Evaluation of <TEX>${\beta}$</TEX>-1,4-Endoglucanases Produced by Bacilli Isolated from Paper and Pulp Mill Effluents Irrigated Soil
Evaluation of ${\beta}$-1,4-Endoglucanases Produced by Bacilli Isolated from Paper and Pulp Mill Effluents Irrigated Soil
Journal of Microbiology and Biotechnology. 2014. Aug, 24(8): 1073-1080
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : November 14, 2013
  • Accepted : April 13, 2014
  • Published : August 28, 2014
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Sangeeta, Pandey
Rameshwar, Tiwari
Surender, Singh
Lata, Nain
Anil Kumar, Saxena

A total of 10 cellulase-producing bacteria were isolated from soil samples irrigated with paper and pulp mill effluents. The sequencing of 16S rRNA gene revealed that all isolates belonged to different species of genus Bacillus . Among the different isolates, B. subtilis IARI-SP-1 exhibited a high degree of β-1,4-endoglucanase (2.5 IU/ml), β-1,4-exoglucanase (0.8 IU/ml), and β-glucosidase (0.084 IU/ml) activity, followed by B. amyloliquefaciens IARI-SP-2. CMC was found to be the best carbon source for production of endo/exoglucanase and β-glucosidase. The β-1,4-endoglucanase gene was amplified from all isolates and their deduced amino acid sequences belonged to glycosyl hydrolase family 5. Among the domains of different isolates, the catalytic domains exhibited the highest homology of 93.7%, whereas the regions of signal, leader, linker, and carbohydrate-binding domain indicated low homology (73-74%). These variations in sequence homology are significant and could contribute to the structure and function of the enzyme.
Earth’s most abundant renewable organic material, the lignocellulosic biomass, is the credible source of bioenergy and commodity chemicals, which can be made economically viable through identification of cellulase-producing bioagents having capability to accelerate the conversion of cellulose to glucose.
Cellulose is a crystalline structure having tightly packed bundles of microfibrils, which prevents penetration of hydrolytic enzymes and its enzymatic conversion to glucose. Owing to the physical nature of the substrate, the enzymatic conversion of cellulose to glucose is slow and economically unviable. The β-1,4 endoglucanase acts randomly along the cellulose chains. It produces small cellulose fragments and generates new sites on which exoglucanases act to produce cellobiose or oligosaccharides. The synergistic action of the enzymes endo-1,4-glucanase (E.C., exo-1,4-glucanase (E.C., and 1,4-β-D glucosidases (E.C. is required for complete hydrolysis of cellulose [8] .
β-1,4-Endoglucanase has many applications in same industries. It is used to remove color from denim to impart a good stonewashing effect [11] . In detergents, β-1,4- endoglucanase is used as an additive for color brightening, softening, and removal of soil particulate matter [21] . Furthermore, β-1,4-endoglucanase decreases the viscosity of β-glucan solution by random cleaving of the glycosidic bond of β-glucan. This process is useful in beer brewing and improving the quality of animal feed [8] . To date, numerous cellulases have been isolated and characterized from bacteria, fungi, and animal species [8] .
The microbial origin cellulase genes have been cloned and the nucleotide sequences of some of the endoglucanse genes have been identified [25] . The study of cellulolytic enzymes at the molecular level revealed that some of the modular features of these enzymes are attributable to their catalytic activity [17] . The sequence comparison indicated that the catalytic cores of cellulase belong to a restricted number of families with significant diversity in their activities [2] . Cellulases produced by two Bacillus strains isolated from hot springs of Zimbabwe exhibited 100% homology with the endoglucanase of Bacillus subtilis and belonged to GH 5 [9] . The endoglucanase of B. licheniformis of the same family has also been reported [3] .
Soils irrigated with untreated pulp and paper mill effluent over the years contain high alkalinity and organic matter. Irrigation with such effluents facilitates growth of lignocellulose degraders in these soils. Hence, these soils can be explored for mining of novel microbes and genes involved in the hydrolysis of cellulose. Studies conducted in the past have also reported a dominance of culturable lignocellulose degraders in such habitats [24 , 27] . Nonetheless, the diversity of cellulolytic bacteria and endoglucanase has not been explored extensively. The aim of this study was to explore the diversity of cellulolytic bacteria and the allelic variation in their endoglucanase genes. The sequences of these genes have been compared with known cellulase genes and their modular structures have been defined. Additionally, characterization of the enzyme was carried out for prediction of its protein based on sequences of genes amplified from different bacteria.
