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The effect of physical and chemical treatments on the esterase activity from Pseudomonas fragi CRDA 037
Food Research International 33 (2000) 767±774
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The eect of physical and chemical treatments on the esterase activity from Pseudomonas fragi CRDA 037 Selim Kermasha a,*, Barbara Bisakowski b, Safwan Ismail a, Andre Morin b a
Department of Food Science and Agricultural Chemistry, McGill University, 21,111 Lakeshore, Ste Anne de Bellevue, PQ, Canada H9X 3V9 b Agriculture and Agri-Food Canada, 3 600, Blvd. Casavant West, St-Hyacinth, PQ, Canada J2S 8E3 Received 14 January 2000; accepted 2 March 2000
Abstract The eect of dierent treatments on the esterase activity of the cellular debris of Pseudomonas fragi, responsible for the production of natural fruity ¯avors in processed products, was investigated. Glass bead homogenization (GBH) decreased activity by 50%, French press homogenization (FPH) produced only a slight decrease, while ultrasonication (US) alone or with GBH increased activity by 25 and 50%, respectively. Treatment with (i) GBH (4 min) and Triton increased activity two-fold, (ii) GBH or US (4 min), Triton and EDTA produced little eect, (iii) FPH (three passes), Triton and EDTA produced a decrease of 30 to 50%, (iv) FP (three passes), US (4 min), Triton and EDTA had no eect, while (v) GBH (4 min), US (4 min), Triton and EDTA showed a 35% decrease in activity. The esterase activity of the cellular debris stored in buer (4 C) and hexane (ÿ80 C) decreased by 80% after 3 days, while that of whole cells stored in hexane (ÿ80 C), and buer at 4 and ÿ80 C remained preserved for 3 days whereas that of the lyophilized whole cells greatly decreased. The esterase activity of whole cells stored in buer containing 15 and 30% glycerol was preserved for three weeks. Crown Copyright # 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Flavors; Fatty acids; Alcohol esters; Esterase; Pseudomonas
1. Introduction Pseudomonas fragi is an aerobic, gram negative, nonpathogenic, short and rod-shaped bacterium (Bassette, Fung & Mantha, 1986; Fairbairn & Low, 1986) that was isolated from milk by Hussong, Long and Hammer (1937). In processed dairy products, the development of fruity ¯avors has been reported to be due to P. fragi responsible for the hydrolysis of milk triglycerides and the esteri®cation of certain low molecular weight fatty acids with ethanol. When grown in skim milk, the P. fragi strain CRDA-037 produced a mixture of 26 dierent odor-active compounds of which 13 were identi®ed as fatty acid ethyl esters (Cormier, Raymond, Champagne & Morin, 1991). The production of a fruity strawberry-like odor by P. fragi is mainly due to the presence of the ethyl butyrate * Corresponding author. Tel.: +1-514-398-7922; fax: +1-514-3988132. E-mail address: [email protected] (S. Kermasha).
and ethyl hexanoate (Morgan 1970; Reddy, Bills, Lindsay, Libbey, Miller & Morgan, 1968) and 3-methylbutanoic acids (Pereira & Morgan, 1958). Raymond, Morin, Cormier, Champagne and Dubeau (1990) reported that dierent concentrations of ethyl butyrate and ethyl hexanoate produced by the cells of P. fragi was related to the activity of lipases and esterases known to be involved in the synthesis of fruity aroma (Hosono, Eilliot & McGugan, 1974; Pereira & Morgan). Although esterase activity was demonstrated in the intact cells of P. fragi, little information is available on the characterization of this activity in vitro. This work is part of ongoing research aimed at the development of a biotechnological approach for the production of natural ¯avors by microbial enzymes (Lamer, Leblanc, Morin & Kermasha, 1996; Kermasha, Bisakowski, Morin & Ismail, 1999). The speci®c objective of this research was to investigate the eect of dierent physical and chemical treatments on the esterase activity of P. fragi. In addition, the esterase activity of P. fragi was investigated with respect to dierent storage conditions.
0963-9969/00/$ - see front matter Crown Copyright # 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S0963-9969(00)00080-6
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2. Materials and methods 2.1. Bacterial strain The gram negative bacteria of P. fragi CRDA 037 was obtained from the Agriculture and Agri-Food Research and Development Center (CRDA, St Hyacinthe, PQ). 2.2. Chemicals Whey powder used for the incubation medium was obtained from Saputo (St Hyacinthe, PQ). Brain Heart Infusion (BHI) and Bacto-Agar were obtained from Difco Laboratories (Detroit, MI). Butyric and valeric acids, as well as phosphate and ethylenediaminetetraacetic acid (EDTA) were purchased from ACP Chemical Inc. (St-LeÂonard, PQ). CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane- sulfonate) was obtained from ICN Biomedicals Inc. (Aurora, OH) while Triton X-100 was from Aldrich Chemical Co. (Milwaukee, WI). Bovine serum albumin (BSA) and potassium phosphate dibasic, anhydrous were obtained from Sigma Chemical Co. (St-Louis, MO). Sodium hydroxide was provided by Fisher Scienti®c (Fair Lawn, NJ) and ethanol (94%) was purchased from the SocieÂte des Alcools du QueÂbec (MontreÂal, PQ). Methyl hexanoate was received from Poly Science Co. (Niles, IL) while potassium dihydrogen orthophosphate and antifoam ``B'' were obtained from BDH Inc. (Toronto, ON). 2.3. Organism and inoculum preparation The reference strain P. fragi CRDA 037 was transferred monthly and kept at 4 C on BHI agar slants covered with sterile mineral oil. The inoculation of P. fragi was carried out according to the procedure described previously by Raymond et al. (1990). Sterilized BHI broth (25 ml) was dispensed into 125 ml Erlenmeyer ¯asks. Three successive subcultures of the bacteria were incubated in BHI broth at 30 C with shaking at 200 rpm for 17 to 24 h in an Environ-Shaker (Lab-line Instruments, Inc., Chicago, IL). A 0.01% (v/v) inoculum was used to initiate each subculture. 2.4. Culture incubation Biomass production of P. fragi was carried out in Erlenmeyer ¯asks of 2 l. The whey medium (6%, w/w) was adjusted to pH 8.0 with 4 N NaOH prior to sterilization (121 C, 10 min). The medium was cooled to room temperature and butyric acid (0.1%, v/v) and food grade ethanol (0.2%, v/v) were added. Antifoam B (0.3%) was supplemented and the medium was adjusted to pH 6.5 with NaOH. The inoculation was performed by the addition of 1% (v/v) of the standardized cellular
