Isolation and characterization of cholesterol degrading bacteria from soap and vegetable oil industrial waste

Indian Journal of Biotechnology Vol 13, October 2014, pp 508-513 Isolation and characterization of cholesterol degrading bacteria from soap and vegetable oil industrial waste S Saranya 1, S Shekinah 1, T Rajagopal 2, J Vijayakumar 2 and P Ponmanickam 1 * 1 Department of Microbiology and 2 Department of Biotechnology, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi 626 124, India Received 18 March 2013 ; revised 4 June 2013; accepted 14 August 2013 The cholesterol degrading bacteria were isolated and characterized from the soap and vegetable oil industrial wastes. Fifteen strains were isolated by the primary screening. Among the fifteen, only three strains were identified as cholesterol oxidase enzyme producers. The morphological and biochemical characters revealed that the isolated microorganisms were Pseudomonas sp., Bacillus sp. and Sterptomyces sp. The isolated microorganisms were grown in a cholesterol medium and their degrading potency of cholesterol was determined by TLC, FTIR and HPLC. The cholesterol oxidase (CHO) extracted from the isolates was partially purified and the molecular mass was determined to be about 55-60 kda. The optimum ph and temperature for CHO activity of the three isolates was evaluated. The present study revealed that the isolates (Pseudomonas sp., Bacillus sp. and Streptomyces sp.) isolated from the industrial wastes had good cholesterol degrading potential and would be considered as good source of CHO for important medical applications. Keywords: Bioconversion, cholesterol degrading bacteria, cholesterol oxidase, soap industrial waste, vegetable oil waste Introduction The degradation (bioconversion) and assimilation of cholesterol by various microorganisms is well known in the natural environments. These microorganisms present in the soil are capable of growing on cholesterol as a sole carbon source. Cholesterol degrading microorganisms generally produce an enzyme known as cholesterol oxidase (CHO). This enzyme catalyzes the conversion of cholesterol into 4-cholestene-3-one 1,2. This reaction is a first step in the microbial degradation of cholesterol and the cholesterol derivatives 3,4. Microbial CHOs are commercially important because of their potential industrial and clinical applications. For instance, CHO is mainly used in the quantification of serum and food cholesterol 2,5,6. It can be used as precursor for the production of pharmaceutical steroids 7. Moreover, it can be used in degradation and lowering the dietary cholesterol 8, providing a diagnostic kit for degrading cholesterol 9,10 and also used as a bio-insecticide 11,12. Hence, research on microbial CHO has received much attention in recent years. Microbial conversions of cholesterol when compared to chemical reactions are more regio- and *Author for correspondence: Tel: +91-4562-254970; Fax: +91-4562-254970 E-mail: pon_manick@yahoo.co.in stereo-selective and have been used in the production of pharmaceutical products for a long time 13,14. The most essential problem in preparing the steroid hormones from sterols is the selective cleavage of the side chain. Chemical approach is not effective because of its low conversion rate and has fallen far short of the economical advantage. This is due to low specificity of the reaction, causing considerable contamination by some undesired by-products 14. These are the main reasons which prompted the use of the microbial degradation of cholesterol in the pharmaceutical industries. CHO producing ability has been reported in many microorganisms. For instance, it has been reported in Rhodococcus sp. 2,4, Pseudomonas sp. 15, Mycobacterium smegmais 14, Bacillus subtilis 16,17, Microbacterium sp. 18, etc. However, knowledge on the isolation and characterization of cholesterol-degrading bacteria from the industrial environment is vey scanty. The present study was aimed to isolate the potent cholesterol degrading bacteria from soap and vegetable oil industrial wastes and evaluate their ability for degradation of cholesterol. Materials and Methods Isolation of Microorganisms Soap industry waste (effluent) and vegetable oil waste (soil) were collected from the local areas of

