Department of Microbiology, Panjab University, Chandigarh ± 160014, India. *Author for correspondence: Tel.: +91-172-541 770, Fax: +91-172-541 409.
World Journal of Microbiology & Biotechnology 16: 277±282, 2000.
Ó 2000 Kluwer Academic Publishers. Printed in the Netherlands.
277
Production, puri®cation and characterization of pectinase from a Bacillus sp. DT7 D.R. Kashyap, S. Chandra, A. Kaul and R. Tewari* Department of Microbiology, Panjab University, Chandigarh ± 160014, India *Author for correspondence: Tel.: +91-172-541 770, Fax: +91-172-541 409 Received 27 September 1999; accepted 25 February 2000
Keywords: Bacillus, pectinase, production, puri®cation
Summary A soil isolate, Bacillus sp. DT7 has been found to produce signi®cant amounts of an extracellular pectinase subsequently characterized as pectin lyase (EC 4.2.2.10). By optimizing growth conditions, Bacillus sp. DT7 produced higher amount of pectin lyase (53 units/ml) than that has been reported in the literature. Using gel ®ltration and ion exchange chromatography, this enzyme was puri®ed and found to have a molecular mass of 106 kDa. The puri®ed enzyme exhibited maximal activity at a temperature of 60 C and pH 8.0. The presence of 100 mM concentrations of CaCl2 and mercaptoethanol signi®cantly enhanced pectinase activity of the puri®ed enzyme. This pectinase has tremendous applications in textile industry, plant tissue maceration and fruit juice wastewater treatments.
Introduction Pectinases constitute a unique group of enzymes which catalyze the degradation of pectic polymers present in the plant cell walls (Fogarty & Kelly 1982). Pectinases are produced by many organisms such as bacteria (Horikoshi 1972; Karbassi & Vaughn 1980), fungi (Aguilar & Huitron 1990) and yeasts (Gainvors & Belarbi 1993). In the industrial sector, acidic pectinases are used in the extraction and clari®cation of fruit juices (Rombouts & Pilnik 1986), whereas alkalophilic pectinases are ®nding immense use in the degumming of ramie ®bers (Cao et al. 1992), retting of ¯ax (Sharma 1987), plant protoplast formation and treatment of euents discharged from fruit processing units (Tanabe et al. 1987). Although the major source of acidic pectinases are fungi, alkaline pectinases are produced from alkalophilic bacteria, mainly Bacillus spp. In this study the investigators have isolated a unique mesophilic Bacillus that produces an alkalophilic and thermotolerant pectinase. In addition, the amount of pectinase produced is higher than that reported in the literature. The enzyme has been puri®ed to homogeneity and has a potential to be used at commercial level. Materials and Methods Materials All chemicals used were of analytical grade. Dinitrosalicylic acid (DNSA), pectin and polygalacturonic acid
(PGA) were procured from Sigma, yeast extract (YE), Luria broth (LB), nutrient broth (NB), brain heart infusion (BHI) and malt extract were supplied by Hi Media Lab., Bombay, India; Biostate peptone and tryptone were supplied by Becton Dickinson Microbiology Systems, Cockeysville, USA. Neurobion was obtained from E-Merck (India) Ltd., Usgaon, Ponda, Goa, India. Culture conditions An overnight-grown Bacillus sp. DT7 was inoculated into 50 ml yeast extract pectin (YEP) medium contained in 250 ml conical ¯asks. The ¯asks were incubated at 37 C on a rotary shaker (150 rev/min) for dierent time intervals. The absorbance (600 nm) of the culture was measured by spectrophotometer (Spectronic 21, Bausch & Lomb, Germany). The culture was centrifuged (7000 g, 10 min, 4 C) and the cell-free supernatant was assayed for pectinase activity as described below. Composition of YEP medium: yeast extract (YE), 10.0 g/l; pectin, 2.5 g/l; pH 7.2. Pectinase activity Polygalacturonase activity was assayed by the colorimetric method of Miller (1959), modi®ed by Aguillar & Huitron (1990). Brie¯y, 100 ll of suitably diluted cellfree supernatant was incubated with 100 ll of substrate (PGA, 1.0%, w/v) at 40 C for 10 min under static conditions. After adding 400 ll of DNSA, the mixture was boiled for 15 min. Mixture was ®nally diluted to 5 ml with deionized water (4.4 ml). The absorbance of
