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Publications &
Presentations
List of presentation and publications
136
A. List of publication No. Title of the Paper Name of the
Journal Author
Year,
Volume & Page
No.
ISSN/ISBN No.
1 Isolation and identification of extracellular cholesterol oxidase producing microorganisms from various sources.
International Journal of
Pharmacy & Life sciences
(IJPLS)
Parekh &
Desai
2012; 3(7)
ISSN
0976-7126
2 Media optimization using orthogonal array technique for cholesterol oxidase production by Streptomyces sp.
International Journal of Applied
Microbiology Science (IJAMS)
Parekh S.N.
and Desai P.B.
2012; 1(2); 32-43
ISSN
2277-6079
3 Extracellular cholesterol oxidase production by Streptomyces sp. isolated from compost.
Proceedings of National
Conference on “Advanced
Trends in Applied
Sciences and Technology”
(ATAST)
Parekh S.N.
and Desai P.B.
2012
ISBN 978- 81-
923514-0-7
4 Isolation and identification of extracellular cholesterol oxidase producing Arthrobacter sp. from waste of regional oil mill.
International Journal of Universal
Pharmacy and Bio Sciences
(IJUPBS)
Parekh S.N.
and Desai P.B.
2013; 2(1)
ISSN 2319-8141
5 Isolation and characterization of extracellular cholesterol oxidase producing Microbacterium sp. from waste of regional oil mill
International Journal of
Advanced Life Sciences (IJALS)
Parekh S.N.
and Desai P.B.
2013; 6(2)
ISSN
2277-758X
6 Purification and characterization of extracellular cholesterol oxidase from Streptomyces sp. RO-10
World Journal of Microbiology
& Biotechnology
(WJMB)
Parekh S.N.
and Desai P.B.
Submitted
List of presentation and publications
137
B. List of presentation
No. Title of the Paper Name of the Event Author & Year
Oral/Poster
1 Extracellular cholesterol oxidase production by Streptomyces sp. isolated from compost.
National Conference on
“Advance Trends in Applied Sciences and Technology”
(ATAST-2012) SRICEAS, Surat
Parekh S.N.
and Desai P.B.
(2012)
Oral
2 Isolation and characterization of extracellular cholesterol oxidase producing Aspergillus sp.
National Symposium
on Entrepreneurshi p in Microbial
Technlogy & 2 nd
National Level Poster
Competition on Applied Sciences & Technololgy. SRICEAS, Surat
Parekh S.N. and
Desai P.B. (2013)
Poster
3 Purification of cholesterol oxidase from Streptomyces sp.
National seminar on “Recent Advances in Applied
Sciences” (RAAS). SANPPGI, Anand
Parekh S.N. and
Desai P.B. (2013)
Poster
Research Article [Parekh & Desai, 3(7): July, 2012]
CODEN (USA): IJPLCP ISSN: 0976-7126
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 7: July: 2012, 1807-1811 1807
INTERNATIONAL JOURNAL OF PHARMACY & LIFE SCIENCES
Isolation and identification of extracellular cholesterol oxidase
producing microorganisms from various sources S. N. Parekh* and P.B. Desai
Department of Microbiology, Shree Ramkrishna Institute of Computer Education and Applied Sciences, Athawalines, Surat, (Gujarat) - India
Abstract Cholesterol oxidase (EC1.1.3.6; CHO) is an enzyme, which catalyzes the oxidation of cholesterol and converts 5-cholesten-3β-ol into 4- cholesten-3-one. The objective of this study is to isolate extracellular cholesterol oxidase (CHO) producing microorganisms to obtain an abundant source of cholesterol oxidase (CHO) for industrial and medicinal needs. Cholesterol oxidase producing bacteria were isolated from waste of regional oil mill, soil and compost. Twenty-five isolates are tested for cholesterol oxidase activity by screening method. As the result of the screening, CHO producer strain was isolated and identified as Streptomyces sp., Arthrobacter sp. and Aspergillus sp. CHO activity of isolates were measured by spectroscopic assay method. Purification and characterization of CHO enzyme is under way.
Key-Words: Cholesterol oxidase, 4-cholesten-3-one, Horseradish peroxidase
Introduction Cholesterol oxidase (CHO) is an enzyme which catalyzes the oxidation of Cholesterol and converts 5-Cholesten-3β- ol into 4-Cholesten 3-one1. Many bacteria can produce this enzyme including members of the genera Arthrobacter, Brevibacterium, Pseudomonas, Nocardia, Rhodococcus, Streptomyces, Corynebacterium and Shizophylum 2, 3. Cholesterol oxidase enzyme has many applications in medicine 4, agriculture, and pharmaceutical 5 and so on. For instance, it can be used for production of diagnostic kits to detect blood Cholesterol 6, biological insecticide 7 and precursors for steroid hormones 8. This enzyme can be secreted from a bacterium in 3 types including intracellular, extracellular and membrane-bound. Due to wide spectrum applications of Cholesterol oxidase, screening and isolation of bacterial strains producing extracellular form of Cholesterol oxidase is of great importance 9. Many microorganisms have been determined which produced extracellular Cholesterol oxidase including Rhodococcus equi, Rhodococcus erythropolis10, Streptomyces sp, Arthrobacter simplex, Brevibacterium sterolicum, Strerptomyces lividanse, Schizophylum commune, Micrococcus sp etc 11, 12,13 .
* Corresponding Author E.mail: [email protected] Mob.: +91 9427576142 Tel.: 0261- 2240172 Fax: 0261- 2240170
Enzymatic properties of cholesterol oxidase from Rhodococcus strains (some of which named formerly as Nocardia) are particularly suitable for use in the analytical determination of cholesterol, in which the hydrogen peroxide formed is used in a chromogenic reaction catalyzed by horseradish peroxidase. In the present study, Streptomyces sp., Arthrobacter sp. and Aspergillus sp was isolated from waste of regional oil mill, soil and compost. The type of CHO enzyme produced by isolates was determined using an enzyme activity assay on supernatant of culture medium. Material and Methods Isolation of microorganisms Cholesterol oxidase producing microorganisms were isolated by following procedure. 1 g of various samples was suspended in 100 ml of distilled water. The suspension was vigorously shaken for 30 min. A volume of 100 µl of supernatant was inoculated in medium (medium A) containing cholesterol as the sole carbon source. A medium contained (g/l): agar, 3.0 %; K2HPO4, 0.25; NH4NO3, 17; MgSO4.H2O 0.25%; FeSO4, 0.001; NaCl, 0.005; cholesterol, 0.1% and Tween 80, 0.5 ml. The pH of medium was adjusted to 7.0. The inoculated plates were incubated at 30oC for 7-12 days. After incubation period was completed, abscission colonies were appeared on the plate surface. For fast growing and generating, larger colonies were sub cultured in secondary medium (medium B) containing cholesterol as the only source of carbon as
Research Article [Parekh & Desai, 3(7): July, 2012]
CODEN (USA): IJPLCP ISSN: 0976-7126
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 7: July: 2012, 1807-1811 1808
well as yeast extract 9, 14 . This medium contained yeast extract, 0.3 g; (NH4)2HPO4, 0.1 g; cholesterol, 0.15; Tween 80, 0.05 ml; pH – 7; agar, 3.0 % and distilled water, 100 ml. Each colony on medium A was cultured in medium B and incubated at 30oC for 24 h. Then, larger colonies generated on medium B were used for further identification. Identification of isolated microorganisms was performed by microbiological examination and biochemical tests 9,11 . Screening of CHO producing organism CHO is able to convert Cholesterol into Cholest-4-en-3-one and hydrogen peroxide. CHO producing colonies were selected on cholesterol oxidase indicator plates. These plates were prepared by adding 1.0 g Cholesterol, 1.0 g Triton X-100, 0.1g o-dianisidine and 1000 Units of peroxidase to 1 liter of agar medium. Bacterial colonies were cultured on these plates and incubated at 30°C. Cholesterol penetrates into bacterial cells where it can be converted into hydrogen peroxide by Cholesterol oxidase. Reagents that exist in the medium react with hydrogen peroxide (H2O2) to form azo compound which turns the color of medium into intense brown color 15, 17, 18. Identification of isolates Identification of isolates was carried out by studying their morphological, cultural, biochemical and molecular characteristics by standard method. Bacterial and fungal isolates were identified by using Bergey’s manual of systematic bacteriology, 2nd edition and llustrated genera of imperfect fungi, 4th edition by Barnet &Hunter respectively. Determination of CHO activity CHO activity was measured by centrifuge the medium at 10,000 rpm for 20 min. at 4oC by modified method based on the study of Allain et.al 4, 12. In this reaction, hydrogen peroxide generated during Cholesterol oxidation process was coupled with 4-aminoantipyrine and phenol by peroxidase to produce quinoneimine dye with maximum absorption in 500nm. The reaction mixture was consisted of 1mM 4-aminoantipyrine , 5 mM phenol, 5 U/ml of horseradish peroxidase and sodium phosphate buffer (20 mM, pH 7.0). 