suspension from the third subculture; the suspension was prepared in sterile BHI and showed an optical density of 0.3 at 600 nm using a Beckman DU-650 spectrophotometer (Beckman, San Ramon, CA). The culture medium (400 ml) of whey protein was then incubated at 11 C for 78 h at 150 rpm. 2.5. Measurement of optical density The course of biomass production was monitored with respect to optical density. Samples (0.5 ml) obtained from the culture medium at dierent time intervals, were diluted with 1.0 ml of deionized water and measured at 600 nm against a blank of deionized water; these absorbance values were then multiplied by the dilution factor (Ingraham, Maaloe & Neidhardt, 1983) to obtain the respective optical density. In addition, the biomass production was also investigated with respect to esterase activity as indicated below. 2.6. Cell harvesting The biomass of P. fragi cells was harvested and separated by centrifugation (12,000 xg, 2 h) at 4 C. The cells were then washed twice with potassium phosphate buer solution (0.05 M, pH 7.0) and centrifuged (39,000 xg, 20 min) at 4 C. 2.7. Cell disruption Twenty-®ve grams of wet weight of biomass were suspended in potassium phosphate buer solution (0.1 M, pH 7.0) to produce a 25% (w/v) cell suspension and subjected to the following methods of homogenization. 2.7.1. French press homogenization The 25% cell suspension (w/v) was subjected to high pressure ranging from 5000 to 8000 psi. A sample volume of 40 to 50 ml was extruded through a narrow hole whose size was controlled by a ball or needle valve. The combination of both pressure release and shear at the hole was responsible for disruption of the cellular walls. The pressure cell used was an Aminco French press cell (SLM Instruments, Inc., Urbana, IL) with a Power Laboratory Press (American Instrument Co., Inc., Silver spring, MD). The cell suspension was passed several times (1±6) through the French press and centrifuged (39,000 xg, 20 min) at 4 C. The supernatant and debris were then assayed for esterase activity. 2.7.2. Glass bead homogenization A 40 ml mixture (1:1, v/v) of the 25% (w/v) cell suspension in potassium phosphate buer solution (0.1 M, pH 7.0) and glass beads (0.10±0.11 mm) was prepared in a
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70 ml vessel. Cell disruption was carried out for dierent time intervals at 4 C, by a gentle stream ¯ow of CO2, using a MSK cell homogenizer (Braun, Melsungen, Germany). The cell suspension was then centrifuged (39,000 xg, 20 min) at 4 C and enzyme activity was determined in both supernatant and debris. 2.7.3. Ultrasonication A 25% cell suspension (w/v) was prepared in potassium phosphate buer solution (0.1 M, pH 7.0) and samples (20 ml) were subjected to ultrasonication at a current of 3.5 for dierent time intervals consisting of 15 s of disruption, following by 15 s of rest using the Ultrasonicator (Sonicator-Ultrasonic XL Heat System, Inc., Farmingadle, NY). The disrupted cells were then centrifuged (39,000 xg, 20 min) at 4 C and the supernatant and debris were assayed for esterase activity. 2.7.4. Combined treatment The cellular suspension (25%, w/v) of P. fragi was disrupted using the combination of two methods; the ®rst treatment consisted of ultrasonication, followed by the second which consisted of either glass bead or French press homogenization. The homogenate was then subjected to centrifugation (39,000 xg, 20 min) at 4 C and both the supernatant and debris were tested for esterase activity. 2.8. Chemical treatment of cellular debris The fresh cellular debris, obtained after subjection to dierent physical treatments and centrifugation, was resuspended (25%, w/v) in potassium phosphate buer solution (0.1 M, pH 7.0) and subjected to the following chemical treatments. 2.8.1. Detergent solutions The detergents Triton X-100 and CHAPS, were prepared in potassium phosphate buer solution (0.1 M, pH 7.0). Triton X-100 was prepared at a wide range of concentrations (1, 2 and 3%, v/v) whereas CHAPS was prepared at concentrations ranging from 10 to 50 mM. Moreover, Triton X-100 was used in the presence of ethylenediaminetetraacetic acid (EDTA) at concentrations of 0, 1, 2.5 and 5 mM. 2.8.2. Cellular debris treatment The detergents with or without EDTA were added to the suspension of the fresh cellular debris (25%, w/v) which was placed in an ice-bath for 60 min with periodical shaking every 10 min. The suspension was centrifuged (39,000 xg, 20 min) at 4 C, and detergent and EDTA were removed from the supernatant by ®ltration using a centricon (Centricon-10 concentrator, Amicon). The esterase assay was carried out on the supernatant and debris.
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2.9. Preservation of whole cells and cellular debris The whole cells and cellular debris were assayed for esterase activity after being subjected to dierent storage conditions: The fresh cells and cellular debris were both subjected to storage at (a) 4 C in aqueous medium and (b) ÿ80 C in hexane, while the fresh cells were either frozen in (c) aqueous medium at ÿ80 C, (d) aqueous medium containing 15% glycerol at ÿ80 C, (e) aqueous medium containing 30% glycerol at ÿ80 C, or (f) lyophilized. 2.10. Protein determination The method of Hartree (1972) was used to determine protein concentration, using bovine serum albumin (Sigma Chemical Co.) as standard. 2.11. Esterase assay The enzymatic assay was carried out by incubating a protein extract (0.3 ml) or a 25% (w/v) suspension of fresh whole cells or cellular debris (0.3 ml), in the presence of a fatty acid and ethyl alcohol (valeric acid 1000 ppm and ethanol 2000 ppm) for 24 h at 150 rpm and 11 C. The ®nal volume of the sample was 3 ml after the addition of potassium phosphate buer solution (0.1 M, pH 7.0). At the end of the reaction, 0.5 ml (300 ppm) of internal standard (methyl heptanoate) was added and the products were extracted using 1.5 ml ethyl ether. The sample was centrifuged (5 min, 3000 rpm) and the ether layer was removed and concentrated from 1 to 0.1 ml using a gentle stream of nitrogen and subjected to gas chromatography analyses. The enzymatic assays were all performed in triplicate (5% RSD 4, where RSD is the relative standard deviation). The fatty acid esters were detected by gas±liquid chromatography using a Varian 3400 equipped with a ¯ame ionization detector. A DB-FFAP (J & W Scienti®c, Folsom, CA) capillary column (30 m 0.32 mm i.d., 0.25 mm) was used. The initial column temperature was 40 C for 4.5 min and then increased at rate of 10 C/min until it reached 240 C where it stayed for 0.5 min. The ¯ow rate of the carrier gas (helium) was 1.5 ml/min while those of hydrogen and air were 30 and 300 ml/min, respectively. The injector temperature was initially at 50 C for 0.5 min, and then rose at 100 C/min until it reached 240 C where it stayed for 22.6 min. The detector temperature was at 240 C. 3. Results and discussion 3.1. Homogenization of whole cells using physical treatments Fig. 1 shows the eect of culture incubation time on the esterase activity of the cells of P. fragi. The results
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S. Kermasha et al. / Food Research International 33 (2000) 767±774 Table 1 Protein concentration of the cellular supernatant of P. fragi obtained after dierent physical and chemical treatments Protein concentration (g/l)
Fig. 1. Measurement of optical density (*) and esterase activity in the cells (&) and supernatant (^) during culture incubation of Pseudomonas fragi.
