Two novel extracellular cholesterol oxidases of Bacillus sp. isolated from fermented flatfish

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1 Biotechnology Letters 24: , Kluwer Academic Publishers. Printed in the Netherlands Two novel extracellular cholesterol oxidases of Bacillus sp. isolated from fermented flatfish Chang-Ho Rhee, Kwan-Pil Kim & Heui-Dong Park Department of Food Science and Technology, Kyungpook National University, 1370 Sankyuk, Taegu , Korea Author for correspondence (Fax: ; Received 19 April 2002; Revisions requested 17 May 2002; Revisions received 14 June 2002; Accepted 17 June 2002 Key words: Bachillus sp., cholesterol oxidase, fermented flatfish, purification Abstract Two novel extracellular cholesterol oxidases designated CO1 and CO2, from Bacillus sp. SFF34, were purified 5.6 and 5.9-fold giving Mr values of 36 and 37 kda. The optimum temperature for the activity was 60 C(CO1) and 40 C (CO2), and the optimum ph was 6.25 (CO1) and 6 (CO2) over 30 min reaction time. The apparent K m values for cholesterol were 6.76 mm (CO1) and 4.50 mm (CO2). Both the enzymes could oxidize 5α-cholestane, 5α-cholestane-3β-ol-7-one, coprostane, dihydrocholesterol, hecogenin, β-sitosterol and stigmasterol. Introduction Cholesterol oxidase (cholesterol: oxygen oxidoreductase, EC ) catalyzes the oxidation of cholesterol to 4-cholesten-3-one in the presence of O 2 (MacLachlan et al. 2000, Sakodinskaya 2000). Its production by microorganisms has aroused considerable interest because of its wide application to many industrial fields. Cholesterol oxidases are used to determine cholesterol in food and blood serum by coupling of the enzyme with peroxidase. They can also be used to produce a precursor for chemical synthesis of steroid hormones (MacLachlan et al. 2000) and to degrade of dietary cholesterol in foods (Watanabe et al. 1986). A high blood cholesterol level is considered to be related to cardiovascular disease and its degradation products may act as co-carcinogens in colon cancer (Paniangvait et al. 1995). Therefore, it has been proposed that bacterial degradation of cholesterol in cholesterolcontaining foods may be useful for human health (Watanabe et al. 1986). Cholesterol oxidase is a key enzyme for the degradation of cholesterol because its products, oxidized derivatives, are more susceptible to further degradation by bacteria (MacLachlan et al. 2000). A variety of microorganisms producing cholesterol oxidase have been reported. The enzymes in various strains including Arthrobacter, Brevibacterium, Corynebacterium, Nocardia, Pseudomonas, Rhodococcus, Schizophyllum and Streptomyces spp. have been purified and characterized (MacLachlan et al. 2000). Previously, we have isolated a novel bacterial strain, Bacillus sp. SFF34, from Korean traditional fermented flatfish, which could produce a high level of extracellular cholesterol oxidase and studied the enzyme production by the strain (Kim et al. 2001). In this study, two extracellular cholesterol oxidases were purified from its culture supernatant and their characteristics were investigated. Materials and methods Microorganisms and growth Bacillus sp. SFF34 (Kim et al. 2001) was grown on a cholesterol medium composed of 1 g cholesterol l 1, 20gglucosel 1, 5 g yeast extract l 1,2gNH 4 NO 3 l 1, 0.2 g K 2 HPO 4 l 1, 0.3 g MgSO 4 7H 2 Ol 1 (ph 7) at 30 C for 36 h with shaking at 150 rpm (Watanabe et al. 1989). The ph of the media was adjusted to 7.

