Expression of Cholesterol Oxidase Gene from Arthrobacter simplex in Escherichia coli and Pichia pastoris

• Main Authors: Hsin-Ho Huang, 黃星賀
• Other Authors: Wen-Hsiung Liu
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/50432532375295101075

碩士 === 國立臺灣大學 === 農業化學研究所 === 91 === Isolating the cholesterol oxidase genes from constitutive cholesterol oxidase-producing strain, Arthrobacter simplex F2-20-8 and inducible cholesterol oxidase-producing strain, A. simplex U-S-A-18 by polymerase chain reaction was conducted in this study according to N-termianl amino acid sequence of cholesterol oxidase secreted from constitutive cholesterol oxidase-producing strain, A. simplex F2-20-8. The length of open reading frames of both cholesterol oxidase genes was 1656 bp after nucleotide sequencing. Although there were some difference in nucleotide sequence between two genes, the deduced amino acid sequences were the same. This indicated that difference in nucleotide sequence between two genes was due to third-base degeneracy. The length of putative signal sequence of A. simplex cholesterol oxidase was 49 amino acids encoded by 147 bp nucleotides. The length of the A. simplex cholesterol oxidase mature peptide was 502 amino acids encoded by 1509 bp nucleotides. The predicted molecular weight was estimated as 54,261 Da by Genetics Computer Group (GCG) program. The gene fragment of A. simplex cholesterol oxidase mature peptide was transformed into E. coli JM109 by pQE31 expression vector. The intracellular activity was 1.1 U / mL after 16 h cultivation using LB medium and inducing by IPTG. The molecular weight of the purified intracellular protein was about 55 kDa on SDS-PAGE. Alignment to the Basic Local Alignment Search Tool (BLAST) database showed that the similarity of A. simplex, Rhodococcus equi and Brevibacterium sterolicum was above 99%. The gene fragment (1509 bp ) of A. simplex cholesterol oxidase mature peptide and the gene fragment (1656 bp) of A. simplex cholesterol oxidase preprotein were transformed and inserted into chromosome of P. pastoris by extracellular expression vector pPICZA. Two recombinants pPC and pPORF exhibiting extracellular cholesterol oxidase activity were isolated, respectively. Recombinants pPC and pPORF were cultivated in a glycerol-containing medium and shaken at 28℃ for 18 h, and then replaced with a 0.5% methanol-containing medium for induction. The former achieved to 1.0 U / mL of extracellular cholesterol oxidase activity at 72 h cultivation and the later achieved the same extracellular cholesterol oxidase activity at 120 h cultivation.