ISOLATION AND PRODUCTION OF EXTRACELLULAR ENZYME FROM MANNANOLYTIC THERMOPHILIC BACTERIUM ORIGINALLY FROM LAMPUNG ISOLATION AND CHARACTERIZATION OF MANNANOLYTIC THERMOPHILIC BACTERIA FROM PALM OIL SHELL AND THEIR MANNANASE ENZYME PRODUCTION PROPERTIES BIOTROPIA NO. 25, 2005 : 1 – 10 SUMARDI1, ANTONIUS SUWANTO2,, MAGGY THENAWIDJAJA3, and TRESNAWATI PURWADARIA4 1Department of Biology, Faculty of Science and Mathematics, University of Lampung, Jl. S. Brojonegoro No. 1, Bandar Lampung 35145, Indonesia 2Department of Biology, Faculty of Science and Mathematics, Bogor Agricultural University, Jl. Raya Pajajaran, Bogor 16144, Indonesia 3Department of Food Science and Human Nutrition, Bogor Agricultural University, Darmaga, 16680, Bogor, Indonesia 4Indonesian Research Institute for Animal Production, P.O. Box 221, Bogor 16002, Indonesia ABSTRACT A mannanolytic thermophilic bacterium (L-07) was isolated from palm oil shell after 2 days of enrichment in liquid medium supplemented with 1% palm kernel meal as mannan source. Sequence analysis of 16S-rRNA indicated that L-07 was similar (98%) to Geobacillus stearothermophilus, a species of thermophilic aerobic bacteria. We found that G. stearothermophilus L-07 produced extracellular β-1,4-mannanases, but no β-manosidase and α-galactosidase activities. The growth of L-07 reached its maximum (3.0 x 106 cell/ml) at 12-20 hours, while the highest β-mannanase activity (0.52 U/ml) was observed in culture medium after 36 hours of cultivation at 60oC. The medium containing locust bean gum was the best for producing extracellular β-1,4-mannanases compared with kolang kaling, konjak, and palm kernel meal. SDS-PAGE and zymogram analysis demonstrated that crude mannanase complex of L-07 from locust bean gum containing medium comprised three active bands with molecular weight of 85, 73 and 50 kDa. Keywords : Extracellular enzyme/mannanase/Geobacillus stearothermophilus INTRODUCTION Hemicelluloses are the second most abundant polysaccharide in nature after cellulose. The major constituents of hemicellulose are the hetero-1,4-β-D-xylans and hetero-1,4-β-D-mannans (galactoglucomannan, galactomannan, and glucoman- nan). The heteroxylans are found mainly in grasses, cereals, and hardwoods (angiosperms). The mannans are more abundant in copra, palm, coffee, and locust bean endosperms (Araujo and Ward 1990). Mannanolytic microbes were found in soil, compost, and animal rumen (Zakaria et al. 1998). Biodegradation of ß-mannans is caused by β-mannanase (1,4-β-D mannan manohidrolase [EC 3.2.1.78]) produced from bacteria and fungi. The enzyme hydrolyses the ß-(1,4)- linkages in backbone of mannan polymer, ∗Corresponding author : asuwanto@indo.net.id 1 producing short chain mannoligosaccharides. Then, these compounds can be further degraded by the action of β-mannosidase (β-D-mannosidase [EC 3.2.1.25]) and a α- galactosidase (EC 3.2.1.22) (Duffaud et al. 1997). Mannan degradation from glucomannan and galactomannan produces manno-oligosaccharide, mannobiose, and mannose. Mannan-degrading enzymes can be used for numerous applications in food, feed, pulp, and paper industries. BIOTROPIA NO. 25, 2005 In the palm oil factory, during the composting process of palm shells containing hemicellulose the temperature rises to 65oC. At such temperature, many thermophilic bacteria are able to develop special properties to survive and prosper in the habitat. The purposes of this study were to isolate the mannanolytic thermophilic bacterium from palm shells, a solid waste of palm oil factory in Lampung, and to study its ß-mannanase production. MATERIALS AND METHODS Materials Palm shell as source of thermophilic bacteria was obtained from palm oil industry in Natar, Lampung at 65oC. Locust bean gum (galactomannan) was obtained from Sigma Chemical