278 the color developed was measured at 530 nm. One unit of enzyme was de®ned as the amount of enzyme which catalyses the formation of 1 lmol of galacturonic acid/ min at ®xed pH. Pectin lyase (PL) activity The PL activity of the given samples was assayed by the method of Pitt (1988). Brie¯y, 1 ml of the suitably diluted enzyme sample was added to 5.0 ml of pectin solution (1% w/v). The volume of the test samples was adjusted to 10.0 ml with distilled water. The samples were incubated at 40 C for 2 h. This was followed by the addition of zinc sulphate (0.6 ml, 9.0% w/v) and sodium hydroxide (0.6 ml, 0.5 M ). The samples were centrifuged (3000 g, 10 min) and 5.0 ml of the clear supernatant was added to a mixture of thiobarbituric acid (3.0 ml, 0.04 M ), HCl (2.5 ml, 0.1 M ) and distilled water (0.5 ml). The mixture was heated in a boiling water bath for 30 min, cooled to room temperature and the absorbance of the coloured solution was measured at 550 nm. One unit of activity was de®ned as the amount of enzyme that caused a change in absorbance of 0.01 under the condition of the assay. Pectinase puri®cation Bacillus sp. DT7 pectinase was puri®ed from one liter YEP culture broth (growth conditions: 37 C, 200 rev/ min, 12 h). Initially, cell-free supernatant was saturated with (NH4 )2 SO4 to two cut os (0±40%, 40±100% saturation). The precipitates were dissolved in the minimum amount of Tris±HCl buer (0.01 M , pH 7.5) and dialysed against the same buffer. Ion exchange chromatography Anion exchanger, DEAE-Sephacel, was packed into a glass column (15 0:55 cm, 10 ml-bed volume). The column was equilibrated with Tris±HCl buer (10 mM , pH 7.5) and a 3.0 ml sample was loaded on to it. The column was washed with Tris±HCl buffer containing 0, 50, 100 and 150 mM NaCl concentrations. Fractions of 2.5 ml volume were collected. The protein content of the fractions was measured spectrophotometrically at 280 nm and the pectinase activity was assayed by the method described earlier. The fractions showing pectinase activity were pooled, concentrated and saved for further analysis. Gel ®ltration chromatography A glass column was packed with Sephadex G 150 (35 1:5 cm, bed volume 60 ml). The concentrated sample was loaded on to this column and elution of the proteins was done using Tris±HCl buer (10 mM , pH 7.5). After the void volume (20 ml), fractions of 1.5 ml volume were collected. The absorbance of the samples for protein content and pectinase activity was done by
D.R. Kashyap et al. the methods described earlier. The pectinase-positive fractions were pooled, concentrated and saved for further analysis. SDS-Polyacrylamide gel electrophoresis (PAGE) Ten percent SDS-PAGE was performed on the puri®ed pectinase-positive sample by the method described by Laemmli (1970) using Bio-Rad electrophoresis apparatus. The gel was run on a constant voltage of 50 V. The gel was stained by the silver staining method of Merril et al. (1981). Results and Discussion Pectinases are increasing in commercial importance. The role of acidic pectinases in fruit juice preparation is well established. Although a few reports have been published recently on the isolation of alkalophiles producing alkaline pectinases, these enzymes have not been characterized in great deal. We have isolated a few bacteria from the soil of a fruit-processing unit which exhibited a large zone of clearance on pectin agar plates, thus indicating the ability of these organisms to produce pectinases. One bacterial colony showing maximum zone of clearance on pectin agar plates was tentatively identi®ed as Bacillus sp. DT7 based on its morphological (Gram-positive, rods, endospore) and biochemical (catalase-positive) properties. Various nutrient media (YE, peptone, LB, malt extract, NB, BHI; concentration 1% w/v) supplemented with pectin (0.25% w/v) were tested for production of pectinase by Bacillus sp. DT7. All the media tested produced varying levels of pectin lyase (EC 4.2.2.10). Maximal activity (15.4 U/ml) was observed in YEP medium followed by peptone medium (10.2 U/ml). These results are in agreement with others where they have reported