Expression, homology modeling and enzymatic characterization of a new β-mannanase belonging to glycoside hydrolase family 1 from Enterobacter aerogenes B19

Background β-mannanase can hydrolyze β-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, β-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. β-mannanase is widespread in various organisms, however, microorganisms are the main source of β-mannanases. Microbial β-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial β-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce β-mannanase, which shows more advantages than traditional microbial fermentation in various aspects. Results A β-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5–8.5. The β-mannanase activity was activated by low concentration of Co2+, Mn2+, Zn2+, Ba2+ and Ca2+. Man1E showed the highest affinity for Locust bean gum (LBG). The Km and Vmax values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 μmol/(mL min), respectively. Conclusions A new type of β-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported β-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.


Background
Mannan, a major hemicellulose, is widely found in plant cell walls, some plant seeds ungi and marine bacteria. It is divided into three classes: linear mannan, glucomannan, galactomannan and galactoglucomannan

Open Access
Microbial Cell Factories *Correspondence: fetbcui@scut.edu.cn School of Biological Science and Bioengineering, South China University of Technology, Guangzhou 510006, China GH113 family is very low. But according to the analysis of three-dimensional structure and catalytic mechanism, the β-mannanases from these three families have TIM (β/α) 8 barrel structure, and follow the same catalytic mechanism (double-substituted reaction mechanism) [16][17][18]. A carbohydrate binding module (CBM) is usually present in the hemicellulose degrading enzyme structure, which is an independent region that constitutes the enzyme. A CBM can bind multiple catalytic active domains (CD) and promote carbohydrates binding to it. Most β-mannanases from the CBM1, CBM6, CBM10, CBM31 and CBM35 families have CBM structures [19,20]. Studies have shown that the binding of CBM and CD can improve structural flexibility of enzyme to make substrate and enzyme better chimeric, thereby increasing the concentration of substrate [21]. A lot of the 3D structures of the catalytic active domains (CD) and family 35 carbohydrate binding modules (CBM) of β-1, 4-mannanases have been resolved (https ://www.rcsb.org), and a few examples are displayed in Additional file 1.
Microbial β mannanases have been extensively studied for their gene expression, biochemical and structural characteristics, and various applications. Microbial β-mannanases have been reported from Bacillus subtilis N16-5 [22], A. oryzae [23], Richteris Trichoderma [24], Aspergillus chalcogenides [25], Penicillium [26] and so on. β-mannanases have showed prospective applications, for instance, pulp decolourizing [27], coffee extract viscosity reducing [28], detergent formulating [29], food quality improving [30,31] and animal feed nutritional value improving [32]. Enterobacter aerogenes B19 is separated from the root soil of rotten stumps, and its sequence of 16S ribosomal RNA gene has been submitted to NCBI database (GenBank accession number: KU500561.1). In the present study, we report a new β-mannanase (Man1E) belonging to CH1 family from E. aerogenes B19 and so far no literatures report β-mannanase from CH1 family. We have carried out the homologous modeling for the catalytic module and CBM of Man1E and molecular docking studies to elucidate the three dimensional structure, active site apparatus and enzyme-substrate interaction of this enzyme. We also have successfully cloned and expressed Man1E gene in Escherichia coli. In addition, we determine biochemical characteristics and substrate specificity of the enzyme.

Cloning and sequence analysis of β-mannanase gene
The β-mannanase gene was successfully amplified from E. aerogenes B19 by PCR. The ORF of the β-mannanase gene was 2196 bp in length, encoding 731 amino acids. The molecular mass (Mw) and isoelectric point (pI) of the β-mannanase deduced by ProtParam were 81.72 kDa and 5.91, respectively. A signal peptide from residues 1 to 24 in the sequence was found by SignalP prediction. The amino acid sequence of Man1E was analyzed by BLASTp and it showed high sequence homology with Enterobacteriaceae bacteria and Cronobacter bacteria, 98.77-99.73% identity with a number of β-mannanases from Enterobacter ludwigii and Enterobacter cloacae strains, and about 90% identity with other bacterial β-mannanases from several Cronobacter dublinensis strains and many Klebsiella aerogenes strains. But so far, the identification and characterization of these β-mannanases through purification and heterologous expression have not been reported yet. Man1E Figure S2. Among these β-1, 4-mannanases, seven highly conserved glycines, which were located at 38, 52, 170, 171, 258, 311 and 337 positions in Man1E, were found. The role of these conserved glycines in β-mannanases has not been reported. A highly conserved histidine residue (at 279 position in Man1E) was found and binded to substrates through imidazole nitrogen [33][34][35]. A highly conserved tryptophan, which was seated at 341 position in Man1E, has been demonstrated to participate in the formation of a hydrophobic platform and bind to substrates in the catalytic region of GH5 family β-1, 4-mannanases [33][34][35]. Two highly conserved Glutamate residues, which were located at 230 and 309 positions in Man1E, were identified as the acid-base catalytic residue and the nucleophilic catalytic residue in all GH5 family and GH26 family β-1, 4-mannanases [33][34][35]. An asparagine residue next to the acid-base catalytic residue existed in β-1, 4-mannanases and binded to substrate. Other highly conserved residues included two arginines, two asparticacids, a tyrosine, a histidine, a phenylalanine, a isoleucine and a lysine, which were located at 104, 207,145, 274, 216, 222, 225, 266 and 303 positions in Man1E, respectively. Two motifs, including an "EF(Y)G" motif and an "IM(F/L) A(S)WE(Q)" motif, were found in β-mannanases. The "EF(Y)G" motif was composed of the nucleophilic catalytic residue and next two residues and its effect of on the nucleophilic residue attacking substrates has been still unclear. The IM(F/L)A(S)WE(Q) motif and its function has not been reported in any literatures.