Company. Other chemicals were analytical grade from Merck Industry. Isolation of Thermophilic Mannanolytic Strains One milligram of palm shells was enriched in a medium containing 0.2% yeast extract; 0.2% trypton, 1.0% palm kernel meal, 0.02% MgSO4, 0.14% KH2PO4, and 0.1% (NH4)2SO4 at 70 oC for two days with agitation (120 rpm). One hundred microliters of each enrichment culture was spread aerobically on the same medium onto agar plates containing 0.3% locust bean gum instead of palm kernel meal. Thermophilic mannanolytic isolates showed a clear zone around the colony after staining with a solution of 0.1% congo red for 15 minutes and destaining through repeated washing with 1 M NaCl. Characterization and Identification of Isolate Morphological properties and taxonomic characteristics of the best isolate were studied according to the methods as described in “Bergey’s Manual of Systematic Bacteriology” (Holt et al. 1994). Identification of the isolate was determined through 16S rRNA sequence analysis. Genomic DNA extraction was performed using the CTAB method. PCR- mediated amplification of the 16S rRNA was performed as described by Marchesi et al. (1998) employing GeneAmp PCR system 2400 (Perkin Elmer). PCR product was purified and sequenced. Cluster analysis was conducted according to a program 2 provided by European Bioinformatics Institute (http://www. Ebi.ac.uk) and phylogenetic tree was constructed using Treecon software (Peer and Watcher 1993). Isolation and characterization of mannanolytic thermophilic bacteria – Sumardi et al. Extracellular Enzyme Production The best bacterial isolate was grown in the medium for mannanase production which contained 0.35% yeast extract, 0.35% trypton, 0.035% MgSO4, 0.245% KH2PO4, 0.175% (NH4)2SO4, 0.2% NaCl, and 0.65% locust bean gum (pH 7.0). Locust bean gum was replaced with other carbon sources (kolang kaling, konjak, palm kernel meal) when the effect of carbon sources on the enzyme production was examined. A 250-ml flask containing 50 ml of the medium was inoculated with a loopful of cells taken from a stock slant and was precultured at 60oC on shaker (120 rpm) for 6-8 hours. The same volume of medium and flasks for enzyme production were inoculated with 2 ml of this culture and cultivated at 60oC for 48 h. Aliquots of the culture medium were sampled at 4 h intervals to determine β-mannanase activity and viable bacterium number. Intracellular Enzyme Extraction G. stearothermophilus L-07 cells were concentrated from 50 ml liquid medium by centrifugation at 3,200 x g for 10 min. at 4oC. The cell pellets were resuspended in 1 ml of ice-cold condition containing 50 mM Phosphat buffer. The cell lysis process used sonication with a Soniprep 150 (USA). The sonicator was set to 16 micron amplitude for 5 minutes. Intracellular enzymes were detected specifically β- mannanase, β-mannosidase, and α-galactosidase. Enzyme Assay β-mannosidase and α-galactosidase activities were determined by monitoring the release of p-nitrophenol from p-nirophenyl B-D-mannopyranoside or p- nitrophenyl α-galactophyranoside (Sigma Chemical Co., USA), respectively. For each assay, 0.9 ml aliquots of 1 mM substrate in 50 mM sodium phosphate buffer (pH 7.0) and 0.1 ml of enzyme were mixed and incubated at 80oC for 30 minutes and the reaction stopped by the addition of 0.1 ml solution of 0.4 M Na2CO3. The release of p-nitrophenyl (PNP) was measured spectrophotometrically by monitoring the changes in absorbance at 405 nm. Control was prepared with the addition of enzyme after Na2CO3 solution. One unit of β-mannosidase or α-galactosidase activity was defined as the amount of enzyme releasing 1 μmol PNP per min. under the specified assay condition. β-mannanase activity was determined by monitoring the release of reducing sugars. The reaction