CC BY
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ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
© КОЛЛЕКТИВ АВТОРОВ, 2012
Jun Li Huang, Ling Xiao Bao, Han Yan Zou, Shu Gang Che, Gui Xue Wang
HIGH-LEVEL PRODUCTION OF A COLD-ACTIVE B-MANNANASE FROM BACILLUS SUBTILIS BS5 AND ITS MOLECULAR CLONING AND EXPRESSION
College of Bioengineering, Chonqing University, Chongqing 400044 Correspondence: huang_junli@126.com
Abstract. Mannanases can be useful in the food, feed, pulp and paper industries. In this research a Bacillus subtilis strain (named Bs5) which produced high-level P-mannanase was isolated. Maximum level of P-mannanase (1231.41 U/ml) was reached when Bacillus .subtilis Bs5 was grown on konjac powder as the carbon source for nine hours at 32 °C. The P-mannanase was a typical cold-active enzyme and its optimal temperature of 35 °C was the lowest among those of the known mannanases from bacteria. In addition, the optimal pH was 5.0 and much wide pH range from 3.0-8.0 was also observed in the P-mannanase. These properties make the P-mannanase more attractive for biotechnological applications. The DNA sequence coding the P-mannanase was cloned and the open reading frame consisted of 1089 bp encoding 362 amino acids. A phylogenetic tree of the P-mannanase based on the similarity of amino acid sequences revealed that the P-mannanase formed a cluster with the P-mannanases of Bacillus subtilis, which was separated from the mannanases of fungi and other bacteria. The P-mannanase gene could be expressed in Escherichia coli and the recombinant P-mannanase was characterized by Western blot. This study provided a new source of carbohydrate hydrolysis enzyme with novel characteristics from Bacillus subtilis.
Keywords: fi-Mannanase, bacillus subtilis, high-level production, cloning and expression
Introduction
Mannans are major components of the hemicellulose in the plants and some algae. Endo-P-1,4-mannanase (P-mannanase, EC 3.2.1.78) is the enzyme that catalyzes the endolytic hydrolysis of P-1,4-mannosidic linkages in mannopolysaccharides such as P-1,4-mannan, glucoman-nan, and galactomannan [1]. Thus, Mannanases can be useful in the processes in the food, feed, pulp and paper [1-3], as well as in the detergent industries [4, 5]. P-Mannanases are known to occur in various kinds of organisms, including plants[6], bacteria [7—11], fungi [12, 13] and molluscs [14—16]. Among these options, production of P-mannanases by microorganisms is more promising due to its low cost, high production rate, and readily controlled conditions.
Although some P-mannanases in bacteria and fungi [1, 17—19] have been isolated and characterized, the microorganisms with highlevel production of P-mannanases are in insufficiency. In addition, only three nucleotide sequences of the P-mannanase gene [20—23] were known until 1993. However, after studies clearly demonstrated the usefulness of mannanases in different industries [1—3], an increasing number of P-mannanase sequences were reported in fungi [24—28], bacteria [10, 23, 29—33], and plants [6].
Bacillus subtilis is a promising producer of P-mannanases [9, 10]. In this study, a bacterial strain which produced high-level P-mannanase (named BsMan in this paper) was isolated from soil. The strain was identified as Bacillus subtilis (named Bs5 in this paper) and was a potential industrial strain for P-mannanase production. Comparative studies of P-mannanases isolated from Bacillus strains so far showed that BsMan had the properties of the shorter period for maximum level of P-mannanase, higher-level production, lower optimal temperature and glycoprotein nature. The DNA coding sequence for BsMan was cloned, over expressed in Escherichia coli and characterized.
Materials and methods
Microorganism and media
Bacillus subtilis Bs5, a highly active P-mannanase producer used in this investigation, was newly isolated from the soil samples of Chongqing, China. The strain was identified by the Institute of
Microbiology, Chinese Academy of Sciences (IMCAS). The medium for mannanase production consisted of the following ingredients (w/v) in distilled water: 1.0% yeast extract, 1.0% beef peptone and 2.0% konjac powder. After growth at 32 °C and 150 rpm for different period, cells were removed by centrifugation (10,000 g). The resulted supernatant was used as crude enzyme preparation.
Escherichia coli JM109 was used as a host for maintenance and propagation of plasmids. E. coli Rosetta-gami (DE3) strain was used as the host for expression of BsMan. These strains were grown in LB medium.
Enzyme assays
BsMan activity was measured by the dinitrosalicylic acid (DNS) method as described [34]. The enzyme activity was assayed by mixing 0.1 ml of crude enzyme solution with 0.9 ml of 0.5% locust bean gum in 50 mM citrate buffer (pH 6.0) at 50 °C for 30 min. One unit (U) of enzyme activity was defined as the amount of enzyme producing 1 ^mol of mannose per minute under the assay conditions. The concentration of soluble proteins was determined according to the method [35] with BSA as a standard.
