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J. Korean Soc. Appl. Biol. Chem. 54(3), 325-331 (2011)

Isolation, Purification and Characterization of a Thermostable β-Mannanase from Paenibacillus sp. DZ3 Muni Rammannagari Subhosh Chandra1†, Yong-Suk Lee1†, In-Hye Park1, Yi Zhou1, Keun-Ki Kim2, and Yong Lark Choi1* 1

Department of Biotechnology, College of Natural Resources and Life Science, Dong-a University 840, HadanDong, Saha-gu, Busan 604-714, Republic of Korea 2 Division of Applied Life Science, Pusan National University, Miryang 629-906, Republic of Korea Received January 7, 2011; Accepted March 22, 2011 Paenibacillus sp. DZ3, newly isolated from Konjack field, produced β-mannanase (900 U/mL) when grown on glucomannan as a carbon source. β-Mannanase was purified 34-fold to homogeneity resulting in final recovery of 15% and specificity of 169 U/mg protein. The molecular mass was approximately 39 kDa as estimated by sodiumdodecylsulfate-polyacrylamide gel electrophoresis. Active band was observed as clear colourless area on zymogram. The optimal temperature and pH for mannanase activity was 60oC and pH 5.0, respectively. The activity was stable up to 60oC at pH 5.0 and remained stable from pH 5.0-7.0. Mannanase was highly specific towards glucomannan and galactomannan, whereas exhibited low activity towards mushroom powder. The Michaelis constant (Km) and maximum velocity (Vmax) for glucomannan substrate were 1.05 mg/mL and 714 U/mg, respectively. These results indicate the enzyme is attractive for industrial applications. Key words: characterization, glucomannan, mannanase, Paenibacillus sp., purification

There is a considerable interest in the biological degradation of lignocelluloses as the most abundant reusable resource in nature and its potential for industrial application [El-Naggar et al., 2006]. The main carbohydrate constituents of lignocellulosic materials (cellulose, mannan, and xylan) consist of chains of β-1,4-linked pyranosyl units, which can be substituted in various forms. The β1,4-glycosidic bonds within the polysaccharide backbones are hydrolyzed by cellulases, mannanases, and xylanases. Cellulose can degrade beta-1,4-bond between glucose and glucose, mannanase can degrade beta-1,4-bond between mannose and mannose, xylanase degrade beta-1,4-bond between xylose and xylose [Sachslehner et al., 1998]. Various mannanases from Streptomyces sp. [Takahashi et al., 1984], Bacillus subtilis [Mendoza et al., 1994b; Zakaria et al., 1998], Sclerotium (Athelia) rolfsii [Sachslehner and Haltrich, 1999], Bacillus stearothermophilus M. R. S. Chandra† and Y. S. Lee† contributed equally. *Corresponding author Phone: +82-51-200-7585; Fax: +82-51-200-6536 E-mail: [email protected] doi:10.3839/jksabc.2011.052

[Zhang et al., 2000], Aspergillus awamori [Kurakake and Komaki, 2001], Trichoderma harzianum [Ferreira and Filho, 2004] and B. subtilis WY34 [Jiang et al., 2006] have been purified and characterized, and some genes from B. subtilis and B. stearothermophilus encoding mannanases were also cloned, sequenced and expressed [Mendoza et al., 1995; Ethier et al., 1998]. The two most important and representative hemicelluloses are the hetero-1,4-β-D-xylans and the hetero-1,4-β-D-mannans. Endo β-D-mannanase (EC 3.2.1.78, mannan endo-1,4-βD-mannosidase) cleaves randomly within the-1,4-β-Dmannan main chain of galactomannan, glucomannan, and mannan [McCleary, 1988]. There has been an increasing interest in the potential application of β-mannanases in the industry, because these enzymes play an important role in the bioconversion of lignocellulosic materials. The coconut residue contains highly concentrated mannan, which can be hydrolyzed by mannan-degrading enzyme system to produce single-cell protein [Hossain et al., 1996]. The other areas of applications include production of animal feed [Lee et al., 2005; Wu et al., 2005] and laundry detergents [McCoy, 2001; Schäfer et al., 2002]. Their effective role in pulp bleaching processes minimized the use of environmentally

