Premium
Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae
Author(s) -
Zhang Min,
Jiang Shaotong,
Zheng Zhi,
Li Xingjiang,
Luo Shuizhong,
Wu Xuefeng
Publication year - 2015
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.201400786
Subject(s) - rhizopus oryzae , biochemistry , xylose , pichia pastoris , cofactor , recombinant dna , catabolite repression , reductase , chemistry , enzyme , biology , gene , fermentation , mutant
Rhizopus oryzae is valuable as a producer of organic acids via lignocellulose catalysis. R. oryzae metabolizes xylose, which is one component of lignocellulose hydrolysate. In this study, a novel NADPH‐dependent xylose reductase gene from R. oryzae AS 3.819 ( Roxr ) was cloned and expressed in Pichia pastoris GS115. Homology alignment suggested that the 320‐residue protein contained domains and active sites belonging to the aldo/keto reductase family. SDS–PAGE demonstrated that the recombinant xylose reductase has a molecular weight of approximately 37 kDa. The optimal catalytic pH and temperature of the purified recombinant protein were 5.8 and 50 °C, respectively. The recombinant protein was stable from pH 4.4 to 6.5 and at temperatures below 42 °C. The recombinant enzyme has bias for D ‐xylose and L ‐arabinose as substrates and NADPH as its coenzyme. Real‐time quantitative reverse transcription PCR tests suggested that native Roxr expression is regulated by a carbon catabolite repression mechanism. Site‐directed mutagenesis at two possible key sites involved in coenzyme binding, Thr 226 → Glu 226 and Val 274 → Asn 274 , were performed, respectively. The coenzyme specificity constants of the resulted Ro XR T226E and Ro XR V274N for NADH increased 18.2‐fold and 2.4‐fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification. J. Basic Microbiol. 2015, 55 , 1–15