show that esterase activity was ®rst detected in the P. fragi cells after 48 h of incubation and that maximal activity was obtained at 78 h. In addition, the results show that maximal optical density was reached at 83 h corresponding to the onset of the stationary phase of growth. Scopes (1994) reported that the harvesting of bacteria and other unicellular organisms is usually desirable during the log phase before the growth rate begins to decrease. These ®ndings are in agreement with those obtained by Raymond, Morin, Claude and Cormier (1991) who reported that the optimal production of odour-active metabolites such as ethyl isovalerate by P. fragi in whey was observed after 72 h at 11 C and 150 rpm. These results are also in agreement with those of Schuepp, Kermasha, Morin and Michaliski (1997) who reported a similar increase in optical density during the growth of P. fragi in the whey medium used in this study. The results (Fig. 1) also indicate the absence of esterase activity in the culture medium. These ®ndings suggest that P. fragi possesses an endo-cellular esterase as opposed to an exo-cellular enzyme. Reddy, Lindsay and Montgomery (1970) and Hosono et al. (1974) also reported that the esterase activity from P. fragi was endo-cellular. The esterases of gram-negative bacteria have been often described as endo-cellular in dierent Enterobacteriaceae (Goullet, 1978, 1980, 1981; Goullet & Picard, 1984, 1985), phytopathogenic bacteria (ElSharkawy & Huisingh, 1971) and P. fragi (Lawrence, Fryer & Reiter, 1967). Table 1 shows the eect of dierent physical treatments on the release of protein from intact cells of P. fragi. The results show that French press homogenization of the cells released the highest concentration of protein into the supernatant, followed by the combined treatment of french press homogenization and ultrasonication; however, the use of the latter treatment produced a small decrease in protein content thereby suggesting protein denaturation during ultrasonication. Table 1 also shows that the use of the combined treatment of glass bead homogenization and ultrasonication
Homogenization method
Duration (min)
Physical treatment
Physical+chemical treatment
Glass bead (GB) French press (FP) Ultrasonication (US) GB+US FP+US
1±4a 3b 1±5a 2,4a±2,3,4a 3b-2a
5.9±15.4 19.6 0.5±10.6 8.2±17.8 17.9
3.5±6.5 5.0±11.0 2.6±4.0 5.0±10.0 5.0±11.0
a
Number of minutes the cellular suspension (25%, w/v) was subjected to glass bead homogenization (alone), ultrasonication (alone), or a combination of glass bead homogenization and ultrasonication. b Number of times the cellular suspension (25%, w/v) was passed through the French press.
produced slightly lower protein concentrations in the supernatant, followed by treatment with glass bead homogenization alone and ultrasonication alone. The results (not shown) indicated that none of these treatments was successful in the liberation of esterase from the P. fragi cells as indicated by the absence of activity in enzyme assays performed using the supernatant possessing protein concentrations ranging between 0.7 and 5 mg protein per ml. These ®ndings suggest that the protein in the supernatant could be due to the release of esterase from the homogenized cells; however, the absence of esterase activity could be due to denaturation of the enzyme during these processes and/or the loss of a cofactor, a phospholipid membrane or the cooperation of another enzyme required for esterase activity. These ®ndings also suggest that the protein in the supernatant was not esterase as the enzyme could have remained strongly bound to the cellular membranes of P. fragi and was therefore only slightly solubilized by these treatments. The eect of dierent physical treatments (Fig. 2) on the esterase activity of the cellular debris of P. fragi was therefore investigated. The results (Fig. 2) demonstrate that glass bead homogenization of the cells decreased the esterase activity of the cellular debris as a function of time. These ®ndings suggest that the enzyme was denatured by the harshness of this homogenization treatment or was inactivated during the process of extraction. The results (Fig. 2) also show that after the cells were homogenized by subsequent passes using the French press, the esterase activity of the cellular debris slightly decreased. Debette and Prensier (1989) reported that the cells of Xanthomonas maltophilia were disrupted after two passes using the French press with high enzymatic recovery in the crude membrane fraction. In contrast, Fig. 2 shows that treatment of whole cells by ultrasonication for 2.5 min produced a 25% increase in activity. The ®ndings also show that the esterase activity of the cellular debris increased by approximately
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Fig. 2. Biogeneration of ethyl valerate by the esterase activity of the cellular debris of Pseudomonas fragi after treatment of cells using glass bead homogenization (^), French press homogenization (*), ultrasonication (&), glass bead homogenization (2 min) in combination with ultrasonication (~), and glass bead homogenization (4 min) in combination with ultrasonication (!).
50% after treatment of whole cells with glass bead homogenization in combination with ultrasonication. The method of ultrasonic irradiation is extensively used to disintegrate biological cells for the release of intracellular compounds (Neppiras & Hughes, 1964). This method of cell disruption is viewed as the use of sonic energy for cell disintegration on the basis of a physical model of cavitating ultrasonic ®elds in which elastic waves are generated by imploding bubbles (Doulah, 1977). The increase in esterase activity could be due to the elimination of numerous cellular components during the homogenization thereby concentrating the enzyme. These ®ndings indicate that there is a strong esterase activity associated with the cellular debris. In addition, the results show that the enzyme may be strongly attached to the membranes in the cellular debris, thereby being a hydrophobic enzyme (Cadwallader, Braddock & Parish, 1992; Debette & Prensier, 1989; Lawrence et al., 1967; van der Werf, Hartmans & van den Tweel, 1995) which cannot be liberated into the supernatant by any of the above treatments used.
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and retained the suspended proteins; however, the results (not shown) indicated that although the retained protein extracts showed a similar protein content to that obtained in Table 1, esterase activity remained undetected which could be due to irreversible denaturation of the enzyme by the detergent. In contrast, a cell bound esterase from Xanthomonas maltophilia was solubilized using Triton X-100 (Debette & Prensier, 1989). In addition, an a-terpineol dehydratase was extracted from the membranes of Pseudomonas gladioli using a solution of Triton and sodium trichloroacetate (Cadwallader et al., 1992). The eect of dierent physical treatments in combination with chemical treatments on the esterase activity of the cellular debris was therefore studied. Fig. 3 shows the esterase activity of cellular debris subjected to glass bead homogenization (4 min) and various concentrations of CHAPS and Triton. The results demonstrate that the esterase activity was higher in the debris treated with detergents than that obtained with the non-treated debris or whole cells. In addition, the ®ndings show that the optimal esterase activity was obtained using 2% (v/v) Triton. Fig. 3 also shows that the addition of CHAPS decreased esterase activity rapidly as a function of concentration, as shown by the absence of esterase activity at 50 mM CHAPS. These results suggest that the enzyme is denatured by CHAPS. Non-ionic detergents such as Triton X-100 are normally non-denaturing in their action (Bjerrum, 1983) whereas ionic detergents such as CHAPS could partially inactivate or denature the enzyme (Debette & Prensier, 1989). The results (Fig. 4A and B) show that after treatment with 4 min of glass bead homogenization or ultrasonication, respectively, in addition to Triton (2%, v/v) and dierent concentrations of EDTA, the esterase activity in the cellular debris either slightly decreased or remained the same in comparison to that of the untreated cellular debris and whole cells. In contrast, Fig. 5 shows that after subjection to French press
3.2. Chemical treatment of cellular debris using detergents The eect of dierent chemical treatments (Table 1) on the release of protein from the cellular debris, obtained after dierent physical treatments, was studied. The results show that the detergents used with or without EDTA had a real eect on the solubilization of membrane proteins as protein contents in the aqueous extracts ranged from 2.6 to 11 mg per ml; however, there was no detection of esterase activity in these protein extracts. The inhibition of esterase activity by Triton and/or EDTA was also investigated. After treatment of the cellular debris with Triton and EDTA, the supernatant containing the protein was passed through a ®lter possessing a cut-o point of 10,000 which allowed the passage of Triton and EDTA
Fig. 3. Esterase activity of the untreated cells and cellular debris obtained after glass bead homogenization (4 min) (&) and treatment with dierent concentrations of Triton (&) and CHAPS ( ).