50 µL of 6 g/L Cholesterol dissolved in dimethyl formamide containing 5% (v/v) Triton X-100 was added to 1ml of reaction mixture, Which was then pre incubated for 3 min. at 30°C. The reaction was initiated by adding 20 µL of enzyme sample and was continued for 5 min at 30°C. The assay mixture was boiled in a water bath for 2 min. to stop the reaction, and then place in an ice bath for 2 min. Absorbance of the reaction solution was monitored at 500 nm. (Systronic 2203, Japan). The assay mixture containing inactivated enzyme was used as the blank. One unite of CHO activity was defined as
the amount of enzyme that converts 1µmol of cholesterol in to 4-cholesten - 3 - one per minute at 30°C. Cholesterol + O2 4- cholesten-3-one + H2O2
2H2O2 + 4 - aminoantipyrene + Phenol Quinoneimine dye + 4H2O
Results and Discussion A 25 samples each from three different sources (waste of regional oil mill, compost and soil) were collected. 20 isolates were obtained from these samples on their capability on growing on isolation medium A containing cholesterol as the sole carbon source. Among them 2 isolates from each sample were found to secrete extracellular CHO were detected by cholesterol oxidase indicator plate. The result of microscopic observation and growth characteristics of these isolates is shown in table-1. Two isolates of waste from regional oil mill RO-3 & RO-10 were identified as Arthrobacter sp. and Streptomyces sp. respectively from their gram staining, morphological, biochemical and colony characteristics. The results of microbiological and biochemical properties of RO-3 is shown in table-2. The cells of RO-3 were irregular rods but eventually presented as coccoid forms as growth continued. The result of growth on medium B is shown in figure-1. Cholesterol oxidase from Streptomyces hygroscopicus and the recombinant enzyme from Brevibacterium sterolicum expressed in Escherichia coli have been characterized for their chemical, physical, and biochemical properties by Giovanni Gadda et al. 16.Isolates C-7 and C-4 from compost was identified as Streptomyces sp. from their gram staining, morphological and colony characteristics. The results of screening for CHO producing organism on cholesterol indicator plate was shown in figure-2. Two fungal isolates from soil S-2 and S-6 were identified as Aspergillus sp. CHO activity of isolates was performed by modified method based on the study of Allain et.al. 4, 12 . Among the six isolates RO-10 shows the highest activity of 1.6 U/ml. Result of activity is shown in table-3. Cholesterol oxidase is an enzyme of great commercial value widely employed by laboratories routinely devoted to the determination of cholesterol in food, serum and other clinical samples. A diversity of micro-organisms, which are capable of producing high levels of this enzyme have been isolated. Our preliminary work led to the conclusion that Streptomyces sp. might be considered as potentially interesting source of extra cellular cholesterol oxidase for clinical and commercial purposes.
Research Article [Parekh & Desai, 3(7): July, 2012]
CODEN (USA): IJPLCP ISSN: 0976-7126
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 7: July: 2012, 1807-1811 1809
Acknowledgements The authors wish to thank management and staff of Shree RamKrishna Institute of Computer Education and Applied Sciences, Surat for providing laboratory facility for this work. References
1. Murooka, Y., Ishizaki, T., Nimi, O. and Maekawa, N., (1986). Cloning and expres- sion of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ 702, Appl Environ Microbiol., 52:. 1382-1385.
2. Yazdi, M. T., Yazdi, Z. T., Zarrini, Gh. and Ghasemian, A., (2008). Purification and characterization of extra-cellular cholesterol oxidase from Rhodococcus sp. PTCC 1633, Biotechnology., 7 (4): 751-6.
3. Fujishiro, K., Uchida, H., Shimokava, K., Nakano, M., Sano, F., Ohta, T., Nakahara, N. and Aisak, K.,Uwajima T., ( 2002) .Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10, FEMS. Microbiol. Lett., 215: 243–248.
4. Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C., (1974). Enzymatic determination of total serum cholesterol, Clin. Chem., 20: 470- 475.
5. Ahmad, S., Garg, S.K. and Johri, B.N., ( 1992). Biotransformation of sterols: selective cleavage of the side chain, Biotechnol., Adv. 10: 1–67.
6. Noma, A. and Nakayama, K., (1976).Comparative studies on cholesterol oxidases from different sources, Clin. Chim. Acta., 73, 487–496.
7. Purcell, J.P., Greenplate, J.T., Jennings, M.G., Ryerse, J.S., Pershing, J.C., Sims S.R., Prinsen, M.J., Corbin, D.R., Tran, M and Sammons, R.D., (1993). Cholesterol oxidase: a potent insecticidal protein active against boll weevil larvae, Biochem. Biophys. Res. Commun., 196: 1406–1413.
8. Bell, K.S., Philp, J.C., Aw, D.W.J. and Christofi, N., (1998). A review of The genus Rhodococcus, Department of Biological Science, Napier University, Edinburgh, UK 6545/01/98.
9. Yazdi1 M. T., Malekzadeh F. , Zarrini1 Gh., Faramarzi M.A. , Kamranpour N. and Khaleghparast, Sh., (2001). Production of cholesterol oxidase by a newly isolated Rhodococcus sp, World journal of microbiology and biotechnology, 17(7): 731-737.
10. Sojo M, Bru R, López-Molina D, García-Carmona F, Argüelles JC, (1997). Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization. Appl Microbiol Biotechnol, 47:583-589.
11. Lee, S. Y., Rhee, H. I., Tae, W. C., Shin, J . C. and Park B. K. ( 1989). Purification and characterization of cholesterol oxidase from Pseudomonas sp. and taxonomic study of the stain, Applied Microbiology and Biotechnology., 31: 542-546.
12. MacLachlan, J., Wotherspoon, A.T.L., Ansell, R.O. and Brooks, C.J.W. ,( 2000). Cholesterol oxidase: sources, physical properties and analytical applications, J. Steroid. Biochem. Mol. Biol,. 72: 169–195.
13. R. Kanchana, Delcy Correia, Sangeeta Sarkar, Prachi Gawde and Aifa Rodrigues, (2011). Production and partial characterization of cholesterol oxidase from Micrococcus Sp. Isolated from Goa, India. International Journal of Applied Biology and Pharmaceutical Technology, 2: 393-398.
14. H. Lashkarian, J. Raheb, K. Shahzamani, H. Shahbani and M. Shamsaram (2010). Extracellular Cholesterol Oxidase from Rhodococcus sp.: Isolation and Molecular Characterization. Iran. Biomed. J, 14: 49-57.
15. Nishiya, Y., Harada, N., Teshima, S., Yamashita, M., Fujii, I., Hirayama, N. and Murooka, Y. (1997).Improvement of thermal stability of Streptomyces cholesterol oxidase by random muta- genesis and a structural interpretation, Protein Engineering., 10: 231–235.
16. Ghasemian, A., Tabatabaei, Y.M., Sepehrizadeh, Z., Tabatabaei, Y.Z. and Zarrini, G.H. ,(2009). Overexpression, one-step purification, and characterization of a type II cholesterol oxidase from a local isolate Rhodococcus sp. PTCC 1633. World J. Microbiol. Biotechnol. 25(5):773-77.
17. Drzyzga, O., J. M. Navarro Llorens, L. Ferna´ndez de las Heras, E. García Ferna´ndez, and J. Perera.. ,(2011).Cholesterol Degradation by Gordonia cholesterolivorans. Applied and Environmental Microbiology, 77 ( 14): 4802–4810.
18. Ferna´ndez de las Heras, L., et al. (2011). ChoG is the main inducible extracellular cholesterol oxidase of Rhodococcus sp. strain CECT3014. Microbiol. Res. 166: 403–418.
