Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23–25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don’t show homology to each other, but all of them are built up from 3–6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70–75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9–1.7mM of KM and 80–120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.

studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation. of T. fusca endomannanase has been investigated by Hilge et al. [27]. This study was the first publishing high resolution 3D structure of a mannan degrading enzyme, and assigned this endomannanase to the glycosyl hydrolase family 5 (GH5).
Recently, two studies have been published focusing on the thermostability of the endomannanase from T. fusca and another endomannanase (StMan) from Streptomyces thermolilacinus. The first study showed that the thermal stability of these enzymes depends on the concentration of calcium ions [28], and the authors also determined the amino acid residues responsible for this phenomenon [29]. Formerly we had already described an intracellular mannobiose cleaving beta-mannosidase (ManB) from T. fusca, which seems to be the terminal part of the mannanase system [22]. Further exploring the genome sequence data, we identified two GH5 hydrolases-endomannanase (man5A) and endoglucanase (cel5A) genes-which are located on the genome upstream of the mannosidase (Fig 1) [30]. Here we report the characterization of the first endomannanase enzyme from T. cellulosilytica, and also a partial characterization of endomannanase from T. halotolerans. We also compared the three endomannanases from three different Thermobifida species namely from T. fusca, T. cellulosilytica and T. halotolerans.

Chemicals
Unless otherwise indicated, all chemicals herein used were analytical-grade and purchased from Sigma-Aldrich Ltd. (Budapest, Hungary).

Microorganisms and culture conditions
Four Thermobifida strains were used in this study. T. fusca TM51 and T. cellulosilytica TB100 were isolated from the hot region of manure compost [31]. T. halotolerans YIM90462 and T. alba CECT3323 (synonym: T. alba ULJB1) were purchased from Japan Collection of Microorganisms (JCM) and Spanish Type Culture Collection (CECT), respectively.
underlined sequences harboring NdeI and XhoI restriction sites. PCR reactions were carried out by using Pfu DNA polymerase (Thermo Fisher Scientific Inc.) for 32 cycles of 30 s at 94°C, 30 s at 60°C, and 3 min at 72°C, preceded by incubation for 5 min at 96°C. PCR-amplified fragments were digested with NdeI and XhoI enzymes (Thermo Fisher Scientific Inc.), ligated into the pET28a plasmid vector by using T4 DNA ligase (Thermo Fisher Scientific Inc.) and used to transform E. coli Top10 competent cells to isolate proper clones which were used for protein expression in E. coli BL21 (DE3) cells.
Selection of endomannanase expressing S. lividans strains from an expression library Genomic DNA from T. halotolerans YIM90462 and T. cellulosilytica TB100 was prepared as previously described [22] and partially digested with serial dilutions of Sau3AI (Thermo Fisher Scientific Inc.) for 1 hour at 37°C. Optimal enzyme concentration yielding the highest proportion of 10 kb DNA fragments was determined by agarose gel electrophoresis (0.6% agarose in TAE buffer) and DNA bands of this size were purified by Qiaquick gel extraction kit (Qiagen) according to manufacturer's instructions. The fragments were then ligated into the Streptomyces vector pIJ699 digested with BamHI restriction endonuclease and treated with alkaline phosphatase to avoid self-ligation. Protoplast preparation, transformation, regeneration and selection of endomannanase-harbouring Streptomyces transformants were carried out as described previously [32]. Transformants were screened for endomannanase activity by growing the clones in 2 ml Luria-Bertani (LB) medium containing 200 μg/ml thiostrepton at 30°C, 200 rpm for 2 days, then culture supernatants were tested on agar plates containing 0.5% (w/v) LBG-mannan. Endomannanase activity was detected by Congo red staining after 30 min incubation at 50°C according to Posta et al. [33].

Phylogenetic analysis of Thermobifida strains and their endomannanases
For the molecular identification of investigated Thermobifida strains PCR amplification of 16S rDNAs were performed as described by Rainey et al. [34]. Partial sequences of the first 500 bp of the 16S rDNA were initiated with the 531r conservative eubacterial primer, almost-complete 16S rDNA sequences were determined by using primers 27f, 531r, 803f and 1492r [35]. 16S rDNA sequence reads and amino acid sequences of the cloned endomannanases were assembled in MEGA6 [36] then aligned by using the ClustalW algorithm. Neighbor-joining trees were constructed in MEGA5, performing 1000 bootstrap replicates.