The advanced structural analysis of Man1E
The secondary structure of Man1E analyzed by PSIPRED program showed that it included 20.11% Helix (147aa), 23.26%, Strand (170aa), and 56.63% Coil. The amino acid sequence of Man1E was analyzed with NCBI CDD Tool (https ://www.ncbi.nlm.nih.gov/cdd) and two CDD matches to Man1E were found, including Glycosyl hydrolase family 1 (116 aa-14 aa) and CBM6-CBM35-CBM36_ like family (611 aa-730 aa) (Additional file 2: Figure S3). The result indicated that Man1E belonged to GH1 family. CBM6-CBM35-CBM36_like family contain carbohydrate binding module family 6 (CBM6), also described as cellulose binding domain family VI, and associated CBMs (CBM35 and CBM36). These CBMs without catalytic function have been found in a series of enzymes. They demonstrate activities on a variety of carbohydrate substrates, such as cellulose, xylan, β-glucan, mannan, agarose, and araban. These domains promote the closer binding of additional catalytic modules with their specific, insoluble substrates. A number of CBMs are related to the domains of glycoside hydrolase (https ://www.ncbi. nlm.nih.gov/Struc ture/cdd/cddsr v.cgi). According to the similarity of amino acid sequences, three-dimensional structures and catalytic mechanism, β-mannanases can be divided into three families: GH5, GH26 and GH113 [1]. But Man1E in this study was found belonging to CH1 family, different from all reported β-mannanases.
The structural model of Man1E was established with SWISS-MODEL server, based on the structures of R. miehei Man5A (PDB code, 4lyp.1.A) [35]. The resultant structure was represented in the Fig. 2a. The rationality of the structure models of Man1E was assessed with SAVES v5.0 tool ((https ://servi cesn.mbi.ucla.edu/SAVES /), the Ramachandran plots were drawn using the PRO-CHECK program (Fig. 2b). For the model, there were 85.1% of total residues (367) in most favoured regions, 14.0% residues in additional allowed regions, 0.9% residues in generously allowed regions and no residues in The amino acid residues linked to the ligand in the predicted catalytic center (e). TRS represents 2-amino-2-hydroxymethyl-propane-1,3-diol disallowed regions. The percentage of total residues (367) in most favoured regions is close to 90%, which is the standard for a good quality model [37]. The result indicated that the model were reasonable and reliable. The overall structure of Man1E showed the typical (α/β) 8barrel motif, like β-mannanases of GH5 and GH26 families [38]. For the surface electrostatic potential, negative charges were a little more than positive charges, which was in accordance with isoelectric point of partial acidity (5.91) of the enzyme. The 3D structure of Man1E contained ten α-helixes, eight β-strands and a number of loops. The four loops that linked α1 and α2, β2 and α3, β5 and β6, and β8 andα11 were composed of 20-45 amino acid residues, whereas the other four loops that linked β3 and α4, β4 and α7, β6 and α8, and β7 and α9 consisted of 10-12 amino acid residues. These loops indicated that the catalytic domin of Man1E possessed considerable flexibility. In addition, a obvious loop at the N-terminus was located at the bottom of the barrel in Man1E, different from T. fusca β-mannanase and L. esculentum β-mannanase which contain two short β-strands at the bottom of the barrel [16,34].