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harmful bleaching chemicals in pulp and paper industry [Buchert et al., 1993; Cuevas et al., 1996]. Mannanases have been used in the food industry for the extraction of vegetable oils from leguminous seeds and the clarification of fruit juices [Gubitz et al., 1996]. The enzyme also can be used in the reduction of the viscosity of extracts during manufacturing of instant coffee, chocolate, and cacao liquor [Belitz and Grosch, 1987; Francoise et al., 1996; Sachslehner et al., 2000] to lower the cost for subsequent evaporation and drying [Wong and Saddler, 1993]. In addition, mannanases have the potential for application in the pharmaceutical industry for the production of physiologically interesting oligosaccharides [Gubitz et al., 1996]. Thus, β-mannanases have wide commercial applications in industries such as paper, pulp, food, feed, as well as pharmaceutical, and energy industries [McCutchen et al., 1996; Suurnakki et al., 1997; Lee et al., 2003; 2005]. This is the first report on purification and characterization of β-mannanase from Paenibacillus sp. DZ3. In the present investigation, a thermostable βmannanase was isolated, purified, and characterized from a Paenibacillus sp. DZ3.

Materials and Methods Microorganism and culture conditions. Paenibacillus sp. DZ3 was newly isolated from the soil samples of Konjack field, Wuhan, China. The Luria Broth medium supplemented with 0.5% glucomannan was used for mannanase production. After 5 days of growth at 37oC and 200 rpm, cells were removed by centrifugation (10,000 g) for 10 min at 4oC. The supernatant was used as crude enzyme preparation. Phylogenetic analysis by 16S rDNA. Polymerase chain reaction (PCR) was performed to amplify the 16S rDNA coding region, using two oligo nucleotide primers, 5'-GAGTTTGATCCTGGCTCAG-3' (position 9 to 27 relative to E. coli 16S rDNA) and 5'-AGAAAGGA GGTGATCCAGCC-3' (position 1,525 to 1,542 relative to E. coli 16S rDNA). PCR was carried out in a thermocycler using the following program: predenaturation for 60 s at 95oC, 30 cycles of denaturation at 95oC for 60 s, annealing at 60oC for 60 s, extension at 72oC for 90 s, and a final extension at 72oC for 10 min. The PCR products were subcloned into pGEM T-easy vector (Promega, St. Louis, MO). Phylogenetic trees were inferred using the ClustalX programme [Thompson et al., 1997]. Enzyme assay. Mannanase activity was assayed by mixing 0.1 mL of enzyme solution with 0.9 mL of 0.5% glucomannan in 50 mM citrate buffer (pH 6.0) at 50oC for 10 min. The released reducing sugar was determined by

the dinitrosalicylic acid (DNS) method using mannose as the standard [Miller, 1959]. One unit (U) of enzyme activity was defined as the amount of enzyme required to produce 1 µmol of reducing sugar per minute under the standard assay conditions. Estimation of protein. The concentration of soluble proteins was determined according to the method of Bradford [1976] using bovine serum albumin as the standard. Purification of the mannanase. The crude extracellular mannanase was obtained by centrifugation of the culture broth at 10,000 g for 10 min at 4oC. The crude supernatant was subjected to 80% (NH4)2SO4 precipitation. The precipitated protein was collected by centrifugation at 10,000 g for 10 min at 4oC and dissolved in 10 mM TrisHCl buffer (pH 7.5). Further purification was done by DEAE ion-exchange chromatography, followed by phenyl sepharose, and Hi-Trap phenyl sepharose chromatography (FPLC, ACTA Prime, Uppsala, Sweden). A 1.0 mL sample from ammonium sulfate precipitation was loaded on DEAE ion-exchange coloumn and equilibrated with 10 mM Tris-HCl [pH 7.5], and the protein was eluted with 10 mM Tris-HCl [pH 7.5] containing 1 M NaCl at a flow rate of 0.5 mL/min. The active fractions were combined. Six milliliters of the solution was applied to a phenyl sepharose coloumn equilibrated with 10 mM TrisHCl [pH 7.5] containing 1 M (NH4)2SO4. The bound protein was eluted with 0-0.5 M (NH4)2SO4 gradient at a flow rate of 1.0 mL/min. The active fractions were combined and subjected to Hi-Trap phenyl sepharose coloumn equilibrated with 10 mM Tris-HCl and 1 M (NH4)2SO4, pH 7.5. The bound mannanase was eluted with 0-0.5 M (NH4)2SO4 gradient at a flow rate of 1.0 mL/ min. The active fractions were combined and used in further experiments as the purified mannanase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and zymogram. SDSPAGE was performed in a 12.0% (w/v) polyacrylamide gel by the method of Laemmli [1970]. Protein bands were visualized by Coomassie brilliant blue R-250 staining. The molecular weight standard used was pre-stained protein size markers (ELPIS, Daejeon, Korea). Zymogram was obtained by co-polymerization of 0.1% (w/v) glucomannan with 12.0% (w/v) polyacrylamide gel using the method of Jiang et al. [2006]. Effects of temperature, pH, and stability on mannanase activity. The effect of temperature on mannanase activity was determined by incubating the purified enzyme with the substrate at temperatures ranging from 30 to 90oC in 50 mM citrate buffer pH 6.0. Thermal stability of the enzyme was determined by assaying for residual enzyme activity after incubation at