Materials and Methods
- Sample Collection
The Century Pulp and Paper Mill, Lalkuan, Uttarakhand (79°E10’E longitude and 29°3’N latitude) is one of the largest paper and pulp mills of India. The untreated effluent of this mill is utilized for irrigation of nearby agricultural lands. The soil samples were collected from these agricultural lands and transported immediately to the laboratory for further processing.
- Isolation and Screening of Cellulase-Producing Microorganisms
Carboxymethyl cellulase (CMCase)-producing bacteria were isolated by using carboxymethyl cellulose (CMC) agar containing 0.5% CMC; 0.1% NaNO 3 ; 0.1% K 2 HPO 4 ; 0.1%, KCl; 0.05% MgSO 4 ·7H 2 O; 0.05% yeast extract; and 1.5% agar, pH 7.0. All colonies were picked and spot inoculated in Reese’s minimal medium (RMM) containing 1% CMC to confirm their cellulase production potential, as described by Pandey et al . [16] . The substrate specificity of the crude enzyme was determined by performing the assay with different substrates; Avicel, CMC, and rice straw. All experiments were conducted in triplicates ( n = 3).
- Molecular Identification of Bacterial Isolates
For molecular characterization of the isolates, 16S rRNA gene sequencing was done. Isolation of genomic DNA was carried out by a standard DNA extraction protocol [20] . The extracted DNA was used as the template for PCR amplification of the 16S rRNA gene using universal 16S rRNA gene primers pA (5’-AGAGTT TGATCCTGGCTCAG-3’) and pH (5’-AAGGAGGTGATCCAG CCGCA -3’) to obtain a product of approximately 1,500 bp [4] . Aliquots of purified 16S rRNA PCR products were digested separately with three different restriction endonucleases ( Alu I, Msp I, and Hae III) in a 2 5 μl reaction volume by using the manufacturer’s recommended buffer and temperature. Restricted DNA was analyzed on 2% agarose gels. Strong and clear bands were scored for similarity and clustering analysis using the NTSYS-2.02e software package, Exeter software ( ). Similarity among the isolates was calculated by Jaccard’s coefficient and a dendrogram was constructed using the UPGMA method [12] . The 16S rRNA gene was sequenced by Xcelris Labs (Ahmedabad, India) using Sanger’s dideoxynucleotide sequencing method. A similarity search for the sequence was carried out using the BLAST program of the National Center of Biotechnology Information ( ).
- Hydrolytic Enzyme Production on Different Cellulosic Substrates
For enzyme production, cellulase-positive isolates were cultured in RMM (KH 2 PO 4 - 2g, (NH 4 ) 2 SO 4 - 1.4 g, KNO 3 - 1.4 g, MgSO 4 ·7H 2 O - 0.3 g, CaCl 2 ·2H 2 O - 0.3 g, FeSO 4 ·7H 2 O - 5 mg, MnSO 4 ·2H 2 O - 1.6 mg, ZnSO 4 ·7H 2 O - 1.4 mg, CoCl 2 ·6H 2 O - 2m g, Agar - 20 g, Distilled water - 1,000 ml) supplemented with CMC (1%), α-cellulose (1%), Avicel PH101 (1%), Sigmacell 101 (1%), or rice straw (1%) as the sole carbon source at 30℃ and pH 7.0 ± 0.2. After 72h of incubation, culture broths were centrifuged at 10,000 × g for 15 min at 4℃. The supernatant was collected and stored at 4℃ for further enzyme assays. The substrate specificity of the crude enzyme was determined by performing the assay with different substrates; Avicel, CMC, and rice straw. All experiments were conducted in triplicates ( n = 3).
- Enzyme Assays
The β-1,4-endoglucanase (CMCase) and β-1,4-exoglucanase (FPase) activity was determined by using CMC and Whatman no 1 filter paper, respectively, as substrate [5] and measuring the amount of reducing sugar [12] . One unit (IU) of filter paper activity or CMCase corresponded to 1 μmole of glucose formed per minute during hydrolysis. The β-glucosidase activity was assayed by measuring the amount of p -nitrophenol released from p -nitrophenyl-β-D-glucopyranoside [26] . One unit (IU) of β-glucosidase activity was defined as the amount of enzyme releasing 1 μmole of p -nitrophenol per milliliter of crude enzyme per minute.