The catalytic module of Man1E
The catalytic center was predicted by bSiteFinder (http:// binfo .shmtu .edu.cn/bsite finde r/) using PaMan5A (PDB code, 3ZIZ) from Podospora anserine as a template, the overlap of the two β-mannases was shown in Fig. 2d. The result showed that several highly conserved regions existed between Man1E and PaMan5A (Fig. 2d). The catalytic elements contained a platform composed of four hydrophobic residues TRP166, TRP168, TYR281 and TRP341, which were exposed to solvent, and four charged hydrophilic residues Asn229, Glu230, Glu309 and Lys374 (Fig. 2e). TRS docking into the catalytic module of Man1E showed the same result as predicted by bSiteFinder (Additional file 1: Figure S1e). The acid-base residue and nucleophilic residue in β-mannanses of CH5 and CH26 families are generally conserved and glutamate (Glu). For example, in BsMan26A (CH26), BCman (CH26), PaMan5A (CH5) and RmMan5A(CH5) the acid-base residue and nucleophilic residue are Glu176 and Glu275 [39], Glu167and Glu266 [40], Glu177 and Glu283 [41], Glu175 and Glu293 [42], respectively. Combined with Fig. 1, the acid-base residue and nucleophilic residue in Man1E were speculated to be Glu230 and Glu309, respectively. Asn229 formed hydrogen bond with the ligand through carbonyl oxygen in the side chain. Glu230 and Glu309 linked to the ligand by hydrogen bond through hydroxyl group of carboxyl group, Glu309 also constituted hydrogen bond with the ligand through carbonyl oxygen of carboxyl group. Lys374 was combined with the ligand through amino-group of the side chain. Like RmMan5B, the active region of Man1E took on a deep slot-like pocket (Fig. 3a, b). Superposition of amino acid residues in the active domain of Man1E and RmMan5B-mannotriose was shown in Fig. 5c. At the − 1 and + 1 subsites, seven superimposed residues were found between RmMan5B and Man1E. In Man1E, these residues included Trp166, Trp168, Asn229, Glu230, His279, Glu309 and Trp341. At the -2 subsite, only Lys residue was found to be superimposed (Lys262 in Man1E). In GH family 5 enzyme structures, there are eight highly conserved residues at the −1 and + 1 subsites [16]. Six out of these eight highly conserved residues were found in the β-mannanases (Figs. 1 and 3c; Trp166, Asn229, Glu230, His279, Glu309 and Trp341 in Man1E). The catalytic region of Man1E-mannotriose possessed a hydrophobic platform extending from the − 1 subsite to the + 1 subsite (Fig. 3d) which consisted of five aromatic amino acid residues. These hydrophobic residues included Trp166, Trp168, Tyr260, Tyr281 and Trp341. Trp341 had the same plane orientation as the pyranose sugar at the − 1 subsite, Trp166 and the pyranose sugar were at the + 1 subsite are in plane direction (Fig. 3c). The similar hydrophobic platform has been found in bacterial GH5 family β-mannanases and GH26 family β-mannanases. For example, the hydrophobic platform in RmMan5B is composed of Trp117, Trp119, Trp257, Trp261, Trp384 and Tyr385, whereas in GH26 family BCman it contains Trp302, Trp298, Trp172, and Trp72 [40]. All of the residues located in the catalytic region of Man1E included Ala375, Lys374, Trp341, Glu309, Glu285, Ala284, Tyr281, His279, Lys262, Tyr260, Glu232, Glu230, Asn229, Trp168, Trp166 and Asp164 (Fig. 3d). The acid-base catalytic glutamate Glu230 and the nucleophilic glutamate Glu309 lay at the loop between β-strand 4 and α-helix 7, and at the loop between β-strand 7 and α-helix 9, respectively. Except for Ala375, Ala284, Tyr260, Glu232 and Asp164, All other residues in the catalytic region of Man1E formed hydrogen bonds directly or indirectly with the substrate (Fig. 3e). At the − 1 subsite, a number of hydrogen bonds were observed. At the − 1 subsite, the acylamino oxygen of Asn229, carboxyl oxygens of Glu230 and Glu309 produced hydrogen bonds with O2 of the sugar. Asn229 also linked to O2 and O3 of the sugar through the amido of acylamino. The indole nitrogens of Trp168 and Trp341 made a polar contact with O3 and O4 of the sugar, respectively. Lys374 and Tyr281 connected with O6 of the sugar through amido and hydroxyl of side chains, respectively. At the + 1 subsite, the acid-base catalytic residue Glu230 generated hydrogen bond with O6 of the sugar. Glu230 also indirectly interacted with Trp166, Asn229, His279 and Glu309 by forming hydrogen bonds with water molecules. The indole nitrogen of Trp166 and the carboxyl oxygen of Glu230 indirectly acted on oxygen of the glucosidic bond through a water molecule (W2). The acylamino oxygen of Asn229, carboxyl oxygen of Glu230, imidazole nitrogen of His279 and carboxyl oxygen of Glu309 indirectly acted on oxygen of the glucosidic bond through a water molecule (W3). At the + 2 subsite, Lys262 formed hydrogen bond with O2 and O3 of the sugar through the amido of side chain. Gu285 was linked to O1 of the sugar by hydrogen bond through the carboxyl of side chain.
Docking analysis can provide very useful information on the key residues involved in the interactions between enzyme and substrate. The noncovalent interactions between the ligand and receptor are important for investigating the subsite of enzyme-substrate binding before the catalytic reaction [43]. In order to reveal the binding subsites and involved residues, molecule docking was performed using mannotriose as the ligand. The docking result showed that mannotriose was properly positioned in the catalytic cavity, thus forming a stable complex with the enzyme. But the conformation of mannotriose produced by molecular docking was obviously different from that obtained by structural superposition (Fig. 4a). The amino acid residues surrounding mannotriose included Arg104, Asp164, Trp166, Trp167, Trp168, Asn229, Glu230, Glu232, Tyr260, Lys262, His279, Tyr281, Ala284, Glu285, Glu309, Trp341 and Lys374 (Fig. 4b). The possible residues involved in substrate binding and catalysis were analyzed from the perspective of polar interactions. As shown in Fig. 4c, there were eight residues, including Trp168, Asn229, Glu230, Lys262, Ala284, Glu285, Glu309 and Lys374, formed hydrogen bonds with mannotriose from − 1 subsite to + 2 subsite (Fig. 4c). Compared with structural superposition, the residues involved in the formation of hydrogen bonds did not include Trp166, His279, Tyr281 and Trp341, but additional Ala284 participated in the formation of hydrogen bond (Figs. 3e and 4c). The eight residues mentioned above not only formed hydrogen bond with mannotriose, but also produced nonpolar contacts. In addition, a large number of nonpolar interactions were formed between Arg104, Trp166, Tyr260, His279, Tyr281, Trp341 and manotriose (Fig. 4d). As could be seen from Fig. 4d, a hydrophobic platform, which was composed of Trp166, Trp168, Tyr260, Tyr281 and Trp341, existed in the catalytic module of Man1E.