SARANYA et al: CHOLESTEROL DEGRADING BACTERIA FROM INDUSTRIAL WASTE 509 Madurai district, Tamil Nadu, India. The cholesteroldegrading bacteria were isolated from the waste samples by an enrichment culture technique 7. The medium used for isolation contained cholesterol as a sole carbon source and 1 L of medium contained: NH 4 NO 3, 1 g; K 2 HPO 4, 250 mg; MgSO 4 7H 2 O, 250 mg; NaCl 5 mg; FeSO 4.7H 2 O, 0.5 mg; and cholesterol, 1 g (Medium-A). The ph was adjusted to 7.0 with 10% NaOH prior to autoclaving. The waste sample was added and the mixture was incubated at 30 C on a shaker at 3000 rpm for 7-12 d. The growing cultures were transferred to the same medium 2 or 3 times. Finally, the broth was plated onto the nutrient agar, consisting of 10% peptone, 1% yeast extract, 0.5% NaCl, and 2% agar (ph 7.0) (Medium-B). Representative colonies were picked up onto an agar slant and were purified by repeated serial plating and used for morphological and biochemical tests for the identification of microbes. Confirmation of CHO Producing Bacteria To confirm the CHO producing strains, a colony staining method was performed on the grown colonies 4. The CHO producing colonies were selected on suitable indicator plates 19. Detection of Cholesterol Degradation After 6 d of incubation at 30 C in a shaker at 80 rpm, the isolates were separated by centrifugation at 3000 rpm and the supernatant was used for purification of the enzyme and detection of degradation products by TLC, FTIR and HPLC. Thin Layer Chromatography For determination of the cholesterol-degrading potency of each bacterium, the culture supernatant was extracted from the medium (Medium-A) with chloroform and was applied to the silica gel plates. These plates were then placed in a chromatography tank containing chloroform:benzene:ethyl acetate (1:3:6). After chromatography, the plates were dried, sprayed with 0.3 M sulphuric acid followed by heating at 110 C until appearance of spots 19. and used to verify cholesterol degradation by the strains isolated. Characterization of CHO Enzyme Isolation The supernatant from the Medium-A centrifuged at 10,000 rpm for 10 minutes at 5 C was used as the extracellular CHO solution 20. Enzyme Activity CHO activity was measured by testing various ph (5 to 9) and temperatures (30 to 65 C) 21. Molecular Mass Determination The molecular mass of purified (ammonium sulphate precipitation) CHO was determined by 10% SDS-PAGE 22. Results and Discussion Fifteen strains of bacteria were isolated from the wastes in the preliminary screening. Colony staining method was carried out to confirm the CHO enzyme producing microorganisms. The CHO indicator plates were used for further confirmation of the isolated bacteria. The CHO producing isolates changed the medium colour into intense brown (Fig. 1). Among 15 strains, only 3 strains were identified as the CHO enzyme producers. These strains were identified and were investigated further. According to the morphological and biochemical tests, these 3 isolates were identified as Pseudomonas sp., Bacillus sp. and Streptomyces sp. (Table 1). Maximum cholesterol degradation by the isolates occurred in the cholesterol medium (Medium-A), where cholesterol (1 g/l) was supplemented with as a sole carbon source for 6 d. Approx, 65-80% of the cholesterol was degraded by the isolates (Table 2). The isolated microorganisms were grown in the cholesterol medium and the growth rate and the cholesterol conversion rate were measured. The generation time of the isolates in the cholesterol medium had been 2 h. The number of viable cells Fourier Transform Infrared Spectroscopy (FTIR) Analysis The culture supernatant from each bacterium (Medium-A) was subjected to FTIR (Schimadzu) in order to identify the degradation of cholesterol. High Performance Liquid Chromatography (HPLC) Analysis The culture supernatant was extracted with methanol, subjected to HPLC (Schimadzu, C18 column) Fig. 1 (a-c) CHO indicator plate: (a) Pseudomonas sp.; (b) Bacillus sp.; & (c) Streptomyces sp.