- Designing of Primers and Amplification of β-1,4-Endoglucanase
Thirty-five sequences of bacterial β-1,4-endoglucanase were retrieved from NCBI GenBank. The amino acid sequences of these genes were aligned using T-Coffee alignment ( ). The alignment of the Bacillus sequences was further used in the primer design for amino acid positions 1 to 5 (MKRSI) and 505 to 509 (GTEPN) to obtain a complete coding region of β-1,4-endoglucanases. The degenerate primers endo-F (5’-ATGAARMGIWSIATH-3’) and endo-R (5’-RTTIGGYTCIGTNCCC- 3’) amplified approximately 1,500 bp of the β-1,4-endoglucanase gene. PCR conditions were optimized by gradient PCR by keeping the reactions at different annealing temperatures ranging from 51℃ to 56℃. The final reaction mixture (25 μl) contained each primer at a concentration of 0.5 mM, each dNTP mix at a concentration of 200 μM, 2.5 U of Taq DNA polymerase, 1.5 mM MgCl 2 , 20 ng of template DNA, and 2.5 μl of 10× PCR buffer. The following thermal profile was used for the PCR: 94℃, 2 min; 35 cycles of 94℃, 1 min; 53℃, 1 min; 72℃, 2 min; 1 cycle of 72℃, 10 min. The PCR product was purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA) and sequenced by Xcleris Lab (India).
- Sequence and Phylogenetic Analysis
Sequence fragments were assembled with the codon code aligner program ( ). The sequenced gene was compared with available sequences from GenBank using the BLASTX program ( ). The deduced amino acid sequence was analyzed with the EXPASY tool ( ). The signal peptide sequence of protein was predicted by the SignalP 3.0 server ( ) and conserved domain analysis was conducted with Pfam ( ). The phylogenetic tree was drawn with Mega 5.0 [22] . In order to statistically evaluate the confidence of branching, bootstrapping was carried out with data resampled 1,000 times. Possible open reading frames (ORFs) were identified with the ORF finder at the NCBI database ( http://www. ). Closely related protein sequences in the databases for the candidate cellulases were identified with BLASTN and BLASTP of NCBI. Module structures of the enzymes were predicted by the simple modular architecture research tool (SMART; ).
- Nucleotide Sequence Accession Numbers
The sequences generated in this study were deposited in NCBI GenBank. The 16S rRNA gene sequences retrieved from the cellulase-producing cultures were assigned accession numbers KF204578- KF204587. The accession numbers of β-1,4-endoglucanase genes obtained from these cultures were KF240847-KF240856.
- Isolation and Screening of Cellulase-Producing Bacteria
A total of 200 bacterial strains, isolated from effluent irrigated soil, were screened for the presence of cellulase-producing activity. Congo red test revealed that, out of 200 isolates, 10 isolates (IARI-SP-1 to IARI-SP-10) produced a clear halo zone around the colony on CMC agar plate and were taken as positive for cellulase activity.
- Effects of Cellulosic Sources on the Production of Cellulases
All the 10 isolates positive for β-1,4-endoglucanase were selected for quantitative estimation of cellulases. IARI-SP-1 was the best cellulase producer with the highest β-1,4-endoglucanase (2.5 IU/ml), β-1,4-exoglucanase (0.8 IU/ml), and β-glucosidase (0.084 IU/ml) activity, followed by IARI-SP-2 ( Fig. 1 ). Among different carbon sources tested, CMC, followed by rice straw, was found to be the best carbon source for the production of endo/exoglucanase and β-glucosidase by all isolates. The results also indicated that all the isolates invariably produced less β-glucosidase on all cellulosic substrates. The crude cellulase preparation of all 10 positive isolates was tested using various cellulosic substrates in order to provide a better understanding of the cellulase enzyme ( Table 1 ). All isolates recorded higher enzyme activity with CMC as the substrate in comparison with Avicel.
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Effects of different cellulosic sources on the production of (A) CMCase (B) FPase, and (C) β-glucosidase by different bacterial isolates.
Substrate specificity of the cellulases from differentBacillusstrains towards different substrates.
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The results are presented as the mean ± SD (standard deviation), n = 3.