Research Article [Parekh & Desai, 3(7): July, 2012]
CODEN (USA): IJPLCP ISSN: 0976-7126
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 7: July: 2012, 1807-1811 1810
Fig. 1: Growth of isolate C-7 and RO-10 on medium B
Fig. 2: Growth on Cholesterol oxidase indicator plates
Research Article [Parekh & Desai, 3(7): July, 2012]
CODEN (USA): IJPLCP ISSN: 0976-7126
Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 7: July: 2012, 1807-1811 1811
Table 1: Morphological and colonial characteristics of isolates from waste of regional oil mill,compost and soil
Sample Isolate No.
Medium Colony / growth characteristics
Morphology Figure
waste of regional oil mill
RO-3
Medium B
Small, creamy white, elevated
colony
Gram positive, short coco bacilli rods.
RO-10 White, cottony,
raised and chalky colony
Gram positive, filamentous organism
Compost C-4 Off white, cottony,
raised and dry colony
Gram positive, filamentous organism
C-7 White, cottony,
raised and chalky colony
Gram positive, filamentous organism
Soil S-2 Green mycelia
colony Conidiophore with septate mycelium
S-3 Brown mycelia
colony Conidiophore with septate mycelium
Table 2: Biochemical and microbiological properties of isolate RO-3. Test Result Test Result
Catalase Positive Motility Non motile Gelatinase Positive Endospore absent
Indole production Negative Lactose Acid & Gas production MR Negative Sucrose Acid & Gas production VP Negative Maltose Acid & Gas production
Citrate utilization Positive Mannitol Acid & Gas production Nitrate reduction Positive Xylose Acid & Gas production
Urease Negative Glucose Acid & Gas production H2S production Negative
Table 3: Extracellular CHO activity of isolates
Isolate Activity (Units/ ml) RO-3 0.91 RO-10 1.6
C-4 1.1 C-7 1.26 S-2 1.03 S-3 0.97
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 32
ISSN-2277-6079
MEDIA OPTIMIZATION USING ORTHOGONAL ARRAY
TECHNIQUE FOR CHOLESTEROL OXIDASE
PRODUCTION BY STREPTOMYCES SP.
Parekh SN*, Desai PB
Department of Microbiology, Shree Ramkrishna Institute of Computer Education and
Applied Sciences, Athawalines, Surat-395 001, Gujarat, India.
* Corresponding Author:
Parekh S.N.
Department of Microbiology,
Shree Ramkrishna Institute of Computer Education and Applied Sciences,
Athawalines, Surat- 395 001, Gujarat, India
E-mail ID : [email protected]
Abstract
The present study focuses on optimization of media components to enhance cholesterol oxidase
(CHO) production by Streptomyces sp. isolated from waste of regional oil mill using Taguchi
orthogonal array design. One factor-at-a-time method was used to investigate the effect of
carbon sources, nitrogen sources and initial pH on biomass growth and enzyme production. A
three-level Taguchi orthogonal array design of L9 (34) was employed to select the effect of
medium components on cholesterol oxidase production. The optimum composition of
fermentation medium as obtained by statistical analysis was starch 2 % w/v, peptone 0.9 %
w/v, yeast extract 0.3 % w/v, Tween 80 0.1 % v/v and employed for cholesterol oxidase
production. The optimized medium exhibited 1.76-fold increase in enzyme activity as compared
to the non-optimized medium (2.82 U/ml and1.6 U/ml, respectively) at shake flask level.
Keywords: Cholesterol oxidase optimization, Streptomyces sp., Taguchi orthogonal array
design.
Sci
ence
In
stin
ct P
ub
lica
tion
s
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 33
Introduction
holesterol oxidase (CHO) is an enzyme which catalyzes the oxidation of Cholesterol and
converts 5-Cholesten-3β- ol into 4-Cholesten 3-one [1]. Many bacteria can produce this
enzyme including members of the genera Arthrobacter, Brevibacterium, Pseudomonas,
Nocardia, Rhodococcus, Streptomyces, Corynebacterium and Shizophylum [2, 3]. Cholesterol
oxidase enzyme has many applications in medicine [4], agriculture, and pharmaceuticals [5]
and so on. For instance, it can be used for production of diagnostic kits to detect blood
Cholesterol [6], as biological insecticide [7] and as precursors of steroid hormones [8]. This
enzyme can be secreted from a bacterium in 3 forms including intracellular, extracellular and
membrane-bound. Due to wide spectrum applications of cholesterol oxidase, screening and
isolation of bacterial strains producing extracellular form of cholesterol oxidase is of great
importance [9]. In the present study, medium optimization for its production by Streptomyces
sp. isolated from waste of regional oil mill was carried out.
Conventional optimization procedures involve altering of one parameter at a time keeping all
other parameters constant, which enables one to assess the impact of those particular
parameters on the process performance. These procedures are time consuming, cumbersome,
require more experimental data sets and cannot provide information about the mutual
interactions of the parameters [10].Alternative to conventional optimization procedures,
design of experiments (DOE) and statistical tools help to gain more information about the
optimization conditions in a few trials [11]. Statistical experimental design methods provide a
systematic and efficient plan for bioprocess optimization considering the interactive effects
among the control factors. Many control factors can be simultaneously studied and optimized
by statistical experimental designs [12, 13]. Among various statistical experimental designs,
Taguchi experimental design offers distinct advantages because many factors can be
examined simultaneously and more quantitative information can be extracted with a few
experimental trials [14, 15]. The basic principle of this method serves as screening filters
which examine the effects of many process variables and identify those factors which have
major effects on process using a few experiments [16].Taguchi method of DOE involves
establishment of large number of experimental situations described as orthogonal arrays to
reduce experimental errors and to enhance their efficiency and reproducibility of the
laboratory experiments [11].
Materials and methods
Isolation of microorganisms
Cholesterol oxidase producing microorganisms were isolated by the following procedure:
1 g samples of waste from regional oil mill of Gujarat state, India were suspended in 100
ml of distilled water, the suspension was vigorously shaken for 30 min, 100 μl volume of
the supernatant was inoculated in a medium (medium A) containing cholesterol as the sole
carbon source. Medium A contained: agar, 3.0 %; K2HPO4 , 0.25 g/l; NH4NO3, 17 g/l;
MgSO4.H2O 0.25 g/l; FeSO4, 0.001 g/l; NaCl, 0.005 g/l; cholesterol, 0.1% and Tween 80,
0.5 ml. The pH of medium was adjusted to 7.0. The inoculated plates were incubated at
30oC for 7-12 days. After incubation period was completed, abscission colonies were
appeared on the plate surface. For generating fast growing, larger colonies sub culturing
was done in a secondary medium (medium B) containing cholesterol along with yeast
extract as the source of carbon [9,17]. This medium contained yeast extract, 0.3 g;
(NH4)2HPO4, 0.1 g; cholesterol, 0.15; Tween 80, 0.05 ml; pH – 7; agar, 3.0 % and distilled
water, 100 ml. Each colony on medium A was cultured in medium B and incubated at 30oC
for 24 h. Then, larger colonies generated on medium B were used for further identification.
C
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 34
Screening of CHO producing organism
CHO is able to convert Cholesterol into Cholest-4-en-3-one and hydrogen peroxide. CHO
producing colonies were selected on cholesterol oxidase indicator plates. These plates were
prepared by adding 1.0 g Cholesterol, 1.0 g Triton X-100, 0.1g o-dianisidine and 1000
Units of peroxidase to 1 liter of agar medium. Selected isolated colonies were cultured on
these plates and incubated at 30°C. Cholesterol penetrates into bacterial cells where it can
be converted into hydrogen peroxide by cholesterol oxidase. Reagents that exist in the
medium then react with hydrogen peroxide (H2O2) to form azo compound which turns the
color of medium into intense brown color [18, 19, 20].
Identification of isolates
Identification of isolates was carried out by studying their morphological, cultural,
biochemical and molecular characteristics. Bacterial and fungal isolates were identified using
procedures as in Bergey’s manual of systematic bacteriology, 2nd
edition and Illustrated
genera of imperfect fungi, 4th
edition by Barnet &Hunter respectively.
