Identification And Functional Analysis Of Cholesterol Oxidase Gene From Streptomyces Virginiae IBL-14

Streptomyces virginiae IBL-14, isolated from activated sludge for treatment of waste from a steroidal drug factory, is a bacterium capable of effective biotransformation and degradation of steroidal compounds, including progesterone and hydrocortisone, etc. Experimental results found that the strain could grow in a basal medium with Cholesterol as the sole carbon source, representing that S. virginiae IBL-14 can degrade Cholesterol. The first step in the biodegradation of Cholesterol was done by Cholesterol oxidase (Cho), which involves coenzyme flavin adenine dinucleotide (FAD). The reaction consists of oxidation and isomerization, that is, oxidizing C3 hydroxy of Cholesterol into C3 carbonyl, and then converting C5 double bonds into C4 double bond and forming a product of 4-Choesten-3-one, acompanying with the release of a by-parduct hydrogen peroxide (H2O2).To identify the biological function of Cholesterol oxidase genes in Streptomyces virginiae IBL-14 and understand their enzymatic mechanism, four putative Cholesterol oxidase genes, named cho-sv01, cho-sv02, cho-sv03 and cho-sv04 were first obtained through a comparsion between the whole genome sequence of S.virginiae IBL-14 and the annotated databases, including SWISSPROT, TREMBL, KEGG, etc. by using BLASTP and with the CDD and COG databases by using Rpsblast. Online software ExPASy analysis showed that the homology between S.virginiae IBL-14 and other streptomyces sp. reaches a high range from 85% to 96%. The four proteins, nemed Cho-sv01, Cho-sv02, Cho-sv03 and Cho-sv04, are putative GMC family members, each having a molecular weight of approximately 50-65 kD and a very small difference in molar ratio of amino acids, and their isoelectric points are higher than 8.0. The phylogenetic relationships of the Chos among different members of Streptomyces sp. were constructed by neighbor-joining (NJ) methods using MEGA5.0. Further analysis showed Cho-sv04 have the closest relationship to ChoA from streptomyces sp. SA-COO, implying that both proteins probably have similar function.Then, the four Cholesterol oxidase genes from S. virginiae IBL-14 were cloned in E.coli BL21(DE3) and the resultant mutants Kcoli BL21(DE3)/pHT304-cho-sv01, E.coli BL21(DE3)/pHT304-cho-sv02,E.coli BL21(DE3)/pHT304-cho-sv03 and E.coli BL21(DE3)/pHT3O4-cho-svO4 were obtained. To purify the four proteins and identify their functions, a soluble expression strategy utilizing low temperature induction for the expression of the four proteins was carried out. The SDS-PAGE analysis showed that the majorities of the four target proteins occurred in the supernatant of the smaples after cells were lysed by sonication.Purification of Cholesterol oxidase Cho-sv04 was implemented via salting-out using (NH4) 2SO4, ultrafiltration and cation exchange chromatography and the final specific activity of the enzyme Cho-sv04 is approximately 1.10 U/mg. After bio-transformation by Cho-sv04 used Cholesterol as substrate, the samples of the substrate and converted products were qualitatively and quantitatively analyzed by HPLC through comparing the retention time and the peak area with corresponding standard compound. The results of HPLC analysis showed that Cho-sv04 can catalyze Cholesterol into 4-Choesten-3-one.For the research of protein structure and function mechanism of cholesterol oxidase (Cho-sv04). We found Cho-sv04 has the greatest similarity with ChoA from streptomyces sp. SA-COO in secondary structure and three-dimensional structure by using Clustalx and Discovery studio 2.5. To build the 3D models of Cho-sv04, the program MODELLER was used with 1N4W and 1COY as templates. The results show that Cho-sv04 has a FAD binding sequence conserved in the amino acid sequence, and a pocket structure, namely an active site. The common key amino acid residues in the active site of the two proteins include Phe82、Leu372、Leu435、 Asn324、Glu364、His450 and Asn488. To comfirm the roles of these amino acid residues, His450 was first selected as a target for site-directed mutagenesis (Leu450 instead of His450). We found that the mutant E.coli BL21(DE3)/pHT304-cho-sv04L450 was unable to catalyze Cholesterol to 4-Choesten-3-one, suggesting that His450 plays an important role in Cho-sv04.