- Molecular Identification
The RFLP analysis of the 16S rRNA gene of all 10 cellulase-producing morphotypes with three different restriction endonucleases ( Alu I, Msp I, and Hae III) indicated variations in the profile. A combined dendrogram constructed based on similarity percentage among the isolates indicated that all 10 cellulase-producing isolates belonged to different clusters ( Fig. 2 ). A partial 16S rRNA gene sequence of about 1,500 bp of all the endoglucanase-positive bacterial isolates was analyzed by BLAST and the sequences were deposited in NCBI GenBank. DNA sequencing and phylogenetic analysis revealed that all positive isolates showed 95-100% similarity with the known sequences in GenBank and belonged to different species of genus Bacillus ( Fig. 3 ).
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Dendrogram showing clustering of 10 isolates generated from RFLP analysis of the 16S rRNA gene by three restriction endonucleases (AluI, MspI, and HaeIII), using the UPGMA algorithim and Jaccard’s coefficient.
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Phylogenetic tree of culturable bacteria based on the 16S rRNA gene sequence isolated from soil irrigated with effluents of century paper mill.
- Phylogenetic Analysis of β-1,4-Endoglucanase Gene
The nucleotide sequences of the cloned β-1,4-endoglucanase genes were compared with those of the cellulase genes registered in the NCBI database, using the BLASTN program. The nucleotide sequences of IARI-SP-1 and IARI-SP-2 indicated 99% and that of IARI-SP-3 showed 98% similarity with β-1,4-endonuclease of B. subtilis strain BS-02. The nucleotide sequences of IARI-SP-4, IARI-SP-8, and IARI-SP-10 indicated 98%, 96%, and 97% similarity, respectively, with B. subtilis strain shu-3. IARI-SP-5, IARI-SP-6, IARI-SP-7, and IARI-SP-9 indicated 98%, 98%, 98%, and 99% identity with B. subtilis strain AH18, B. subtilis strain DR, Uncultured bacterium clone celWS14, and B. megaterium strain AP25, respectively ( Table 2 ). Based on amino acid sequences of the 10 identified ORFs, it was observed that all sequences are closely related to glycosyl hydrolase family 5 (GH5). The comparison of deduced amino acid sequences of the 10 bacterial isolates and B. subtilis strain BS-02 (GenBank Accession No. JF965375) reveals a close evolutionary relationship among the isolates ( Fig. 4 ). The analysis of the deduced amino acid sequences of the cellulases from our 10 isolates using the pfam program of Expasy revealed a modular enzyme composed of two discrete domains in the following order: catalytic domain (CD) (Q-48 through S-301) of the glucosyl hydrolase family 5/A2 (endoglucanase, E.C., and carbohydrate-binding domain (CBD) (V-356 through G-437) of family IIIa. Similar to the modular organization of many Bacillus endoglucanases, the CDs of these enzymes were located in the N-terminal region and the CBDs in the C-terminal region. There were 7 to 8 amino acid residue substitutions in the signal peptide region (1 through 29), and 22 amino acid residues were conserved among all of the cellulases, resulting in 79% homology. The leader region (30 through 47) consisted of 18 residues, and 16 amino acid residues among them were conserved, resulting in 89% homology. The CD consisted of 254 residues (48 through 301), and 238 amino acid residues were conserved among the cellulases, resulting in 93.7% homology. In the linker region (302 through 355) consisting of 54 residues, 40 amino acid residues were conserved among the cellulases, resulting in 74% homology. In the CBD (356 through 437) consisting of 83 residues, 57 amino acid residues were conserved among the cellulases, resulting in 68.6% homology. The CD showed the highest 93.7% homology, whereas the regions of signal, leader, linker, and CBD regions showed low homology. The β-1,4-endoglucanase gene encoded a signal peptide at the N-terminal end of the protein. The active site 131-H, 169-E, and 257-E are conserved among all isolates except IARI-SP-4, in which Q is present at position 257. IARI-SP-1 and IARI-SP-2, being the highest cellulase producers found in this study, were found to be 100% homologous for the β-1,4-endoglucanase gene.
Phylogenetic affiliations of the β-1,4-endoglucanase gene, isolated from differentBacillussp., with their nearest neighbor.
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Phylogenetic affiliations of the β-1,4-endoglucanase gene, isolated from different Bacillus sp., with their nearest neighbor.
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Alignment of deduced amino acid sequences of endoglucanases of the 10 isolates with B. subtilis strain shu-3. Region 1-29, signal peptide; Region 48-301, cellulase catalytic domain (glucosyl hydrolase family 5/A2); Region 302-355, linker region; Region 356-437, cellulose-binding domain (CBD family IIIa); Sites 131-H, 169-E, and 257-E, active sites (proton donor or nucleophile).
Soils perpetually irrigated with paper and pulp effluent represents a unique niche with a predominance of cellulose-degrading microorganisms, owing to their high organic matter content. The predominance of Bacillus and Bacillus -derived genera as dominant cellulase producers in the present study confirms the earlier reports from such environments [6 , 18] . Genus Bacillus , being endospore formers, are tolerant to the harsh conditions (high pH) prevailing in such soils, which makes them the major microflora of such environments [15] .
Enzyme assays indicated that all isolates invariably produced less β-glucosidase on different cellulosic substrates tested. However, all isolates had greater catalytic activity for CMC in comparison with other cellulosic substrates, which revealed that enzymes produced by all isolates could be categorized as endoglucanase with significant amount of exoglucanase activity. Many reports suggest that the cellulase systems from bacilli are incomplete and could not act on microcrystalline cellulose [1] , but others do confirm the exoglucanase activity in certain bacilli [9] . The exoglucanase activity of the crude enzymes might be due to the presence of a CBD in the β-1,4-endoglucanase. Earlier studies have indicated that the CBD plays crucial roles in crystalline cellulose hydrolysis, by binding to the cellulose surface and enhancing the sacchrification ability of endoglucanses [14 , 23] . In the present study, rice straw was found to be a good carbon source for production of cellulase and hence can be evaluated as a substrate for the large-scale production of cellulase, replacing costly substrates like CMC. There are several studies on cellulose degradation by Bacillus sp., and large variations in enzyme activity under both optimized and non-optimized conditions have been reported [3 , 7 , 9 , 18] . For example, Li et al . [7] have reported that in a Bacillus sp., maximum cellulase activity (0.26 U/ml) was detected when the culture was grown in Luria broth supplemented with 1% CMC at 37℃ for 24 h. Our data on the β-1,4-exoglucanase, β-1,4-endoglucanase, and β-glucosidase activities of all 10 isolates were generated from crude culture supernatants, but were encouraging as all 10 bacterial isolates exhibited considerably higher endoglucanase activities (ranging from 1.1 to 2.5 IU/ml) than earlier reported [18 , 19 , 28] . The maximum CMCase activity in the alkaline range may be due to the alkaline condition prevailing in the soil. The nucleotide sequencing and BLAST search of β-1,4-endoglucanase revealed limited variations, indicating a close evolutionary relationship among the isolates. The high proportion of cellulase enzyme belonging to the GH5 family among bacilli found in our study is in agreement with earlier reports [3 , 9] . The CDs of these enzymes were located in the N-terminal region and the CBDs in the C-terminal region, like for endoglucanases of many other Bacillus species earlier reported. Currently, the CDs of polysaccharides are grouped into at least 15 of the more than 80 known glycosyl hydrolase families, whereas CBDs fall into at least 13 families. Two basic residues at positions 2 and 3 (lysine and arginine) in the hydrophilic leader region were followed by a hydrophobic core of 18 amino acid residues rich in leucine and isoleucine, which is consistent with the findings of Mezes et al . [10] . The β-1,4- endoglucanase gene encoded a signal peptide at the N-terminal end of the protein and was characterized by a short, hydrophilic, basic region along with a subsequent long, hydrophobic region. The presence of a signal peptide suggests higher extracellular cellulase activity observed for various strains screened during the present study.
The soils irrigated with pulp and paper mill effluent specifically enrich cellulolytic bacilli that exhibit both endo-and exoglucanase activities. The analysis of the β-1,4-endoglucanase gene isolated from Bacillus sp. provides a new insight into the potential capacity of the microbial community to hydrolyze the lignocellulosic biomass. Although all sequences found in the present study belonged to the GH5 family, sequence analysis revealed significant variations among them. These variations need to be further exploited to correlate the efficiency of the enzyme, and its ability to hydrolyze crystalline cellulose and also to be secreted out of the cell in large amounts. Research is under way to clone and overexpress the endoglucanase gene in a suitable expression vector so that the product could be utilized in the biofuel program being carried out at the Institute.
The authors acknowledge funding from NAIP (70-24) and ICAR National Fund for Basic, Strategic and Frontier Application Research in Agriculture and sponsoring the research project. The first author thanks the Indian Agricultural Research Institute for providing fellowship during the doctoral program.
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