Production, Purification and Cloning of Bacterial Cholesterol Oxidases and their Application in Medical Bioremediation

Abstract

In living cells, the recycling process is hampered by accumulation of metabolic waste, resulting to triggers the aging process. The accumulated recalcitrant compounds are difficult to be degraded by endogenous system and have deleterious effect on cell leading to progression of several diseases. Some aging storage diseases (ASD) such as atherosclerosis and cancer are affiliated with accumulation of cholesterol and 7- ketocholesterol (7-KC). There are several therapies for reversing this accumulation and recently focus is on a new approach in which human's natural catabolic machinery is boosted with foreign enzymes. Removal of these compounds by exogenous enzymes of microbial origin to relief the disease associated conditions is termed as medical bioremediation. No doubt there are proven challenges to use the foreign enzymes in therapeutic trials such as delivery, efficacy, and immunogenicity problems. To overcome all that there is a need to engineer the therapeutic proteins with desired characteristics. The goal of this research is to screen and isolate the cholesterol and 7-KC degrading bacteria from diverse environmental samples and identification and purification of candidate enzyme involved in the degradation and have therapeutic potential. Total of twelve bacterial strains were isolated with capability to utilize the cholesterol as a source of energy in M9 cholesterol agar plate from different environmental samples. They were characterized taxonomically by morphological, biochemical and 16S rRNA sequencing and were identified as Bacillus subtilis LSB, Bacillus safensis W1, Stenotrophomonas pavanii GS2, Psychrobacter sp. GS3, Moretella sp. GS6, Vibrio sp. GS7, Moretella sp. GS9, Shewanella pealeana GSS, Klebsiella sp. WS, Vibrio sp. DS and Psychrobacter sp. UP. Bacterial isolates were tested for their degradation potential in shake flask experiments. Two of the bacterial strains B. safensis W1 and B. pumilus W8 were selected for degradation optimization studies due to their best growth in the presence of cholesterol. Optimum pH, temperature and cholesterol concentration was found to be 7, 35℃ and 1g/L respectively for both the strains. It was also observed that MgSO₄ and CaCl₂ favors the cholesterol degradation. Later, under optimized conditions degradation potential of all the strains were tested for cholesterol and some selected strains for 7-keto cholesterol and their reduction was analyzed by HPLC. The results indicated 58-86% cholesterol reduction, with xii maximum reduction by B. pumilus W8. While 7-KC degradation was 70, 90, 86 and 92% by B. safensis W1, B. pumilus W8, Moretella sp. GS6 and S. pealeana GSS, respectively. Furthermore, cholesterol metabolites generated by some of the isolates including B. subtilis LSB, B. safensis W1, Psychrobacter sp. UP, Vibrio sp. DS, Psychrobacter sp. GS3 and Moretella sp. GS6 were detected on GCMS. Several significant peaks were detected and identified as 7-oxo-cholesterol, 2-dodecene-1-yl-Succinic anhydride, 26,27-Dinorergosta 5,23-dien-3-ol and some of common fatty acids including hexadecenoic acid and octadecanoic acid. All the isolates were screened for production of cholesterol oxidase ChO and found positive. Two isolates B. safensis W1 and B. subtilis LSB showing promising results for cholesterol oxidase production (0.140 and 0.336 U/mL activity respectively) were selected for further studies. Extracellular flavin adenine dinucleotide (FAD) containing ChO was purified from both of the selected isolates by passing through Diethylaminoethyl sepharose (DEAE- Sepharose) column. Cholesterol oxidase with a molecular weight of 58 kDa and 22 kDa was purified from B. safensis W1 and B. subtilis LSB, respectively. The ChO gene from Chromobacterium DS1 strain was synthesized by Integrated DNA technology (IDT) and cloned in pET21 vector. Expression of recombinant cholesterol oxidase was carried out in E. coli strain BLR and origami in terrific broth (TB) and super broth (SB) at three different post induction temperatures (20, 30, 37℃). Good expression was seen in TB media at low temperature (20℃) and longer incubation (16 hours). Protein solubility was enhanced by using 0.1mM of Isopropyl β-d-1-thiogalactopyranoside (IPTG as inducer. Recombinant protein (Molecular weight 58 kDa) was purified at room temperature (25℃) by passing through NpuC trapped affinity resin from BLR and Origami with activity of 26 U/mg and 33 U/mg respectively. Finally, cholesterol oxidase was engineered with one amino acid change by replacing the threonine with phenylalanine at position one and significant effect on elusion profile was seen with pronounced increased activity (41 U/mg). To understand the biochemical properties of recombinant cholesterol oxidase effect of several factors such as pH (3, 5, 7, 9, 11), temperature (20-100℃), solvents (methanol, ethanol, isopropanol, dimethyl sulfoxide (DMSO), ethyl acetate, acetone) and detergents (tween 80, triton-x-100, sodium cholate and sodium dodecyl sulphate (SDS) were evaluated. Protein was more active at pH 6 followed by 7 and active in broad range of xiii temperature (40-80℃) with optimum activity at 60℃. Moreover, protein was found to be active in the presence of all the solvents tested except of ethyl acetate and acetone where maximum activity was seen in the presence of methanol and tween 80. The recombinant cholesterol oxidase sequence when analyzed on immune epitope database (IEDB) for prediction of B and T cells epitopes showed potential immunogenic response and could be pegylated to be used in therapeutic trials. Recently it has been reported that cholesterol play an important role in development of cancer and cholesterol lowering agents could be used as anticancer therapy. In view of these reports when the activity of purified ChO against various cancer cells including colon carcinoma (HT-29), prostate cancer (DU 145) and breast cancer (MCF-7) cells was tested, result showed maximum cell inhibition of all the tested cell types with 13U /mL enzyme concentration. Maximum inhibition was recorded of DU-145 cells by recombinant ChO (96.6%), followed by HT-29 and MCF-7. Purified cholesterol oxidase of Bacillus safensis W1 and Bacillus subtilis LSB also showed inhibition from 53-92.2% with tested cell lines. From present research work it is concluded that bacterial isolates from environmental samples can degrade cholesterol and 7-keto cholesterol. The potential therapeutic enzyme cholesterol oxidase has been identified and successfully purified and cloned as model enzyme that can pave the way towards exogenous enzymatic therapy termed as medical bioremediation. Furthermore, cell toxicity of ChO has been found against HT-29, DU-145 and MCF-7 cancer cell lines. The cytotoxic potential of the cloned and purified enzymes comparable with Taxol as control showed high potential and further research is needed to reduce the immunogenicity and increase the safe delivery towards the targeted sites. In future this enzyme may become first choice of therapeutics for diseases considered the first (cardiovascular diseases) and second (cancer) leading cause of death worldwide.