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homogenization (three passes) in combination with different concentrations of Triton and EDTA, the esterase activity of the treated cellular debris decreased by 30 to 50% with respect to that of the non-treated one. Fig. 6A shows that there was a decrease of 35% in esterase activity after the cellular debris was treated with a combination of glass bead homogenization (4 min) and ultrasonication (4 min) and the addition of 2% (v/ v) Triton and dierent concentrations of EDTA. However, the results (Fig. 6B) also show that the treatment
Fig. 4. (A) Esterase activity of the untreated cells and cellular debris obtained after glass bead homogenization (4 min) (&) and treatment with 2% Triton and dierent concentrations of EDTA (&). (B) Esterase activity of the untreated cells and cellular debris obtained after ultrasonication (4 min) (&) and treatment with 2% Triton and dierent concentrations of EDTA (&).
Fig. 5. Esterase activity of the cellular debris obtained after French press homogenization (three passes) (&) and treatment with dierent concentrations of EDTA and Triton at 1% ( ), 2% ( ), and 3% (&).
of cellular debris with a combination of French press (three passes), ultrasonication (4 min) and the addition of 2% (v/v) Triton and dierent concentrations of EDTA produced little change in the esterase activity. These ®ndings suggest that the chemical treatments can be used to concentrate the enzyme in the cellular debris, as indicated by the increase in activity obtained by treatment of the debris with glass bead homogenization and 2% Triton; however depending on the detergent used, the use of chemical treatments may also partially denature the enzyme as shown by the decrease in esterase activity after treatment with glass bead homogenization and CHAPS.
Fig. 6. (A) Esterase activity of the untreated cells and cellular debris obtained after glass bead homogenization (4 min), ultrasonication (4 min) (&) and treatment with 2% Triton and dierent concentrations of EDTA (&); (B) esterase activity of the untreated cells and cellular debris obtained after French press homogenization (three passes), ultrasonication (4 min) (&) and treatment with 2% Triton and dierent concentrations of EDTA (&).
Fig. 7. Esterase activity of cellular debris after storage in potassium phosphate buer solution (0.1 M, pH 7.0) at 4 C (&) and in hexane at ÿ80 C (*).
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3.3. Eect of storage on the esterase activity of P. fragi
4. Conclusions
Fig. 7 shows that the esterase activity of the cellular debris, during storage in aqueous medium (4 C) and hexane (ÿ80 C), decreased gradually with time. The results also show that the enzymatic activity decreased by 50 and 80% after 1 and 3 days of storage, respectively. Fig. 8 indicates that cells stored for 3 days in hexane (ÿ80 C) retained 70 to 80% of the residual activity while those stored in potassium phosphate buer solution (pH 7.0, 0.1 M) at 4 and ÿ80 C retained only 60 to 70%; however, the results also indicate that the dierence in activity is not signi®cant as shown by the 80% decrease in the esterase activity after 8 days of storage. These ®ndings show that there is little dierence in activity retention between the organic solvent medium and the aqueous media at 4 and ÿ80 C when used as a storage environment for the long-term preservation of the whole cells; however, these media can be eectively used for temporary storage of the cells up to 3 days. Fig. 8 also indicates that lyophilization of the cells greatly aects esterase activity as indicated by the 50 and 75% decrease after 1 and 3 days of storage, respectively. Meanwhile, Fig. 8 shows that the presence of glycerol in the potassium phosphate buer solution (pH 7.0, 0.1 M) can be used to stabilize esterase activity for 2 to 3 weeks. The results demonstrate that the residual relative activity was greater than 55 and 60% after 3 weeks storage in the potassium phosphate buer solution (pH 7.0, 0.1 M) containing 15 and 30% glycerol, respectively. The decrease in enzymatic activity with respect to storage time could be due to the degradation of the enzyme by proteases in the aqueous medium (4 C), the denaturation of the protein in the aqueous medium or hexane (ÿ80 C) due to the low temperature, or the denaturation of the enzyme due to its contact with the non-polar solvent hexane (Scopes, 1994).
Although the physical and chemical treatments used throughout this study were not able to completely dissociate the esterase from the cellular membranes of P. fragi, the enzyme maintained its activity in the cellular debris. Among the dierent treatments, ultrasonication used alone or in combination with other treatments resulted in a relatively good recovery of enzyme activity in the cellular debris. From a practical viewpoint, the use of ultrasonication was also an appropriate method for cell disruption.
Fig. 8. Esterase activity of whole cells after storage in potassium phosphate buer solution (0.1 M, pH 7.0) at 4 C (~), at ÿ80 C (*), containing 15% (&) and 30% (*) glycerol; in hexane at ÿ80 C (X), and lyophilized (^).
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Goullet, P., & Picard, B. (1985). A two dimensional electrophoretic pro®le for bacterial esterases. Electrophoresis, 6, 132±135. Hartree, E. P. (1972). Determination of protein: a modi®cation of Lowry method that gives a linear photometric response. Analytical Biochemistry, 48, 422±427. Hosono, A., Eilliot, J. A., & McGugan, W. A. (1974). Production of ethylesters by some lactic acid and psychrotrophic bacteria. Journal of Dairy Science, 57, 535±539. Hussong, R. V., Long, H. F., & Hammer, B. W. (1937). Classi®cation of organisms important in dairy products. II. Pseudomonas fragi research bulletin. Iowa Agriculture Experimental Station, 225, 117±136. Ingraham, J. L., Maaloe, O., & Neidhardt, F. C. (1983). Growth of cells and cultures. In Growth and bacterial cell (pp. 227±265). Maine: Sinauer Associates Inc. Kermasha, S., Bisakowski, B., Morin, A., & Ismail, S. (1999). Biogeneration of short-chain fatty acid alcohol esters by Pseudomonas fragi CRDA 037. Biocatalysis and Biotransformation, 17, 269±282. Lamer, S., Leblanc, D., Morin, A., & Kermasha, S. (1996). Biogeneration of ethyl valerate by whole cells of Pseudomonas fragi CRDA 037 in aqueous medium. Biotechnology Techniques, 10, 475±478. Lawrence, R. C., Fryer, T. F., & Reiter, B. (1967). The production and characterization of lipases from Micrococcus and a pseudomonad. Journal of General Microbiology, 48, 401±418. Morgan, M. E. (1970). Microbial ¯avor defects in dairy products and methods for their stimulation II. Fruity ¯avor. Journal of Dairy Science, 53, 273±275. Neppiras, E. A., & Hughes, D. E. (1964). Some experiments on the disintegration of yeast by high intensity ultrasound. Biotechnology and Bioengineering, 6, 247±270.