510 INDIAN J BIOTECHNOL, OCTOBER 2014 Table 1 Biochemical characterization of isolated organisms No. Characteristics Pseudomonas sp. Bacillus sp. Streptomyces sp. 1 Gram staining Gram -ve rods Gram +ve rods Gram +ve rods 2 Motility + + - 3 Indole - - - 4 Methyl red - + + 5 Voges-Proskauer - - - 6 Citrate + - + 7 Catalase + + + 8 Oxidase + + - 9 Nitrate + + - 10 Urease - - - 11 Starch - + - hydrolysis 12 Blood agar β-hemolysis - - Table 2 Growth of Pseudomonas sp., Bacillus sp. and Streptomyces sp. on cholesterol medium No. Day Pseudomonas sp. (CFU/mL) 1 2 3 4 5 6 7 8 9 10 11 12 13 0 1 2 3 4 5 6 7 8 9 10 11 12 5 10 5 4 10 7 5 10 8 8 10 9 7 10 8 5 10 7 4 10 5 Bacillus sp. (CFU/mL) 2 10 7 3 10 8 8 10 9 4 10 8 3 10 7 2 10 6 4 10 5 Streptomyces sp. (CFU/mL) 2 10 5 4 10 6 3 10 7 2 10 8 5 10 9 5 10 9 6 10 9 5 10 9 5 10 8 2 10 7 2 10 5 decreased almost exponentially with prolonged incubation. The concentration of cholesterol in the culturing medium decreased slightly after 6 d of incubation. If the incubation was prolonged beyond 6 d, the viability of the isolates decreased but the concentration of the cholesterol remained the same. This indicates that the cholesterol degradation ability was lost due to the prolonged incubation. Different approaches were used to determine the cholesterol utilization by the isolates. In the present study, TLC, FTIR and HPLC were used. The TLC analysis was carried out in order to rule out the possibility of accumulation of cholesterol in the growth media rather than degradation and to determine whether the cholesterol level decreased or not during the growth period. The cholesterol fraction was purified by TLC. Fig. 2 TLC of cholesterol degradation: Lane 1, Pseudomonas sp.; lane 2, Bacillus sp.; lane 3, Streptomyces sp.; & lane 4, Cholesterol. The metabolites involved in the cholesterol degradation by the isolates were detected by their Rf value (Fig. 2). The present study revealed that R f value ranged between 0.5-0.8. The three isolates displayed only one spot. Johnson and Somkuti 23 reported that a single spot indicates that the isolates degrade cholesterol without accumulating appreciable amounts of the cholesterol intermediate products. The R f value of 4-cholesten-3-one was reported as 0.6-0.8 and that for cholesterol 0.5 19. The present observations also showed somewhat the same R f value in this range. Most probably the metabolite produced by the test microorganisms could be 4-cholesten-3-one. The isolated microorganisms appeared to have degraded the side chains in the cholesterol molecule, thereby releasing some metabolites. The FTIR analysis was used to identify the cholesterol metabolites generated during the degradation process and the results revealed the frequency from 1710-1665 cm -1 (Fig. 3). It indicates the C=O stretch bond and the ketone functional group. Thus, the FTIR analysis indicates that cholesterol after its degradation generated the metabolites 4-cholestene-3-one, cholest- 4-ene-3,17-dione and androst-1,4-diene-3,17-dione. These metabolites by their chemical nature are the ketonic derivatives of the cholesterol 17. After degradation, the functional groups of the cholesterol also degraded, thereby giving rise to the ketones (C=O stretch bond). The data obtained from the FTIR analysis indicates that the main metabolite generated could be 4-cholestene-3-one.

