Expression and characterization of a thermostable sarcosine oxidase (SOX) from Bacillus sp. in Escherichia coli.
A heat-stable sarcosine oxidase produced by Bacillus sp. BSD-8 (SOX) had been studied and its complete gene sequence, which contained 1,164 bp nucleotides and encoded a protein of 387 amino acids, was obtained by DNA Walking method. The sox gene was cloned and functionally overexpressed in E. coli and the recombinant SOX (rSOX) was purified to homogeneity, its properties was studied and compared with the wild type of SOX. The rSOX as well as SOX was stable at 60 degrees C and at pH 7.0 approximately 10.0, respectively. The optimal temperature for this enzyme was 60 degrees C and at pH 8.5, it showed its highest activity. The Km and Kcat of the enzyme was 3.1 mM and 20.3/s, respectively. The difference between the properties of the SOX and rSOX was that the SOX contained noncovalent FAD, whereas the rSOX contained covalent FAD. The study also showed that an increased number of alanine residues in the rSOX might have some contribution in the enzymatic thermostability.
Guo
Kangping
K
College of Life Sciences, Zhejiang University, No. 268 , Kaixuan Road, Hangzhou 310029, People's Republic of China.
Ma
Xiaohang
X
Sun
Guiqin
G
Zhao
Yuhua
Y
Li
Xia
X
Zhao
Weifeng
W
Kai
Lei
L
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008967:Molecular Biology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050879:Sarcosine Oxidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D050879:Sarcosine Oxidase