of 188 mM.