the combination of YE with pectin to be the best medium for pectinase production (Moran & Starr 1969; Dave & Vaughn 1971; Garibaldi & Bateman 1971). To optimize the amount of YE and pectin in the medium for pectinase production, dierent amount of YE (0.25±4.0% w/v) and pectin (0±0.5% w/v) were tested. Maximal activity (15.82 U/ml) was observed at 1.0% YE and 0.25% pectin in the medium. Further increase of YE and pectin in the medium caused a slight decrease in cell growth as well as in pectinase activity. Although most workers have also used YEP medium for pectinase production, the concentrations of yeast extract and pectin used by them is higher than ours (Kelly & Fogarty 1978; Aguillar & Huitron 1990; Fonseca & Said 1995). It was also observed that pectinase production by Bacillus sp. DT7 is inducible in nature, as no pectinase activity was noticed in the absence of pectin in the growth medium. The pectinase production by Bacillus sp. DT7 was tested by adjusting the pH of the YEP medium between pH 5.0±9.0. Maximal pectinase production (15.72 U/ml)
Bacillus sp. DT7 pectinase was observed around neutral pH (7.2), which coincided with maximal cell growth (0.71, OD600nm ). Shifting the pH of YEP medium either to acidic (5.0±6.5) or alkaline (8.0±9.0) resulted in decreased production of pectinase. A decrease of nearly 50% in pectinase activity was observed when the pH of the YEP medium was adjusted either at pH 5.0 or pH 9.0. This decrease in the pectinase production was attributed to decrease in the growth of organism at these pH values. The incubation temperature was also found to in¯uence the pectinase production. Of the four incubation temperatures tested (25, 30, 37 and 40 C) maximal growth of Bacillus sp. DT7 as well as maximum pectinase production was observed at 37 C incubation. It also seems that aeration has a signi®cant in¯uence on the pectinase production by this organism. Although culture grown under static condition did show pectinase activity (9.5 U/ml), aeration not only increased pectinase production but also reduced the time for pectinase production. Maximal enzyme activity observed at 150, 200 and 250 rev/min was 15.7, 16.6 and 18.0 U/ml after 18, 12 and 8 h of incubation respectively. The time required for the production of pectinase by Bacillus sp. DT7 is remarkably less as compared to microbial pectinases reported in literature (Nasumo & Starr 1967; Dave & Vaughn 1971; Kelly & Fogarty 1978; Acuna-Arguelles et al. 1995). The eect of various salts and vitamins on pectinase production was also studied. The addition of either (0.05% w/v) or MgSO4 7H2 O CaCl2 (fused) (0.05% w/v) to YE medium resulted in a signi®cant increase (more than three-fold) in pectinase production. Interestingly, this increase was unrelated to the increased growth of the cells. Compared to 16.5 U/ml of pectinase produced by Bacillus sp. DT7 culture in YEP medium, supplementation of Salt-I and Salt-II solutions to the medium resulted in the production of 27.0 and 34.0 U/ml of pectinase respectively (Table 1). A similar increase in pectinase production by Bacillus sp. DT7 was observed by the addition of a multivitamin solution (Neurobion, ®nal concentration 1 ll=ml). The addition of Neurobion caused a slight decrease in the growth of Bacillus sp. DT7 but there was 61% increase in pectinase production. Various combinations of salts and vitamins were also tested for pectinase production in YEP medium. However it was observed that addition of CaCl2 (0.5% w/v) was sucient to produce maximum pectinase, as supplementation of other salts/vitamins to YEP+CaCl2 (YEPC) medium did not enhance pectinase production. The relationship of growth of Bacillus sp. DT7 to pectinase production was studied in YEPC medium at 37 C over 36 h under shaking conditions (200 rev/min). The culture achieved maximal growth (1.2, OD600nm ) after 12 h of incubation (Figure 1). However, further incubation caused a decrease in the culture absorbance. After 36 h the absorbance was reduced to half (0.53, OD600nm ) of the maximum value. This decrease in growth suggested that bacterial cells were lysing after 12 h of incubation.
