Affiliation:
1. BioNgene Co. Ltd., Jongro-Ku, Seoul
2. Bolak Co. Ltd., Hwasung-Si
3. Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin-Si, Kyunggi-Do, Korea
Abstract
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°C and 10.0 and 40°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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
43 articles.
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