Determination of CHO activity
CHO activity was measured in the supernatant of the medium obtained by centrifugation
(10,000 rpm for 20 min. at 4oC) using modified method based on the study of Allain et.al
[4, 21]. In this reaction, hydrogen peroxide generated during Cholesterol oxidation process
was coupled with 4-aminoantipyrine and phenol by peroxidase to produce quinoneimine
dye with a maximum absorption at 500nm. The reaction mixture consisted of 1mM 4-
aminoantipyrine , 5 mM phenol, 5 U/ml horseradish peroxidase and sodium phosphate
buffer (20 mM, pH 7.0). 50 μL of 6 g/L Cholesterol dissolved in dimethyl formamide
containing 5% (v/v) Triton X-100 was added to 1ml of the reaction mixture, which was
then pre incubated for 3 min. at 30°C. The reaction was initiated by adding 20 μL of the
enzyme sample and continuing it for 5 min at 30°C. The assay mixture was boiled in a
water bath for 2 min. to stop the reaction, and then placed in an ice bath for another 2 min.
Absorbance of the reaction solution was monitored at 500 nm (Systronic 2203, Japan). The
assay mixture containing inactivated enzyme was used as the blank. One unite of CHO
activity was defined as the amount of enzyme that converts 1μmol of cholesterol in to 4-
cholesten - 3 - one per minute at 30°C.
Optimization of fermentation medium using one factor-at-a-time method
The one factor-at-a-time method was used to determine the effect of fermentation time,
inoculums age and concentration, medium components (carbon and nitrogen source) and
pH on biomass and CHO production. The study was carried out in 250 ml Erlenmeyer
flasks containing 100 ml. liquid medium B, on a rotary shaker at 200 rpm at 30oC for 72
hrs. The medium was inoculated with 10% (v/v) of 48 hrs. old culture grown in the same
medium. In the present study, Dry cell weight (DCW) was determined by centrifugation of
fermentation broth at 10,000 rpm for 15 min. and washing twice with distilled water; the
recovered biomass was dried to a constant weight at 80oC for 24 hrs.
In order to investigate the optimum fermentation time for CHO production, a series of
flasks were inoculated and harvested for 24 to 120 hrs. at time interval of 12 hrs. The
parameters monitored were pH, biomass and CHO activity. The effect of inoculums
concentration and age were monitored by inoculating medium with different concentrations
(3,5,7,10,12,15 % v/v) of inoculums of different ages (12,24,36,48,60,72 hrs ). 1 gm% of
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 35
Glucose, Lactose, Sucrose, Maltose, Glycerol and Starch, were studied as an alternative
source of carbon. Cholesterol (0.002%) as an inducer of CHO was added, in suspended
form in 1ml of 5% Tween 80 solution, to the medium B. Cells were cultivated in the
medium B containing various organic and inorganic nitrogen sources, including meat
extract, yeast extract, malt extract, peptone, ammonium sulfate, ammonium phosphate and
ammonium nitrate. In the study, yeast extract and ammonium phosphate present in the basal
medium at a concentration of 0.3% (w/v) and 0.1% (w/v) respectively, were replaced with
different nitrogen source at a concentration of 0.5% (w/v). To investigate the effect of
initial pH of medium on CHO production, fermentation runs were carried out by adjusting
initial pH of the medium B in the pH range of 5 to 8.5, and analyzing the samples for CHO
production.
Taguchi orthogonal array design
An L9 (34) orthogonal array method was used for screening the most significant fermentation
parameters influencing CHO production. The design for the L9 orthogonal array was
developed and analyzed using “MINITAB 15” software (Pennsylvania State University,
University Park, Pennsylvania). The levels of the factors studied and the layout of the L9
Taguchi’s orthogonal array are shown in Tables 1 and 2. The experimental results were
analyzed to extract independently the main effects of the factors. The controlling factors were
identified, with the magnitude of effects qualified and the statistically significant effects
determined. Accordingly, the optimal conditions were determined by combining the levels of
factors that had the highest main effect value. All experiments were performed in triplicates.
The validation of data was done by using optimized parameters of fermentation media
components and levels in the shake flask.
Table 1. Factors and their levels employed in the Taguchi’s experimental design for
CHO production by Streptomyces sp.
No. Factor Level 1 Level 2 Level 3
1 Starch (gm%) 0.5 2.0 3.5
2 Peptone (gm %) 0.3 0.6 0.9
3 Yeast extract (gm %) 0.3 0.6 0.9
4 Tween 80 (%v/v) 0.05 0.1 0.15
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 36
Table 2. L9 (34) orthogonal array of Taguchi experimental design and corresponding
CHO production by Streptomyces sp.
Run Starch
(gm %)
Peptone
(gm %)
Yeast extract
(gm %)
Tween 80
(%v/v)
CHO Activity (U/ml)
Experimental Predicted
1 0.5 (1) 0.3 (1) 0.3 (1) 0.05 (1) 0.59 ± 0.01 0.57
2 0.5 (1) 0.6 (2) 0.6 (2) 0.1 (2) 0.81 ± 0.02 0.78
3 0.5 (1) 0.9 (3) 0.9 (3) 0.15 (3) 1.4 ± 0.03 1.41
4 2 (2) 0.3 (1) 0.6 (2) 0.15 (3) 1.25 ± 0.01 1.23
5 2 (2) 0.6 (2) 0.9 (2) 0.05 (1) 2.02 ± 0.07 2.0
6 2 (2) 0.9 (3) 0.3 (1) 0.1 (2) 2.82 ± 0.04 2.83
7 3.5 (3) 0.3 (1) 0.9 (3) 0.1 (2) 0.75 ± 0.04 0.73
8 3.5 (3) 0.6 (2) 0.3 (1) 0.15 (3) 0.89 ± 0.05 0.90
9 3.5 (3) 0.9 (3) 0.6 (2) 0.05 (1) 1.73 ± 0.05 1.76
Values in the parenthesis indicate the level.
Results and Discussion
Isolation and screening of CHO producing microorganisms.
Samples from waste of regional oil mill were collected. 20 isolates were obtained from these
samples depending on their capability to grow on isolation medium A containing cholesterol
as the sole carbon source. Among them one of the isolates RO-10 was found to secrete
extracellular CHO, detected by cholesterol oxidase indicator plate. The result of growth on
medium B and indicator plate is shown in Figure -1. RO-10 was identified as Streptomyces
sp. from the results of microscopic observation, growth characteristics and 16sRNA
sequencing as shown in Table 3.
Fig. 1. Growth of RO-10 on medium B (i) and indicator plate (ii).
(i) (ii)
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 37
Table 3. Characteristics of RO-10.
Isolate No. RO-10
Medium Medium B
Colony characteristics White, cottony, submerged, and chalky colony
Gram reaction, morphology
& figure
Gram positive, filamentous organism
16 sRNA Sequence
>RO10
GTACTCCACCAGGCGGGGGAACWTAAATGCGTTAGYTGCGGCACGGACGACGTGGA
ATGTCGCCCCACACCTAGTTCCCAAACGTTTACGGGCGTGGACTACCAGGGTATCTA
AATCMTGTTCGCTCCCCCACGCTTTCGCTCCTCAGCGTCAGTAATCGGCCCCAGAGAT
CCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATCTTCACCCGCTACACCAGG
AAATTCCGATCTCCCCTACCGAACTCTAGCCCTGCCCGTATCGAAATGCAGACCCCG
GGGKTAAAGCCCCGGGCTTTCACATCCGACGCGACAAGCCGCCTACGAGCTCTTTAC
GCCCAATAATTCCGGACAACGCTTTGCGCCCTACGTATTACCGCGGCTGCTGGCACGT
AGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTGCGCTTCTTCCCTGCTGAAAGA
GGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGC
CCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTC
CCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTAGGCCATT
ACCCCACCAACAAGCTGATAGGCCGCGGGCTCATCCTGCACCGCCGGAGCTTTCCAC
ACACAGACCATGCGGTCGTGTGTCATATCCGGTATTAGACCCCGTTTCCAGGGCTTGT
CCCAGAGTGCAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCACC
CCGAAAGGGCTTCATCGTTCGACTTGCATGTGTTAAGCACGCCGCCAGCGTTCGTCCT
GAGCCAG
Optimization of fermentation medium One-factor-at-a-time method was used to optimize fermentation medium component and the
effect of other parameters on CHO production were also studied.Figure-2 shows the results of
optimum time required for enzyme production in medium B by Streptomyces sp. During this
study enzyme activity, DCW and pH were monitored at 12 hrs interval. When cells began to
lyse after 96 hrs the enzyme activity rose sharply, this is due to the secretion of intracellular
CHO by the strain. This was confirmed by rupturing the cells after 72 hrs fermentation and
assaying for CHO activity. Highest enzyme activity of 1.6 U/ml was obtained after 72 hrs
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 38
fermentation. So, all the experiments were carried out for 72 hrs Figure-3 shows the effect of
inoculums age on CHO production. 48 hrs old inoculums showed highest enzyme activity
with 0.95gm% DCW, while 72 hrs old inoculums exhibited low enzyme activity. Figure-4
shows the effect of different inoculums concentration on CHO production. Inoculums
concentration of more than 10% v/v showed highest enzyme activity.Figure-5 shows the
effect of different carbon sources on CHO production. Starch and glycerol gave CHO activity
of 1.68 and 1.3 U/ml respectively. 1% glucose and maltose yielded 0.97 U/ml enzyme
activities. The two carbon sources showed low activity of CHO among the screened carbon
sources. Figure-6 determines the effect of various nitrogen sources on enzyme production.