mixture, containing 0.5% locust bean gum, 50 mM sodium phosphate buffer (pH 7.0), suitably diluted enzyme solution in a total volume of 1 ml, was incubated at 80oC for 30 minutes. The reducing sugar content was determined by dinitrosalisylic acid method. One unit of enzyme was defined as the 3 amount of enzyme producing 1 μmol of mannose per minute under the given assay condition. BIOTROPIA NO. 25, 2005 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Zymogram SDS-PAGE was performed in 8% polyacrylamide gels. Protein bands were visualized after staining with silver nitrate. Zymogram was prepared using 0.1% locust bean gum co-polymerized with polyacrylamide. After separation, SDS was removed by method of Spindler and Rapp (1997). Then, the gel was equilibrated through several washings and finally immersed in 50 mM sodium acetate buffer (pH 6.0), followed by incubation at 65oC for 35 minutes, staining with 0.1% congo red for 15 minutes, and destaining in 1 M NaCl. RESULTS AND DISCUSSION We found 15 bacterial isolates from palm oil shell. Then the bacteria were screened for mannanase activity on selected medium agar plate containing locust bean gum at 70oC with pH 7.0. Only isolate L-07 showed mannanase activity. The isolate L-07 could grow at 55-70oC and showed mannanase activity as high as 3.1 U/mg. a. b. Figure 1. Bacterial isolate L-07. a. Gram-positive rod-shaped cells were cultured at 65oC for 1 day. b. Cells produced endospore in terminal position after 5 days of incubation. Table 1 shows the morphological and physiological characteristics of L-07. L- 07 was gram-positive (Figure 1.a), aerobic, catalase-positive, and endospore- forming bacterium (Figure 1.b). It was non-motile and rod-shaped (1-1.2 x 5-5.7 μm). Based on these characteristics L-07 belonged to the genus Bacillus. 4 Table 1. Morphological and physiological characteristics of L07 isolate Isolation and characterization of mannanolytic thermophilic bacteria – Sumardi et al. Characteristics Strain L-07 Shape rod Endospora shape oval/cylindrical Endospora position terminal Gram stain positive Catalase positive Motility negative Growth at 37oC negative Growth 55 – 75oC Optimum growth temperature 60-70oC Cell size (μm) 1,2x5,7 Nitrate reduction positive Indole production negative Urease positive Voges proskauer test negative Utilization as sole carbon source of Glucose positive Mannose positive Xylose positive Sucrose positive Arabinose positive Ramnose negative Citrate negative Lactose negative Mannitol negative Sorbitol negative Adonitol negative Rafinose negative Hydrolysis of mannan positive Hydrolysis of carboxymethyl cellulose positive Hydrolysis of starch negative Hydrolysis of xylan negative Then, the total sequence of 1315 bp of 16S rRNA gene of L-07 showed 98% similarity to that of Geobacillus stearothermophilus. The constructed phylogenetic tree for L-07 is shown in Figure 2. G. stearothermophilus (formerly Bacillus stearothermophilus) was a highly resistant thermophilic organism. Major strains of the genus Geobacillus live in geothermal areas, such as the oil field subsurface and hydrothermal vents (Nazina et al. 2001; and Euzeby 2005). At this time, no study has reported on the presence of mannanolytic thermophilic bacteria from palm shell. As comparison Aurora et al (2003) showed that Bacillus pumilus DYP2, a mannanolytic mesophilic bacteria, was isolated from West Sumatra copra soil sample, Indonesia. Another bacterium, B. stearothermophilus ATCC 12016 has been previously reported to produce thermostable β-mannanase (Ethier et al. 1998). 