Purification of a thermostable β-mannanase from Paenibacillus sp. DZ3

various temperatures without the substrate for 1 h in 50 mM citrate buffer pH 6.0. The optimal pH of mannanase activity was examined at pH 3.0-11.0 under standard assay conditions using various buffers: 50 mM citrate buffer (pH 3.0-5.0), 50 mM sodium phosphate buffer (pH 6.0 and 7.0), 50 mM Tris-HCl (pH 8.0 and 9.0), and 50 mM glycine-NaOH buffer (pH 10.0 and 11.0). The pH stability of the mannanase was determined using the same buffer system in the range of pH 3.0-11.0. After incubation of the enzyme solution at various pH values for 1 h at 4oC without substrate, the remaining enzyme activity was measured. Effects of metal ions and reagents. The effect of various metal ions and reagents on mannanase activity was determined by assaying for residual activity after incubating the enzyme with 1 mM of various metal ions and reagents dissolved in 50 mM citrate buffer (pH 6.0) for 10 min at 50oC. Substrate specificity and kinetic parameters. The substrate specificity of the mannanase was determined by incubating the enzyme with 5 mg/mL of each substrate in 50 mM citrate buffer (pH 6.0) at 60oC for 10 min. Amount of released reducing sugars was estimated using the DNS method described above. Kinetic parameters, the Michaelis constant (Km) and maximum velocity (Vmax) were determined in 50 mM citrate buffer containing 0.2-1.0 mg/mL substrates, after incubation with purified mannanase at pH 6.0 and 50oC for 10 min. The data were plotted according to the Lineweaver-Burk method. Each data was an average of three independent experiments, and every experiment included three samples. N-terminal amino acid sequence. The purified βmannanase was transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The N-terminal amino acid sequence was determined by automated Edman degradation using PROCISE 491 HT protein sequencer (Applied Biosystems, Carlsbad, CA).

Results and Discussion Strain properties and identification. The strain used in the present study, Paenibacillus sp. DZ3, was isolated from Konjack field, China. Numerous glucomannandegrading bacteria formed a clear zone around their colonies on the isolation plates, among which, DZ3 showed the largest clear zone. Therefore, DZ3 was selected as a potential strain for the present study. This strain was a Gram-positive rod-shaped bacterium. The partial nucleotide sequence of the 16S rDNA gene,

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Fig. 1. Phylogenetic tree based on 16S rDNA sequences, showing the positions of strain DZ3 and other Paenibacillus species.