Effect of pH and temperature on BsMan activity
The effect of pH on BsMan activity was investigated at the pH range of 3.0-8.0 using 50 mM of sodium various buffers: citrate buffer (pH 3.06.0) and phosphate buffer (pH 6.0-8.0), at 50 °C with locust bean gum as the substrate. Substrate solution was prepared in the respective buffers and the crude enzyme was incubated in various buffers from pH 3.0 to 8.0 at 50 °C for 4 h, and then the residual activity was assayed.
To investigate the effect of temperature on BsMan activity, the crude enzyme solution was incubated with the substrate at temperatures ranging from 20-60 °C in 50mM citrate buffer at pH 6.0. Thermal stability of the enzyme was determined by assaying for residual enzyme activity after incubation at various temperatures for 15 min in 50 mM citrate buffer (pH 6.0).
DNA isolation and manipulation
Genomic DNA from B. subtilis Bs5 was isolated by the method [36]. Isolation of recombinant plasmid for DNA sequence analysis was performed by using the Qiagen Midi-Prep System (Qiagen,
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Fig. 1. Time course of BsMan production by Bacillus subtilis Bs5 at 32°C for 48 hours. Data were obtained from three independent experiments each with triplicates.
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Fig. 2. Effect of pH on BsMan activity (A) and stability (B). The influence of pH on BsMan activity was determined at 50 °C using 50 mM of different buffers. The remaining activity was measured after incubation for 4 h at 50°C over various pH. Data were obtained from three
independent experiments each with triplicates.
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Fig. 3. Effect of temperature on BsMan activity (A) and stability (B). The optimal temperature was measured at different temperatures. For determination of thermal stability, the residual activity of the treated mannanase was measured after 15 min pre-incubation at different temperatures at pH 6.0. Data were obtained from three independent experiments each with triplicates.
Inc., Chatsworth, Calif.). Restriction enzymes were purchased from Stratagene (Stratagene Cloning Systems, La Jolla, Calif.) and were used according to the manufacturer's instructions.
Cloning and expression of the BsMan
Primers design was performed according to the conserved DNA sequences of P-mannanase gene from Bacillus spp. The primers were designed using the Primer Express 1.0 (PE Applied Biosystems, Foster City, Calif.): BsMan-F: 5-CGCGGATCCTTGTTTAAGAAACATAC-3 BsMan-R: 5-CCCAAGCTTTCACTCAACGATTGGC-3. The underlined nucleotides in the primers are the restriction enzyme sites for BamHI and Hindni, respectively. The PCR reaction was performed as follows: the reaction mix (25 ^l) consisted of 1*PCR buffer [10 mM TrisHCl (pH 8.8), 50 mM KCl], 1.5 mM MgCl2, 0.2 ^M of each BsMan-F and BsMan-R primer, 0.1 mM dNTPs, 1 U ofpfu (Promega, Germany) and 50 ng of DNA template. The amplification was carried out in a Veriti Thermo cycler (Applied Biosystems, USA). After a denaturation step (95 °C for 5 min), 30 cycles of amplification (94 °C for 30 s, 60 °C for 1 min and 72 °C for 1.5 min) and 10 min at 72 °C were performed.
The purified PCR product was cloned into pMD19-T vector (TaKaRa, China) for sequencing and into pET-32a (Invitrogen, USA) vector between BamHI and HindIII sites for expressing recombinant BsMan, respectively. The generated recombinant plasmid was transformed into DE3 and the positive clone was used to produce BsMan.
Fig. 4. PCR amplification of BsMan gene from Bacillus subtilis Bs5. M: 100 bp ladder; 1: PCR product; 2: Negative control.
Fig. 5. Phylogenetic relationship of the BsMan from B. subtilis Bs5 with other P-mannanases. The neighbor joining tree was constructed by MEGA3.1 and PROML Bootstrap support was assessed by 1000 repetitive resampling of the data set and construction of the phylogeny.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 0.1% (w/v) SDS, 12% (w/v) polyacrylamide gel according to the method [37]. After the electrophoresis, the gel was stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 in 50% (v/v) methanol, 10% (v/v) acetic acid, and destained with 5% (v/v) methanol, 7% (v/v) acetic acid. For Western blot detection, the cell extracts of BsMan expressing strains were resolved on 12% SDS-PAGE gels and blotted onto nitrocellulose membranes. The membranes were probed with rabbit anti-His antibodies (Clontech) and the signals were visualized with alkaline phosphatase-coupled goat anti-rabbit antibodies (Sigma), and colorimetric detection with BCIP/ NBT substrate (Roche Applied Science) according to the instructions of the manufacturer.
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Fig. 6. The recombinant BsMan of total extract from E. coli cells (A) Proteins were loaded on SDS—10% polyacrylamide gel and stained with Coomassie brilliant blue. Lanes: M, Protein molecular mass markers; 1~4: Expression of BsMan induced by 0.1 mM IPTG added when the bacteria was cultured for 2, 4, 6 and 8 h, respectively; 5: Expression of BsMan without IPTG; 6: Expression of pET-32a induced by IPTG (B) Western blot of the recombinant BsMan. 1~2: Expression of BsMan induced by 0.1 mM IPTG added when the bacteria was cultured for 6 and 8 h, respectively; 3: Expression of pET-32a induced by IPTG.