5 Figure 2. Phylogenetic tree construction for L-07 isolate. Bootstrap values are shown at each node tree. When G. stearothermophilus L-07 produced thermophilic mannanase, the enzyme was observed primarily in culture media. The other two enzymes, α-1,6 galactosidase and β-1,4 mannosidase were not detected in extracellular enzyme preparation (Table 2). These enzymes might act to provide simple sugar to Geobacillus stearothermophilus L-07 growing on locust bean gum as base media. Regarding the cellular localization of these three activities, we speculate that the mannan backbone was cleaved by endo-acting β-1,4 mannanase prior to the transport of smaller galactomannans to the site of the other two enzymes. The other two enzymes, exo-acting α-1,6 galactosidase and β-1,4 mannosidase, are found in the cell membrane. As comparison, Duffaud et al. (1997) showed that extracellular β-1,4 mannanase of Thermotoga neopalitana 5068 was sevenfold greater than the cell extract. The other two enzymes, α-1,6 galactosidase and β-1,4 mannosidase were found in the cell the extract. Table 2. The specific activity of extracellular and intracellular mannanolytic enzymes from Geobacillus stearothermophilus L-07 cultured at 60oC for 36 hours Specific activity (U/mg) No Kind of enzyme Intracellular Extracellular 1 β-1,4-mananase 0.02 ± 0.002 3.10 ± 0.050 2 β-1,4-manosidase 0.03 ± 0.003 0.00 ± 0.000 3 α-1,6-galactosidase 24.1 ± 0.060 0.00 ± 0.000 72 93 92 100 100 Pyrococcus furiosus subsp. woesei Alicyclobacillus acidocaldarius MIH321 G. stearothermophylus BGSC 9A21 L-07 10% BIOTROPIA NO. 25, 2005 E. coli Thermus aquaticus YT-1 C. botulinun KYTO-F L. delbrueckii subsp. bulgaricus ATCC11842 B.subtilis WL-7 100 50 B. thermoleovorans CCR11 85 6 We also observed the growth and extracellular β-mannanase activity of G. stearothermophilus L-07 as shown in Figure 3. After 12-20 hours of observation strain L-07 reached its maximum growth, while the highest β-1,4-mannanase activity was found in culture medium after 36 hours of cultivation. β-1,4-mannanase may be associated with the cell membrane. So, after G. stearothermophilus cells died β-1,4-mannanase leaked out from membrane and increased the activities. Besides for mannan degradation to produce manno-oligosaccharide, this thermo- stable enzyme can be used in the bleaching process (Ethier et al. 1998). The other β-mannanase from Bacillus sp. was active at 70oC. The enzyme was stable when incubated for 1 h at ≤ 60oC (Ooi and Kikuchi 1995). While the β- mannanase from Rhodothermus marinus retained 87% of its initial activity after 1 h at 90oC (Politz et al. 2000). 4,5 5 5,5 6 6,5 7 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (h) L og c el l n um be r/ m l 0 0,1 0,2 0,3 0,4 0,5 0,6 M an na na se a ct iv ity (U /m l) Figure 3. Growth curve and extracellular mannanase activity of Geobacillus stearothermophilus L-07. Strain L-07 was cultured at 60oC for 48 hours in the enzyme production medium. -■- logarithmic viable cell number and -ο- mannanase activity. We studied the effect on the kind of carbon source on β-1,4 mannanase production. The bacteria produced active β-1,4 mannanase when they were grown in a kind of carbon source as medium (Table 3). Table 3. Effect of carbon sources on the mannanase production. G. stearothermophilus L-07 was cultured at 60oC for 36 hours. No Carbon sources Relative activity (%) 1 None 2 ± 0.01 Locust bean gum 2 100 ± 0.04 Kolang kaling (endosperm from Arenga pinata) 46 ± 0.06 3 Konjak 4 13 ± 0.05 Palm kernel meal 0 ± 0.00 5 Isolation and characterization of mannanolytic thermophilic bacteria – Sumardi et al. 7 The enzyme activity in the medium containing locust bean gum was the highest, followed by that in kolang kaling, and konjak. No activity was observed in palm kernel meal. Thus, the best induction medium consisted of locust bean gum. It may be pointed out that the mannanase activity is preferably induced by polysaccharides containing mannose or galactose as monomeric unit, and the induction is strongly increased by