279 Table 1. Eect of dierent salts and vitamins on pectinase production by Bacillus sp. DT7 in YEP growth medium. Salts/vitamins
Pectinase activity (U/ml)
Culture absorbance (600 nm)
No salt/vitamin Salt-I solution Salt-II CaCl2 (0.05%) MgSO4 (0.05%) Neurobion (1 ll/ml) Salt-I + Neurobion (1 ll/ml) CaCl2 + Neurobion (1 ll/ml) CaCl2 + Salt-I CaCl2 + Salt-I + Neurobion (1 ll/ml) MgSO4 + CaCl2
16.5 27.0 33.9 54.7 52.4 27.0 47.6 54.7 44.7 53.1 50.6
0.75 0.87 0.83 0.70 0.66 0.70 0.78 0.68 0.65 0.72 0.79
Salt-I composition: CuCl2 á 2H2O (0.0015%), MnCl2 á 4H2O (0.015%), NaMoO4 á 2H2O (0.002%), ZnSO4 (0.003%), Fe-citrate (0.025%), EDTA á 2H2O (0.014%), H3BO3 (0.003%), CoCl2 á 2H2O (0.0025%). Salt-II composition: FeSO4 (1 mM ), MnCl2 (10 mM ), KCl (25% w/v), MgSO4 á 7H2O (1.0 mM ).
Pectinase production was directly proportional to culture growth upto 12 h of incubation. Further incubation resulted in decreased biomass, but pectinase activity kept on increasing. Maximal pectinase activity (53 U/ml) was observed after 24 h of incubation. However, further incubation led to a slight decrease in the pectinase activity. Pectinase production by this bacterium is higher than by most other microbes e.g. B. polymyxa (Nagel & Vaughn 1961), Bacillus sp. RK9 (Kelly & Fogarty 1978), Xanthomonas compestris (Nasumo & Starr 1967), Penicillium frequentans (Borin et al. 1996); Aspergillus niger, A. nodulans and Rhizopus stolonifer (Siessere & Said 1989). A shift in the pH of the growth medium was also observed at dierent intervals of incubation. Initially, a gradual decrease in the pH of the medium was seen. After 6 h of incubation the pH
Figure 1. Growth curve and pectinase production by Bacillus sp. DT7 in YEPC medium.
280
D.R. Kashyap et al.
value had fallen to 6.4 from an initial value of 7.2. Subsequently, the pH value of the medium increased with increase in the incubation period, reaching a maximum (pH 8.1) after 36 h of incubation. Puri®cation of pectinase The pectinase from Bacillus sp. DT7 was puri®ed to homogeneity using various steps. Initially, the enzyme was partially puri®ed by addition of solid ammonium sulphate to the cell-free supernatant. Pectinase activity was found in the 40±100% salt saturation fraction. This fraction was loaded onto the anion exchanger, DEAESephacel equilibrated with 20 mM Tris±HCl (pH 7.5) buffer. The proteins were eluted with a NaCl gradient (0±2 M ). Most of the protein was present in two major peaks, but pectinase activity was detected only in the ®rst fraction (Figure 2). Using DEAE-Sephacel chromatography, 67.2-fold puri®cation of the enzyme was achieved and its speci®c activity was found to be 730.3 U/mg of protein. Fractions showing pectinase activity were pooled, concentrated using a Centricon P-10 unit, and loaded onto Sephadex G150 column. Protein was eluted with Tris±HCl buffer (pH 7.5). As pectinase activity was detected in fractions 21±30 (Figure 3), these fractions were pooled, concentrated and dialysed against 0.01 M Tris±HCl buffer (pH 8.0). This phase of puri®cation yielded a 131.8-fold increase in the puri®cation of pectinase and its speci®c activity was 1433 U/mg protein. The detection of pectinase activity in fractions 21±30 suggested that this enzyme is of high molecular weight. When this puri®ed pectinase was electrophoresed on 10% SDS-PAGE, a single band was observed, indicating the complete puri®cation of the enzyme. Using standard protein markers the size of the puri®ed enzyme was found to be around 106 kDa
Figure 2. Puri®cation of pectinase by ion exchange chromatography using DEAE-Sephacel.