Inorganic nitrogen source gave poor enzyme activity as compared to organic nitrogen source.
Peptone and yeast extract increased the enzyme production among the organic nitrogen
sources. Figure-7 shows the effect of initial pH of the medium B on CHO production.
Streptomyces sp. exhibitewd high yield of enzyme when initial pH of the medium B was kept
at 7.5. Yazdi et. al. [9] Studied the effect of different initial pH on Rhodococcus sp. and
concluded pH 8 as optimal for the production of CHO, where as Lee et. al [22] found pH 7 as
optimal for enzyme production from Rhodococcus sp.
Fig.2. Effect of fermentation time on CHO production DCM & ph.
Fig. 3. Effects of inoculums age on CHO production and DCW.
24 36 48 60 72 84 96 108 120 12120 1120
12 24 36 48 60 72 72
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 39
Fig. 4. Effect of inoculums concentration on CHO production and DCW.
Fig. 5. Effect of different carbon source on CHO production and DCW.
Fig. 6. Effect of different nitrogen source on CHO production and DCW.
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 40
Fig.7. Effect of initial pH on CHO production and DCW.
Once the best carbon and nitrogen sources were selected by conventional one-factor-at-a-time
method, the medium was subjected to screening of the most significant parameters for CHO
production using the L9 orthogonal array. There was hardly to observe any variation between
software prediction and experimental values for CHO production. The responses for means
(larger is better) and for signal to noise (S/N) ratios obtained using the L9 orthogonal array
are shown in Table 4. The last two rows in the tables show delta values and ranks for the
system. Rank and delta values help in assessing which factors have the greatest effect on the
response characteristic of interest. Delta measures the size of the effect by taking the
difference between the highest and lowest characteristic average for a factor. A higher delta
value indicates a greater effect of that component. Rank orders the factors from the greatest
effect (on the basis of the delta values) to the least effect on the response characteristic. The
order in which the individual components affected the fermentation process were peptone >
starch > yeast extract > Tween 80 suggesting that starch and peptone had a major effect,
while Tween 80 had the least effect on CHO production by Streptomyces sp. The
experimental data revealed that selected level 2 values of starch and Tween 80, level 3
values of peptone and level 1 values of yeast extract in the medium are optimal for enzyme
production (Figure 8).
The optimum conditions for maximum CHO production was achieved using the medium
starch 2.0 gm%, peptone 0.9 gm%, yeast extract 0.3 gm%, Tween 80 0.1 % v/v at pH 7.5
with 12 % v/v inoculums. The validation of data was done at flask level with 100 ml medium
in which the activity observed was 2.82 U/ml, which was in good agreement with the
predicted results (2.83 U/ml) of software. This showed a significant 1.76-folds increase in
CHO activity in optimized medium than in non-optimized medium (1.6 U/ml). Figure-9
shows the batch profile of Streptomyces sp. with optimized medium.
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 41
Fig. 8. Impact of selected fermentation factors and their assigned level on CHO
production by Streptomyces sp. X-axis represents assigned levels of selected
factor and Y-axis represents CHO (U/ml).
Table 4. Response table for means and S/N ratio.
Level
Starch Peptone Yeast extract Tween 80
Mean S/N Mean S/N Mean S/N Mean S/N
1 0.9244 -1.3208 0.8467 -1.9179 1.4356 1.0878 1.4511 2.0416
2 2.0267 5.6453 1.2367 1.0363 1.2622 1.5536 1.4511 1.4191
3 1.1367 0.4569 2.0044 5.6629 1.3900 2.1400 1.1856 1.3206
Delta 1.1022 6.9661 1.1578 7.5808 0.1733 1.0522 0.2656 0.7209
Rank 2 1 3 4
Parekh et al.
International Journal of Applied Microbiology Science 2012; 1(2); 32-43 42
Fig. 9. Batch profile of Streptomyces sp. with optimized medium.
Conclusion
Using one-factor-at-a-time and orthogonal array method, the most effective components were
selected to design a simple yet very effective medium for remarkably improved CHO
production. The selected orthogonal array was L9 and optimum factors for CHO production
were found to be peptone, starch and yeast extract. The optimum medium condition derived
was: starch 2.0 gm%, peptone 0.9 gm%, yeast extract 0.3 gm% , Tween 80 0.1 % v/v at pH
7.5 with 12 % v/v inoculums. At this optimum condition, the yield of CHO production by
Streptomyces sp. was found to be 2.82 U / ml.
References
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gene in Streptomyces lividans with plasmid pIJ 702. Appl Environ Microbiol 1986; 52:1382-1385.
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243–248.
[4] Allain CC, Poon LS, Chan CSG, et al. Enzymatic determination of total serum cholesterol. Clin Chem
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International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 2(1): January-February2013
INTERNATIONAL JOURNAL OF UNIVERSAL
PHARMACY AND BIO SCIENCES
Bio Sciences Research Article……!!!
Received: 18-02-2013; Accepted: 22-02-2013
ISOLATION AND IDENTIFICATION OF EXTRACELLULAR
CHOLESTEROL OXIDASE PRODUCING ARTHROBACTER SP. FROM
WASTE OF REGIONAL OIL MILL
Parekh S.N*1. and Desai P.B.
2
1,2 Department of Microbiology, Shree Ramkrishna Institute of Computer Education and
Applied Sciences, Athawalines, Surat-395 001, Gujarat, India.
KEYWORDS:
Cholesterol Oxidase, 4-
Cholesten-3-one,
Indicator plate,
Horseradish Peroxidase.
For Correspondence:
Parekh S.N*
Address: Department of
Microbiology, Shree
Ramkrishna Institute of
Computer Education and
Applied Sciences,
Athawalines, Surat-395
001, Gujarat, India.
Email ID:
ABSTRACT
Cholesterol oxidase (EC1.1.3.6; CHO) is an enzyme, which
catalyzes the oxidation of cholesterol and converts 5-
cholesten-3β-ol into 4- cholesten-3-one. The objective of this
study is to isolate extracellular cholesterol oxidase (CHO)
producing microorganisms to obtain an abundant source of
cholesterol oxidase (CHO) for industrial and medicinal needs.
Cholesterol oxidase producing Arthrobacter sp. was isolated
from waste of regional oil mill. Twenty-five isolates were
tested for cholesterol oxidase activity by screening method.
As a result of the screening, isolate number RO-3 was
identified as Arthrobacter sp. by morphological, biochemical
and molecular methods. CHO activity of isolates was
measured by spectroscopic assay method.
