Molecular Biology Study Of Cholesterol Oxidase Gene Of Streptomyces Griseus ATCC14811

Cholesterol oxidase (E C 1. 1. 3. 6) catalyzes the oxidation of cholesterol to 4-cholesten-3-one with the reduction of oxygen to hydrogen peroxide. This enzyme is industrially important and useful for the clinical determination of total or free serum cholesterol by coupling with related enzymes, the determination of the steric configuration of 3 3 -hydroxysteroids and the preparation of hormone. Streptomyces species are especially attractive as a host to express useful genes by means of a recombinant DNA technology because the organism produces a large variety of medically important antibiotics and extracellular enzymes. The techniques for their cultivation on an industrial scale and product isolation are well understood. The plasmid vectors for expression of foreign genes in Streptomyces species have been developed. Air of this advanced development provides efficient methods for protein engineering.In this study, a new two-step procedure has been developed for the detection of cholesterol oxidase activity in Streptomyces strains. Streptomyces griseus ATCC14811 was confirmed to produce cholesterol oxidase and it also showed homologies with the cholesterol oxidase gene (choA) from Streptomyces sp. strain SA-COO by Southern hybridization.A cosmid-based gene library of Streptomyces griseus ATCC14811 was constructed using pHZ1357, a Streptomyces-E.coli bifunctional vector carrying two cohesive sites. Two positive clones, pHZl 140 and pHZl 141, were obtained by colony hybridization applying choA. as heterologous probe and subsequently show to code for desired enzymatic activity. After a serial of subcloning coupled with Southern hybridization and enzymatic activity assay, the functional S. griseus ATCC14811 cholesterol oxidase gene (choG)was localized onto 2.3kb EcoRI-Sall fragment. The 2.3kb fragment was then cloned to pBluescript II SK(+). The resulting plasmid, pHZ1754, was subjected to progressive unidirectional deletions using ExonucleaseIII provided in the Erase-a-base system for DNA sequencing. The nucleotide (nt) sequence of the insert in pHZ1754 is 2299bps in size. Computer assisted analysis of the sequence revealed an open reading frame (ORF) with a G+C content of 70.3% that would encode a protein of 552 amino acids (aa). The nt sequence comparision revealed that the ORF in the sequenced region exhibits 85% DNA sequence homology with the cholesterol oxidase gene choA of Streptomyces sp. strain SA-COO. An aa sequence comparison of the ChoG with ChoA revealed that levels of sequence identity and sequence similarity were 79% and 87%, respectively, whereas 53% sequence identity and 70% aa sequence similarity to that of ChoB of Brevibacterilum sterolicum were observed. N-terminal aa sequence of ChoG has the characteristics of a signal sequence, including basic amino acids near the amino terminus and a hydrophobic core. The precursor ofcholesterol oxidase of ATCC14811 was predicted with a calculated Mr of 60020.The cholesterol oxidase produced by Streptomyces griseus ATCC14811 was purified from the culture broth by procedures including steps for preparation of crude enzyme solution, ammonium sulfate fraction and column chromatography on DEAE-cellulose. Optimum pH and temperature for the enzyme activity were approximately pH8.0 and 45 , respectively. The enzyme retained full activity after being treated at room temperature for 1 hour at pH between 4.0 and 11.5. The enzyme can be incubated at 50 for 4h with only less 50 percent loss of activity and is stable in the frozen state. When Streptomyces griseus ATCC14811 was cultured in 10.3% sucrose YEME liquid medium, production of extracellular cholesterol oxidase increased for 5 days before decrease.