DNA sequencing and computer analysis
16SrDNA sequences and DNA fragments up-and downstream of endomannanases subcloned into pUC19 were determined with a DNA sequencer (ABI Prism 310, Perkin Elmer Co., USA). DNA and protein endomannanase sequences were analyzed by using the BLAST server [37] and the MEGA6 software package [36]. Amino acid sequence and domain structure of ManB were determined by Swiss-Prot, EMBL and NCBI database queries and by using the Pfam [38] and InterPro [39] bioinformatics servers. Phylogenetic trees were reconstructed by the maximum likelihood method [40] by using the MEGA6 software package.

Expression and purification of endomannanases
Recombinant His-tagged endomannanases were over-expressed in E.coli BL21 (DE3) cells. Transformants were grown at 37°C with 200 rpm aeration in 500 ml of LB medium containing 50 μg/ml kanamycin until optical density measured at 600 nm (OD 600 ) reached 0.6-1.0. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM final concentration), followed by overnight agitation at 20°C. Cells were harvested and disrupted by sonication and the lysate was centrifuged at 2,360 x g for 20 min at 4°C and supernatant was loaded on a 5 ml Hi Trap column (GE Healthcare) for immobilized metal ion affinity chromatography (IMAC) purification. Protein elution was performed with a 0-500 mM imidazole gradient in 300 mM NaCl, 20 mM sodium phosphate buffer, pH 7.2 and protein concentration of pooled fractions was determined by Bradford method using BSA as protein standard [41].
The molecular mass of the enzymes was estimated by SDS-PAGE analysis. Endomannanase zymography was done according to Posta et al. [33], with minor modification: instead of carboxymethyl-cellulose 0,1% LBG mannan was added to the gel.
Endomannanase activities were determined on polysaccharide substrate by measuring liberated reducing sugars according Somogyi-Nelson method [42]. Dilution series of mannose stock solution were used for the determination of the reducing sugar calibration curves. Michaelis-Menten kinetic parameters of the endomannanases were estimated for LBG-mannan substrate in 10 different concentrations between 0-4 mg/ml (0, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg/ml) at 50°C, in 50 mM sodium phosphate buffer, pH 7.5. The final volume of the enzyme reactions was 0.5 ml containing 1.4 μg/ml of Man5ATc, Man5ATh and of Man5-ATf, respectively. Reactions were initiated by adding enzyme samples for the pre-incubated LBG-mannan substrate solutions at 50°C and then the incubation continued for further 5 minutes. All the measurements were carried out in triplicates. The initial rate of the enzyme catalysis was expressed in mM of reducing sugar liberated in one minute as it was determined from the calibration curve. The Michaelis-Menten kinetic constants were calculated by Origin 8.0 software program (OriginLab, Northampton, MA) using the Hill equation when the number of the occupied binding site was equal to one.
The effect of temperature on endomannanase activity was determined in 50 mM sodium phosphate buffer, pH 7.5 at different temperature ranging from 40-90°C and using 2 mg/ml LBG-mannan substrate in 0.5 ml reaction volume. Enzyme concentrations were the same as in case of pH optimum determination. For both pH and temperature dependence studies the Somogyi-Nelson method was applied for the determination of the liberated reducing sugar concentration. Then, initial rates were calculated and converted into relative rates and plotted against pH and temperature.
Thermal stability of the three endomannanases was assessed at 70°C in either 100 mM Triethanolamine/HCl or 50 mM MOPS/NaOH buffer. For all the three enzymes, the effects of Ca 2+ on thermal denaturation were investigated. In case of Man5ATh (50μg/ml), the effect of ion strength on thermal stability (NaCl at 0.2; 0.6; 1.0; 1.2; 1.6M concentrations) in MOPS/ NaOH buffer (50mM, pH 7.5) with 5 mM Ca 2+ was also determined at 70°C. The pH of the buffers was corrected by the temperature effect.
Man5ATc enzyme (40 μg/ml) in 0.5 ml of 100 mM Triethanolamine/HCl buffer, pH 7.5, was incubated at 70°C in the presence and in the absence of Ca 2+ . In case of Man5ATf the same measurements in 100mM Triethanolamine/HCl buffer, pH 7.5 were performed as it was described for the Man5ATc enzyme. For all the three enzymes the rate of thermal unfolding was also followed when Ca 2+ was replaced by 2mM EGTA at 70°C.
In all cases, from the incubated endomannanase solutions, time course aliquots (10 μl) were withdrawn, cooled on ice for at least 30 min and then assayed for endomannanase activity at 50°C in the presence of 2 mg/ml LGB-mannan in 50 mM phosphate buffer by Somogyi-Nelson method. The residual activity was calculated as a fraction of the initial activity and plotted against time. The data were fitted to one-step transition mechanism between two states, the native and the denatured ones (E!E D ). The single step is assumed irreversible and to follow first order kinetics. In this mechanism, the thermal inactivation rate constant (min -1 ) was assessed from a single exponential decay curve, d[E]/t = -k D [E]. The active enzyme concentration can be expressed as the enzyme activity, A t , after heat treatment for a given period of time and the initial activity, A 0 . The integration of the equation then gives lnA t /A 0 = k D , where A t / A 0 is the residual activity. ORIGIN software was used for the data analysis and graphic representation. Half-life t 1/2 is derived by the following equation t 1/2 = ln2/k D .