Pereira, J. N., & Morgan, M. E. (1958). Identity of esters produced in milk cultures of Pseudomonas fragi. Journal of Dairy Science, 42, 1201±1205. Raymond, Y., Morin, A., Claude, P., & Cormier, F. (1991). Enhancement of fruity aroma production of Pseudomonas fragi grown on skim milk, whey and whey permeate supplemented with C3-C7 fatty acids. Applied Microbiology and Biotechnology, 34, 524± 527. Raymond, Y., Morin, A., Cormier, F., Champagne, C. P., & Dubeau, H. (1990). Physical factors in¯uencing the production of strawberry aroma by Pseudomonas fragi grown on skim milk. Biotechnology Letters, 12, 931±936. Reddy, M. C., Bills, D. D., Lindsay, R. C., Libbey, L. M., Miller, A., & Morgan, M. E. (1968). Ester production by Pseudomonas fragi. I. Identi®cation and quanti®cation of some esters produced in milk cultures. Journal of Dairy Science, 51, 656±659. Reddy, M. C., Lindsay, R. C., & Montgomery, M. W. (1970). Ester production by Pseudomonas fragi. V. Demonstration of esterase activity. Applied Microbiology, 20, 555±557. Schuepp, C., Kermasha, S., Morin, A., & Michaliski, M.-C. (1997). Partial puri®cation and characterization of lipases from Pseudomonas fragi CRDA 037. Process Biochemistry, 32, 225±232. Scopes, R. K. (1994). In Protein puri®cation principle and practice (pp. 22±43). New York: Springer-Verlag van der Werf, M. J., Hartmans, S., & van den Tweel, W. J. J. (1995). Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by Triton X-100 for ecient production of d-malate. Applied Microbiology and Biotechnology, 43, 590±594.
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The eect of physical and chemical treatments on the esterase activity from Pseudomonas fragi CRDA 037 Selim Kermasha a,*, Barbara Bisakowski b, Safwan Ismail a, Andre Morin b a
Department of Food Science and Agricultural Chemistry, McGill University, 21,111 Lakeshore, Ste Anne de Bellevue, PQ, Canada H9X 3V9 b Agriculture and Agri-Food Canada, 3 600, Blvd. Casavant West, St-Hyacinth, PQ, Canada J2S 8E3 Received 14 January 2000; accepted 2 March 2000
Abstract The eect of dierent treatments on the esterase activity of the cellular debris of Pseudomonas fragi, responsible for the production of natural fruity ¯avors in processed products, was investigated. Glass bead homogenization (GBH) decreased activity by 50%, French press homogenization (FPH) produced only a slight decrease, while ultrasonication (US) alone or with GBH increased activity by 25 and 50%, respectively. Treatment with (i) GBH (4 min) and Triton increased activity two-fold, (ii) GBH or US (4 min), Triton and EDTA produced little eect, (iii) FPH (three passes), Triton and EDTA produced a decrease of 30 to 50%, (iv) FP (three passes), US (4 min), Triton and EDTA had no eect, while (v) GBH (4 min), US (4 min), Triton and EDTA showed a 35% decrease in activity. The esterase activity of the cellular debris stored in buer (4 C) and hexane (ÿ80 C) decreased by 80% after 3 days, while that of whole cells stored in hexane (ÿ80 C), and buer at 4 and ÿ80 C remained preserved for 3 days whereas that of the lyophilized whole cells greatly decreased. The esterase activity of whole cells stored in buer containing 15 and 30% glycerol was preserved for three weeks. Crown Copyright # 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Flavors; Fatty acids; Alcohol esters; Esterase; Pseudomonas
1. Introduction Pseudomonas fragi is an aerobic, gram negative, nonpathogenic, short and rod-shaped bacterium (Bassette, Fung & Mantha, 1986; Fairbairn & Low, 1986) that was isolated from milk by Hussong, Long and Hammer (1937). In processed dairy products, the development of fruity ¯avors has been reported to be due to P. fragi responsible for the hydrolysis of milk triglycerides and the esteri®cation of certain low molecular weight fatty acids with ethanol. When grown in skim milk, the P. fragi strain CRDA-037 produced a mixture of 26 dierent odor-active compounds of which 13 were identi®ed as fatty acid ethyl esters (Cormier, Raymond, Champagne & Morin, 1991). The production of a fruity strawberry-like odor by P. fragi is mainly due to the presence of the ethyl butyrate * Corresponding author. Tel.: +1-514-398-7922; fax: +1-514-3988132. E-mail address: [email protected] (S. Kermasha).
and ethyl hexanoate (Morgan 1970; Reddy, Bills, Lindsay, Libbey, Miller & Morgan, 1968) and 3-methylbutanoic acids (Pereira & Morgan, 1958). Raymond, Morin, Cormier, Champagne and Dubeau (1990) reported that dierent concentrations of ethyl butyrate and ethyl hexanoate produced by the cells of P. fragi was related to the activity of lipases and esterases known to be involved in the synthesis of fruity aroma (Hosono, Eilliot & McGugan, 1974; Pereira & Morgan). Although esterase activity was demonstrated in the intact cells of P. fragi, little information is available on the characterization of this activity in vitro. This work is part of ongoing research aimed at the development of a biotechnological approach for the production of natural ¯avors by microbial enzymes (Lamer, Leblanc, Morin & Kermasha, 1996; Kermasha, Bisakowski, Morin & Ismail, 1999). The speci®c objective of this research was to investigate the eect of dierent physical and chemical treatments on the esterase activity of P. fragi. In addition, the esterase activity of P. fragi was investigated with respect to dierent storage conditions.