RESULTS AND DISCUSSION Production of BsMan
The newly isolated B. subtilis Bs5 produced highlevel extracellular mannanase during growth in the medium (1.0% yeast extract and 2.0% konjac powder, Ph 6.0) at 32 °C (Fig. 1). Maximum level of BsMan (1234.41 U/ml) was reached after growth of nine hours of B. subtilis Bs5 under the above culture conditions.
To our knowledge, this is the first report on the high-level production of P-mannanase from B. subtilis in the shortest period, which produced a mannanase after growth of nine hours. Other B. subtilis strains isolated so far produced maximumlevel of mannanases in longer period [38, 39]. Mannanase production by B. .subtilis WY34 has been reported even after growth of four days [9].
Enzyme properties of BsMan
The optimum pH for BsMan activity was measured to be pH 5.0 by the standard method using locust bean gum (Fig. 2A). More than 60% of the maximum activity was maintained at pH 3.0-8.0 (Fig. 2B). The optimum temperature for BsMan activity was 35 °C (Fig. 3A) and more than 60% of the maximum activity was retained even at 45 °C (Fig. 3B). The enzyme became drastically inactive at temperatures above 50 °C (Fig. 3B).
Several mannanases of B. subtilis strains have been characterized. Although their molecular masses are similar to BsMan, the enzymatic properties are different. The optimal pH for BsMan was pH 5.0 (Fig. 1A), different from pH 6.0 from B. subtilis WY34 [9] and pH 7.0 from B. subtilis KU-1 [38] and Bacillus sp. [40]. Like mannanases isolated from other B. subtilis strains [11, 38], BsMan was also stable over a wide pH range, within pH 3.0-8.0 (Fig. 2B). BsMan shows biochemical features of typical cold-active enzymes. The optimum temperature for BsMan activity is 35 °C (Fig. 3A), which is the lowest among the known P-mannanases of bacteria [8, 9, 11, 23, 29, 32, 40, 41]. P-Mannanases from bacteria and most fungi are most active at temperatures higher than 40 °C [16]. BsMan activity exhibited narrow temperature range (Fig. 3B), however the temperature under which BsMan was produced (32°C) was close to its optimal temperature (35 °C), which makes this enzyme more attractive for industrial applications.
The p-mannanase gene (BsMan) of Bacillus subtilis Bs5 and its expression
A unique DNA fragment of approxima-H n 0 tely 1100 bp was amplified from B. subtilis Bs5 by BsMan-F/BsMan-R (Fig. 4). DNA sequence analysis of the P-mannanase gene (BsMan in this paper) revealed that BsMan consists of 1089 nucleotides coding for a protein with 362 amino acid residues. The nucleotide sequence of BsMan was deposited in GenBank under the accession number HM143944. The deduced amino acid sequence includes a signal peptide of 26 amino acids, which was identified by the SignalP3.0 program (http://www.cbs.dtu. dk/services/). The first 10 N-terminal amino acid sequence (HTVSPVNPNA) behind the signal peptide of the deduced amino acid showed high homology with the N-terminal region of the P-mannanase from B. subtilis WY34 [9]. No O-glycosylation site and one N-glycosylation site was observed in the amino acid sequence in the analysis with NetOGlyc3.1 and NetNGly1.0 (http://www. cbs.dtu.dk/services/).
A phylogenetic tree of the P-manna-nases based on the similarity of amino acid sequences reveals that BsMan forms
a cluster with the ß-mannanases of the Bacillus spp, which was separated from the mannanases of fungi and other bacteria (Fig. 5). The deduced amino acid sequence of BsMan is aligned well with that from Bacillus subilis (accession number: P55278).
The gene expression of BsMan was performed in the E. coli expression system. From the SDS-PAGE shown in Fig. 6A, the best induction of BsMan was obtained when 0.1 mM IPTG was added at 0.4 OD and the bacteria were cultured at 30 °C for 8 h. The molecular mass of the enzyme was estimated to be 41 kDa and the theoretical pi was 5.94, predicted by ExPASy (http://expasy.org/tools/protparam. html). BsMan was expressed in E.coli and the recombinant BsMan appeared as a single protein band on SDS—PAGE gel with a molecular mass of approx. 55 kDa and characterized by Western blot (Fig. 6B).
Conclusions
In this study we described a newly isolated Bacillus subtilis strain which produced high-level mannanase of 1231.41 U/ml when grown on konjac powder as the carbon source at 32°C and pH 6.0. BsMan is a novel cold-active endo-ß-1, 4-Dmannan hydrolase that we identified from Bacillus subtilis Bs5. It could offer a new enzyme source for mannan-hydrolysis industries which need especially a low temperature condition such as the food and the animal feed industries.
Acknowledgement. The authors acknowledge the research grant provided by the Fundamental Research Funds for the Central Universities (CDJZR10 23 00 21).
Note. The authors declare that they have no financial relationship with the organization that sponsored the research.
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