2 1386 Preparation of bacterial culture supernatant The culture supernatant was obtained by centrifugation of the culture broth at g for 30 min and stored at 20 C for further experiments. Enzyme assay Protein contents were determined at 595 nm using Coomassie Brilliant Blue G-250 with BSA as a standard by the method of Bradford. Cholesterol oxidase activity was measured at 240 nm by the method of Richmond (1973). The reaction mixture was composed of 3 ml of 0.1 M sodium phosphate buffer (ph 7)/0.05% Triton X-100, 0.05 ml 6 mm cholesterol in 2-propanol and 0.05 ml enzyme. The enzyme reaction was carried out at 30 C for 30 min followed by the measurement of the increase of absorbancy at 240 nm. The molar absorption (ɛ) of 4-cholesten-3- one was lm 1 cm 1. Cholesterol oxidase activity was calculated as [( A reaction volume 0.082)/volume of enzyme used] = A 5.1 units ml 1. The enzyme unit of cholesterol oxidase was defined as the amount of enzyme oxidizing 1 µmole of cholesterol to 4-cholesten-3-one per min at 30 C. Purification of cholesterol oxidase The cholesterol oxidase was purified from the culture supernatant using four steps; ammonium sulfate fractionation, DEAE anion-exchange chromatography, Sephacryl S200HR and Sepharose CL-6B gel filtrations. All the purification steps were carried out at 4 C. Molecular weight determination Molecular weight of the purified cholesterol oxidase was determined by gel filtration using a Sepharose CL- 6B column (Andrews 1964) and SDS-PAGE using a 15% polyacrylamide-sds gel (Laemmli 1970). Analysis of cholesterol oxidase reaction product Lipid was extracted from the enzyme reaction mixture for analysis of cholesterol oxidation products by the method of Marmer & Maxwell (1981) and analysed using a GC (Hewlett Packard 6890A, Wilmington, USA) by the method of Pie et al. (1991). The enzyme reaction mixture was mixed for 1 h with 80 ml of chloroform/ethyl ether (1:1, v/v), in which 0.5 ml 5αcholestane (2 mg ml 1 chloroform) was added as an Fig. 1. SDS-polyacrylamide gel electrophoresis of the purified cholesterol oxidases of Bacillus sp. SFF34. The two purified cholesterol oxidases, CO1 (lane A) and CO2 (lane B), were resolved in a 15% polyacrylamide-sds gel. After the electrophoresis, the gels were stained with a Coomissie Brilliant Blue R-250 solution for the protein staining. Lane M represents molecular weght marker; serum albumin (66 kda), ovalbumin (45 kda), glyceraldehydes-3-phosphate dehydrogenase (36 kda), bovine carbonic anhydrase (29 kda), trypsinogen (24 kda), trypsin inhibitor (20 kda), α-lactoalbumin (14.2 kda) and bovine lung aprotinin (6.5 kda) from the top of the gel. internal standard. The mixture was filtered and rinsed with ethanol/chloroform (1:1, v/v). The organic solvent was, then, evaporated under vacuum, and the extract was resuspended in 15 ml diethyl ether. Lipid extracts were filtered and mixed with 20 ml 0.5 M KOH in methanol, which was saponified at 60 C for 1 h. After 20 ml distilled water was added, unsaponified fractions were extracted twice with 25 ml diethyl ether. The pooled organic fractions were rinsed with distilled water and used for the GC analysis using a flame ionization detector and a fused silica capillary DB5 column (J & W scientific, Folsom, USA). The conditions for the GC operation were as following: flow rate, 1.5 ml min 1 ; pressure, 1.5 atm; oven temperature, 280 C; injector temperature, 290 C; detector temperature, 300 C. Helium was used as carrier gas.