Results and Discussion
Endomannanase cloning based on genome sequences PCR production of endomannanase genes from four thermobifida strains were probed by homologous and degenerated primers. The primer design was based on the complete genome sequence of T. fusca TM51. 1362 bp DNA fragments were synthesized when T. fusca and T. alba genomic DNA served as template, but in case of T. halotolerans and T. cellulosilytica no PCR products were obtained in spite of extensive PCR optimization experiments. The obtained PCR products were named man5ATf and man5ATa and since they showed 99% DNA and 100% aminoacid (AA) homology investigations were limited to man5ATf which was cloned into the pET28a expression vector.

Cloning endomannanases from expression libraries
To capture endomannanase genes of T. halotolerans YIM90462 and T. cellulosilytica TB100, expression libraries were generated in Streptomyces lividans TK24 strain. Genomic DNAs were partially digested with Sau3AI endonuclease and DNA fragments of 10 kb were cloned into the pIJ699 vector. After transformation, thiostrepton resistant clones were selected. Clones of each library were screened on LBG containing agar plates. Endomannanase over-producing colonies were detected by Congo red staining method.
Plasmids were isolated from the mannanase positive clones and the inserts were fully sequenced after subcloning. Sequence analyses revealed a 1368 bp man5ATh gene from T. halotolerans and a 1320 bp man5ATc gene from T. cellulosilytica. Specific primers were designed for the identified endomannanase genes and used in PCR reactions to obtain DNA fragments having NdeI and XhoI cloning sites for cloning into the corresponding sites of pET28a vector. By this method 6His tag for affinity purification was introduced on the N-terminus of both genes.

Phylogenetic analysis of thermobifida endomannanases
The four investigated strains were taxonomically characterized by 16S rDNA sequence analysis. The obtained phylogenetic tree indicates that T. alba CECT3323 clashes to one cluster with type strain T. fusca ATCC27730 T and T. fusca TM51, and is clearly separated from type strain T. alba (Fig 2). When the endoxylanase of T. alba CECT3323 was published [26] the strain was characterized by phenotypic characteristics and so far there are data on hydrolases only from this strain. The nucleotide sequence alignment between XylA of T. alba CECT3323 (Z81013) and T. fusca YX xylanase (AAZ56956) revealed 100% homology in the catalytic and carbohydrate binding module [26,43]. Accordingly, the Man5ATa enzyme of T. alba CECT3323 shows 100% identity to the Man5ATf enzyme of T. fusca TM51 (Fig 2). Based on enzyme identities and molecular taxonomy results we concluded that the CECT3323 strain belongs to T. fusca species therefore the Man5ATa enzyme was excluded from further biochemical analysis.
Similar linker regions also can be found in cellulases of T. fusca YX and their role has been investigated by posttranslational modification [20]. A protease was identified cleaving the Cel9A intact enzyme along the linker sequence producing catalytic and CBM domains [44]. The substrate specificity of the enzyme without the CBM domain was changed; its activity increased towards shorter oligosaccharide fractions. The genome of thermobifida strains is relatively small, and instead of producing a larger enzyme set this posttranslational mechanism makes their polysaccharide degrading capability more diverse and effective. The different linker sequences in homologous enzymes of thermobifida species (that populate same niches) most probably provides a control over posttranslational modifications to make thermobifidas more competitive in the race for available substrates.