The carbohydrate binding module of Man1E
Man1E possessed a noncatalytic carbohydrate binding module (CBM) at its C-terminal. Noncatalytic CBMs exist in most of glycoside hydrolases. When glycoside hydrolases attack generally unapproachable macromolecular substrates, CBMs may increase the concentration of the additional enzymes surrounding the substrate, thus getting involved in a twofold to fivefold increase in the activity of endocutting enzymes [44]. The overall structure of Man1ECBM was shown in Fig. 5a. It was composed of four pairs of antiparallel β-strands, a short α-helix and several loops. The loop between β1 and β2 contained 27 amino acid residues, other loops between two β-strands or between α-helix and β-strand consisted of 5-8 residues. The eight β-strands formed a hydrophobic cavity in which a number of Val, Phe and Ile residues were located. The homology structure of ManE1CBM was PaCBM35. The main differences between Man1ECBM and PaCBM35 were that PaCBM35 harbored eleven β-strands and no α-helix (Fig. 5b). Man1ECBM also diaplayed higher homology in structure with C. thermocellum CBM35 (PDB code 2W1W) (Fig. 5c). Three additional β-strands and the different length and quantity of the loops occurred in ManE1CBM compared with C. thermocellum CBM35. Calcium ion was not found in ManE1CBM, but both PaCBM35 and C. thermocellum CBM35 bind to a calcium ion (Fig. 5b, c). Overlap of Man1ECBM and PaCBM35 showed that 11 amino acid residues formed a "Bending Channel", in which substrates were likely to be bound. These residues included Trp57, Gly58, Ser59, Lys60, Lys61, Ser73, Ile74, Asp107, Try108, Gly109 and Tyr110, containing the WGY motif (Fig. 5d).
CBMs are divided into more than 50 families based on sequence in cazy database [45]. Because of their modular features, they are often studied independently [46]. In order to investigate the residues involved in binding substrate, molecule docking was conducted using mannopentaose as the ligand. The docking result showed that mannopentaose was appropriately located in a groove in the surface of Man1ECBM (Fig. 6a). The possible residues involved in substrate binding included eight residues, namely, Asn8, Ser34, Asn67, Gly68, Asn97, Thr98, Ser100 and Lys103 (Fig. 6b). The result of molecular docking was quite different from that of superposition analysis (Figs. 5d and 6b). The reason for this difference may be explained that CBM35 undergoes significant conformational change upon ligand binding, and the binding specificity may mainly depend on the curvature of the binding site and the size of the binding groove [47]. Whereas, in molecular docking procedures, the receptor molecules are assumed to be rigid. It is likely a more accurate method to determine the binding residues by comparing the surfaces of the free and bound structures of CBM35 with magnitude of the chemical shift changes from the substrate [47].

