TY - JOUR
T1 - A new member of the short-chain dehydrogenases/reductases superfamily
T2 - Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis
AU - Ye, Qi
AU - Yan, Ming
AU - Yao, Zhong
AU - Xu, Lin
AU - Cao, Hou
AU - Li, Zhengjiang
AU - Chen, Yong
AU - Li, Shuya
AU - Bai, Jianxin
AU - Xiong, Jian
AU - Ying, Hanjie
AU - Ouyang, Pingkai
PY - 2009/12
Y1 - 2009/12
N2 - A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS-PAGE and only required NADPH as an electron donor. The Km value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding Vmax was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.
AB - A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS-PAGE and only required NADPH as an electron donor. The Km value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding Vmax was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.
KW - Carbonyl reductase
KW - Pichia stipitis
KW - Short-chain dehydrogenases/reductases
KW - Substrate specificity
UR - http://www.scopus.com/inward/record.url?scp=68649096334&partnerID=8YFLogxK
U2 - 10.1016/j.biortech.2009.06.014
DO - 10.1016/j.biortech.2009.06.014
M3 - 文章
C2 - 19574038
AN - SCOPUS:68649096334
SN - 0960-8524
VL - 100
SP - 6022
EP - 6027
JO - Bioresource Technology
JF - Bioresource Technology
IS - 23
ER -