SARANYA et al: CHOLESTEROL DEGRADING BACTERIA FROM INDUSTRIAL WASTE 511 Fig. 3 (a-c) FTIR analyses of cholesterol degradation by Pseudomonas sp. (a), Bacillus sp. (b) and Streptomyces sp. (c). The culture supernatant of 3 isolates was analyzed by HPLC. The chromatograms with the standard (cholesterol+methanol; Fig. 4a) and the test material (methanolic extract of culture supernatant of 3 isolates; Figs 4b-d) were compared, which showed a less prominent peak of cholesterol from the test samples, indicating a cholesterol transformation and its loss in the test sample chromatogram, meaning its disappearance. The cholesterol peak showed a retention time of 2.257 against the standard. The trends observed from the retention time from 2-4 (Pseudomonas sp. and Bacillus sp.) and 2-3 (Streptomyces sp.), indicated cholesterol transformation. This finding is in agreement with that of Andhale and Sambrani 17 who suggested that a biotransformation of cholesterol was mediated by B. subtilis isolated from the soil from an industrial area. The specific activity of CHOs isolated from the culture supernatant of Pseudomonas sp., Bacillus sp. and Streptomyces sp. isolates was recorded to be 2.3, 1.9 and 1.7 U mg -1, respectively. On further purification by ammonium sulphate precipitation, the specific activity of CHOs of the respective isolates was improved to 2.1, 1.6 and 1.5 U mg -1. The effect of ph on the activity of the CHO enzyme was assessed (Fig. 5). The optimum ph for the enzyme from Pseudomonas sp. was found to be in the range of ph 7.0-7.5; while the optimum ph values for for Bacillus sp. and Streptomyces were 6.5 and Fig. 4 (a-d) HPLC analysis of cholesterol (a) degradation by Pseudomonas sp. (b), Bacillus sp. (c) and Streptomyces sp. (d).

512 INDIAN J BIOTECHNOL, OCTOBER 2014 Fig. 5 (a-c) Effect of various ph on CHO enzyme activity of Pseudomonas sp. (a), Bacillus sp. (b) and Streptomyces sp. (c). Fig. 6 (a-c) Effect of various temperatures on CHO enzyme activity of Pseudomonas sp. (a), Bacillus sp. (b) and Streptomyces sp. (c). 6.5-7.0, respectively. The present observation is in agreement with those of several reports. Optimum ph for CHO derived from Nocardia and Rhodococcus was reported to be 7.5 24 and Rhodococcus sp. PTCC 1633 to be 7.0-7.5 2. The effect of temperature on the activity of CHO was also assessed (Fig. 6). The optimum temperature for Pseudomonas sp. and Streptomyces sp. was 50 C; while for Bacillus sp., it was 45 C. According to Yazdi et al 2, CHO enzyme was found active at 40ºC, while others 4 reported 35ºC. The purity of the enzyme was confirmed by the SDS-PAGE. In the present study, the mol wt of the partially purified CHO was found to be approx 55-60 kda (Fig. 7). A similar trend has been observed in several other studies 2,3,7,13,25. The present study revealed that the cholesterol degrading microorganisms from the industrial waste are endowed with the ability to degrade cholesterol and are the very good source for CHO, which will be exploited for many biomedical applications. Acknowledgement The authors are thankful to the Management and the Principal, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi, Tamil Nadu, India for providing facilities and encouragement to carry out this research work. Fig. 7 SDS-PAGE of partially purified CHO from the isolates: M, Mol wt marker; lane 1, Pseudomonas sp.; lane 2, Bacillus sp.; & lane 3, Streptomyces sp. [Bands appeared between the mol wt 55-60 kda) References 1 Lee S-Y, Rhee H-I, Tae W-C, Shin J-C & Park B-K, Purification and characterization of cholesterol oxidase from Pseudomonas sp. and taxonomic study of the strain, Appl Micribiol Biotechnol, 31 (1989) 542-546. 2 Yazdi M T, Yazdi Z T, Ghasemian A, Zarrini G, Olyaee N H et al, Purification and characterization of extra-cellular cholesterol oxidase from Rhodococcus sp. PTCC 1633, Biotechnology, 7 (2008) 751-756. 3 Sojo M, Bru R, Lopez-Molina D, Garcia-Carmona F & Arguelles J C, Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization, Appl Micribiol Biotechnol, 47 (1997) 583-589. 4 Lashkarian H, Raheb J, Shahzamani K, Shahbani H & Shamsara M, Extracellular cholesterol oxidase from Rhodococcus sp.: Isolation and molecular characterization, Iran Biomed J, 14 (2010) 49-57.