heterogeneous polysaccharides containing mannose and galactose. Locust bean gum consists of 88% galactomannan (Whistler and BeMiller 1973). The composition may support the increase of mannanase production. So far, there was no accurate information on mannan composition in kolang kaling and palm kernel meal. However, they are found in palm seeds where galactomannans are major part of their dry weight. BIOTROPIA NO. 25, 2005 Konjak mannan consist of 50-60% glucomannan containing mannose and glucose. As comparison, Torrie et al. (1990) reported that the highest mannanase production was found from mold of Trichoderma harzianum E58 in the medium containing locust bean gum (26.7 U/mg), followed by konjak (glucomannan) (7.5 U/mg). Zakaria et al. (1998) showed that the enzyme activity from Flavobacterium sp. in the medium containing guar gum was the highest, followed by locust bean gum. In another research, Hossain et al. (1996), showed that Bacillus sp. KK01 in coconut meal resulted in mannanase activity of 0.04 U/mg. In non-mannan medium, 2% relative activity of mannanase was still found. In spite of non-mannan carbon source addition, the base medium might contain small amount of mannan from yeast extract. The mannan from yeast extract could induce mannanase production. There was no mannanase activity in media containing mannan from palm kernel meal. When G. stearothermophilus L-07 was isolated by enrichment in liquid medium supplemented with 1% palm kernel meal as mannan source, the bacterium might survive due to synergism with other bacteria. Furthermore, G. stearo- thermophilus L-07 could not survive alone if it was cultured in palm kernel meal medium. According to Sumardi (2005), the palm kernel meal produced inhibiting substance after sterilization using an autoclave. Thus, G. stearothermophilus L-07 could neither grow nor produce β-mannanase. The enzyme from G. stearothermophilus L-07 in the locust bean gum was further run on SDS-PAGE and analyzed by zymogram assay towards locust bean gum. Three bands were observed at molecular mass of approximately 85, 73, and 50 kDa, respectively (Figure 4). The presence of three isozymes indicated that at least three mannanase encoded genes are present in L-07. Other G. stearothermophilus strains have been previously reported to produce mannanase with molecular mass of 76 kDa and active at 70oC (Ethier et al. 1998). 8 M Cr Z Isolation and characterization of mannanolytic thermophilic bacteria – Sumardi et al. kDa 85 kDa 73 kDa 50 kDa 94 67 43 30 20 14 Figure 4. Zymogram of crude mannanase from G. stearothermophilus L-07. Lane M indicates the molecular size standard used in this study. Lane Cr indicates crude enzyme mannanase separated in SDS-PAGE. Lane Z indicates bands of mannanase activity detected by zymogram. CONCLUSIONS We isolated G. stearothermophilus L-07 from palm shell, a solid waste of palm oil factory in Lampung. The bacterium produced extracellular β-1,4- mannanase in locust bean gum medium, while β-mannosidase and α-galactosidase were not detected. The optimum enzyme activity was observed after 36 hours of incubation at 60oC. The mannanase comprised three active bands with molecular weight of 85, 73, and 50 kDa. 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Biochem., 62: 655-660. 10 http://www.bacterio.net/ INTRODUCTION Isolation of Thermophilic Mannanolytic Strains Characterization and Identification of Isolate Extracellular Enzyme Production Enzyme Assay RESULTS AND DISCUSSION We found 15 bacterial isolates from palm oil shell. Then the bacteria were screened for mannanase activity on selected medium agar plate containing locust bean gum at 70oC with pH 7.0. Only isolate L-07 showed mannanase activity. The isolate L-07 could grow at 55-70oC and showed mannanase activity as high as 3.1 U/mg.