0963-9969/00/$ - see front matter Crown Copyright # 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S0963-9969(00)00080-6
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2. Materials and methods 2.1. Bacterial strain The gram negative bacteria of P. fragi CRDA 037 was obtained from the Agriculture and Agri-Food Research and Development Center (CRDA, St Hyacinthe, PQ). 2.2. Chemicals Whey powder used for the incubation medium was obtained from Saputo (St Hyacinthe, PQ). Brain Heart Infusion (BHI) and Bacto-Agar were obtained from Difco Laboratories (Detroit, MI). Butyric and valeric acids, as well as phosphate and ethylenediaminetetraacetic acid (EDTA) were purchased from ACP Chemical Inc. (St-LeÂonard, PQ). CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane- sulfonate) was obtained from ICN Biomedicals Inc. (Aurora, OH) while Triton X-100 was from Aldrich Chemical Co. (Milwaukee, WI). Bovine serum albumin (BSA) and potassium phosphate dibasic, anhydrous were obtained from Sigma Chemical Co. (St-Louis, MO). Sodium hydroxide was provided by Fisher Scienti®c (Fair Lawn, NJ) and ethanol (94%) was purchased from the SocieÂte des Alcools du QueÂbec (MontreÂal, PQ). Methyl hexanoate was received from Poly Science Co. (Niles, IL) while potassium dihydrogen orthophosphate and antifoam ``B'' were obtained from BDH Inc. (Toronto, ON). 2.3. Organism and inoculum preparation The reference strain P. fragi CRDA 037 was transferred monthly and kept at 4 C on BHI agar slants covered with sterile mineral oil. The inoculation of P. fragi was carried out according to the procedure described previously by Raymond et al. (1990). Sterilized BHI broth (25 ml) was dispensed into 125 ml Erlenmeyer ¯asks. Three successive subcultures of the bacteria were incubated in BHI broth at 30 C with shaking at 200 rpm for 17 to 24 h in an Environ-Shaker (Lab-line Instruments, Inc., Chicago, IL). A 0.01% (v/v) inoculum was used to initiate each subculture. 2.4. Culture incubation Biomass production of P. fragi was carried out in Erlenmeyer ¯asks of 2 l. The whey medium (6%, w/w) was adjusted to pH 8.0 with 4 N NaOH prior to sterilization (121 C, 10 min). The medium was cooled to room temperature and butyric acid (0.1%, v/v) and food grade ethanol (0.2%, v/v) were added. Antifoam B (0.3%) was supplemented and the medium was adjusted to pH 6.5 with NaOH. The inoculation was performed by the addition of 1% (v/v) of the standardized cellular
suspension from the third subculture; the suspension was prepared in sterile BHI and showed an optical density of 0.3 at 600 nm using a Beckman DU-650 spectrophotometer (Beckman, San Ramon, CA). The culture medium (400 ml) of whey protein was then incubated at 11 C for 78 h at 150 rpm. 2.5. Measurement of optical density The course of biomass production was monitored with respect to optical density. Samples (0.5 ml) obtained from the culture medium at dierent time intervals, were diluted with 1.0 ml of deionized water and measured at 600 nm against a blank of deionized water; these absorbance values were then multiplied by the dilution factor (Ingraham, Maaloe & Neidhardt, 1983) to obtain the respective optical density. In addition, the biomass production was also investigated with respect to esterase activity as indicated below. 2.6. Cell harvesting The biomass of P. fragi cells was harvested and separated by centrifugation (12,000 xg, 2 h) at 4 C. The cells were then washed twice with potassium phosphate buer solution (0.05 M, pH 7.0) and centrifuged (39,000 xg, 20 min) at 4 C. 2.7. Cell disruption Twenty-®ve grams of wet weight of biomass were suspended in potassium phosphate buer solution (0.1 M, pH 7.0) to produce a 25% (w/v) cell suspension and subjected to the following methods of homogenization. 2.7.1. French press homogenization The 25% cell suspension (w/v) was subjected to high pressure ranging from 5000 to 8000 psi. A sample volume of 40 to 50 ml was extruded through a narrow hole whose size was controlled by a ball or needle valve. The combination of both pressure release and shear at the hole was responsible for disruption of the cellular walls. The pressure cell used was an Aminco French press cell (SLM Instruments, Inc., Urbana, IL) with a Power Laboratory Press (American Instrument Co., Inc., Silver spring, MD). The cell suspension was passed several times (1±6) through the French press and centrifuged (39,000 xg, 20 min) at 4 C. The supernatant and debris were then assayed for esterase activity. 2.7.2. Glass bead homogenization A 40 ml mixture (1:1, v/v) of the 25% (w/v) cell suspension in potassium phosphate buer solution (0.1 M, pH 7.0) and glass beads (0.10±0.11 mm) was prepared in a
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70 ml vessel. Cell disruption was carried out for dierent time intervals at 4 C, by a gentle stream ¯ow of CO2, using a MSK cell homogenizer (Braun, Melsungen, Germany). The cell suspension was then centrifuged (39,000 xg, 20 min) at 4 C and enzyme activity was determined in both supernatant and debris. 2.7.3. Ultrasonication A 25% cell suspension (w/v) was prepared in potassium phosphate buer solution (0.1 M, pH 7.0) and samples (20 ml) were subjected to ultrasonication at a current of 3.5 for dierent time intervals consisting of 15 s of disruption, following by 15 s of rest using the Ultrasonicator (Sonicator-Ultrasonic XL Heat System, Inc., Farmingadle, NY). The disrupted cells were then centrifuged (39,000 xg, 20 min) at 4 C and the supernatant and debris were assayed for esterase activity. 2.7.4. Combined treatment The cellular suspension (25%, w/v) of P. fragi was disrupted using the combination of two methods; the ®rst treatment consisted of ultrasonication, followed by the second which consisted of either glass bead or French press homogenization. The homogenate was then subjected to centrifugation (39,000 xg, 20 min) at 4 C and both the supernatant and debris were tested for esterase activity. 2.8. Chemical treatment of cellular debris The fresh cellular debris, obtained after subjection to dierent physical treatments and centrifugation, was resuspended (25%, w/v) in potassium phosphate buer solution (0.1 M, pH 7.0) and subjected to the following chemical treatments. 2.8.1. Detergent solutions The detergents Triton X-100 and CHAPS, were prepared in potassium phosphate buer solution (0.1 M, pH 7.0). Triton X-100 was prepared at a wide range of concentrations (1, 2 and 3%, v/v) whereas CHAPS was prepared at concentrations ranging from 10 to 50 mM. Moreover, Triton X-100 was used in the presence of ethylenediaminetetraacetic acid (EDTA) at concentrations of 0, 1, 2.5 and 5 mM. 2.8.2. Cellular debris treatment The detergents with or without EDTA were added to the suspension of the fresh cellular debris (25%, w/v) which was placed in an ice-bath for 60 min with periodical shaking every 10 min. The suspension was centrifuged (39,000 xg, 20 min) at 4 C, and detergent and EDTA were removed from the supernatant by ®ltration using a centricon (Centricon-10 concentrator, Amicon). The esterase assay was carried out on the supernatant and debris.