Gene expression in Escherichia coli
The appropriate E. coli BL21(DE3) harboring pET-28a(+) -Man1E strains were inoculated in LB medium and cultured for 24 h at 37 °C, 200 rpm in shake flasks. The cells and the supernatant were collected separately for β-mannanase assays. Comparative analysis for the proteins of the E. coli BL21(DE3) host strains and the recombinant strains by SDS-PAGE displayed that a number of the recombinant proteins existed in insoluble form (Fig. 7a, Lane 4) and some in an soluble form (Fig. 7a, Lane 5) in the cells. The molecular weight of recombinant β-mannanase was about 82.5 kDa, which was corresponding to the predicted value of Man1E (Fig. 7a). It is reported that the Mws of microbial β mannanase are between 17.7 and 130 kDa [27,[48][49][50] [55]. Only a few literatures have reported β-mannanases from Klebsiella-Enterobacter group, which possess the Mws with 43 kDa, 45 kDa, 90 kDa (shown Fig. 4 The analysis of the catalytic domin performed by molecular docking. a The comparison of two conformations of mannotriose, red stick represented the conformation from RmMan5B-mannotriose complex, cyan stick represented the conformation from the molecular docking between Man1E and mannotriose. b Amino acid residues in the catalytic module of Man1E-mannotriose complex. c Diagrammatic sketch of polar contacts between mannotriose and amino acid residues at subsites − 1 to + 2. d Diagrammatic sketch of any contacts (within 4 Å) between mannotriose and amino acid residues at subsites − 1 to + 2 in Table 1). 82.5 kDa of β-mannanase in this study was reported for the first time. The single factor experiment was used for investigating the effect of different factors on soluble expression of the recombinant β-mannanase.
The total soluble expression of Man1E reached maximum at 20 °C (Fig. 7c), the mannanase activity was 608.11 U/mL. High induction temperatures displayed negative effects on the accumulation of soluble β-mannanase. The soluble expression and activity of Man1E increased with the increase of IPTG concentration in a range of 0.2-0.6 mM, the mannanase activity was 720.81 U/mL at 0.6 mM IPTG (Fig. 7d). Man1E presented higher expression after IPTG induction for16 h (Fig. 7e), the mannanase activity was 642.25 U/ mL, however, there was no significant difference in soluble expression and activity between 12 and 16 h. By comparison, the optimal inductive temperature and time for Man1E gene expression similar to those of β-mannanase genes from Bacillus circulans NT 6.7 (for 16 h at 18 °C) [56] and Pantoea agglomerans A021 (for 15 h at 18 °C) [57]. However, for the expression of three β mannanase genes, the IPTG concentrations were different, which were 0.6 mm, 1.0 mm and 0.05-0.15 mm, respectively.