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International Standard Serial Number (ISSN): 2319-8141
1. INTRODUCTION :
Cholesterol oxidase (CHO) is an enzyme which catalyzes the oxidation of Cholesterol and
converts 5-Cholesten-3β- ol into 4-Cholesten 3-one [1]. Many bacteria can produce this
enzyme including members of the genera Arthrobacter, Brevibacterium, Pseudomonas,
Nocardia, Rhodococcus, Streptomyces, Corynebacterium and Shizophylum [2,3]. Cholesterol
oxidase enzyme has many applications in medicine [4]., agriculture, and pharmaceutical [5]
and so on. For instance, it can be used for production of diagnostic kits to detect blood
Cholesterol [6], biological insecticide [7] and precursors for steroid hormones [8]. This
enzyme can be secreted from a bacterium in 3 types including intracellular, extracellular and
membrane-bound. Due to wide spectrum applications of Cholesterol oxidase, screening and
isolation of bacterial strains producing extracellular form of Cholesterol oxidase is of great
importance [9]. Many microorganisms have been determined which produced extracellular
Cholesterol oxidase including Rhodococcus equi, Rhodococcus erythropolis [10],
Streptomyces sp, Arthrobacter simplex, Brevibacterium sterolicum, Strerptomyces lividanse,
Schizophylum commune, Micrococcus sp etc [11,12,13]. Enzymatic properties of cholesterol
oxidase from Rhodococcus strains (some of which named formerly as Nocardia) are
particularly suitable for use in the analytical determination of cholesterol, in which the
hydrogen peroxide formed is used in a chromogenic reaction catalyzed by horseradish
peroxidase. In the present study, Arthrobacter sp. was isolated from waste of regional oil mil.
The type of CHO enzyme produced by isolate was determined using an enzyme activity
assay on supernatant of culture medium.
2. MATERIALS AND METHODS:
2.1 Isolation of microorganisms:
Cholesterol oxidase producing microorganisms were isolated by following procedure. 1 g
of samples were suspended in 100 ml of distilled water. The suspension was vigorously
shaken for 30 min. A volume of 100 μl of supernatant was inoculated in medium (medium
A) containing cholesterol as the sole carbon source. A medium contained (g/l): agar, 3.0 %;
K2HPO4, 0.25; NH4NO3, 17; MgSO4.H2O 0.25%; FeSO4, 0.001; NaCl, 0.005; cholesterol,
0.1% and Tween 80, 0.5 ml. The pH of medium was adjusted to 7.0. The inoculated plates
were incubated at 30oC for 7-12 days. After incubation period was completed, abscission
colonies were appeared on the plate surface. For fast growing and generating, larger
colonies were sub cultured in secondary medium (medium B) containing cholesterol as the
only source of carbon as well as yeast extract [9,14] .
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International Standard Serial Number (ISSN): 2319-8141
This medium contained yeast extract, 0.3 g; (NH4)2HPO4, 0.1 g; cholesterol, 0.15; Tween
80, 0.05 ml; pH – 7; agar, 3.0 % and distilled water, 100 ml. Each colony on medium A
was cultured in medium B and incubated at 30oC for 24 h. Then, larger colonies generated
on medium B were used for further identification [9, 11].
2.2 Screening of CHO producing organism
CHO is able to convert Cholesterol into Cholest-4-en-3-one and hydrogen peroxide. CHO
producing colonies were selected on cholesterol oxidase indicator plates. These plates were
prepared by adding 1.0 g Cholesterol, 1.0 g Triton X-100, 0.1g o-dianisidine and 1000
Units of peroxidase to 1 liter of agar medium. Bacterial colonies were cultured on these
plates and incubated at 30°C. Cholesterol penetrates into bacterial cells where it can be
converted into hydrogen peroxide by Cholesterol oxidase. Reagents that exist in the
medium react with hydrogen peroxide (H2O2) to form azo compound which turns the color
of medium into intense brown color [15, 16, 17].
2.3 Identification of isolates
Primary identification of isolates was carried out by studying their morphological, cultural
and biochemical characteristics by standard method. The phylogenetic analysis of isolate was
carried out on the basis of 16S ribosomal DNA (rDNA) partial sequences. The results of
primary identification and 16S rDNA sequences were used to identify the isolates. Bergey’s
manual of systematic bacteriology, 2nd
edition was used to identify bacterial isolates.
Taxonomic studies of the isolates was carried out by 16S rDNA partial sequencing at
Microbial culture collection, National center for cell science, Pune university campus, Pune
400011, India. The 16S rDNA was amplified using 8F 5’-AGAGTTTGATCCTGGCTCAG-
3’ and 907R 5’- CGTCAATTCMTTTRAGTTT-3’ as forward and reverse primer
respectively.
The nucleotide sequence of related organisms used for alignment and for calculating the
homology level was obtained from the NCBI (National Center for Biotechnology
Information) database and ClustalW2 programme was used to align the sequences. The
phylogenetic tree was constructed using MEGA 5.1 software using neighbor-joining method
(Tamura et al., 2011) [18, 19]. Bootstrap analysis was carried out to assess the reliability of
phylogenetic tree by the same software.16S rDNA sequence of isolate was deposited in
NCBI database and received accession number.
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2.4 Determination of CHO activity
CHO activity was measured by centrifuge the medium at 10,000 rpm for 20 min. at 4oC by
modified method based on the study of Allain et.al [4,12]. In this reaction, hydrogen
peroxide generated during Cholesterol oxidation process was coupled with 4-
aminoantipyrine and phenol by peroxidase to produce quinoneimine dye with maximum
absorption in 500nm. The reaction mixture was consisted of 1mM 4-aminoantipyrine , 5
mM phenol, 5 U/ml of horseradish peroxidase and sodium phosphate buffer (20 mM, pH
7.0). 50 μL of 6 g/L Cholesterol dissolved in dimethyl formamide containing 5% (v/v)
Triton X-100 was added to 1ml of reaction mixture, Which was then pre incubated for 3
min. at 30°C. The reaction was initiated by adding 20 μL of enzyme sample and was
continued for 5 min at 30°C. The assay mixture was boiled in a water bath for 2 min. to
stop the reaction, and then place in an ice bath for 2 min. Absorbance of the reaction
solution was monitored at 500 nm. (Systronic 2203, Japan). The assay mixture containing
inactivated enzyme was used as the blank. One unite of CHO activity was defined as the
amount of enzyme that converts 1μmol of cholesterol in to 4-cholesten - 3 - one per minute
at 30°C.
Cholesterol + O2 4- cholesten-3-one + H2O2
2H2O2 + 4 - aminoantipyrene + Phenol Quinoneimine dye + 4H2O
3. RESULTS AND DISCUSSION
25 samples of waste of regional oil mill were collected. 20 isolates were obtained from these
samples on their capability on growing on isolation medium A containing cholesterol as the
sole carbon source. Figure 1 shows the growth of isolate RO-3 on medium A and B. RO-3
was found to secrete extracellular CHO were detected by cholesterol oxidase indicator plate
(Fig.2). The result of microscopic observation and growth characteristics of these isolates is
shown in table-1. The results of biochemical and enzymatic properties were shown in table 2.
The cells of RO-3 were gram positive, irregular rods but eventually presented as coccoid
forms as growth continued changed. The partial sequence of 16S rDNA (834 bp) was shown
in table 3 of RO-3. Which shows 99% identity with 16S rDNA partial sequence of
Arthrobacter polychromogenes DSM 20136 and Arthrobacter scleromae YH-2001 (NCBI
accession No. NR 026192.1 & NR 041824.1 respectively) (Fig. 3).
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RO-3 was identified as Arthrobacter sp. from their gram staining, morphological,
biochemical and molecular characteristics. Extra cellular CHO activity of RO-3 was found to
be 0.91 units/ml and measured by harvesting the cells grown in the medium B on rotary
shaker at 150 rpm and 30oC for 72 hrs. Cholesterol oxidase from Streptomyces hygroscopicus
and the recombinant enzyme from Brevibacterium sterolicum expressed in Escherichia coli
have been characterized for their chemical, physical, and biochemical properties by Giovanni
Gadda et al. [20]. The Optimum activity and stability of CHO from Streptomyces fradiae and
Brevibacterium sp.were rerpoted at 50°C and 53°C for 30 min. respectively by Yazdi M. et
al., 2001 and Fujishiro et al., 2002 [21,3].
Figure 1 Growth of isolate RO-3 on medium A and B.
Figure 2: Growth of RO-3 on Cholesterol oxidase indicator plates
Table 1: Morphological and colonial characteristics of RO-03.
Sample Isolate
No.
Medium Colony / growth
characteristics
Morphology Figure
waste of
regional
oil mill
RO-3
Medium B
Small, circular,
slightly elevated,
smooth opaque, non
pigmented colony
Gram positive, motile,
short coco bacilli rods
arranged in irregular
clumps.
Table 2: Biochemical and enzymatic properties of RO-3.