Heterologous expression and purification of endomannanases
Man5ATc, Man5ATf and Man5ATh endomannanases were expressed in E. coli BL21 (DE) and the yield from 1 L culture after IMAC affinity chromatography purification was as follows: Man5ATh 25 mg, Man5ATc 33 mg and Man5ATf 45 mg. SDS PAGE analysis of expressed proteins indicated 48-50 kDa size that was in good agreement with theoretical molecular weights (Fig 4). Isoelectric points were predicted using the algorithm of Kozlowski (2013, http://isoelectric.ovh.org/). The most acidic protein is Man5ATh (pI 4.102) while Man5ATf and Man5ATc are less acidic with almost identical values (pI 4.519 and 4.567, respectively).

-endomannanases
Substrate specificity measurements. The GH5 endomannanase family includes several types of hydrolases, among others endocellulases, glucosidases, xylanases and mannanases. Substrate specificities of expressed endomannanases were tested with carboxymethyl-cellulose (CMC), crystalline and micro-crystalline cellulose (MN300, Avicel), beech wood xylan, pNpmannopiranozid and locust bean gum (LBG). All the investigated Man5A were active only on LBG. This narrow substrate specificity indicates that the axial OH group at C2 on the pyranose ring is essential in ligand binding at the active site and suggests a potential biotechnological application of the enzymes in the production of oligomannan prebiotics [5].
Kinetic studies. Endomannanase activities of the three GH5 glycoside hydrolases were investigated on LBG-mannan substrate. The applied substrate consists of a β-(l,4)-linked mannan backbone with single α-(l,6)-linked galactose side chains. The endomannanase activity of the three GH5 glycoside hydrolases were not affected by the presence of chelating agents such as EDTA or EGTA suggesting that the catalytic effect of the enzymes did not depend on metal ions.
The temperature optima of the Thermobifida endomannanases are in the range of 70-75°C (70°C for Man5ATc and Man5ATh; 75°C for Man5ATf) at given assay conditions (Fig 5) and this value classifies them to high temperature optimum enzymes. Endomannanases from the eubacterial Caldibacillus cellulovorans [45] and the archaeon Thermotoga neapolitana [46] have significantly higher temperature optimum: 85°C. The CBM domain free catalytic domain of endomannanase of T. fusca KW3 was also characterized as thermophilic enzyme (80°C) but it can't be compared to our values as a significant portion of the enzyme was deleted [27]. The recently described endomannanase from T. fusca BCRC19214 which was expressed in Yarrowia lipolytica has a very similar thermal optimum at the 75-80°C range [47].
The functional pH range of Man5ATc and Man5ATh endomannanases completely overlaps in the range of 5.5-9.0 (where more than 50% of maximum activity was detected) with pH optimum of 7.0 and 7.5 for Man5ATc and Man5ATh, respectively. Man5ATf possesses the wildest working pH range of 5.5-9.7 with pH optimum of 8.0 (Fig 5). With these slightly basic values thermobifida Man5A enzymes form a distinct sub-group in the microbial GH5 endomannanase family. Endomannanases of bacilli have more basic values around pH 9 [48][49][50] and most of the prokaryotic endomannanase enzymes from Streptomyces lividans, Clostridium cellulovorans, Vibrio sp. and Geobacillus stearothermophilus have activity maximum at neutral pH [6,[51][52][53]. Fungal GH5 endomannanases are adapted to acidic environment [54][55][56]. Michaelis-Menten kinetic parameters determined at pH 7.5 and 50°C are listed in Table 1. Kinetic performances expressed in catalytic constants (k cat ) are similar for all the three investigated mannanases with the value of 100±20 sec -1 . Slight difference found in the individual Michaelis-Menten constants, Man5ATc has the highest affinity toward the carob-mannan substrate with K M value of 0.84 mg/ml (Table 1). Endomannanases from Aspergillus niger BK01  and from Bacillus sp. MG-33 have higher affinity toward locust bean galactomannan with Km value of 0.6 mg/ml and 0.16 mg/ml, respectively [57,58]. The majority of the endomannanases from either fungi or bacilli taxa exhibited considerable lower affinity for this type of mannan like endomannanases from Bacillus licheniformis and from Penicillium oxalicum GZ-2 with Km values of 14.9 mg/ml and 7.6 mg/ml, respectively [59,60]. Thermostability studies. Denaturation kinetics of the three mannanases were studied at 70°C in the presence and in the absence of Ca 2+ in order to investigate the thermostability of the enzymes and the influence of this metal ion on thermal unfolding. For all the three endomannanases the unfolding kinetics obeys a single step exponential decay. The heat inactivation constants (k D ) and the calculated half-lifes (t 1/2 ) determined in the different conditions were summarized in Table 2. The unfolding kinetics of T. fusca and T. halotolerans enzymes were affected by Ca 2+ as their thermal stability has increased significantly: doubled their life-times when the enzyme solutions contained 5mM of metal ion. The stabilizing effect of Ca 2+ ion against thermal denaturation has also been detected in the case of the catalytic domain of T. fusca endomannanase by Kumagai et al. at different temperatures with comparable results [28]. The key residues in calcium binding have been identified in case of T. fusca mannanase [29] and the same motif (Asp-264, Glu-265, and Asp-266) was present also in the other investigated mannanases (Man5ATc and Man5ATh). Interestingly, the thermal denaturation of Man5ATc was not influenced by Ca 2+ despite the existence of the putative Ca 2+ binding motive ( Table 2). The effect of salt on thermal denaturation of Man5ATh was investigated in the presence of NaCl in the concentration range of 0-1.6M. The unfolding kinetics were not affected significantly by the presence of NaCl when its concentration was not higher than 0.8M. However, when the NaCl concentration was increased to 1.6 M, the speed of unfolding tripled compared to the level when there was no salt in the system (Table 3). These results suggest that Man5ATh is a moderately halophilic protein, which is in a good agreement with the original habitat (a salt mine) of T. halotolerans YIM90462 strain [17]. In our measurements, T. fusca β-(l,4)-endomannanase was proved to possess the greatest thermostability among the investigated enzymes with a life-time of 123 min -1 at 70°C in the presence of Ca 2+ (Fig 6).
Alterations in mannanase stabilities can be explained with differences in environmental factors of niches populated by investigated thermobifida strains. T. fusca TM51 was isolated from moderately basic (pH 8.5) compost [31] and its temperature optimum is 60°C. The other two strains, T. halotolerans and T. cellulosilytica have considerably lower temperature optimum (50-55°C). The half-life value of Man5ATf for 70°C (123 min) places the enzyme into an elite group of highly stable mannanases together with robust enzymes from the archaeon Thermotoga thermarum (half life 120 min at 90°C) and the eubacterium Caldibacillus cellulovorans (half life 48 min at 85°C, and no loss in activity after 24 h at 70°C) [45,61].

Conclusion
Endomannanase genes from thermobifida strains were isolated whose recombinantly expressed polysaccharide degrading enzymes have been partially characterized. The molecular taxonomy investigations carried out clearly indicated that T. alba CECT3323 strain was previously miss-classified and in the reality it is a T. fusca strain, therefore at present time there are no data on T. alba hydrolases.
Investigated endomannanases from T. fusca, T. cellulosilytica and T. halotolerans belong to GH5 hydrolases. They have modular architecture and have polysaccharide binding site at the C-terminus. The AA homology between them is 82-84% and their characteristics are very similar regarding the kinetic parameters although the pH and temperature optima are slightly different. The differences in thermal stability of the three enzymes are more pronounced: the lifetime of Man5ATf is four-five times higher at 70°C compared to the other two enzymes. Man5-ATh of T. halotolerans moderately salt tolerant, its thermal stability is preserved up to 0.8M of NaCl concentration. These parameters coincided well with environmental parameters of niches where these thermobifida strains were isolated from.
Despite the high sequence similarity of the investigated mannanases they exhibit different temperature stability, and this can be a starting point for further structural-functional investigations and for industrial applications to produce biologically active, oligomannan prebiotics.