The purification and Biochemical characterization of Man1E
The crude extract of intracellular proteins from the recombinant strains was applied to affinity chromatography on a Ni-NTA column. The eluant was analyzed on SDS-PAGE gel and the result proved that the enzyme could achieve electrophoretic purity through this onestep purification scheme, and the Mw value of the purified β-mannanase was about 82.5 kDa (Fig. 7b), which was accordance with the expected value.
The purified Man1E was incubated in a temperature range of 30 °C to 70 °C to determine the effect of temperature on β-mannanase activity. Man1E performed the highest activity at 55 °C. The β-mannanase activity increased gradually from 30 to 55 °C, but displayed a decreasing trend when the temperature was above 55 °C (Fig. 8a). Reportedly, the optimum temperature of β mannanases from different strains varied from 45 to 85 °C [3]. For instance, Man5 from Thermotoga maritime displays the optimum temperature of 90 °C, which is the highest temperature reported so far [58]. Whereas the optimal temperature of β mannanases from E. ludwigii MY271 [59], B. subtilis YH12 [60], B. subtilis NM-39 [61] was 55 °C, the same as that of Man1E. The optimum temperature value of Man1E is close to those of β-mannanases from Enterobacter sp. strain N18 [62] and Klebsiella oxytoca CW23 [63]. The purified Mann1E was stable below 60 °C, the enzyme still remained above 60% activity after being incubated for 1 h at 60 °C, especially below 55 °C retained more than 85% of activity after being incubated for 1 h (Fig. 8b), which shows similarity to the relevant enzymes from E. ludwigii MY271 and Penicillium occitanis Pol6 [64], and inferior to the corresponding enzymes from Enterobacter sp. strain N18 and K. pneumoniae SS11. However, The thermal stability of purified Mann1E was better than β-mannanases from K. oxytoca KUB-CW2-3 [65] and Aspergillus sulphureusis [66].
The activity determination at pH values in the range of 3.0-9.0 showed that Man1E exhibited the maximal activity at pH 6.5. Below or above the optimum pH, the activity declined to about 21.3% at pH 4.0 and 37.5% at pH 9.0 (Fig. 8c). It has been reported that the optimal pH value of β-mannanases from B. subtilis YH12, B. subtilis G1 [67], Bacillus stearothermophilus [68] is 6.5, which is the same as that of Man1E, whereas β-mannanases from E. ludwigii MY271 and K. oxytoca CW23 [65] show the highest activity at neutral pH. β-mannanases from Enterobacter sp. strain N18, K. pneumoniae SS11 and Enterobacter asburiae SD26 [69] show optimum catalytic capacity at pH 7.5, 9.0 and 6.0, respectively. In addition, it is known that microbial β-mannanases have the maximum activity in a wide pH range of 4.0 to 10.0 [70]. Man1E was stable over a wide pH range, it remained more than 60% activity at pH 4.0-9.0, remarkably more than 80% activity at pH 4.0-7.0. Even at low pH values of 3.0 and 3.5, Man1E still displayed 38.1% and 55.3% activity respectively (Fig. 8d). The pH stability of Man1E is similar to the reported β-mannanase from Klebsiella-Enterobacter group strains ( Table 1). The good pH stability of Man1E makes it attractive for industrial applications, for example, it can still function as feed additive in animal gastrointestinal tract and be used in pulp bleaching.
The effects of different metal ions on Man1E activity were shown in Table 2. Compared with the control sample without any metal ions, the purified Man1E activity was activated by Co 2+ , Mn 2+ , Zn 2+ , Ba 2+ and Ca 2+ . The presence of 2 mM Co 2+ and Mn 2+ could increase the activity by 35.6% and 26.9% respectively. However, the enzyme activity was significantly inhibited by 2 mM Cu 2+ that decreased the relative activity by 34.7%. The enzyme activity was moderately inhibited by K + and Mg 2+ . The enzyme activity showed little change in the presence of Na + , Ni 2+ or Fe 2+ . The activation of Co 2+ , Ba 2+ and Ca 2+ on Man1E activity is similar to the β-mannanase from K. pneumoniae SS11, but the effects of Zn 2+ , Mg 2+ Na + , Ni 2+ and Fe 2+ are completely different between the two β-mannanases. Mn 2+ , Co 2+ , Ca 2+ and Ba 2+ could enhance the enzyme activity of Man1E and β-mannanase from Paenibacillus sp. CH-3 [71], but Mg 2+ and Zn 2+ just exhibit the opposite effects on the two enzymes. Cu 2+ exerts a strong inhibitory effect on many β-mannanases, for example, the β-mannanases from E. ludwigii MY271, B. circulans NT 6.7 [72], Paenibacillus cookie [73] and Bacillus sp. MK-2.

Kinetic parameters of Man1E
LBG, konjac powder, guar gum, xylan, soluble starch and CMC-Na are used as substrates for evaluating the activity of Man1E. No activity was detected for xylan, soluble starch and CMC-Na (data not shown). Man1E exhibited high activity on LBG, Konjac powder and guar gum, and the catalytic kinetic parameters were determined by Lineweaver-Burk diagram. Man1E showed the highest affinity for LBG, followed successively by Konjac powder and guar gum. The K m values for LBG, Konjac powder and guar gum were 3.09 ± 0.16, 6.07 ± 0.30 and 11.53 ± 0.45 mg/mL, respectively (Additional file 2: Figure S4). The V max of Man1E was 909.10 ± 3.85, 666.67 ± 2.30, and 312.50 ± 4.11 μmol/(mL min) toward LBG, Konjac powder and guar gum, respectively (Additional file 2: Figure S4). The K m value of Man1E for LBG is similar to the β-mannanase from Enterobacter sp. strain N18, but greater than β-mannanase from K. pneumoniae SS11 ( Table 1). The V max of Man1E is equivalent to β-mannanase from K. pneumoniae SS11, but lower than β-mannanase from E. asburiae SD26 (Table 1). Table 3 showed a summary of the kinetic parameters of Man1E.