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2.9. Preservation of whole cells and cellular debris The whole cells and cellular debris were assayed for esterase activity after being subjected to dierent storage conditions: The fresh cells and cellular debris were both subjected to storage at (a) 4 C in aqueous medium and (b) ÿ80 C in hexane, while the fresh cells were either frozen in (c) aqueous medium at ÿ80 C, (d) aqueous medium containing 15% glycerol at ÿ80 C, (e) aqueous medium containing 30% glycerol at ÿ80 C, or (f) lyophilized. 2.10. Protein determination The method of Hartree (1972) was used to determine protein concentration, using bovine serum albumin (Sigma Chemical Co.) as standard. 2.11. Esterase assay The enzymatic assay was carried out by incubating a protein extract (0.3 ml) or a 25% (w/v) suspension of fresh whole cells or cellular debris (0.3 ml), in the presence of a fatty acid and ethyl alcohol (valeric acid 1000 ppm and ethanol 2000 ppm) for 24 h at 150 rpm and 11 C. The ®nal volume of the sample was 3 ml after the addition of potassium phosphate buer solution (0.1 M, pH 7.0). At the end of the reaction, 0.5 ml (300 ppm) of internal standard (methyl heptanoate) was added and the products were extracted using 1.5 ml ethyl ether. The sample was centrifuged (5 min, 3000 rpm) and the ether layer was removed and concentrated from 1 to 0.1 ml using a gentle stream of nitrogen and subjected to gas chromatography analyses. The enzymatic assays were all performed in triplicate (5% RSD 4, where RSD is the relative standard deviation). The fatty acid esters were detected by gas±liquid chromatography using a Varian 3400 equipped with a ¯ame ionization detector. A DB-FFAP (J & W Scienti®c, Folsom, CA) capillary column (30 m 0.32 mm i.d., 0.25 mm) was used. The initial column temperature was 40 C for 4.5 min and then increased at rate of 10 C/min until it reached 240 C where it stayed for 0.5 min. The ¯ow rate of the carrier gas (helium) was 1.5 ml/min while those of hydrogen and air were 30 and 300 ml/min, respectively. The injector temperature was initially at 50 C for 0.5 min, and then rose at 100 C/min until it reached 240 C where it stayed for 22.6 min. The detector temperature was at 240 C. 3. Results and discussion 3.1. Homogenization of whole cells using physical treatments Fig. 1 shows the eect of culture incubation time on the esterase activity of the cells of P. fragi. The results
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S. Kermasha et al. / Food Research International 33 (2000) 767±774 Table 1 Protein concentration of the cellular supernatant of P. fragi obtained after dierent physical and chemical treatments Protein concentration (g/l)
Fig. 1. Measurement of optical density (*) and esterase activity in the cells (&) and supernatant (^) during culture incubation of Pseudomonas fragi.
show that esterase activity was ®rst detected in the P. fragi cells after 48 h of incubation and that maximal activity was obtained at 78 h. In addition, the results show that maximal optical density was reached at 83 h corresponding to the onset of the stationary phase of growth. Scopes (1994) reported that the harvesting of bacteria and other unicellular organisms is usually desirable during the log phase before the growth rate begins to decrease. These ®ndings are in agreement with those obtained by Raymond, Morin, Claude and Cormier (1991) who reported that the optimal production of odour-active metabolites such as ethyl isovalerate by P. fragi in whey was observed after 72 h at 11 C and 150 rpm. These results are also in agreement with those of Schuepp, Kermasha, Morin and Michaliski (1997) who reported a similar increase in optical density during the growth of P. fragi in the whey medium used in this study. The results (Fig. 1) also indicate the absence of esterase activity in the culture medium. These ®ndings suggest that P. fragi possesses an endo-cellular esterase as opposed to an exo-cellular enzyme. Reddy, Lindsay and Montgomery (1970) and Hosono et al. (1974) also reported that the esterase activity from P. fragi was endo-cellular. The esterases of gram-negative bacteria have been often described as endo-cellular in dierent Enterobacteriaceae (Goullet, 1978, 1980, 1981; Goullet & Picard, 1984, 1985), phytopathogenic bacteria (ElSharkawy & Huisingh, 1971) and P. fragi (Lawrence, Fryer & Reiter, 1967). Table 1 shows the eect of dierent physical treatments on the release of protein from intact cells of P. fragi. The results show that French press homogenization of the cells released the highest concentration of protein into the supernatant, followed by the combined treatment of french press homogenization and ultrasonication; however, the use of the latter treatment produced a small decrease in protein content thereby suggesting protein denaturation during ultrasonication. Table 1 also shows that the use of the combined treatment of glass bead homogenization and ultrasonication
Homogenization method
Duration (min)
Physical treatment
Physical+chemical treatment
Glass bead (GB) French press (FP) Ultrasonication (US) GB+US FP+US
1±4a 3b 1±5a 2,4a±2,3,4a 3b-2a
5.9±15.4 19.6 0.5±10.6 8.2±17.8 17.9
3.5±6.5 5.0±11.0 2.6±4.0 5.0±10.0 5.0±11.0
a
Number of minutes the cellular suspension (25%, w/v) was subjected to glass bead homogenization (alone), ultrasonication (alone), or a combination of glass bead homogenization and ultrasonication. b Number of times the cellular suspension (25%, w/v) was passed through the French press.
produced slightly lower protein concentrations in the supernatant, followed by treatment with glass bead homogenization alone and ultrasonication alone. The results (not shown) indicated that none of these treatments was successful in the liberation of esterase from the P. fragi cells as indicated by the absence of activity in enzyme assays performed using the supernatant possessing protein concentrations ranging between 0.7 and 5 mg protein per ml. These ®ndings suggest that the protein in the supernatant could be due to the release of esterase from the homogenized cells; however, the absence of esterase activity could be due to denaturation of the enzyme during these processes and/or the loss of a cofactor, a phospholipid membrane or the cooperation of another enzyme required for esterase activity. These ®ndings also suggest that the protein in the supernatant was not esterase as the enzyme could have remained strongly bound to the cellular membranes of P. fragi and was therefore only slightly solubilized by these treatments. The eect of dierent physical treatments (Fig. 2) on the esterase activity of the cellular debris of P. fragi was therefore investigated. The results (Fig. 2) demonstrate that glass bead homogenization of the cells decreased the esterase activity of the cellular debris as a function of time. These ®ndings suggest that the enzyme was denatured by the harshness of this homogenization treatment or was inactivated during the process of extraction. The results (Fig. 2) also show that after the cells were homogenized by subsequent passes using the French press, the esterase activity of the cellular debris slightly decreased. Debette and Prensier (1989) reported that the cells of Xanthomonas maltophilia were disrupted after two passes using the French press with high enzymatic recovery in the crude membrane fraction. In contrast, Fig. 2 shows that treatment of whole cells by ultrasonication for 2.5 min produced a 25% increase in activity. The ®ndings also show that the esterase activity of the cellular debris increased by approximately
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Fig. 2. Biogeneration of ethyl valerate by the esterase activity of the cellular debris of Pseudomonas fragi after treatment of cells using glass bead homogenization (^), French press homogenization (*), ultrasonication (&), glass bead homogenization (2 min) in combination with ultrasonication (~), and glass bead homogenization (4 min) in combination with ultrasonication (!).
50% after treatment of whole cells with glass bead homogenization in combination with ultrasonication. The method of ultrasonic irradiation is extensively used to disintegrate biological cells for the release of intracellular compounds (Neppiras & Hughes, 1964). This method of cell disruption is viewed as the use of sonic energy for cell disintegration on the basis of a physical model of cavitating ultrasonic ®elds in which elastic waves are generated by imploding bubbles (Doulah, 1977). The increase in esterase activity could be due to the elimination of numerous cellular components during the homogenization thereby concentrating the enzyme. These ®ndings indicate that there is a strong esterase activity associated with the cellular debris. In addition, the results show that the enzyme may be strongly attached to the membranes in the cellular debris, thereby being a hydrophobic enzyme (Cadwallader, Braddock & Parish, 1992; Debette & Prensier, 1989; Lawrence et al., 1967; van der Werf, Hartmans & van den Tweel, 1995) which cannot be liberated into the supernatant by any of the above treatments used.