Test Result Test Result
Indole production Negative Maltose fermentation Positive
Methyl Red test Negative Mannitol fermentation Positive
Vogus Proskaur test Negative Xylose fermentation Positive
Citrate utilization test Positive Glucose fermentation Positive
Nitrate reduction reduction test Positive Catalase Positive Urease production test Negative Gelatinase Positive H2S production test Negative Oxidase Positive Lactose fermentation Positive Amylase and Lipase Negative Sucrose fermentation Positive Caseinase Positive
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Table 3: Partial sequence of 16S rDNA of RO-3
Isolate
No.
16S rDNA Sequence
RO-3 >RO3
CATTTGAGTTTTAGCCTTGCGGCCGTACTCCCCCAGGCGGGGCACTTAATGCGTTAGCTACGGCGCGGA
AAACGTGGAATGTCCCCCCACACCTAGTGCCCAACGTTTACGGCATGGACTACCAGGGTATCTAATCCT
GTTCGCTCCCCATGCTTTCGCTCCTCAGCGTCAGTTAATGCCCAGAGACCTGCCTTCGCCATCGGTGTTC
CTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTACATCACTCTAGTCTGCCC
GTACCCACCGCAGATCCGGAGTTGAGCCCCGGACTTTCACGGCAGACGCGACAAACCGCCTACGAGCT
CTTTACGCCCAATAATTCCGGATAACGCTTGCGCCCTACGYMTTACCGCGGCTGCTGGCACGTAGTTAG
CCGGCGCTTCTTCTGCAGGTACCGTCACTTTCGCTTCTTCCCTACTGAAAGAGGTTTACAACCCGAAGGC
CGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCC
CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCTCTCAGGCCGGCTACCCGTCGTC
GCCTTGGTAAGCCATTACCTCACCAACAAGCTGATAGGCCGCGAGTCCATCCAAAAACCACAATAAAG
CTTTCCACCCCCCACCATGCGATGAGGAGTCATATCCGGTATTAGACCCAGTTTCCCAGGCTTATCCCAG
AGTTAAAGGGCAGGTTACTCACGTGTTACTCACCCGTTTCGCCACTAATCCAGGAGCAAGCTCCCATCA
TCG
Figure 3: Phylogenetic tree showing position of isolate RO-3 with related organisms
based on 16S rDNA sequences. Scale bar represent 0.02 substitutions per nucleotide
position. E. coli was used as the out group. Bootstrap values are shown as percentages
on branches. Sequence accession numbers are listed in parentheses. The 16S rDNA
sequence of strain RO-3 was deposited in the NCBI database under the accession no.
KC415767.
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4. CONCLUSION:
Cholesterol oxidase is an enzyme of great commercial value widely employed by laboratories
routinely devoted to the determination of cholesterol in food, serum and other clinical
samples. A diversity of micro-organisms, which are capable of producing high levels of this
enzyme have been reported. Our preliminary work led to the conclusion that Arthrobacter sp.
isolated from waste of regional oil mill might be considered as potentially interesting source
of extra cellular cholesterol oxidase for clinical and commercial purpose.
5. REFERENCES:
1. Murooka, Y., Ishizaki, T., Nimi, O. and Maekawa, N., (1986). Cloning and expres-
sion of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with
plasmid pIJ 702, Appl Environ Microbiol., 52:. 1382-1385.
2. Yazdi, M. T., Yazdi, Z. T., Zarrini, Gh. and Ghasemian, A., (2008). Purification and
characterization of extra-cellular cholesterol oxidase from Rhodococcus sp. PTCC
1633, Biotechnology., 7 (4): 751-6.
3. Fujishiro, K., Uchida, H., Shimokava, K., Nakano, M., Sano, F., Ohta, T., Nakahara,
N. and Aisak, K.,Uwajima T., ( 2002) .Purification and properties of a new
Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10,
FEMS. Microbiol. Lett., 215: 243–248.
4. Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C., (1974).
Enzymatic determination of total serum cholesterol, Clin. Chem., 20: 470- 475.
5. Ahmad, S., Garg, S.K. and Johri, B.N., ( 1992). Biotransformation of sterols: selective
cleavage of the side chain, Biotechnol., Adv. 10: 1–67.
6. Noma, A. and Nakayama, K., (1976).Comparative studies on cholesterol oxidases
from different sources, Clin. Chim. Acta., 73, 487–496.
7. Purcell, J.P., Greenplate, J.T., Jennings, M.G., Ryerse, J.S., Pershing, J.C., Sims S.R.,
Prinsen, M.J., Corbin, D.R., Tran, M and Sammons, R.D., (1993). Cholesterol
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`
Introduction
Cholesterol oxidase (EC1.1.3.6; CHO) catalyses
the first step of cholesterol degradation to 4-Charleston-
3-one with the reduction of oxygen at C-3 of hydrogen
peroxides. CHO is one of the key enzymes in microbial
sterol metabolism. The interest in this enzyme is due to
its industrial importance and its wide utilization in the
determination of cholesterol in blood serum and food
(Parra et al., 2007), and to its assumed potential in the
manufacture of diets with reduced cholesterol (Chenfeng
et al., 2002). Indeed, dietary cholesterol degradation is
considered as a means of protection against athero
sclerosis. Other possible applications of CHO are in the
production of precursors for the chemical synthesis of
pharmaceutical steroid hormones (Alexander et al.,
1995), and for its insecticidal activity that is vital for pest
control strategies employing transgenic crops (Shen
et al. 1997). CHO has been isolated from several microbial
sources, including species of Arthrobacter (Wenhsiung
et al., 1988), Bacillus (Kim et al., 2002), Brevibacterium
(Pornpen et al. 2006), Nocardia (Sojo et al., 1997),
Pseudomonas (Isobe et al., 2003), Rhodococcus (Elalami
et al., 1999), Streptomyces (Pornpen et al., 2006) and
Schizophyllum (Fukuyama et al., 1979). In this study,
CHO producing organisms were isolated from the waste
of a regional oil mill and characterize by microbiological
methods and evolutionary relationship with relevant
organisms were established.
Materials and Methods
Isolation of microorganisms
Cholesterol oxidase producing microorganisms
were isolated by following procedure : 1 gm of samples
Abstract
Cholesterol oxidase (EC1.1.3.6; CHO) is an enzyme, which catalyzes the oxidation of cholesterol and converts 5-cholesten-3β-ol into 4-cholesten-3-one. The objective of this study is to isolate extracellular cholesterol oxidase producing microorganisms to obtain an abundant source of cholesterol oxidase for industrial and medicinal needs. Cholesterol oxidase producing Microbacterium sp. was isolated from waste of regional oil mill. Twenty-five isolates were tested for cholesterol oxidase activity by screening method. As a result of the screening, isolate number RO-5 was identified as Microbacterium sp. by morphological, biochemical and molecular methods. A phylogenetic tree for the isolate was constructed by neighbor-joining method. Enzyme activity was measured by spectroscopic analysis.
Keywords: Microbacterium sp., enzyme, Cholesterol oxidase, 4-cholesten-3-one, phylogenetic, indicator plate and horseradish peroxidase,
Isolation and characterization of extracellular cholesterol oxidase producing Microbacterium sp. from waste of regional oil mill
S.N. Parekh and P.B. Desai
Department of Microbiology, Shree Ramkrishna Institute of Computer Education and Applied Sciences, Athawalines, Surat-395 001, Gujarat, India
Email : [email protected]
Corresponding Author S.N. Parekh
Department of Microbiology, Shree Ramkrishna Institute of Computer Education and Applied Sciences,
Athawalines, Surat-395 001, Gujarat, India
Email : [email protected] Article History
Received on 26 December, 2012; Revised in revised form 24 January, 2012; Accepted 8 February, 2013
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Page 2
was suspended in 100 ml of distilled water. The
suspension was vigorously shaken for 30 min. A volume
of 100 µl of supernatant was inoculated in medium
(Medium - A) containing cholesterol as the sole carbon
source. A medium contained (gm/l): agar, 3.0 %;
K2HPO4, 0.25; NH4NO3, 17; MgSO4. H2O 0.25%; FeSO4,
0.001; NaCL, 0.005; cholesterol, 0.1% and Tween 80,
0.5 ml. The pH of the medium was adjusted to 7.0. The
inoculated plates were incubated at 30oC for 7- 12 days.