Analysis on hydrolysis products from LBG and KGM
TLC analysis of the hydrolysates showed that the purified Man1E could degrade LBG and produced mannanoligosaccharides, which proved Man1E to be an endo-β-mannanase. However, there were obvious differences in the enzymatic degradation products between LBG and KGM. Degradation products of LBG contained mannose to mannohexaose and its components were uniform, while degradation products of KGM was mainly composed of mannobiose, mannopentaose and mannoheptose, and also contained a small amount of mannose and mannotriose (Fig. 9). The hydrolysis of LBG by Man1E was similar to those of β-mannanases from Bacillus licheniformis and B. subtilis [74].

Conclusions
To summarise, we have cloned, expressed, and characterized a highly active Man1E from E. aerogenes. The amino acid sequence and the molecular mass of Man1E demonstrated significant differences by comparison with all reported β-mannanases in literature, which include several β-mannanases of the Klebsiella-Enterobacter group strains. Unlike previous reports that β-mannanases belong to four families: CH5, CH26, CH133 and CH134, Man1E belonged to CH1 family. Three dimensional structures of the catalytic module and CBM of Man1E were established using homology modeling method, respectively. Important amino acid residues located in the catalytic module and CBM were analyzed by superposition of Man1E and other β-mannanases and molecular docking. The purified recombinant protein showed higher β-mannanase activity. The general properties of the purified enzyme were carefully studied. The enzyme displayed an optimal activity at pH 6.5 and 55 °C. The activity was stable over a broad pH range from acidic to alkaline (3.5-8.5) and below 60 °C, especially below 55 °C.
In addition, different metal ions exhibited varied effects on the mannanase activity, Co 2+ and Mn 2+ showed better activation. The purified enzyme had a lower K m value to locust bean gum galactomannan, indicating a higher affinity toward locust bean gum galactomannan. The enzyme was an endo-β-mannanase, which displayed excellent catalytic effect on the hydrolysis of locust bean gum galactomannan and Konjac powder glucomannan for the production of oligosaccharides. In future work, it is expected to clarify amino acid sites that play key roles in catalysis and substrate binding, so that Man1E is directionally performed molecular modification to improve its catalytic efficiency and substrate specificity.

Strains, plasmids, and media
Enterobacter aerogenes B19 used in this work is now preserved in our laboratory. Escherichia coli BL21(DE3) and pET28a(+) acted as the host strain and expression vector, respectively. Escherichia coli DH5α was cultured for the amplification of plasmids. Escherichia strains were cultivated in Luria-Bertani (LB) medium at 37 °C and 200 r/min in shake flask. Luria-Bertani (LB) medium was composed of 1% NaCl, 0.5% yeast extract, 1% tryptone.
Mannotriose was used as a substrate molecule (ligand1) and docked into the Man1E catalytic module (receptor1) in the structure model using an AutoDock Tools 4.2.5 (http://autod ock.scrip ps.edu) to detect the interactions between the ligand1 and the receptor1 and the key residues binding to the ligand. Mannopentaose (ligand2) was docked into the Man1ECBM (receptor2) using an Auto-Dock4 Tools 4.2.5 to investigate the interactions between the ligand2 and the receptor2 and the key residues involved in ligand binding. Molecular visualization and graph drawing were conducted using PyMOL software.

Expression of Man1E in Escherichia coli BL21(DE3)
The recombinant pMD19-T -Man1E plasmids from the correctly sequenced positive clones and expression vectors pET-28a(+) were digested with NdeI and XhoI, and then linked by DNA ligase to construct the recombinant expression pET-28a(+) -Man1E plasmids. The pET-28a(+) -Man1E plasmids were transformed into Positive transformants were screened on LB plate containing 50 μg/mL Kanamycin. At the same time, the pET-28a(+) -Man1E recombinant plasmids were extracted from the positive transformants and then digested by NdeI and XhoI, the Man1E fragment was further identified by DNA sequencing. The suitable transformants were inoculated in LB medium and cultured for 24 h at 37 °C and 200 r/min. After culture, the cells and supernatant were collected separately for analyzing the recombinant proteins.