Figure 3. Elution pro®le of protein and pectinase activity on Sephadex G 150.
(Figure 4) which is much higher than that of pectinase from Fusarium oxysporium f. sp. melonis (58 kDa) (Martinez et al. 1991); Penicillium frequentans (20 kDa) (Borin et al. 1996); and Bacillus stearothermophillus (24 kDa) (Karbassi & Vaughn 1980) reported in the literature. The eect of various physical and chemical agents on the activity of puri®ed pectinase was also checked. The
Figure 4. SDS-PAGE of pectinase on 10% gel at 50 V. Lane 1: molecular weight protein standards, lane 2: ammonium sulphate precipitated protein, lane 3: DEAE-Sephacel elute and lane 4: Sephadex G 150.
Bacillus sp. DT7 pectinase pH seemed to in¯uence the pectinase activity. Most fungal pectinases are stable under low pH. However pectinase from Bacillus sp. DT7 was maximally stable under alkaline conditions of pH 7.5±8.5. The enzyme retained nearly 50% of its activity at neutral pH. There was near complete loss of pectinase activity at pH values less than 6.0 and more than 9.0. This range of pH stability is close to that reported for Bacillus pumilus (pH 8.0±8.5) by Dave & Vaughn (1971) but is lower than the optimal range (pH 8.9±9.4) reported for pectinase of Bacillus polymyxa (Nagel & Vaughn 1961); pH 9.0 for Bacillus stearothermophillus (Karbassi & Vaughn 1980); and pH 10.0 reported for Bacillus sp. RK9 (Fogarty & Kelly 1983). The temperature stability pro®le of pectinase activity revealed that the enzyme is maximally active at moderately high temperatures ranging from 40 to 60 C with highest activity (63 U/ml) detected at 60 C incubation temperature for 1 h. This temperature stability of Bacillus sp. DT7 is higher than the values reported by Nagel & Hasegawa (1968) for the crude enzyme of a Bacillus sp. identi®ed as isolate no. 5 (below 30 C) and by Nagel &Vaughn (1961) for the crude enzyme of B. polymyxa (45 C) but slightly less than the values reported by Horikoshi (1972) for Bacillus No. P-4-N (65 C) and by Karbassi & Vaughn (1980) for B. stearothermophilus (70 C). A further increase in the reaction temperature caused signi®cant drop in the pectinase activity. Similarly, temperatures lower than 40 C resulted in decrease in the pectinase activity. The puri®ed enzyme was sensitive to some of the salts tested at a concentration of 1.0 mM . The presence of CaCl2 acted as a stimulator of pectinase activity resulting in an increase of 50% (approx.) in the enzyme activity, whereas MgSO4 was found to have no effect. HgCl2 , EDTA and FeCl3 decreased pectinase activity by 98.1, 83.0 and 30.5% respectively. Similar results have already been reported using various organisms (Starr & Nasumo 1967; Nagel & Hasegawa 1968; Dave & Vaughn 1971; Horikoshi 1972; Kelly & Fogarty 1978). Of the ®ve chemical agents tested at a concentration of 1.0 mM , mercaptoethanol increased the pectinase activity by 1.5-fold. However urea, ascorbic acid and cysteine had no effect on pectinase activity. Only glycine decreased its activity by 18%. The high yield of pectinase from Bacillus sp. DT7 along with its alkalophilic and thermotolerant properties suggest that this strain is better pectinase producer than the earlier reported microbes. In addition to these properties, some additional features like enhanced production with the addition of CaCl2; shorter period of incubation for pectinase production, lesser amount of YE and pectin in the growth medium, and stimulation of pectinase activity by mercaptoethanol indicate the potential of this organism to be used at commercial level for degumming of ramie, retting of ¯ax, pretreatment of waste water from fruit juice-processing industries.
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