After incubation period was completed, abscission
colonies were appearing on the plate surface. For fast
growing and generating, larger colonies were sub cultured
in secondary medium (Medium - B) containing cholesterol
as the only source of carbon as well as yeast extract
(Yazdi1 et al., 2001, Lashkarian, 2010). This medium
contained yeast extract, 0.3 gm; (NH4)2HPO4, 0.1 gm;
cholesterol, 0.15; Tween 80, 0.05 ml; pH – 7; agar, 3.0
% and distilled water, 100 ml. Each colony on medium
A was cultured in medium B and incubated at 30oC for
24 hrs. Then, larger colonies generated on medium B were
used for further identification (Yazdi1 et al., 2001).
Screening of CHO is producing organism
CHO is able to convert Cholesterol into Cholest -
4-en-3-one and hydrogen peroxide. CHO producing
colonies were selected on cholesterol oxidase indicator
plates. These plates were prepared by adding 1.0 gM.
Cholesterol, 1.0 GM. O-dianisidine and 1000 Units of
peroxide to 1 liter of agar medium. Bacterial colonies
were cultured on these plates and incubated at 30°C.
Cholesterol penetrates into bacterial cells where it can
be converted into hydrogen peroxide by Cholesterol
oxidase. Reagents that exist in the medium react with
hydrogen peroxide (H2O2) to form azo compound which
turns the color of medium into intense brown color
(Ghasemian et al., 2009 and Drzyzga, 2011).
Identification of isolates
Primary identification of isolates was carried out
by studying their morphological, cultural and biochemical
characteristics by standard method. The phylogenetic
analysis of isolate was carried out on the basis of 16S
ribosomal DNA (RDNA) partial sequences. The results
of primary identification and 16S rDNA sequences
were used to identify the isolates. Bergey’s manual of
systematic bacteriology, 2nd edition was used to identify
bacterial isolates.
Taxonomic studies of the isolates was carried
out by 16S rDNA partial sequencing at Microbial culture
collection, National center for cell science, Pune university
campus, Pune 400011, India. The 16S rDNA was amplified
using 8F 5’ AGAGTTTGATCCTGGCTCAG-3’ and
907R 5’- CGTCAATTCMTTTRAGTTT-3’ as forward
and reverse primer respectively. The nucleotide sequence
of related organisms used for alignment and for
calculating the homology level was obtained from the
NCBI (National Center for Biotech- nology Information)
database and ClustalW2 programme- was used to align
the sequences. The phylogenetic tree was constructed
using MEGA 5.1 software using neighbor-joining
method (Tamura et al., 2011). Bootstrap analysis was
carried out to assess the reliability of phylogenetic tree
by the same software. 16S rDNA sequence of isolate
was deposited in NCBI database and received accession
number.
Determination of CHO activity
CHO activity was measured by centrifuge the
medium at 10,000 rpm for 20 min. at 4oC by modified
method based on the study of Allain et al. (1974) and
MacLachlan et al. (2000). In this reaction, hydrogen
peroxide generated during Cholesterol oxidation process
was coupled with 4-aminoantipyrine and phenol by
peroxidase to produce quinoneimine dye with maximum
absorption in 500nm. The reaction mixture was consisted
of 1mM 4-aminoantipyrine , 5 mM phenol, 5 U/ml of
horseradish peroxide and sodium phosphate buffer (20
mm, pH 7.0). 50 µL of 6 g/L Cholesterol dissolved in
dimethyl formamide containing 5% (v/v) Triton X-100
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was added to 1ml of reaction mixture, Which was then
pre-incubated for 3 min. at 30°C. The reaction was
initiated by adding 50 µL of enzyme sample and was
continued for 5 min at 30°C. The assay mixture was
boiled in a water bath for 2 min. to stop the reaction,
and then place in an ice bath for 2 min. Absorbance of
the reaction solution was monitored at 500 nm.
(Systronic 2203, Japan). The assay mixture containing
inactivated enzyme was used as the blank. One unite of
CHO activity was defined as the amount of enzyme that
converts 1µmol of cholesterol in to 4-cholesten - 3 -
one per minute at 30°C.
Results and Discussion
69 samples of waste of regional oil mill were
collected. 20 isolates were obtained from these samples
on their capability on growing on isolation medium A
containing cholesterol as the sole carbon source.
Figure - 1 shows the growth of isolate RO-5 on medium
A and B. RO-5 was found to secrete extracellular CHO
were detected by cholesterol oxidase indicator plate
(Fig.-2). The result of microscopic observation and
growth characteristics of these isolates is shown in table-1.
The results of biochemical and enzymatic properties
were shown in table - 2. The cells of RO-5 were gram
positive short rods. The partial sequence of 16S rDNA
(835 bp) is shown in table 3 of RO-5. Which shows
99% identity with 16S rDNA partial sequence of
Microbacterium paraoxydans CF 36 and Microbacterium
maritypicum DSM 12512 (NCBI accession No. NR
025548 & NR 042351 respectively) (Fig.- 3) . RO-5 was
identified as Microbacterium sp. from their gram staining,
morphological, biochemical and molecular characteristics.
Extra cellular CHO activity of RO-5 was found to be
1.02 units/ml and measured by harvesting the cells
grown in the medium B on rotary shaker at 150 rpm
and 30oC for 72 hrs. In contrast, Chengtao et al. (2005)
reported extracellular CHO production of Rhodococcus sp.
R14-2 is about 1.5 U/ml where as CHO production from
Rhodococcus sp. GKI (Elalami et al. 1999) is 0.38
U/ml after 90 h cultivation.
Table – 1. Morphological and colonial characteristics of RO-5
Sample Isolate No. Medium Colony / growth
characteristics Morphology Figure
Waste of regional oil
mill RO-5
Medium B
Small, round, raised, glistening, opaque, lemon yellow color pigmented colony
Gram positive, non motile, Short rods.
Table – 2. Biochemical and enzymatic properties of RO-5.
Test Result Test Result
Indole production Negative Maltose fermentation Acid
Methyl Red test Negative Mannitol fermentation Negative
Vogus Proskaur test Negative Xylose fermentation Negative
Citrate utilization test Positive Glucose fermentation Acid
Nitrate reduction reduction test Negative Catalase Positive
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Fig – 2. Growth of RO-5 on Cholesterol oxidase indicator plates
Urease production test Negative Gelatinase Negative
H2S production test Positive Oxidase Positive
Lactose fermentation Positive Amylase and Lipase Negative
Sucrose fermentation Positive Caseinase Negative
Table - 3. Partial sequence of 16S rDNA of RO-5
Isolate No.
16S rDNA Sequence
RO-5 >RO-5 TGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGTGAACACGGAGCTT GCTCTGTGGGATCAGTGGCGAACGGGTGAGTAACACGTGAGCAACCTGCCCCTGACTCTGGGATAAGCGC TGGAAACGGCGTCTAATACTGGATATGTGACGTGACCGCATGGTCTGCGTTTGGAAAGATTTTTCGGTTG GGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGTCGACGGGTAGCCGGC CTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAA TATTGCACAATGGGCGGAAGCCTGATGCAGCAACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACC TCTTTTAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAAAAGCGCCGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTC GCGTCTGCTGTGAAATCCCGAGGCTCAACCTCGGGCCTGCAGTGGGTACGGGCAGACTAGAGTGCGGTAG GGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAG ATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGGGTGGGGAGCAAACAGGCTTAGATACCCTGGTAGT CCACCCCGTAAACGTTGGGAACTAGTTGTGGGGTCCATTCCACGGATTCCGTGACGCAGCTAACG
Fig – 1. Growth of isolate RO-5 on medium A and B.
A B
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Conclusion
Cholesterol oxidase is an enzyme of great
commercial value widely employed by laboratories
routinely devoted to the determination of cholesterol in
food, serum and other clinical samples. A diversity of
micro-organisms, which are capable of producing high
levels of this enzyme have been reported.
work led to the conclusion that Microbacterium
isolated from waste of regional oil mill might be a
promising source of extra cellular cholesterol oxidase
for clinical and commercial purpose.
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Corresponding Author : S.N. Parekh, Department of Microbiology, Shree Ramkrishna Institute of Computer Education and Applied Sciences, Athawalines, Surat - 395 001, Gujarat, India. Email : [email protected], ©2013, IJALS. All Rights Reserved.