corresponding to the region from 9 to 1,525 bp was also determined in E. coli [David, 1991; Kunst et al., 1997]. The similarities of the 16S rDNA nucleotide sequences were 93% for Paenibacillus lentimorbus and Paenibacillus popilliae, 92% for Paenibacillus curdlanolyticus, 91% for Paenibacillus apiarius; 90% for Paenibacillus thiaminolyticus; 89% for Paenibacillus kobensis, Paenibacillus chondroitinus, Paenibacillus alginolyticus, Paenibacillus larvae sub sp. pulvifaciens, and Paenibacillus validus, and 87% for Paenibacillus alvei. The phylogenetic tree based on the analysis of the 16S rDNA sequences are shown in Fig. 1. The isolate was identified as Paenibacillus sp. DZ3 based on the morphological, biochemical, and physiological characteristics and phylogenetic analysis. Paenibacillus sp. DZ3 was not identical to other Paenibacillus species. Therefore, the culture was used for further experimental studies. Production of the mannanase. The newly isolated Paenibacillus sp. DZ3 produced maximum level of extracellular mannanase during growth on 0.5% glucomannan at 37oC. Maximum level of mannanase (900 U/mL) was reached after 5 days. To the best of our knowledge, this is the first report on the production of β-mannanase from Paenibacillus sp. DZ3, which produces mannanase at 37oC. Similarly, B. subtilis NM-39 produced mannanase at 37oC with locust bean gum as the substrate [Mendoza et al., 1994a]. Other B. subtilis strains isolated so far produced maximum mannanases at below 50oC [Jiang et al., 2006]. Some B. subtilis strains can produce mannanases at up to 45oC [Khanongnuch et al., 1998; Zakaria et al., 1998]. Normally, microbial mannanases appear extracellularly in multiple forms [Stalbrand et al., 1993; Gubitz et al., 1996; Hossain et al., 1996]. Five β-mannanases were secreted by Sclerotium rolfsii when cultivated in glucomannan [Gubitz et al., 1996]. Bacillus sp. KK01

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Table 1. Purification of mannanase from the isolated Paenibacillus sp. DZ 3 Purification step Crude supernatant 80% (NH4)2SO4 precipitation DEAE Phenyl Sepharose Hi-Trap Phenyl Sepharose

Total activitya (U)

Total proteinb (mg)

Specific activity (U/mg)

Yield (%)

Purification (fold)

900 770 700 300 135

180 60 30 7 0.8

5.0 12.83 23.33 42.86 169

100 86 77.77 33.33 15

1 2.56 4.66 8.57 34

a

Activity was measured in 50 mM citrate buffer (pH 6.0) at 50oC using 0.5% (w/v) glucomannan as a substrate by the DNS method. b The protein was measured using the method of Bradford [1976], using bovine serum albumin (BSA) as the standard.

Fig. 2. SDS-PAGE (A), lane M, pre-stained protein size markers (ELPIS); lane 1, DEAE, lane 2, Phenylsepharose, lane 3, Hi-Trap phenyl sepharose and Zymogram (B).

produced four β-mannanases in the culture medium [Hossain et al., 1996]. However, in the present study Paenibacillus sp. DZ3 produce only one β-mannanase. Similar observation was found in B. subtilis WY34 [Jiang et al., 2006]. Purification of the mannanase. The purification results are summarized in Table 1. Overall recovery of 15% and a 34-fold purification of the mannanase were obtained (Fig. 2A). The specific activity determined using glucomannan as substrate was 169 U/mg protein. The enzyme was also confirmed to be mannanase by zymogram analysis by congored staining (Fig. 2B). The apparent molecular mass of mannanase (39.0 kDa) from Paenibacillus sp. DZ3 is similar to that of 39.0 kDa for B. subtilis KU-1 [Zakaria et al., 1998]. N-terminal amino acid sequence of β-mannanase from Paenibacillus sp. DZ3 was D-M-A-E-I-E-K-F-D-K. Temperature and pH optima and stabilities. The optimal temperature for β-mannanase activity was 60oC (Fig. 3A). The enzyme was stable up to 60oC, but about 80% of its activity was lost at 80oC after 1 h of incubation (Fig. 3B). The optimal pH for β-mannanase activity was pH 5.0 (Fig. 4A). The enzyme showed stability within the pH range of 5.0-7.0 (Fig. 4B).

Fig. 3. Effect of temperature on mannanase activity (A) and stability (B). For determination of thermal stability, the residual activity of the treated mannanase was measured 1 h after pre-incubation at different temperatures at pH 6.0.

The mannanase was optimally active at 60oC compared to 50-55oC from B. subtilis strains NM-39 [Mendoza et al., 1994b], KU-1 [Zakaria et al., 1998], and 5H [Khanongnuch et al., 1998]. Similar observation was found in B. licheniformis; at 60oC the activity was optimal [Zhang et al., 2000]. Alkaliphilic Bacillus sp. strain JAMB-750 showed optimal activity at 55oC [Takeda et al., 2004]. The mannanase in present investigation was

Purification of a thermostable β-mannanase from Paenibacillus sp. DZ3

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Fig. 5. Effect of substrate concentration on β-mannanase activity. The Km and Vmax values of double reciprocal Lineweaver-Burke plot.