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and retained the suspended proteins; however, the results (not shown) indicated that although the retained protein extracts showed a similar protein content to that obtained in Table 1, esterase activity remained undetected which could be due to irreversible denaturation of the enzyme by the detergent. In contrast, a cell bound esterase from Xanthomonas maltophilia was solubilized using Triton X-100 (Debette & Prensier, 1989). In addition, an a-terpineol dehydratase was extracted from the membranes of Pseudomonas gladioli using a solution of Triton and sodium trichloroacetate (Cadwallader et al., 1992). The eect of dierent physical treatments in combination with chemical treatments on the esterase activity of the cellular debris was therefore studied. Fig. 3 shows the esterase activity of cellular debris subjected to glass bead homogenization (4 min) and various concentrations of CHAPS and Triton. The results demonstrate that the esterase activity was higher in the debris treated with detergents than that obtained with the non-treated debris or whole cells. In addition, the ®ndings show that the optimal esterase activity was obtained using 2% (v/v) Triton. Fig. 3 also shows that the addition of CHAPS decreased esterase activity rapidly as a function of concentration, as shown by the absence of esterase activity at 50 mM CHAPS. These results suggest that the enzyme is denatured by CHAPS. Non-ionic detergents such as Triton X-100 are normally non-denaturing in their action (Bjerrum, 1983) whereas ionic detergents such as CHAPS could partially inactivate or denature the enzyme (Debette & Prensier, 1989). The results (Fig. 4A and B) show that after treatment with 4 min of glass bead homogenization or ultrasonication, respectively, in addition to Triton (2%, v/v) and dierent concentrations of EDTA, the esterase activity in the cellular debris either slightly decreased or remained the same in comparison to that of the untreated cellular debris and whole cells. In contrast, Fig. 5 shows that after subjection to French press
3.2. Chemical treatment of cellular debris using detergents The eect of dierent chemical treatments (Table 1) on the release of protein from the cellular debris, obtained after dierent physical treatments, was studied. The results show that the detergents used with or without EDTA had a real eect on the solubilization of membrane proteins as protein contents in the aqueous extracts ranged from 2.6 to 11 mg per ml; however, there was no detection of esterase activity in these protein extracts. The inhibition of esterase activity by Triton and/or EDTA was also investigated. After treatment of the cellular debris with Triton and EDTA, the supernatant containing the protein was passed through a ®lter possessing a cut-o point of 10,000 which allowed the passage of Triton and EDTA
Fig. 3. Esterase activity of the untreated cells and cellular debris obtained after glass bead homogenization (4 min) (&) and treatment with dierent concentrations of Triton (&) and CHAPS ( ).
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homogenization (three passes) in combination with different concentrations of Triton and EDTA, the esterase activity of the treated cellular debris decreased by 30 to 50% with respect to that of the non-treated one. Fig. 6A shows that there was a decrease of 35% in esterase activity after the cellular debris was treated with a combination of glass bead homogenization (4 min) and ultrasonication (4 min) and the addition of 2% (v/ v) Triton and dierent concentrations of EDTA. However, the results (Fig. 6B) also show that the treatment
Fig. 4. (A) Esterase activity of the untreated cells and cellular debris obtained after glass bead homogenization (4 min) (&) and treatment with 2% Triton and dierent concentrations of EDTA (&). (B) Esterase activity of the untreated cells and cellular debris obtained after ultrasonication (4 min) (&) and treatment with 2% Triton and dierent concentrations of EDTA (&).
Fig. 5. Esterase activity of the cellular debris obtained after French press homogenization (three passes) (&) and treatment with dierent concentrations of EDTA and Triton at 1% ( ), 2% ( ), and 3% (&).
of cellular debris with a combination of French press (three passes), ultrasonication (4 min) and the addition of 2% (v/v) Triton and dierent concentrations of EDTA produced little change in the esterase activity. These ®ndings suggest that the chemical treatments can be used to concentrate the enzyme in the cellular debris, as indicated by the increase in activity obtained by treatment of the debris with glass bead homogenization and 2% Triton; however depending on the detergent used, the use of chemical treatments may also partially denature the enzyme as shown by the decrease in esterase activity after treatment with glass bead homogenization and CHAPS.
Fig. 6. (A) Esterase activity of the untreated cells and cellular debris obtained after glass bead homogenization (4 min), ultrasonication (4 min) (&) and treatment with 2% Triton and dierent concentrations of EDTA (&); (B) esterase activity of the untreated cells and cellular debris obtained after French press homogenization (three passes), ultrasonication (4 min) (&) and treatment with 2% Triton and dierent concentrations of EDTA (&).
Fig. 7. Esterase activity of cellular debris after storage in potassium phosphate buer solution (0.1 M, pH 7.0) at 4 C (&) and in hexane at ÿ80 C (*).
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3.3. Eect of storage on the esterase activity of P. fragi
4. Conclusions
Fig. 7 shows that the esterase activity of the cellular debris, during storage in aqueous medium (4 C) and hexane (ÿ80 C), decreased gradually with time. The results also show that the enzymatic activity decreased by 50 and 80% after 1 and 3 days of storage, respectively. Fig. 8 indicates that cells stored for 3 days in hexane (ÿ80 C) retained 70 to 80% of the residual activity while those stored in potassium phosphate buer solution (pH 7.0, 0.1 M) at 4 and ÿ80 C retained only 60 to 70%; however, the results also indicate that the dierence in activity is not signi®cant as shown by the 80% decrease in the esterase activity after 8 days of storage. These ®ndings show that there is little dierence in activity retention between the organic solvent medium and the aqueous media at 4 and ÿ80 C when used as a storage environment for the long-term preservation of the whole cells; however, these media can be eectively used for temporary storage of the cells up to 3 days. Fig. 8 also indicates that lyophilization of the cells greatly aects esterase activity as indicated by the 50 and 75% decrease after 1 and 3 days of storage, respectively. Meanwhile, Fig. 8 shows that the presence of glycerol in the potassium phosphate buer solution (pH 7.0, 0.1 M) can be used to stabilize esterase activity for 2 to 3 weeks. The results demonstrate that the residual relative activity was greater than 55 and 60% after 3 weeks storage in the potassium phosphate buer solution (pH 7.0, 0.1 M) containing 15 and 30% glycerol, respectively. The decrease in enzymatic activity with respect to storage time could be due to the degradation of the enzyme by proteases in the aqueous medium (4 C), the denaturation of the protein in the aqueous medium or hexane (ÿ80 C) due to the low temperature, or the denaturation of the enzyme due to its contact with the non-polar solvent hexane (Scopes, 1994).
Although the physical and chemical treatments used throughout this study were not able to completely dissociate the esterase from the cellular membranes of P. fragi, the enzyme maintained its activity in the cellular debris. Among the dierent treatments, ultrasonication used alone or in combination with other treatments resulted in a relatively good recovery of enzyme activity in the cellular debris. From a practical viewpoint, the use of ultrasonication was also an appropriate method for cell disruption.
Fig. 8. Esterase activity of whole cells after storage in potassium phosphate buer solution (0.1 M, pH 7.0) at 4 C (~), at ÿ80 C (*), containing 15% (&) and 30% (*) glycerol; in hexane at ÿ80 C (X), and lyophilized (^).
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