3 1387 Table 1. Summary for the purification of two extracellular cholesterol oxidases from Bacillus sp. SFF34. Purification step Total Total Specific Yield Purification protein activity activity (%) (fold) (mg) (U) (U mg 1 ) Culture supernatant Ammonium sulfate CO1 DEAE cellulose Sephacryl S200HR Sepharose CL-6B CO2 DEAE cellulose Sephacryl S200HR Sepharose CL-6B Ammonium sulfate precipitation was carried out from 20% to 60% saturation. The precipitants were collected and resuspended in 50 mm phosphate buffer (ph 7). After dialysis against the same buffer for 24 h, the dialysed solution was applied to a DEAE-cellulose column (2 by 40 cm), equilibrated with the same buffer. Cholesterol oxidases were eluted with a linear gradient of 0 to 0.5 M NaCl at a flow rate of 40 ml h 1 to collect 5 ml of each fraction, which were divided into two fractions (designated CO1 and CO2). Each cholesterol oxidase fraction was pooled and concentrated using an ultrafiltration kit with an Amicon YM10 membrane (Millipore Co., Waltham, USA). The concentrated fraction was then applied to Sephacryl S200HR column (1.8 by 80 cm), equilibrated with 50 mm phosphate buffer (ph 7). The enzyme was eluted at a flow rate of 15 ml h 1 to collect 3 ml of each fraction. The active fractions were pooled and concentrated using the ultrafiltration kit and applied to a Sepharose CL-6B column (1 by 80 cm), equilibrated with the same buffer. Cholesterol oxidase was eluted at a flow rate of 25 ml h 1 to collect 3 ml of each fraction. The active fractions were pooled and used as the purified cholesterol oxidase for their characterization. The enzyme unit of the cholesterol oxidase activity was defined as the amount of enzyme oxidizing 1 µmole of cholesterol per min at 30 C under the conditions of this study. Results and discussion Purification of cholesterol oxidases The purification steps employed to purify the cholesterol oxidase are summarized in Table 1. DEAEcellulose column chromatography of the culture supernatant resulted in the separation of the enzyme to two fractions, which were designated as CO1 and CO2 (data not shown). The CO1 and CO2 fractions of the enzyme were purified 5.6-fold and 5.9-fold, resulting in 59.4 mg and 40.5 mg of purified proteins, respectively. Their purification yield was 36.1% (CO1) and 25.6% (CO2), and their specific activity was 7.6 U mg 1 protein (CO1) or 8 U mg 1 protein (CO2). The apparent K m values were 6.76 mm (CO1) and 4.50 mm (CO2) against cholesterol (data not shown). Bacterial cholesterol oxidases can be divided into three different groups; intracellular, membrane-bound and extracellular types (MacLachlan et al. 2000). Among them, the extracellular type enzyme has been studied more intensively and well documented, because it is easier to purify. Two cholesterol oxidases were detected in Rhodococcus erythropolis; a celllinked type and an extracellular type (MacLachlan et al. 2000). In addition, three forms of the enzyme were detected in Nocardia rhodochrous (MacLachlan et al. 2000). It is interesting to notice that Bacillus sp. SFF34 produced two different cholesterol oxidases, both of which belong to extracellular type. Molecular weight When the purified cholesterol oxidase fractions were resolved in a 15% polyacrylamide-sds gel, both CO1 and CO2 fractions showed a single band (Figure 1). Molecular weights were determined to be 36 kda for CO1 fraction and 37 kda for CO2 fraction from SDS- PAGE. They were found to be the same on a Sepharose CL6-B column (data not shown). These results suggest that the two purified cholesterol oxidases are monomer type. Several cholesterol oxidases have been purified from bacteria. Their molecular weights were generally

4 1388 Table 2. Substrate specificity of the two purified cholesterol oxidases from Bacillus sp. SFF34. Substrate Relative activity (%) CO1 CO2 Cholesterol Anderostene-3, 17-dione 9 2 1, 4-Anderostadiene-3, 17-dione α-Anderostane-3, 17-dione 0 2 5α-Cholestane α-Cholestane-3β-ol-7-one Coprostane Cholesterol-5α, 6β-epoxide 9 10 Dihydrocholesterol Hecogenin Lanosterol 4 5 β-sitosterol Stigmasterol The enzyme activity was expressed as relative activity to those of the CO1 at 60 C (26.9 U mg 1 ) and the CO2 at 40 C (12.3 U mg 1 ). No activity was observed against cholestane-3β, 5α, 6β-triol, campesterol, dehydroisoandrostane and epiandrostenone by both the two purified cholesterol oxidase fractions (CO1 and CO2). Fig. 2. Effect of temperature on the activity (A) and stability (B) of the purified cholesterol oxidases from Bacillus sp. SFF34. The enzyme activity was assayed from 25 Cto80 C, and expressed as relative activity to those of the CO1 at 60 C (26.9 U mg 1 ) andtheco2at40 C (12.3 U mg 1 ) (A). For the enzyme stability test, the residual enzyme activity was assayed after the enzyme was incubated for 1 h in a temperature range from 25 Cto80 C(B). Closed circles and open squares indicate the purified CO1 and CO2 fractions, respectively. in the range of 52 kda to 61 kda, which are much bigger than the purified enzyme in this study. A small cholesterol oxidase with 31 kda of Mr value has been also purified from Brevibacterium sterolicum, which is similar to the purified CO1 and CO2 fractions in the molecular size (Uwajima et al. 1973). Effect of temperature and ph on the activity and stability The optimum temperatures for the activity of the purified two fractions were 60 C for CO1 and 40 C for CO2, respectively (Figure 2A). Both the fractions wereobservedtobestableupto60 C and their residual activities were about 90% after treatment at 60 C for 1 h. No significant loss of activity was observed at 40 C for 1 h (Figure 2B). The maximal activity of CO1 was observed at ph 6.5 with 83% activity at ph 5 and 77% at ph 9. The optimum ph was 6 for CO2 with 78% activity at ph 5 and 75% at ph 8. The CO1 fraction was the most stable at ph 6 but CO2 fraction was at ph 5 (data not shown). Substrate specificity Specificity of the two purified cholesterol oxidases to various sterols was investigated and shown as percentage of that of cholesterol oxidation (Table 2). Although there were significant differences in the relative activity, their substrate specificity showed similar patterns to each other. Both the enzymes could oxidize 5α-cholestane, 5α-cholestane-3β-ol-7-one, coprostane, dihydrocholesterol, hecogenin, β-sitosterol and stigmasterol in a significantly high level, which was over 55% activity compared to that against cholesterol. However, no significant oxidizing activity (less than 10% activity compared to that against cholesterol) was observed with 4-anderostene-3, 17-dione, 1, 4-anderostadiene-3, 17-dione, 5α-anderostane-3, 17-dione, campesterol, cholestane-3β, 5α, 6β-triol, cholesterol-5α, 6β-epoxide, dehydroisoandrostane, epiandrostenone and lanosterol. Identification of cholesterol oxidase reaction product GC analysis of the reaction mixture with the two cholesterol oxidases showed that a peak newly ap-

5 1389 peared at 11.2 min of retention time, which is consistent with the retention time of the standard 4- cholesten-3-one (data not shown). Cholesterol oxidase is the enzyme to convert cholesterol to 4-cholesten- 3-one (MacLachlan et al. 2000, Sakodinskaya 2000). This reaction has been reported to occur by two different functional domains of the enzyme; one for dehydrogenation to produce the intermediate 5-cholestane- 3-one and one for 5-6 to 4-5isomerizationtoform to 4-cholestane-3-one (Yamashita et al. 1998). References Andrews P (1964) Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochem. J. 91: Kim KP, Rhee CH, Park HD (2001) Isolation and characterization of cholesterol degradation bacteria from Korean traditional salt fermented flat fish. Kor. J. Postharvest Sci. Technol. 8: Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: MacLachlan J, Wotherspoon ATL, Ansell RO, Brooks CJW (2000) Cholesterol oxidase: sources, physical properties and analytical applications. J. Steroid Biochem. Mol. Biol. 72: Marmer WN, Maxwell RJ (1981) Dry column method for the quantitative extraction and simultaneous class separation of lipids from muscle tissue. Lipids 16: Paniangvait P, King AJ, Jones AD, German BG (1995) Cholesterol oxides in foods of animal origin. J. Food Sci. 60: Pie JE, Spahis K, Christine S (1991) Cholesterol oxidation in meat products during cooking and frozen storage. J. Agric. Food Chem. 39: Richmond W (1973) Preparation and properties of a cholesterol oxidase from Norcardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin. Chem. 19: Sakodinskaya IK (2000) Crown ether activates cholesterol oxidase in low water media. Biotechnol. Lett. 22: Uwajima T, Yagi H, Nakamura A, Terada O (1973) Isolation and crystallization of extracellular 3β-hydroxy steroid oxidase of Brevibacterium sterolicum nov sp. Agric. Biol. Chem. 37: Watanabe K, Aihara H, Nakagawa Y, Sasaki T (1989) Properties of the purified extracellular cholesterol oxidase from Rhodococcus equi No. 23. J. Agric. Food Chem. 37: Watanabe K, Shimizu H, Aihara H, Nakamura R, Suzuki KI, Momagata K (1986) Isolation and identification of cholesteroldegradation Rhodococcus strains food of animal origin and their cholesterol oxidase activities. J. Gen. Appl. Microbiol. 32: Yamashita M, Toyama M, Ono H, Fuji I, Hirayama N, Murooka Y (1998) Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase. Protein Eng. 11: