Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6613-2
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Cloning, molecular modeling, and docking analysis of alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 Prakram Singh Chauhan 1 & Satya Prakash Tripathi 2 & Abhays T. Sangamwar 2 & Neena Puri 3 & Prince Sharma 1 & Naveen Gupta 1
Received: 8 January 2015 / Revised: 9 April 2015 / Accepted: 15 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract An alkali-thermostable β-mannanase gene from Bacillus nealsonii PN-11 was cloned by functional screening of E. coli cells transformed with pSMART/HaeIII genomic library. The ORF encoding mannanase consisted of 1100 bp, corresponding to protein of 369 amino acids and has a catalytic domain belonging to glycoside hydrolase family 5. Cloned mannanase was smaller in size than the native mannanase by 10 kDa. This change in molecular mass could be because of difference in the glycosylation. The tertiary structure of the β-mannanase (MANPN11) was designed and it showed a classical (α/β) TIM-like barrel motif. Active site of MANPN11 was represented by 8 amino acid residues viz., Glu152, Trp189, His217, Tyr219, Glu247, Trp276, Trp285, and Tyr287. Model surface charge of MANPN11 predicted that surface near active site was mostly negative, and the opposite side was positive which might be responsible for the stability of the enzymes at high pH. Stability of MANP N11 at alkaline pH was further supported by the formation of a
Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6613-2) contains supplementary material, which is available to authorized users. * Prince Sharma [email protected] * Naveen Gupta [email protected] 1
Department of Microbiology, BMS Block, Panjab University South Campus, Chandigarh, India
2
Department of Pharmacoinformatics, National Institute of Pharmaceutical Science Education and Research, S.A.S, Nagar, Mohali, Punjab, India
3
Department of Industrial Microbiology, Guru Nanak Khalsa College, Yamunanagar, Haryana, India
hydrophobic pocket near active site of the enzyme. To understand the ability of MANPN11 to bind with different substrates, docking studies were performed and found that mannopentose fitted properly into active site and form stable enzyme substrate complex. Keywords Bacillus nealsonii . Alkali-thermostable enzyme . Cloning . β-Mannanasegene . Homologymodeling . Docking
Introduction β-Mannanase (endo-1,4-β-D-mannanase, EC 3.2.1.78) is a hydrolase that catalyzes the random hydrolysis of β-D-1,4mannosidic linkages in the main chain of mannans and releases linear/branched oligosaccharides of various lengths (Chauhan et al. 2012, 2014a). Microbial mannanases are mainly extracellular and can act in wide range of pH and temperature because of which they have found applications in pulp, pharmaceutical, food, feed, and coffee industry (Chauhan et al. 2012, 2014a). Most important application of alkaline mannanases is in pulp and paper industry. Treating the pulp with β-mannanase in combination with xylanase improves lignin extraction. In doing so, we can reduce hazardous wastes which may harm the environment (George et al. 2014a, b; Sondhi et al. 2014). Nucleotide as well as amino acid sequence analysis gives an important insight into the structure of any enzyme at molecular level (Zhao et al. 2011). In the absence of high cost of analysis through atomic resolution, X-ray diffraction, and nuclear magnetic resonance spectroscopy (Santos et al. 2012), amino acid sequence provides most of the information required for determining and characterizing the molecule’s function and physical and chemical properties. Computationally based characterization of the features of proteins in completely
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sequenced proteomes is an important task in the search for knowledge of protein function (Singh et al. 2011). Previously, we isolated, optimized, purified, and characterized an extracellular alkali-thermostable β-mannanase (MANPN11) from the Bacillus nealsonii PN-11 having role in pulp biobleaching, reduction in viscosity of coffee extract, as well as in production of mannooligosaccharides having prebiotic potential (Chauhan et al. 2014b, c, d, e). Because of the industrial importance of this mannanase, it was important to understand structural functional relationship at molecular level. In this regard, the present study describes the cloning and detailed sequence analysis of MANPN11. The study also aims at adopting homology modeling and docking studies to elucidate the three dimensional structure, active site machinery and enzyme-substrate interaction of this enzyme. Analysis has also been done to identify the probable regions for stability of the enzyme in extreme conditions.
Material and methods Strains and vector The bacterial strain Bacillus nealsonii PN-11 which produce an alkali-thermostable β-mannanase (MANPN11) was isolated earlier in our laboratory (Chauhan et al. 2014b) (Microbial Type Culture Collection (MTCC) No. 11401; Chandigarh, India). Electrocompetent E. coli DH10β was used as the host for gene cloning and expression linearized (blunt ends) and dephosphorylated pSMART LC-Amp plasmid (Lucigen Corporation, USA) was used as the cloning vector.
Media Luria-Bertani medium (LB) containing 1 % (w/v) bactotryptone, 0.5 % (w/v) yeast extract, and 1 % (w/v) NaCl, at a pH of 7–7.5, was used for cultivation of E. coli and Bacillus nealsonii PN-11. LB agar contained additionally 2 % (w/v) agar. One hundred micrograms of ampicillin per milliliter was added for growing the transformants.
Chemicals and reagents Locust bean gum (LBG), guar gum (GG), bovine serum albumin (BSA), sephadex-150, and DEAE-cellulose were purchased from Sigma-Aldrich Co. (St. Louis, USA). HaeIII and HindIII enzymes were purchased from New England Biolabs (USA). Ampicillin and dinitrosalicylic acid (DNSA) were purchased from HiMedia, India. All other chemicals and reagents used in this study were of analytical grade.
Computational software All computational experiments were carried out using MODELLER 9.12, SYBYL7.1, GLIDE ver 5.7 and PyMOL molecular modeling packages on a Sun workstation with Red hat enterprise linux 3 and Silicon graphics fuel workstation with IRIX 6.5 operating system. Generation of genomic library Genomic DNA was prepared as described by Ausubel et al. (2006). The DNA was partially digested by blunt-end tetra cutter enzyme HaeIII and run on a preparative agarose gel along with λ/HindIII marker. The agarose gel piece containing desired fragments (2–6 kb) was excised, and DNA was extracted using the QIAquick Gel Extraction Kit (QIAGEN). Ligation was done using pSMART vector at 22 °C for 4 h. The electrocompetent E. coli cells suspension was mixed with ligation mix and electroporated. Then, 100 μl of transformed cells were plated on Luria agar plates containing ampicillin (100 μg ml−1). The plates were incubated at 37 °C for 72 h. Transformants were screened on Luria agar+ampicillin plate containing (locust bean gum 0.5 %) by replica plate method. The transformants showing zone of clearance after staining with congo red were selected. Plasmid was isolated and gene inserts were confirmed by restriction analysis. Sequence analysis of the insert The insert was sequenced using standard sequencing primers of pSMART vector (Lucigen Corporation, USA) and then doing step sequencing. The nucleotide sequence was analyzed using the NCBI ORF Finder tool (http://www.ncbi.him.nih. gov/gorf/gorf/gorf.html). The nucleotide sequence of βmannanase from Bacillus nealsonii PN-11 was submitted in GenBank under accession number KF425325. Nucleotide sequence and derived amino acid sequences were aligned using the BLASTn and BLASTp programs (http://www.ncbi.nlm. nih.gov/BLAST/), respectively. Putative N-glycosylation sites were predicted by the NetNGlyc 1.0 program (http://www. cbs.dtu.dk/services/NetNGlyc). InterProScan (http://www. ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi and http://www. ebi.ac.uk/Tools/pfa/iprscan/) and MultAlin (Corpet 1988) were used to search the conserved domains and execute multiple alignments. Protein purification and molecular weight determination E. coli DH10β cells harboring mannanase gene from Bacillus nealsonii PN-11 were grown in Luria broth containing 100 μg ml−1 ampicillin. Cell-free supernatant was used as enzyme and mannanase was purified to homogeneity by using combination of ammonium sulfate precipitation (60–80 %),
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gel filtration (Sephadex G-150), and ion exchange chromatography (DEAE-Cellulose). Activity of the enzyme was determined by activity staining (zymography). All the purification steps and zymographic analysis were carried out according to the method of Chauhan et al. (2014d). Glycosylation of proteins was checked by staining with periodic acid Schiff (PAS) as reported by Zacharius et al. (1969).
Active site and surface charge analysis The information about the active site of mannanase was obtained from Catalytics site atlas (CSA) database of European bioinformatics institute (http://www.ebi.ac.uk/thorntonsrv/ databases/CSA/) and surface charge analysis was performed with Discovery studio 2.5 (Accelrys, USA) using the βmannanase from Bacillus sp. JAMB-602 (1WKY) as the template.
Enzyme assay Molecular docking study of modeled mannanase Mannanase activity was assayed by measuring the amount of reducing sugars released by the enzyme using locust bean gum as a mannan substrate (Chauhan et al. 2014b). One unit of mannanase activity was defined as the micromoles of a mannose release by 1 ml of enzyme in 1 min under standard assay conditions.
Mannanase homology modeling Homology modeling of MANPN11 was performed using the crystal structure of Bacillus sp. JAMB-602 AMN5A mannanase (PDB code 1WKY) as template (Akita et al. 2004). Homology modeling was carried out using MODELLER 9.12. Construction of protein models by homology modeling techniques involved different steps: sequence alignment between target (MANPN11) and the template sequence (AMN5A); building an initial crude model; and refining the model and evaluating the quality of final homology model using different structure validation tools.
The 3D structures of the selected ligands mannose, mannobiose, mannotriose, mannotetraose, and mannopentaose (Fig. S1) which are known to be effective substrate for mannanase enzyme were built in Sybyl7.1 software package. The 3D structures were minimized by Powell method. The optimized 3D structures of the ligands were further prepared for molecular docking studies using LigPrep module of Maestro 9.3. The best modeled structure of MANPN11 was prepared using Protein Preparation Wizard tool of Maestro 9.3. Molecular docking of substrates was performed using Glide software included in Schrodinger Suit 9.0.01 (Schrodinger, Inc. Portland, www.schrodinger.com). Glide is a ligand docking program for predicting protein-ligand binding modes and ranking ligands. Receptor Grid Generation Wizard of GLIDE 5.7 was used to generate grid of 20 Å around predicted active site using the default values of all the parameters; ten poses were generated for each substrate.
Results Model structure refinement and validation Cloning and sequence analysis Initially, 100 models of MANPN11 were generated using MODELLER, out of them model having the best discrete optimized protein energy score (DOPE) and molecular PDF score (molpdf) was selected for structure refinement and validation. Loop refinement module of MODELLER was used to refine the loops of the model and its quality was further evaluated by Ramachandran plot in the SAVES and PROCHECK validation package. In order to further improve the structure quality, it was subjected to Molecular operating environment (MOE) refinement using energy refine module in MOE program. The MOE report of the MANPN11 model having best packing quality was generated showing its stereochemical significance which includes planarity, chirality, phi/psi preferences, chi angles, non-bonded contact distances, unsatisfied donors, and acceptors. In this report, the value of the final MANPN11 model was compared with the high resolution structures from PDB and its statistical significance is presented.
Bacillus nealsonii PN-11 produces an important alkalithermostable mannanase which has application in pulp, food, and other industries (Chauhan et al. 2014b, c, d, e). To study the enzyme at molecular level, which can be helpful for further improvement of enzyme properties, β-mannanase gene from Bacillus nealsonii PN-11 was cloned by functional screening of E. coli cells transformed with pSMART/HaeIII genomic library. Out of the 5 × 103 transformants, a mannanasepositive transformant was selected; on sequencing, it was found to have an insert size of 3174 bp. The NCBI ORF finder revealed the presence of eight ORFs in the same orientation. The proposed mannanase enzyme coding ORF (ORF 2) contained 1100 bp yielding a protein of 369 amino acids and theoretical molecular mass of 41 kDa. Consensus sequences for promoter -10 (TAATA), -35 (TTGA CA), and ribosome-binding site (GGAGG) were located upstream of the start codon. Based on sequence similarity results,
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the deduced amino acid sequence of mannanase (MANPN11) from Bacillus nealsonii PN-11 shared highest similarity with the β-mannanase from Bacillus sp. JAMB-602 (69 %, BAD99527.1) followed by Paenibacillus sp. CH-3 (66 %, AEX60762.1), Vibrio sp. MA-138 (45 %, BAA25188.1), Pseudomonas sp. ND137 (40 %, BAB79290.2), Clostridium butyricum (64 %, WP003428823.1) which indicated that MANPN11 was a typical GH5 β-mannanase (Fig. 1). Interproscan analysis of MANPN11 showed the presence of only one domain in the protein which was similar to that of typical catalytic domain (33-319 AA) of glycoside hydrolase family 5. However, on comparison, closest similar mannanase (Bacillus sp. JAMB-602) showed the presence of two domains, one catalytic domain of GH family-5 on N-terminal and other carbohydrate binding domain on C-terminal (Fig. S2), indicating the absence of carbohydrate-binding domain in MANPN11. The sequence showed the presence of two glutamic acid residues, E152 and E247, which are conserved in all member of GH family 5 and have been postulated to participate in catalysis. Further sequence analysis indicated the probable N-glycosylation site at 179 and 342 amino acid. Theoretical pI of MANPN11 was found to be 5.35. Protein purification and molecular weight determination Cloned mannanase was purified, and the results of the purification are summarized in Table 1. It was observed that after the final purification step, the enzyme was purified to 58.84fold with a recovery of 5.09 %. The specific activity determined using locust bean gum as substrate was 107.69 U mg−1 protein. The SDS-PAGE and activity staining of the purified MANPN11 gave a single band with a molecular mass of Fig. 1 Amino acid sequence alignment of MANPN11 with five closest GH 5 β-mannanases using the program Multalin. Residues participating in catalytic action as nucleophile or proton donor E152 and E247 are marked by square column
41 kDa which was smaller in size than the native mannanase of Bacillus nealsonii PN-11 (Chauhan et al. 2014d) by 10 kDa (Fig. 2). However, when native and cloned mannanase were compared both the enzymes were found to have similar characteristics (unpublished data). In PAS analysis native mannanase was stained indicating the presence of carbohydrate moieties whereas cloned mannanase was not stained (Fig. S3). Mannanase homology modeling The homology model structure of the MANPN11 was obtained from the MODELLER software. One hundred different MANPN11 models were generated, and the 78th model was chosen as the best model on the basis of most stabilized form taking into account minimum DOPE score and molpdf score. After structural refinement [using molecular operating environment (MOE)] geometric quality of backbone conformations, residue interactions, residue contacts, and energy profile of the modeled MANPN11 structure were well within the restrictions established for reliable structures. The quality of the MANPN11 homology model before and after MOE refinement in the closed state was assessed by Ramachandran plot and is depicted in Fig. S4. Residues (85.9 %) in core region, 13.7 % in the allowed region, 0.4 % in the generously allowed region, and no residues in the disallowed region showed good stereochemical structural quality of the MANP N11 model. This indicated that in refined model, backbone dihedral angles phi (φ) and psi (ψ) occupy reasonable accurate position in the 3D model (Fig. S4). G-factor for MANP N11 model was also computed, which is a measure of stereochemical property of the model (dihedrals, bond lengths, and
Appl Microbiol Biotechnol Table 1
Summary of the purification procedures for cloned MANPN11 from E. coli
Purification steps
Volume (ml)
Activity (U ml−1)
Protein conc. (mg ml−1)
Total activity (U)
Total protein (mg)
Specific activity (U mg−1)
Purification fold
Recovery (%)
Crude (NH)2SO4 precipitation Sepphadex-150 chromatography DEAE-cellulose ion exchange chromatography
250 12.0 2.0
5.5 38.9 68.0
3.0 19.0 7.5
1375 466.8 136.0
750 228.0 15.0
1.83 2.04 9.06
1.00 1.11 4.95
100 33.94 9.84
1.0
70.0
0.65
70.0
0.65
107.69
58.84
5.09
angles), positive G-value indicates normal and negative value indicates abnormal stereochemistry of the model. Final refined MANPN11 model had an overall G-factor of 0.12 showing its normal stereochemistry. As observed values for structural parameters were closed to the expected value it depicted the good stereochemistry and model structure quality (Table S1). Overall quality factor of the model was 90.87 %. The final refined homology modeled structure of MANPN11 [classical (α/β)8 TIM-like barrel motif] is shown in Fig. 3.
JAMB-602) (Fig. 4a). Amino acids of active site along with some other amino acids Arg50, Trp59, Asn151, and His217 formed a hydrophobic pocket which might be a factor contributing to the alkaline stability of the enzyme (Fig. 4b). Analysis of the model surface charge of MANPN11 at pH 7.4 (internal cellular pH of the E.coli in which MANPN11 was expressed) using Discovery Studio 2.5 MODELER predicted that the surface near active site cleft was mostly negative (Fig. 5a), and the opposite side was positive (Fig. 5b). This may also relate to the stability of the enzyme in alkaline solutions.
Active site and surface charge analysis
Molecular docking study of modeled mannanase
The information about the active site was obtained through superimposing 3D model structure of the MANPN11 with that of template structure of mannanase from Bacillus sp. JAMB602 (having maximum similarity with MANPN11), which provided accuracy of homology between two structures and also helped in positioning the conserved active site residues. Active site of MANPN11 was represented by 8 amino acid residues viz., Glu152, Trp189, His217, Tyr219, Glu247, Trp276, Trp285, and Tyr287 which corresponded to Glu158, Trp195, His223, Tyr225, Glu253, Trp282, Trp291, and Tyr293 of template protein (mannanase from Bacillus
To find out the best substrate, docking studies were carried out with modeled MANPN11 using different substrate viz., mannose, mannobiose, mannotriose, mannotetraose, and mannopentaose. It was found that mannopentaose (M5), fitted appropriately into the active site and formed stable enzyme substrate complex (Fig. 6). This was confirmed by minimum Glide score (−6.23) for mannopentaose (Table 2). The neighboring amino acids, which possibly formed hydrogen bonds and stacking interactions with the bound M5 at individual subsites are shown in Fig. 7a, b. Most of the aromatic residues in each subsite potentially formed both hydrogen bonds and stacking interactions. The mannose residue at subsite +2 formed hydrogen bond between its OH3 and OH6 positions to the side chains of Arg117 and Asp284, respectively. The hydroxyl groups (OH1, OH2, and OH6) of mannose residue at subsite −1 formed hydrogen bond with side chain of Trp189, Trp285, and Lys251, respectively. At subsite −2 hydrogen bonds are formed between hydroxyl group (OH5 and OH6) of mannose residue and carboxylic group of Glu152 and Glu247.
Discussion
Fig. 2 Comparison between molecular weight of cloned and native mannanase: a SDS-PAGE: lane 1, molecular weight marker; lane 2, cloned mannanase; lane 3, native mannanase. b Zymographic analysis: lane 1, cloned mannanase; lane 2, native mannanase
Cloning of the gene helps in amplification and provides information about its sequence, function, and evolutionary relationship, which helps in modification of any protein. More than 50 % of the industrially important enzymes are now produced from genetically engineered microorganisms
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Fig. 3 Tertiary structure of MANPN11 generated by using the GH5 βmannanase from Bacillus sp. JAMB-602 (1WKY) (having maximum similarity with MANPN11) as the template. The conserved catalytic residues of GH 5 β-mannanase, acid/base catalyst Glu152 and nucleophile Glu247, are indicated
(Ghosh et al. 2013; Lv et al. 2013). Several reports have been published in the past decade on the isolation and manipulation of microbial mannanase genes with the aim of enzyme overproduction, studying the structure of proteins and alter the enzymes properties to increase their commercial applicability (Chauhan et al. 2012; Gálvez et al. 2013).
Fig. 4 a Superimposition of active-site residues of modeled mannanase and template (1WKY). Green and red color sticks represent modeled and template proteins, respectively. b Model of the solvent-accessible surface of MANPN11. The colored circle of residues indicates the shallow-dishshaped active center of MANPN11. The catalytic groups Glu152 and Glu247 are in yellow. Different residues are gathered round the active site to provide a hydrophobic platform for the binding of mannan (color figure online)
Bacillus nealsonii PN-11 produces an important alkalithermostable mannanase (MANPN11) which have diverse industrial application in pulp, food and other industries (Chauhan et al. 2014b, c, d, e). In this regard, MANPN11 was cloned and expressed in E. coli, BLAST analysis showed that it was smaller in size than the closest homologus mannanases. It is known that mannanases have two domains: catalytic domain on N-terminal and carbohydrate-binding domain on C-terminal (Hatada et al. 2005). Analysis showed that mannanase from Bacillus nealsonii PN-11 was having a catalytic domain but no carbohydrate-binding domain (CBD), which can be the reason for its smaller size. A number of mannanases are there in which CBD is not present (Hatada et al. 2005; He et al. 2008). Similarity of MANPN11 to GH family 5 and subfamily 8 correlated with its alkaline characteristics as it is known that mannanase of this subfamily are alkaline mannanases (Zhao et al. 2011). Higher optimal pH (8.8) of MANPN11 activity than its pI relates to its alkaline stability. It is known that if pH of activity is greater than pI then there is an excess of OH– ions which react with the –COOH to form excess –COO– ions and water which might stabilize the enzymes in alkaline solutions (Mamo et al. 2009). SDS-PAGE as well as zymography analysis of the cloned mannanase showed that it was smaller in size than the native mannanase of Bacillus nealsonii PN-11 by 10 kDa. However, when native and cloned mannanases were characterized they were found to be similar with respect to their all characteristic. This indicated that cloned protein was most probably same as that of native with some change in its molecular mass. This change in molecular mass could be because of post translational modification such as glycosylation, phosphorylation, sulfation, or deamination (Yoshida et al. 1998). PAS analysis showed that native mannnanase was having carbohydrate moieties which were not seen in cloned enzyme. This indicated that glycosylation of native mannanase might have been the reason for its higher molecular mass than the cloned enzyme. Mannanase might have been glycosylated in Grampositive Bacillus nealsonii PN-11 after its translation but not in Gram-negative E. coli 10β. This was further confirmed by the sequence analysis which showed possible glycosylation sites (179 and 342) in the sequence. Glycosylation of proteins is a known phenomenon in Gram-positive organisms (Gálvez et al. 2013; Hakamada et al. 2014; He et al. 2008; Wang et al. 2014); however, glycosylation is not common in Gramnegative organisms (Dell et al. 2011). Tertiary structure of the MANPN11 was designed by MODELLER software and further refined by homology modeling which showed a classical (α/β) TIM-like barrel motif for glycoside hydrolase families (Stoll et al. 2005). Analysis of the model surface charge of MANPN11 predicted that surface near active site was mostly negative, and the opposite side was positive. This type of charge distribution is
Appl Microbiol Biotechnol Table 2 Glide docking score for binding of mannose, mannobiose, mannotriose, manotetraose, and mannopentaose with MANPN11 derived by molecular docking studies Substrates
Glide docking score (kcal mol−1)
Mannose Mannobiose Mannotriose Mannotetraose Mannopentaose
−2.87 −3.02 −3.91 −5.88 −6.23
of hydrophobic pocket is known to bring change in the polarity, charge distribution, and/or hydrogen bonding in the microenvironment of the active site of these mannanases, which might in turn affect the protonation state of key catalytic amino acids, and subsequently determine the pH optimum of the enzyme (Coughlan et al. 2001). Changing the microenvironment of the active site by site-directed mutagenesis has been shown to increase the pH optimum of glucoamylase from Aspergillus awamori (Fang and Ford 1998). Docking study provide valuable information about the residues, which are involved in enzyme substrate interactions. The hydrogen bonding and stacking interactions are very
Fig. 5 Charge distribution on the surface of a three-dimensional model of MANPN11 using Discovery Studio 2.5 MODELER. Charges on the surface were calculated at pH 7.4. Negative charges in red and positive charges are depicted in blue. a The surface showing the substrate cleft with the active site. b The opposite side surface (color figure online)
known to be responsible for the stability of the enzymes at high pH (Shirai et al. 2001). The stability of MANPN11 at alkaline pH was further supported by the formation of a hydrophobic pocket near the active site of the enzyme. This type
Fig. 6 Best fitted docked conformation of mannopentaose (M5) into active site cavity of MANPN11
Fig. 7 The binding interaction of modeled MANPN11 mannanase and mannopentaose a ligand–protein interactions as obtained by docking studies. Orange and green color sticks represent the active site residues whereas mannopentaose is represented by multicolored sticks. b Schematic representation of mannanase-M5 complex showed possible hydrogen bond (—) and stacking interaction in the individual subsites (color figure online)
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important aspects to study the subsites in substrate binding site of enzymes because both are actually involved in binding mechanism between enzyme and units of substrate before the enzymatic hydrolysis reaction. These studies are also aimed to determine the potential amino acid residues that formed bond with substrate units at individual subsite of enzyme (Kumar et al. 2013). Among the 1000 model/ confirmation generated by GLIDE server, optimal interaction, and the best GLIDE score were used as criteria to describe the best confirmation. The mannose rings of mannopentaose molecule fitted into the active site. This analysis indicated that mannanase from Bacillus nealsonii PN-11 was an endomannanase because smaller polysaccharides such as mannobiose and mannotriose were not able to properly interact with the enzyme. These results correlated with the experimental findings where end-product analysis by high pressure anion exchange chromatography (HPAEC) showed that this mannanase was an endomannanase (Chauhan et al. 2014d) In conclusion, cloning and sequence analysis of MANP N11 helped to understand its primary, secondary and tertiary structure. It showed that in this mannanase CBD is missing therefore it can be used as a system to study the role of such domain in enzyme catalysis. It also helped to understand the possible reasons for its stability at high temperature and high pH. This knowledge can be helpful for the further improvement of this commercially important enzyme.
Acknowledgments Prakram Singh Chauhan is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing a Senior Research Fellowship. Conflict of interest interest.
The authors declare no competing financial
References Akita M, Takeda N, Hirasawa K, Sakai H, Kawamoto M, Yamamoto M, Grant WD, Hatada Y, Ito S, Horikoshi K (2004) Crystallization and preliminary X-ray study of alkaline mannanase from an alkaliphilic Bacillus isolate. Acta Crystallogr D Biol Crystallogr 60:1490–1492 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidmann JD, Smith JA, Struhl K (2006) Current Protocols in Molecular Biology, Vol 1, Greene Publishing Association, Wiley-Interscience, New York Chauhan PS, Puri N, Sharma P, Gupta N (2012) Mannanases: microbial sources, production, properties and potential biotechnological applications. Appl Microbiol Biotechnol 93:1817–1830 Chauhan PS, George N, Sondhi S, Puri N, Gupta N (2014a) An overview of purification strategies for microbial mannanases. Int J Pharm Bio Sci 5:176–192 Chauhan PS, Soni SK, Sharma P, Saini A, Gupta N (2014b) A mannanase from Bacillus nealsonii PN-11: statistical optimization of production and application in biobleaching of pulp in combination with xylanase. Int J Pharma Bio Sci 5:237–251 Chauhan PS, Bharadwaj A, Puri N, Gupta N (2014c) Optimization of medium composition for alkali-thermostable mannanase production
by Bacillus nealsonii PN-11 in submerged fermentation. Int J Curr Microbiol Appl Sci 3:1033–1045 Chauhan PS, Sharma P, Puri N, Gupta N (2014d) Purification and characterization of an alkali-thermostable β-Mannanase from Bacillus nealsonii PN-11 and its application in manno-oligosaccharides preparation having prebiotic potential. Eur Food Res Technol 238:927– 936 Chauhan PS, Sharma P, Puri N, Gupta N (2014e) A process for reduction in viscosity of coffee extract by enzymatic hydrolysis of mannan. Bioprocess Biosyst Eng 3:1459–67 Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890 Coughlan S, Wang XG, Britton KL, Stillman TJ, Rice DW, Chiaraluce R, Consalvi V, Scandurra R, Engel PC (2001) Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme’s unusual pH dependence. Biochim Biophys Acta 1544:10–17 Dell A, Galadari A, Sastre F, Hitchen P (2011) Similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes. Int J Microbiol. doi:10.1155/2010/148178 Fang TY, Ford C (1998) Protein engineering of Aspergillus awamori glucoamylase to increase its pH optimum. Protein Eng 11:383–388 Gálvez CA, Ospina JV, Sanchez PG, Zornosa RA (2013) Cloning and biochemical characterization of an endo-1,4-β-mannanase from the coffee berry borer Hypothenemus hampei. BMC Res Notes 6:333. doi:10.1186/1756-0500-6-333 George N, Chauhan PS, Puri N, Gupta N (2014a) Statistical optimization of process parameters for production of alkaline protease from Vibrio metschnikovii NG155 having application in leather industry. Int J Pharma Bio Sci 5:509–517 George N, Chauhan PS, Kumar V, Puri N, Gupta N (2014b) An approach to ecofriendly leather: lime and sulphide free dehairing of skin and hide using bacterial alkaline protease at industrial scale. J Clean Prod 79:249–257 Ghosh A, Luís AS, Brás JLA, Fontes CMGA, Goyal A (2013) Th e r m o s t a b l e r ec o m b i n a n t β ( 1 → 4 ) - m a n n a n a s e f r o m C. thermocellum: biochemical characterization and mannooligosaccharides production. J Agric Food Chem 61:12333–12344 Hakamada Y, Ohkubo Y, Ohashi S (2014) Purification and characterization of β-mannanase from Reinekea sp. KIT-YO10 with transglycosylation activity. Biosci Biotechnol Biochem. doi:10. 1080/09168451.2014.895658 Hatada Y, Takeda N, Hirasawa K, Ohta Y, Usami R, Yoshida Y, Ito S, Horikoshi K (2005) Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JAMB-750, its expression in B. subtilis and characterization of the recombinant enzyme. Extremophiles 9:497–500 He X, Liu N, Zhang Z, Zhang B, Ma Y (2008) Inducible and constitutive expression of a novel thermo stable alkaline β-mannanase from alkalophilic Bacillus sp. N16-5 in Pichia pastoris and characterization of the recombinant enzyme. Enzyme Microb Technol 43:13–18 Kumar L, Dutt D, Tapas S, Kumar P (2013) Purification and biochemical characterization, homology modeling and active side binding mode interaction of thermo-alkali-stable β-1,4 endoxylanase from Coprinus cinerus LK-D-NCIM-1369. Biocat Agric Biotechnol 2: 267–277 Lv J, Chen Y, Pei H, Yang W, Li Z, Dong B, Cao Y (2013) Cloning, expression, and characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris. Appl Biochem Biotechnol 169:2326–2340. doi:10.1007/s12010-013-0156-8 Mamo G, Thunnissen M, Kaul R, Mattiasson B (2009) An alkaline active xylanase: insights into mechanisms of high pH catalytic adaptation. Biochimie 91:1187–1196 Santos CR, Paiva JH, Meza AN, Cota J, Alvarez TM, Ruller R, Prade RA, Squina FM, Murakami MT (2012) Molecular insights into
Appl Microbiol Biotechnol substrate specificity and thermal stability of a bacterial GH5CBM27 endo-1,4-β-D-mannanase. J Struct Biol 177:469–76 Shirai T, Ishida H, Noda J, Yamane T, Ozaki K, Hakamada Y, Ito S (2001) Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme. J Mol Biol 310:1079–1087 Singh RP, Rai AR, Choudhury KR, Dubey RC (2011) Homology modeling and sequence analysis of a highly thermostable endo-(1,4)-betamannanase from the marine bacterium Rhodothermus marinus. Int J App Sci Environ Sanit 6:485–494 Sondhi S, Sharma P, George N, Chauhan PS, Puri N, Gupta N (2014) A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 having potential to biobleach softwood pulp. 3Biotech. doi:10.1007/s13205-014-0207-z Stoll D, LeNours J, Anderson L, Stalbrand H, Loleggio L (2005) The structure and characterization of a modular endo-β-1,4-mannanase from Cellulomonas fimi. Biochemistry 44:12700–12708
Wang Y, Vilaplana F, Brumer H, Aspeborg H (2014) Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5-7) mannanase from Arabidopsis thaliana. Planta 239: 653–665 Yoshida S, Sako Y, Uchida A (1998) Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for an enzyme from Bacillus circulans K-1 that degrades guar gum. Biosci Biotechnol Biochem 62:514–520 Zacharius RM, Zell TE, Morrison JH, Woodlock JJ (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem 30:148–152 Zhao Y, Zhang Y, Cao Y, Qi J, Mao L, Xue Y, Gao F, Peng H, Wang X, Gao GF, Ma Y (2011) Structural analysis of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5: implications for adaptation to alkaline conditions. Plos One 6(1):e14608. doi:10.1371/journal. pone.0014608
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Cloning, molecular modeling, and docking analysis of alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 Prakram Singh Chauhan 1 & Satya Prakash Tripathi 2 & Abhays T. Sangamwar 2 & Neena Puri 3 & Prince Sharma 1 & Naveen Gupta 1
Received: 8 January 2015 / Revised: 9 April 2015 / Accepted: 15 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract An alkali-thermostable β-mannanase gene from Bacillus nealsonii PN-11 was cloned by functional screening of E. coli cells transformed with pSMART/HaeIII genomic library. The ORF encoding mannanase consisted of 1100 bp, corresponding to protein of 369 amino acids and has a catalytic domain belonging to glycoside hydrolase family 5. Cloned mannanase was smaller in size than the native mannanase by 10 kDa. This change in molecular mass could be because of difference in the glycosylation. The tertiary structure of the β-mannanase (MANPN11) was designed and it showed a classical (α/β) TIM-like barrel motif. Active site of MANPN11 was represented by 8 amino acid residues viz., Glu152, Trp189, His217, Tyr219, Glu247, Trp276, Trp285, and Tyr287. Model surface charge of MANPN11 predicted that surface near active site was mostly negative, and the opposite side was positive which might be responsible for the stability of the enzymes at high pH. Stability of MANP N11 at alkaline pH was further supported by the formation of a
Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6613-2) contains supplementary material, which is available to authorized users. * Prince Sharma [email protected] * Naveen Gupta [email protected] 1
Department of Microbiology, BMS Block, Panjab University South Campus, Chandigarh, India
2
Department of Pharmacoinformatics, National Institute of Pharmaceutical Science Education and Research, S.A.S, Nagar, Mohali, Punjab, India
3
Department of Industrial Microbiology, Guru Nanak Khalsa College, Yamunanagar, Haryana, India
hydrophobic pocket near active site of the enzyme. To understand the ability of MANPN11 to bind with different substrates, docking studies were performed and found that mannopentose fitted properly into active site and form stable enzyme substrate complex. Keywords Bacillus nealsonii . Alkali-thermostable enzyme . Cloning . β-Mannanasegene . Homologymodeling . Docking
Introduction β-Mannanase (endo-1,4-β-D-mannanase, EC 3.2.1.78) is a hydrolase that catalyzes the random hydrolysis of β-D-1,4mannosidic linkages in the main chain of mannans and releases linear/branched oligosaccharides of various lengths (Chauhan et al. 2012, 2014a). Microbial mannanases are mainly extracellular and can act in wide range of pH and temperature because of which they have found applications in pulp, pharmaceutical, food, feed, and coffee industry (Chauhan et al. 2012, 2014a). Most important application of alkaline mannanases is in pulp and paper industry. Treating the pulp with β-mannanase in combination with xylanase improves lignin extraction. In doing so, we can reduce hazardous wastes which may harm the environment (George et al. 2014a, b; Sondhi et al. 2014). Nucleotide as well as amino acid sequence analysis gives an important insight into the structure of any enzyme at molecular level (Zhao et al. 2011). In the absence of high cost of analysis through atomic resolution, X-ray diffraction, and nuclear magnetic resonance spectroscopy (Santos et al. 2012), amino acid sequence provides most of the information required for determining and characterizing the molecule’s function and physical and chemical properties. Computationally based characterization of the features of proteins in completely
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sequenced proteomes is an important task in the search for knowledge of protein function (Singh et al. 2011). Previously, we isolated, optimized, purified, and characterized an extracellular alkali-thermostable β-mannanase (MANPN11) from the Bacillus nealsonii PN-11 having role in pulp biobleaching, reduction in viscosity of coffee extract, as well as in production of mannooligosaccharides having prebiotic potential (Chauhan et al. 2014b, c, d, e). Because of the industrial importance of this mannanase, it was important to understand structural functional relationship at molecular level. In this regard, the present study describes the cloning and detailed sequence analysis of MANPN11. The study also aims at adopting homology modeling and docking studies to elucidate the three dimensional structure, active site machinery and enzyme-substrate interaction of this enzyme. Analysis has also been done to identify the probable regions for stability of the enzyme in extreme conditions.
Material and methods Strains and vector The bacterial strain Bacillus nealsonii PN-11 which produce an alkali-thermostable β-mannanase (MANPN11) was isolated earlier in our laboratory (Chauhan et al. 2014b) (Microbial Type Culture Collection (MTCC) No. 11401; Chandigarh, India). Electrocompetent E. coli DH10β was used as the host for gene cloning and expression linearized (blunt ends) and dephosphorylated pSMART LC-Amp plasmid (Lucigen Corporation, USA) was used as the cloning vector.
Media Luria-Bertani medium (LB) containing 1 % (w/v) bactotryptone, 0.5 % (w/v) yeast extract, and 1 % (w/v) NaCl, at a pH of 7–7.5, was used for cultivation of E. coli and Bacillus nealsonii PN-11. LB agar contained additionally 2 % (w/v) agar. One hundred micrograms of ampicillin per milliliter was added for growing the transformants.
Chemicals and reagents Locust bean gum (LBG), guar gum (GG), bovine serum albumin (BSA), sephadex-150, and DEAE-cellulose were purchased from Sigma-Aldrich Co. (St. Louis, USA). HaeIII and HindIII enzymes were purchased from New England Biolabs (USA). Ampicillin and dinitrosalicylic acid (DNSA) were purchased from HiMedia, India. All other chemicals and reagents used in this study were of analytical grade.
Computational software All computational experiments were carried out using MODELLER 9.12, SYBYL7.1, GLIDE ver 5.7 and PyMOL molecular modeling packages on a Sun workstation with Red hat enterprise linux 3 and Silicon graphics fuel workstation with IRIX 6.5 operating system. Generation of genomic library Genomic DNA was prepared as described by Ausubel et al. (2006). The DNA was partially digested by blunt-end tetra cutter enzyme HaeIII and run on a preparative agarose gel along with λ/HindIII marker. The agarose gel piece containing desired fragments (2–6 kb) was excised, and DNA was extracted using the QIAquick Gel Extraction Kit (QIAGEN). Ligation was done using pSMART vector at 22 °C for 4 h. The electrocompetent E. coli cells suspension was mixed with ligation mix and electroporated. Then, 100 μl of transformed cells were plated on Luria agar plates containing ampicillin (100 μg ml−1). The plates were incubated at 37 °C for 72 h. Transformants were screened on Luria agar+ampicillin plate containing (locust bean gum 0.5 %) by replica plate method. The transformants showing zone of clearance after staining with congo red were selected. Plasmid was isolated and gene inserts were confirmed by restriction analysis. Sequence analysis of the insert The insert was sequenced using standard sequencing primers of pSMART vector (Lucigen Corporation, USA) and then doing step sequencing. The nucleotide sequence was analyzed using the NCBI ORF Finder tool (http://www.ncbi.him.nih. gov/gorf/gorf/gorf.html). The nucleotide sequence of βmannanase from Bacillus nealsonii PN-11 was submitted in GenBank under accession number KF425325. Nucleotide sequence and derived amino acid sequences were aligned using the BLASTn and BLASTp programs (http://www.ncbi.nlm. nih.gov/BLAST/), respectively. Putative N-glycosylation sites were predicted by the NetNGlyc 1.0 program (http://www. cbs.dtu.dk/services/NetNGlyc). InterProScan (http://www. ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi and http://www. ebi.ac.uk/Tools/pfa/iprscan/) and MultAlin (Corpet 1988) were used to search the conserved domains and execute multiple alignments. Protein purification and molecular weight determination E. coli DH10β cells harboring mannanase gene from Bacillus nealsonii PN-11 were grown in Luria broth containing 100 μg ml−1 ampicillin. Cell-free supernatant was used as enzyme and mannanase was purified to homogeneity by using combination of ammonium sulfate precipitation (60–80 %),
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gel filtration (Sephadex G-150), and ion exchange chromatography (DEAE-Cellulose). Activity of the enzyme was determined by activity staining (zymography). All the purification steps and zymographic analysis were carried out according to the method of Chauhan et al. (2014d). Glycosylation of proteins was checked by staining with periodic acid Schiff (PAS) as reported by Zacharius et al. (1969).
Active site and surface charge analysis The information about the active site of mannanase was obtained from Catalytics site atlas (CSA) database of European bioinformatics institute (http://www.ebi.ac.uk/thorntonsrv/ databases/CSA/) and surface charge analysis was performed with Discovery studio 2.5 (Accelrys, USA) using the βmannanase from Bacillus sp. JAMB-602 (1WKY) as the template.
Enzyme assay Molecular docking study of modeled mannanase Mannanase activity was assayed by measuring the amount of reducing sugars released by the enzyme using locust bean gum as a mannan substrate (Chauhan et al. 2014b). One unit of mannanase activity was defined as the micromoles of a mannose release by 1 ml of enzyme in 1 min under standard assay conditions.
Mannanase homology modeling Homology modeling of MANPN11 was performed using the crystal structure of Bacillus sp. JAMB-602 AMN5A mannanase (PDB code 1WKY) as template (Akita et al. 2004). Homology modeling was carried out using MODELLER 9.12. Construction of protein models by homology modeling techniques involved different steps: sequence alignment between target (MANPN11) and the template sequence (AMN5A); building an initial crude model; and refining the model and evaluating the quality of final homology model using different structure validation tools.
The 3D structures of the selected ligands mannose, mannobiose, mannotriose, mannotetraose, and mannopentaose (Fig. S1) which are known to be effective substrate for mannanase enzyme were built in Sybyl7.1 software package. The 3D structures were minimized by Powell method. The optimized 3D structures of the ligands were further prepared for molecular docking studies using LigPrep module of Maestro 9.3. The best modeled structure of MANPN11 was prepared using Protein Preparation Wizard tool of Maestro 9.3. Molecular docking of substrates was performed using Glide software included in Schrodinger Suit 9.0.01 (Schrodinger, Inc. Portland, www.schrodinger.com). Glide is a ligand docking program for predicting protein-ligand binding modes and ranking ligands. Receptor Grid Generation Wizard of GLIDE 5.7 was used to generate grid of 20 Å around predicted active site using the default values of all the parameters; ten poses were generated for each substrate.
Results Model structure refinement and validation Cloning and sequence analysis Initially, 100 models of MANPN11 were generated using MODELLER, out of them model having the best discrete optimized protein energy score (DOPE) and molecular PDF score (molpdf) was selected for structure refinement and validation. Loop refinement module of MODELLER was used to refine the loops of the model and its quality was further evaluated by Ramachandran plot in the SAVES and PROCHECK validation package. In order to further improve the structure quality, it was subjected to Molecular operating environment (MOE) refinement using energy refine module in MOE program. The MOE report of the MANPN11 model having best packing quality was generated showing its stereochemical significance which includes planarity, chirality, phi/psi preferences, chi angles, non-bonded contact distances, unsatisfied donors, and acceptors. In this report, the value of the final MANPN11 model was compared with the high resolution structures from PDB and its statistical significance is presented.
Bacillus nealsonii PN-11 produces an important alkalithermostable mannanase which has application in pulp, food, and other industries (Chauhan et al. 2014b, c, d, e). To study the enzyme at molecular level, which can be helpful for further improvement of enzyme properties, β-mannanase gene from Bacillus nealsonii PN-11 was cloned by functional screening of E. coli cells transformed with pSMART/HaeIII genomic library. Out of the 5 × 103 transformants, a mannanasepositive transformant was selected; on sequencing, it was found to have an insert size of 3174 bp. The NCBI ORF finder revealed the presence of eight ORFs in the same orientation. The proposed mannanase enzyme coding ORF (ORF 2) contained 1100 bp yielding a protein of 369 amino acids and theoretical molecular mass of 41 kDa. Consensus sequences for promoter -10 (TAATA), -35 (TTGA CA), and ribosome-binding site (GGAGG) were located upstream of the start codon. Based on sequence similarity results,
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the deduced amino acid sequence of mannanase (MANPN11) from Bacillus nealsonii PN-11 shared highest similarity with the β-mannanase from Bacillus sp. JAMB-602 (69 %, BAD99527.1) followed by Paenibacillus sp. CH-3 (66 %, AEX60762.1), Vibrio sp. MA-138 (45 %, BAA25188.1), Pseudomonas sp. ND137 (40 %, BAB79290.2), Clostridium butyricum (64 %, WP003428823.1) which indicated that MANPN11 was a typical GH5 β-mannanase (Fig. 1). Interproscan analysis of MANPN11 showed the presence of only one domain in the protein which was similar to that of typical catalytic domain (33-319 AA) of glycoside hydrolase family 5. However, on comparison, closest similar mannanase (Bacillus sp. JAMB-602) showed the presence of two domains, one catalytic domain of GH family-5 on N-terminal and other carbohydrate binding domain on C-terminal (Fig. S2), indicating the absence of carbohydrate-binding domain in MANPN11. The sequence showed the presence of two glutamic acid residues, E152 and E247, which are conserved in all member of GH family 5 and have been postulated to participate in catalysis. Further sequence analysis indicated the probable N-glycosylation site at 179 and 342 amino acid. Theoretical pI of MANPN11 was found to be 5.35. Protein purification and molecular weight determination Cloned mannanase was purified, and the results of the purification are summarized in Table 1. It was observed that after the final purification step, the enzyme was purified to 58.84fold with a recovery of 5.09 %. The specific activity determined using locust bean gum as substrate was 107.69 U mg−1 protein. The SDS-PAGE and activity staining of the purified MANPN11 gave a single band with a molecular mass of Fig. 1 Amino acid sequence alignment of MANPN11 with five closest GH 5 β-mannanases using the program Multalin. Residues participating in catalytic action as nucleophile or proton donor E152 and E247 are marked by square column
41 kDa which was smaller in size than the native mannanase of Bacillus nealsonii PN-11 (Chauhan et al. 2014d) by 10 kDa (Fig. 2). However, when native and cloned mannanase were compared both the enzymes were found to have similar characteristics (unpublished data). In PAS analysis native mannanase was stained indicating the presence of carbohydrate moieties whereas cloned mannanase was not stained (Fig. S3). Mannanase homology modeling The homology model structure of the MANPN11 was obtained from the MODELLER software. One hundred different MANPN11 models were generated, and the 78th model was chosen as the best model on the basis of most stabilized form taking into account minimum DOPE score and molpdf score. After structural refinement [using molecular operating environment (MOE)] geometric quality of backbone conformations, residue interactions, residue contacts, and energy profile of the modeled MANPN11 structure were well within the restrictions established for reliable structures. The quality of the MANPN11 homology model before and after MOE refinement in the closed state was assessed by Ramachandran plot and is depicted in Fig. S4. Residues (85.9 %) in core region, 13.7 % in the allowed region, 0.4 % in the generously allowed region, and no residues in the disallowed region showed good stereochemical structural quality of the MANP N11 model. This indicated that in refined model, backbone dihedral angles phi (φ) and psi (ψ) occupy reasonable accurate position in the 3D model (Fig. S4). G-factor for MANP N11 model was also computed, which is a measure of stereochemical property of the model (dihedrals, bond lengths, and
Appl Microbiol Biotechnol Table 1
Summary of the purification procedures for cloned MANPN11 from E. coli
Purification steps
Volume (ml)
Activity (U ml−1)
Protein conc. (mg ml−1)
Total activity (U)
Total protein (mg)
Specific activity (U mg−1)
Purification fold
Recovery (%)
Crude (NH)2SO4 precipitation Sepphadex-150 chromatography DEAE-cellulose ion exchange chromatography
250 12.0 2.0
5.5 38.9 68.0
3.0 19.0 7.5
1375 466.8 136.0
750 228.0 15.0
1.83 2.04 9.06
1.00 1.11 4.95
100 33.94 9.84
1.0
70.0
0.65
70.0
0.65
107.69
58.84
5.09
angles), positive G-value indicates normal and negative value indicates abnormal stereochemistry of the model. Final refined MANPN11 model had an overall G-factor of 0.12 showing its normal stereochemistry. As observed values for structural parameters were closed to the expected value it depicted the good stereochemistry and model structure quality (Table S1). Overall quality factor of the model was 90.87 %. The final refined homology modeled structure of MANPN11 [classical (α/β)8 TIM-like barrel motif] is shown in Fig. 3.
JAMB-602) (Fig. 4a). Amino acids of active site along with some other amino acids Arg50, Trp59, Asn151, and His217 formed a hydrophobic pocket which might be a factor contributing to the alkaline stability of the enzyme (Fig. 4b). Analysis of the model surface charge of MANPN11 at pH 7.4 (internal cellular pH of the E.coli in which MANPN11 was expressed) using Discovery Studio 2.5 MODELER predicted that the surface near active site cleft was mostly negative (Fig. 5a), and the opposite side was positive (Fig. 5b). This may also relate to the stability of the enzyme in alkaline solutions.
Active site and surface charge analysis
Molecular docking study of modeled mannanase
The information about the active site was obtained through superimposing 3D model structure of the MANPN11 with that of template structure of mannanase from Bacillus sp. JAMB602 (having maximum similarity with MANPN11), which provided accuracy of homology between two structures and also helped in positioning the conserved active site residues. Active site of MANPN11 was represented by 8 amino acid residues viz., Glu152, Trp189, His217, Tyr219, Glu247, Trp276, Trp285, and Tyr287 which corresponded to Glu158, Trp195, His223, Tyr225, Glu253, Trp282, Trp291, and Tyr293 of template protein (mannanase from Bacillus
To find out the best substrate, docking studies were carried out with modeled MANPN11 using different substrate viz., mannose, mannobiose, mannotriose, mannotetraose, and mannopentaose. It was found that mannopentaose (M5), fitted appropriately into the active site and formed stable enzyme substrate complex (Fig. 6). This was confirmed by minimum Glide score (−6.23) for mannopentaose (Table 2). The neighboring amino acids, which possibly formed hydrogen bonds and stacking interactions with the bound M5 at individual subsites are shown in Fig. 7a, b. Most of the aromatic residues in each subsite potentially formed both hydrogen bonds and stacking interactions. The mannose residue at subsite +2 formed hydrogen bond between its OH3 and OH6 positions to the side chains of Arg117 and Asp284, respectively. The hydroxyl groups (OH1, OH2, and OH6) of mannose residue at subsite −1 formed hydrogen bond with side chain of Trp189, Trp285, and Lys251, respectively. At subsite −2 hydrogen bonds are formed between hydroxyl group (OH5 and OH6) of mannose residue and carboxylic group of Glu152 and Glu247.
Discussion
Fig. 2 Comparison between molecular weight of cloned and native mannanase: a SDS-PAGE: lane 1, molecular weight marker; lane 2, cloned mannanase; lane 3, native mannanase. b Zymographic analysis: lane 1, cloned mannanase; lane 2, native mannanase
Cloning of the gene helps in amplification and provides information about its sequence, function, and evolutionary relationship, which helps in modification of any protein. More than 50 % of the industrially important enzymes are now produced from genetically engineered microorganisms
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Fig. 3 Tertiary structure of MANPN11 generated by using the GH5 βmannanase from Bacillus sp. JAMB-602 (1WKY) (having maximum similarity with MANPN11) as the template. The conserved catalytic residues of GH 5 β-mannanase, acid/base catalyst Glu152 and nucleophile Glu247, are indicated
(Ghosh et al. 2013; Lv et al. 2013). Several reports have been published in the past decade on the isolation and manipulation of microbial mannanase genes with the aim of enzyme overproduction, studying the structure of proteins and alter the enzymes properties to increase their commercial applicability (Chauhan et al. 2012; Gálvez et al. 2013).
Fig. 4 a Superimposition of active-site residues of modeled mannanase and template (1WKY). Green and red color sticks represent modeled and template proteins, respectively. b Model of the solvent-accessible surface of MANPN11. The colored circle of residues indicates the shallow-dishshaped active center of MANPN11. The catalytic groups Glu152 and Glu247 are in yellow. Different residues are gathered round the active site to provide a hydrophobic platform for the binding of mannan (color figure online)
Bacillus nealsonii PN-11 produces an important alkalithermostable mannanase (MANPN11) which have diverse industrial application in pulp, food and other industries (Chauhan et al. 2014b, c, d, e). In this regard, MANPN11 was cloned and expressed in E. coli, BLAST analysis showed that it was smaller in size than the closest homologus mannanases. It is known that mannanases have two domains: catalytic domain on N-terminal and carbohydrate-binding domain on C-terminal (Hatada et al. 2005). Analysis showed that mannanase from Bacillus nealsonii PN-11 was having a catalytic domain but no carbohydrate-binding domain (CBD), which can be the reason for its smaller size. A number of mannanases are there in which CBD is not present (Hatada et al. 2005; He et al. 2008). Similarity of MANPN11 to GH family 5 and subfamily 8 correlated with its alkaline characteristics as it is known that mannanase of this subfamily are alkaline mannanases (Zhao et al. 2011). Higher optimal pH (8.8) of MANPN11 activity than its pI relates to its alkaline stability. It is known that if pH of activity is greater than pI then there is an excess of OH– ions which react with the –COOH to form excess –COO– ions and water which might stabilize the enzymes in alkaline solutions (Mamo et al. 2009). SDS-PAGE as well as zymography analysis of the cloned mannanase showed that it was smaller in size than the native mannanase of Bacillus nealsonii PN-11 by 10 kDa. However, when native and cloned mannanases were characterized they were found to be similar with respect to their all characteristic. This indicated that cloned protein was most probably same as that of native with some change in its molecular mass. This change in molecular mass could be because of post translational modification such as glycosylation, phosphorylation, sulfation, or deamination (Yoshida et al. 1998). PAS analysis showed that native mannnanase was having carbohydrate moieties which were not seen in cloned enzyme. This indicated that glycosylation of native mannanase might have been the reason for its higher molecular mass than the cloned enzyme. Mannanase might have been glycosylated in Grampositive Bacillus nealsonii PN-11 after its translation but not in Gram-negative E. coli 10β. This was further confirmed by the sequence analysis which showed possible glycosylation sites (179 and 342) in the sequence. Glycosylation of proteins is a known phenomenon in Gram-positive organisms (Gálvez et al. 2013; Hakamada et al. 2014; He et al. 2008; Wang et al. 2014); however, glycosylation is not common in Gramnegative organisms (Dell et al. 2011). Tertiary structure of the MANPN11 was designed by MODELLER software and further refined by homology modeling which showed a classical (α/β) TIM-like barrel motif for glycoside hydrolase families (Stoll et al. 2005). Analysis of the model surface charge of MANPN11 predicted that surface near active site was mostly negative, and the opposite side was positive. This type of charge distribution is
Appl Microbiol Biotechnol Table 2 Glide docking score for binding of mannose, mannobiose, mannotriose, manotetraose, and mannopentaose with MANPN11 derived by molecular docking studies Substrates
Glide docking score (kcal mol−1)
Mannose Mannobiose Mannotriose Mannotetraose Mannopentaose
−2.87 −3.02 −3.91 −5.88 −6.23
of hydrophobic pocket is known to bring change in the polarity, charge distribution, and/or hydrogen bonding in the microenvironment of the active site of these mannanases, which might in turn affect the protonation state of key catalytic amino acids, and subsequently determine the pH optimum of the enzyme (Coughlan et al. 2001). Changing the microenvironment of the active site by site-directed mutagenesis has been shown to increase the pH optimum of glucoamylase from Aspergillus awamori (Fang and Ford 1998). Docking study provide valuable information about the residues, which are involved in enzyme substrate interactions. The hydrogen bonding and stacking interactions are very
Fig. 5 Charge distribution on the surface of a three-dimensional model of MANPN11 using Discovery Studio 2.5 MODELER. Charges on the surface were calculated at pH 7.4. Negative charges in red and positive charges are depicted in blue. a The surface showing the substrate cleft with the active site. b The opposite side surface (color figure online)
known to be responsible for the stability of the enzymes at high pH (Shirai et al. 2001). The stability of MANPN11 at alkaline pH was further supported by the formation of a hydrophobic pocket near the active site of the enzyme. This type
Fig. 6 Best fitted docked conformation of mannopentaose (M5) into active site cavity of MANPN11
Fig. 7 The binding interaction of modeled MANPN11 mannanase and mannopentaose a ligand–protein interactions as obtained by docking studies. Orange and green color sticks represent the active site residues whereas mannopentaose is represented by multicolored sticks. b Schematic representation of mannanase-M5 complex showed possible hydrogen bond (—) and stacking interaction in the individual subsites (color figure online)
Appl Microbiol Biotechnol
important aspects to study the subsites in substrate binding site of enzymes because both are actually involved in binding mechanism between enzyme and units of substrate before the enzymatic hydrolysis reaction. These studies are also aimed to determine the potential amino acid residues that formed bond with substrate units at individual subsite of enzyme (Kumar et al. 2013). Among the 1000 model/ confirmation generated by GLIDE server, optimal interaction, and the best GLIDE score were used as criteria to describe the best confirmation. The mannose rings of mannopentaose molecule fitted into the active site. This analysis indicated that mannanase from Bacillus nealsonii PN-11 was an endomannanase because smaller polysaccharides such as mannobiose and mannotriose were not able to properly interact with the enzyme. These results correlated with the experimental findings where end-product analysis by high pressure anion exchange chromatography (HPAEC) showed that this mannanase was an endomannanase (Chauhan et al. 2014d) In conclusion, cloning and sequence analysis of MANP N11 helped to understand its primary, secondary and tertiary structure. It showed that in this mannanase CBD is missing therefore it can be used as a system to study the role of such domain in enzyme catalysis. It also helped to understand the possible reasons for its stability at high temperature and high pH. This knowledge can be helpful for the further improvement of this commercially important enzyme.
Acknowledgments Prakram Singh Chauhan is thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing a Senior Research Fellowship. Conflict of interest interest.
The authors declare no competing financial
References Akita M, Takeda N, Hirasawa K, Sakai H, Kawamoto M, Yamamoto M, Grant WD, Hatada Y, Ito S, Horikoshi K (2004) Crystallization and preliminary X-ray study of alkaline mannanase from an alkaliphilic Bacillus isolate. Acta Crystallogr D Biol Crystallogr 60:1490–1492 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidmann JD, Smith JA, Struhl K (2006) Current Protocols in Molecular Biology, Vol 1, Greene Publishing Association, Wiley-Interscience, New York Chauhan PS, Puri N, Sharma P, Gupta N (2012) Mannanases: microbial sources, production, properties and potential biotechnological applications. Appl Microbiol Biotechnol 93:1817–1830 Chauhan PS, George N, Sondhi S, Puri N, Gupta N (2014a) An overview of purification strategies for microbial mannanases. Int J Pharm Bio Sci 5:176–192 Chauhan PS, Soni SK, Sharma P, Saini A, Gupta N (2014b) A mannanase from Bacillus nealsonii PN-11: statistical optimization of production and application in biobleaching of pulp in combination with xylanase. Int J Pharma Bio Sci 5:237–251 Chauhan PS, Bharadwaj A, Puri N, Gupta N (2014c) Optimization of medium composition for alkali-thermostable mannanase production
by Bacillus nealsonii PN-11 in submerged fermentation. Int J Curr Microbiol Appl Sci 3:1033–1045 Chauhan PS, Sharma P, Puri N, Gupta N (2014d) Purification and characterization of an alkali-thermostable β-Mannanase from Bacillus nealsonii PN-11 and its application in manno-oligosaccharides preparation having prebiotic potential. Eur Food Res Technol 238:927– 936 Chauhan PS, Sharma P, Puri N, Gupta N (2014e) A process for reduction in viscosity of coffee extract by enzymatic hydrolysis of mannan. Bioprocess Biosyst Eng 3:1459–67 Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890 Coughlan S, Wang XG, Britton KL, Stillman TJ, Rice DW, Chiaraluce R, Consalvi V, Scandurra R, Engel PC (2001) Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme’s unusual pH dependence. Biochim Biophys Acta 1544:10–17 Dell A, Galadari A, Sastre F, Hitchen P (2011) Similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes. Int J Microbiol. doi:10.1155/2010/148178 Fang TY, Ford C (1998) Protein engineering of Aspergillus awamori glucoamylase to increase its pH optimum. Protein Eng 11:383–388 Gálvez CA, Ospina JV, Sanchez PG, Zornosa RA (2013) Cloning and biochemical characterization of an endo-1,4-β-mannanase from the coffee berry borer Hypothenemus hampei. BMC Res Notes 6:333. doi:10.1186/1756-0500-6-333 George N, Chauhan PS, Puri N, Gupta N (2014a) Statistical optimization of process parameters for production of alkaline protease from Vibrio metschnikovii NG155 having application in leather industry. Int J Pharma Bio Sci 5:509–517 George N, Chauhan PS, Kumar V, Puri N, Gupta N (2014b) An approach to ecofriendly leather: lime and sulphide free dehairing of skin and hide using bacterial alkaline protease at industrial scale. J Clean Prod 79:249–257 Ghosh A, Luís AS, Brás JLA, Fontes CMGA, Goyal A (2013) Th e r m o s t a b l e r ec o m b i n a n t β ( 1 → 4 ) - m a n n a n a s e f r o m C. thermocellum: biochemical characterization and mannooligosaccharides production. J Agric Food Chem 61:12333–12344 Hakamada Y, Ohkubo Y, Ohashi S (2014) Purification and characterization of β-mannanase from Reinekea sp. KIT-YO10 with transglycosylation activity. Biosci Biotechnol Biochem. doi:10. 1080/09168451.2014.895658 Hatada Y, Takeda N, Hirasawa K, Ohta Y, Usami R, Yoshida Y, Ito S, Horikoshi K (2005) Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JAMB-750, its expression in B. subtilis and characterization of the recombinant enzyme. Extremophiles 9:497–500 He X, Liu N, Zhang Z, Zhang B, Ma Y (2008) Inducible and constitutive expression of a novel thermo stable alkaline β-mannanase from alkalophilic Bacillus sp. N16-5 in Pichia pastoris and characterization of the recombinant enzyme. Enzyme Microb Technol 43:13–18 Kumar L, Dutt D, Tapas S, Kumar P (2013) Purification and biochemical characterization, homology modeling and active side binding mode interaction of thermo-alkali-stable β-1,4 endoxylanase from Coprinus cinerus LK-D-NCIM-1369. Biocat Agric Biotechnol 2: 267–277 Lv J, Chen Y, Pei H, Yang W, Li Z, Dong B, Cao Y (2013) Cloning, expression, and characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris. Appl Biochem Biotechnol 169:2326–2340. doi:10.1007/s12010-013-0156-8 Mamo G, Thunnissen M, Kaul R, Mattiasson B (2009) An alkaline active xylanase: insights into mechanisms of high pH catalytic adaptation. Biochimie 91:1187–1196 Santos CR, Paiva JH, Meza AN, Cota J, Alvarez TM, Ruller R, Prade RA, Squina FM, Murakami MT (2012) Molecular insights into
Appl Microbiol Biotechnol substrate specificity and thermal stability of a bacterial GH5CBM27 endo-1,4-β-D-mannanase. J Struct Biol 177:469–76 Shirai T, Ishida H, Noda J, Yamane T, Ozaki K, Hakamada Y, Ito S (2001) Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme. J Mol Biol 310:1079–1087 Singh RP, Rai AR, Choudhury KR, Dubey RC (2011) Homology modeling and sequence analysis of a highly thermostable endo-(1,4)-betamannanase from the marine bacterium Rhodothermus marinus. Int J App Sci Environ Sanit 6:485–494 Sondhi S, Sharma P, George N, Chauhan PS, Puri N, Gupta N (2014) A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 having potential to biobleach softwood pulp. 3Biotech. doi:10.1007/s13205-014-0207-z Stoll D, LeNours J, Anderson L, Stalbrand H, Loleggio L (2005) The structure and characterization of a modular endo-β-1,4-mannanase from Cellulomonas fimi. Biochemistry 44:12700–12708
Wang Y, Vilaplana F, Brumer H, Aspeborg H (2014) Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5-7) mannanase from Arabidopsis thaliana. Planta 239: 653–665 Yoshida S, Sako Y, Uchida A (1998) Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for an enzyme from Bacillus circulans K-1 that degrades guar gum. Biosci Biotechnol Biochem 62:514–520 Zacharius RM, Zell TE, Morrison JH, Woodlock JJ (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem 30:148–152 Zhao Y, Zhang Y, Cao Y, Qi J, Mao L, Xue Y, Gao F, Peng H, Wang X, Gao GF, Ma Y (2011) Structural analysis of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5: implications for adaptation to alkaline conditions. Plos One 6(1):e14608. doi:10.1371/journal. pone.0014608