Fig. 4. Effects of pH on mannanase activity (A) stability (B). The effect of pH on mannanase activity monitored at 60oC using 50 mM of different buffers. remaining activity was measured after incubation for 1 4oC at various pH’s.

and was The h at

stable up to 60oC and retained about 20% of its activity at 80oC after 1 h of incubation. This thermostability is comparable to those reported for several mannanases from the thermophilic fungus and significantly higher than those of other B. subtilis strains [Mendoza et al., 1994b; Ooi and Kikuchi, 1995; Khanongnuch et al., 1998; Zakaria et al., 1998; Puchart et al., 2004; Jiang et al., 2006]. The thermal stability of the purified mannanase makes this enzyme more attractive for use in industrial applications. The optimal pH for the mannanase was pH 5.0 (Fig. 4A). Similarly, optimal pH 5.0 was also observed from B. subtilis NM-39 [Mendoza et al., 1994b]. Other Bacillus strains showed different optimal pH’s; the optimal pH of mannanase was pH 6.0 from B. subtilis WY4 [Jiang et al., 2006], pH 7.0 from B. subtilis KU-1 [Zakaria et al., 1998], Bacillus sp. [Ooi and Kikuchi, 1995], and pH 10.0 from Bacillus sp. strain JAMB-750 [Takeda et al., 2004]. Just as mannanases isolated from some B. subtilis strains [Mendoza et al., 1994b; Zakaria et al., 1998], the mannanase of the present

study was also stable over a wide pH range. Effects of metal ions and chemical reagents. The enzyme activity was stable in the presence of Fe2+, but was slightly activated by Cu2+ and Li+. The enzyme activity was strongly inhibited by Hg2+, Zn2+, Ni2+, Mg2+, and Ca2+. The mannanase from B. subtilis NM-39 was strongly inhibited by Ca2+ and slightly by Ag2+, and was not affected by most of the other metal ions [Mendoza et al., 1994b]. The enzyme activity was strongly inhibited by EDTA, Hg2+, SDS and Co2+. The mannanase activity was strongly inhibited by Ag+, Hg2+, and Fe3+ from B. subtilis 5H [Khanongnuch et al., 1998]. The mannanase was strongly inhibited by Hg2+, Cr2+, Ag+, Mn2+ and Cu2+ from B. subtilis KU-1 [Zakaria et al., 1998]. The enzyme activity 60-100% was inactivated by Fe3+, Fe2+, Pb2+, Hg2+ and Cd2+, whereas, in Mn2+, Ca2+, Ni2+, Mg2+, and Co2+, no effects were found [Takeda et al., 2004]. Substrate specificity and kinetic parameters. The relative activity of the purified mannanase on various substrates was determined. The mannanase exhibited high activity towards glucomannan (100%), galactomannan (86%), and starch (25%), but showed low activity towards mushroom powder (8%). MANB48 from B. circulans CGMCC 1416 was most effective at hydrolyzing locust bean and guar gums, with relative hydrolysis percentages of 100 and 26, respectively, but showed no activity for soluble starch and carboxymethyl cellulose (CMC) [Li et al., 2008]. Talbot and Sygusch [1990] reported that mannanase from B. stearothermophilus has no activity towards cellulosic substrates. Results of the present study, on the other hand, showed slight enzyme activity towards cellulosic substrates. This indicates the enzyme plays a significant role towards the enhancement of the industrial solubilization of lignocellulose. The kinetic parameters were also determined for glucomannan. The Km and Vmax values were 1.05 mg/mL and 714 U/mg, respectively (Fig. 5). The determined Vmax for glucomannan

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was higher than that of B. subtilis WY34 mannanase [Jiang et al., 2006]. In conclusion, there has been no report on purification and characterization of β-mannanase from Paenibacillus sp. DZ3. Therefore, in the present study, β-mannanase from Paenibacillus sp. DZ3 was isolated, purified, and characterized. This thermostable β-mannanase retained about 20% of its activity at 80oC after 1 h of incubation. Other reports showed no mannanase activity against cellulosic substrates [Ferreira and Filho, 2004], but the present study revealed that mannanase has slight activity against cellulosic substrates. The mannanase showed differences from the mannanases of other species. The results of present study collectively suggest locally isolated Paenibacillus sp. DZ3 and/or its extracellular enzyme could play a vital role in industrial applications. Acknowledgment. This work was supported by a grant from the Dong-A University Research Fund in Republic of Korea.

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