SARANYA et al: CHOLESTEROL DEGRADING BACTERIA FROM INDUSTRIAL WASTE 513 5 Richmond W, Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum, Clin Chem, 19 (1973) 1350-1356. 6 Yazdi M T, Malekzadeh F, Zarrini G, Faramarzi M A, Kamranpour N et al, Production of cholesterol oxidase by a newly isolated Rhodococcus sp., World J Microbiol Biotechnol, 17 (2001) 731-737. 7 Watanabe K, Aihara H, Nakagama Y, Nakamura R & Sasaki T, Properties of the purified extracellular cholesterol oxidase from Rhodococcus equi No. 23, J Agri Food Chem, 37 (1989) 1178-1182. 8 Kaunitz H, Cholesterol and repair processes in arteriosclerosis, Lipids, 13 (1978) 373-374. 9 Noma A & Nakayama K, Comparative studies on cholesterol oxidases from different sources, Clin Chim Acta, 73 (1976) 487-496. 10 Bholay A D, Gadekar D J, Sawant S K & Sonawane S M, Bacterial extracellular cholesterol oxidase and its pharmaceutical perspectives, Int J Curr Microbiol Appl Sci, 2 (2013) 19-28. 11 Purcell J P, Greenplate J T, Jennings M G, Ryerse J S, Pershing J C et al, Cholesterol oxidase: A potent insecticidal protein active against boll weevil larvae, Biochem Biophys Res Commun, 196 (1993) 1406-1413. 12 García J L, Uhía I & Galán B, Catabolism and biotechnological applications of cholesterol degrading bacteria, Microb Biotechnol, 5 (2012), 679-699. 13 Ahmad S, Garg S K & Johri B N, Biotransformation of sterols: Selective cleavage of the side chain, Biotechnol Adv, 10 (1992) 61-67. 14 Naghibi F, Yazdi M T, Sahebharani M & Daloii M R N, Microbial transformation of cholesterol by Mycobacterium smegmatis, J Sci (Iran), 13 (2002) 103-106. 15 Doukyu N & Aono R, Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomonas sp. strain ST-200, Appl Environ Microbiol, 64 (1998) 1929-1932. 16 Kim K-P, Rhee I-K & Park H-D, Degradation of cholesterol by Bacillus subtilis SFF34 in flatfish during fermentation, J Microbiol, 41 (2003) 284-288. 17 Andhale M S & Sambrani S A, Cholesterol biotransformation in monophasic systems by solvent tolerant Bacillus subtilis AF333249, Indian J Biotechnol, 5 (2006) 389-393. 18 Parekh S N & Desai P B, Isolation and characterization of extracellular cholesterol oxidase producing Microbacterium sp. from waste of regional oil mill, Int J Adv Life Sci, 6 (2013) 87-92. 19 Yazdi M T, Malekzadeh F, Khatami H & Kamranpour N, Cholesterol degrading bacteria: Isolation, characterization and bioconversion, World J Microbiol Biotechnol, 16 (2000) 103-105. 20 Salva T J G, Liserre A M, Moretto A L, Zullo M A T, Ventrucci G et al, Some enzymatic properties of cholesterol oxidase produced by Brevibacterium sp., Rev Microbiol, 30 (1999) 315-323. 21 Rhee C-H, Kim K-P & Park H-D, Two novel extracellular cholesterol oxidases of Bacillus sp. isolated from fermented flat fish, Biotechnol Lett, 24 (2002) 1385-1389. 22 Laemmli U, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond), 227 (1970) 680-685. 23 Cheetham P S, Dunxill P & Lilly M D, The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous, Biochem J, 20 (1982) 512-521. 24 Sasaki I, Goto H, Yamamoto R, Tanaka H, Takami K I et al, Hydrophobic-ionic chromatography: Its application to microbial glucose oxidase, hyaluronidase, cholesterol oxidase and cholesterol esterase, J Biochem, 91 (1982) 1555-1561. 25 Mac Lachlan J, Wotherspoon A T L, Ansell R O & Brooks C J W, Cholesterol oxidase: Sources, physical properties and analytical applications, J Steroid Biochem Mol Biol, 72 (2000) 169-195.