Purification of recombinant Man1E
The fermentation broth was centrifuged at 11,000×g and 4 °C for 30 min, the precipitate were harvested and washed with distilled water for three times. The cells was resuspended in 20 mL of 100 mM phosphate buffer (pH 6.5) and the resuspending was ultrasonically disrupted in an ice bath for 20 min. The cell disruption liquid was centrifuged for 30 min at 11,000×g and 4 °C. The resultant supernatant was collected and passed through a nickelchelate column (1 × 5 cm). After equilibration with 100 mM phosphate buffer (pH 6.5), a linear imidazole gradient of 20-250 mM was used to elute the column, Active components were collected and merged, then the solution was dialyzed with 100 mM phosphate buffer (pH 6.5), and stored at 4 °C. The purified protein was analyzed through SDS-PAGE and enzyme activity determination.

Enzyme activity and protein analysis
The activity of β mannanase was determined by using 3,5-dinitrosalicylic acid (DNS) reagent to measure the amount of reducing sugar released from the substrate. The standard reaction solution consisted of 0.1 mL enzyme liquid with proper dilution and 0.9 mL 0.5% (w/v) substrate solution, which was prepared by dissolving locust bean gum into 100 mM phosphate buffer (pH 6.5). The mixture was incubated at 50 °C for 10 min, then the reaction solution was treated with 1.0 mL DNS reagent to terminate the reaction and boiled for 10 min. The reducing sugar content was determined by measuring its absorbance at 540 nm, taking mannose (sigma) as the standard control to make the standard curve, the relationship formula between the absorbance and the mannose content was obtained. One unit (U) of β-mannanase activity was set to the required enzyme amount that released 1 μmol of reducing sugar (calculated by d-mannose) per minute. Protein content was determined according to Bradford's method with bovine serum albumin as the standard [75]. The homogeneity and molecular weight of Man1E was investigated by 10% SDS-PAGE as clarified by Laemmli [76]. Protein bands on SDS-PAGE gel were stained with Coomassie brilliant blue R-250. The low molecular weight calibration kit (TaKaRa, China) was used as the molecular weight standard, which included phosphorylase b (97.4 kDa), albumin (66.2 kDa), ovalbumin (43 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa).

Effects of pH, temperature, and metal ions on β-mannanase activity
The optimal catalytic temperature of Man1E was analyzed in a range of 30 and 70 °C in phosphate buffer (pH 6.5). The thermostability of the purified Man1E was investigated by determining the residual activity after the enzyme was preincubated in phosphate buffer (pH 6.5) at different temperatures ranging from 50 °C to 65 °C for 0, 5, 10, 15, 30, 45 and 60 min, respectively. The optimal pH for β-mannanase activity was examined by preparing enzyme solution with different pH buffers which included disodium hydrogen phosphate-citric acid buffer (pH 3.0-6.0), phosphate buffer (pH6.0-8.0), and Tris-HCl buffer (pH 8.0-9.0), the temperature and the substrate used were 50 °C and 0.5% (w/v) locust bean gum. To determine the pH stability, the enzyme was preincubated for 60 min at 40 °C in different pH buffers (pH 3.0-9.0), and the residual activity was measured under the standard conditions. To obtain the effects of metal ions on the purified Man1E, the enzyme was preincubated for 5 min at 40 °C in phosphate buffer (pH 6.5) containing 1 mM single metal ion which contained Na + , K + , Mn 2+ , Mg 2+ , Zn 2+ , Ca 2+ , Cu 2+ , Ni 2+ , Co 2+ , Ba 2+ , Fe 2+ , separately and then enzyme activity was determined at 50 °C and pH 6.5. The enzyme reaction without any metal ions was used as the control group.

Kinetic parameters of Man1E
The kinetic parameters were measured using locust bean gum (LBG), guar gum, and KGM as the substrates, respectively. The Michaelis-Menten constant (K m ) and rate of reaction (V max ) were calculated from the Lineweaver-Burk plot.

Analysis of enzymatic hydrolysis products
0.5% (w/V) LBG, KGM were mixed with proper dilution Man1E (final concentration 1.5 U/mL), and the total system volume was 1 mL. The mixture was incubated at pH 6.5 and 55 °C for 8 h, then boiled for 5 min to stop the reaction and the hydrolysates were obtained. The hydrolysates were centrifuged at 12,000×g for 10 min. Supernatants were analyzed by using thin layer chromatography (TLC). At room temperature, each sample (0.5 μL) was loaded on the thin layer plate silica gel G). After the plate was dried, it was put into the developing agent composed of n-butanol, acetic acid