1060
S. KAWANO et al.
Table 6. Comparison of Biochemical Properties of (R)-Specific Alcohol Dehydrogenases
Leifsonia sp.
ADHa
Lactobacillus kefir
ADHb
Lactobacillus brevis
ADHc
Pseudomonas sp.
ADHd
Property
AFPDH
Classification
Coenzyme
Molecular mass
Short-chain ADH
NADH
110 kDa,
Short-chain ADH
NADPH
105 kDa,
Short-chain ADH
NADPH
105 kDa,
Short-chain ADH
NADH
NADH
59.9 kDa,
homodimer
none
162 kDa,
homotetramer
NDe
homotetramer
none
homotetramer
)
homotetramer
2þ
Mg2 (Mn
þ
2þ
Mg (Mn
ꢀ
2þ
)
Metal requirement
Temperature optimum
pH-optimum
ꢀ
ꢀ
ꢀ
50 C
50 C
37 C
65 C
ND
reduction
oxidation
Substrate specificity
pH 5.0–5.5
pH 8.5
pH 6.0
pH 9.5
pH 7.0
pH 8.0
pH 7.0
ND
pH 6.5
>pH 10
(
(
(
R)-1-(2-Pyridyl)-ethanol
R)-1-Phenylethanol
S)-1-Phenyl-2,2,2-trifluoroethanol
>99%e.e.
>99%e.e.
>99%e.e.
ND
99%e.e.
>99%e.e.
>97%e.e.
>99%e.e.
>99%e.e.
ND
ND
ND
ND
94%e.e.
92%e.e.
aData from references 18 and 19
bData from references 5, 20, and 21
cData from reference 5
dData from references 22 and 23
eND, not determined
dehydrogenase28) have been reported. Recombinant cells
that can overproduce two enzymes, a reducing enzyme
for substrates and an NAD(P)H regenerating enzyme,
should be powerful tools for the industrial syntheses of
chiral alcohols.2 While some NADH-dependent alco-
hol dehydrogenases can catalyze both asymmetric
WL, Poel T-J, Thomas RC, Voorman RL, Stefanski KJ, Stehle
RG, Tarpley WG, and Morris J, J. Med. Chem., 41, 1357–1360
(
1998).
7
)
)
Kawano S, Horikawa M, Yasohara Y, and Hasegawa J, Biosci.
Biotechnol. Biochem., 67, 809–814 (2003).
6)
8
Wishka DG, Grber DR, Seest EP, Dolak LA, Han F, Watt W,
and Morris J, J. Org. Chem., 63, 7851–7859 (1998).
þ
reduction of the substrate and the reduction of NAD
via cheap alcohols, in these cases, a second enzyme for
9) Matsuda H, Asamuma G, Shin T, Shiono M, and Kikuyama S,
European Patent Application 911335 (Apr. 28, 1999).
10) Bradford MM, Anal. Biochem., 72, 248–254 (1976).
2
1,23)
NADH regeneration is not required.
AFPDH can
1
1) Kizaki N, Sawa I, Yano M, Yasohara Y, and Hasegawa J,
Biosci. Biotechnol. Biochem., 69, 79–86 (2005).
also oxidize 2-propanol to acetone, so reduction of
carbonyl compounds to chiral alcohols by this enzyme
may be accomplished by the use of 2-propanol as
NADH regenerator. Both of these reactions are rever-
sible. Hence there is a call for an additional device to
remove acetone from the reaction mixture so that the
reduction can go to completion. As explained above, the
reactions catalyzed by glucose dehydrogenase and
formate dehydrogenase are irreversible. Hence the
application of these enzymes to the coenzyme regener-
ation makes it easy to achieve a high yield for the
reduction.
1
2) Wermuth B, J. Biol. Chem., 256, 1206–1213 (1981).
3) Tsukube H, Shinoda S, Uenishi J, Hiraoka T, Imakoga T, and
Yonemitsu O, J. Org. Chem., 63, 3884–3894 (1998).
1
14) Britt AJ, Bruce NC, and Lowe CR, FEMS Microbiol. Lett., 93,
9–56 (1992).
4
1
5) Isobe K and Wakao N, J. Biosci. Bioeng., 96, 387–393 (2003).
6) Karanewsky DS, Badia MC, Ciosek Jr CP, Robl JA, Sofia MJ,
Simpkins LM, DeLange B, Harrity TW, Biller SA, and Gordon
EM, J. Med. Chem., 33, 2952–2956 (1990).
1
1
1
7) Wittman M, Carboni J, Attar R, Balasubramanian B, Balimane
P, Brassil P, Beaulieu P, Chang C, Clarke W, Dell J, Eummer J,
Frennesson D, Gottardis M, Greer A, Hansel S, Hurlburt W,
Jacobson B, Krishnananthan S, Lee FY, Li A, Lin T-A, Liu P,
Ouellet C, Sang X, Saulnier MG, Stoffan K, Sun Y, Velaparthi
U, Wong H, Yang Z, Zimmermann K, Zoeckler M, and Vyas D,
J. Med. Chem., 48, 5639–5643 (2005).
Without limitation to the above mentioned 1-
pyridyl)ethanol derivatives, various other versatile
(
optically active compounds can be produced by suitable
application of AFPDH. Further detailed studies, includ-
ing the gene cloning and expression of AFPDH and
practical application in chiral alcohol production, are
scheduled to be reported in a subsequent paper.
8) Nie Y, Xu Y, Mu XQ, Wang HY, Yang M, and Xiao R, Appl.
Environ. Microbiol., 73, 3759–3764 (2007).
19) Kira I and Onishi N, Biosci. Biotechnol. Biochem., 73, 1640–
646 (2009).
0) Inoue K, Makiko Y, and Itoh N, Appl. Environ. Microbiol., 71,
633–3641 (2005).
1
2
2
3
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