Mannan endo-1,4-β-mannosidase from Kitasatospora sp. isolated in Indonesia and its potential for production of mannooligosaccharides from mannan polymers

Mannan endo-1,4-β-mannosidase (commonly known as β-mannanase) catalyzes a random cleavage of the β-d-1,4-mannopyranosyl linkage in mannan polymers. The enzyme has been utilized in biofuel production from lignocellulose biomass, as well as in production of mannooligosaccharides (MOS) for applications in feed and food industries. We aimed to obtain a β-mannanase, for such mannan polymer utilization, from actinomycetes strains isolated in Indonesia. Strains exhibiting high mannanase activity were screened, and one strain belonging to the genus Kitasatospora was selected. We obtained a β-mannanase from this strain, and an amino acid sequence of this Kitasatospora β-mannanase showed a 58–71% similarity with the amino acid sequences of Streptomyces β-mannanases. The Kitasatospora β-mannanase showed a significant level of activity (944 U/mg) against locust bean gum (0.5% w/v) and a potential for oligosaccharide production from various mannan polymers. The β-mannanase might be beneficial particularly in the enzymatic production of MOS for applications of mannan utilization. Electronic supplementary material The online version of this article (doi:10.1186/s13568-017-0401-6) contains supplementary material, which is available to authorized users.

Mannan polymers are widely distributed in plants as part of the hemicellulose fraction in hardwoods, softwoods, seeds of leguminous plants, and beans (Dhawan and Kaur 2007). The use of β-mannanase is one method to the extensive degradation of these mannan polymers for application in biofuel production from lignocellulose biomass (Ishii et al. 2016), as well as in production of mannooligosaccharides (MOS) (Yamabhai et al. 2016). MOS are non-digestible oligosaccharides that have potential applications in dietary fiber and prebiotics (Asano et al. 2003;Jian et al. 2013). Unfortunately, most of the MOS products are derived from sugar polymers present in the cell wall of Saccharomyces cerevisiae yeast. Some researchers have studied the prebiotic effect of MOS from plants and showed the potential use to improve human health, such as promoting the growth of intestinal beneficial microflora, decreasing enteric pathogenic bacteria, and reduction of dietary fat absorption (for example, Van Zyl et al. 2010;Jian et al. 2013;Chauhan et al. 2014). The supply of mannan polymers must meet the market demand for MOS.
Application of β-mannanase to produce MOS might be greatly beneficial if a cheap mannan-rich biomass is used as the substrate. Interestingly, Indonesia has abundant sources of biomass that contain high levels of heteromannan, such as palm kernel cake, copra cake, porang potato from the bulbs of porang (Amorphophallus onchophyllus), and suweg potato from Amorphophallus campanulatus. This fact suggests that the production of MOS based on a cheap mannan-rich biomass can be exactly realized. Therefore, the potential of mannan polymers from these biomasses in Indonesia has received an industrial focus, such as the food and feed industry.
β-Mannanase could be naturally produced by actinomycetes. Actinomycetes produce extracellular enzymes that can decompose different types of substrates, and some enzymes from actinomycetes can be employed as important enzyme accessories in industrial processes, such as cellulase, xylanase, pectinase, protease, and chitinase (Prakash et al. 2013). There are several reports related to the cloning and expression of β-mannanase genes from actinomycetes. Fortunately, Indonesia is also rich in terrestrial microbes, particularly in actinomycetes. Several scientists have reported that actinomycetes isolated from Indonesian soils and leaf-litters represent new species and demonstrate genus diversity (for example, Otoguro et al. 2009Otoguro et al. , 2011Lisdiyanti et al. 2010;Yamamura et al. 2010). Extensive exploration of industrially useful enzymes has been conducted from these actinomycetes, such as inulin fructotransferase (Pudjiraharti et al. 2011(Pudjiraharti et al. , 2014. Accordingly, there are two impacts using β-mannanases from these actinomycetes for the industrial production of MOS; the first is the possibility to obtain many kinds of β-mannanases with varied enzymatic properties due to the variety of actinomycetes strains, and the second is the high potential production of various MOS from mannan polymers due to the readiness of many kinds of β-mannanases with a broad substrate specificity. Herein, we report the screening and cloning of a β-mannanase from actinomycetes isolated in Indonesia to explore its possible utilization as mentioned above. We selected one strain (ID04-0555) from the genus Kitasatospora, which represents the first trial, as a mannan-degrading enzyme producer. After cloning a β-mannanase gene from this strain, we found that the enzyme demonstrated the release of various types of oligosaccharides from the mannan polymers (particularly substrate derived from raw mannan polymers), indicating its beneficial potential in the production of MOS from raw biomass.

Screening of actinomycetes strains for a mannan degrading enzyme from actinomycetes
500 strains were cultured on ISP2 agar medium. A single colony was pre-cultured in ISP2 broth at 28 °C, 190 rpm for 3 days, then inoculated into 10 mL modified ISP2 broth which contained 0.4% yeast extract, 1.0% malt extract, and 0.5% each of mannan biomass (LBG, porang potato, copra cake, palm kernel cake, and suweg potato) in 100 mL Erlenmeyer flasks. The fermentation was carried out on a rotary shaker at 28 °C and 190 rpm for 5 days. Sampling was carried out every 24 h. Culture supernatants were collected by centrifugation at 5000 rpm for 10 min. The analysis of β-mannanase activity against mannan substrates was measured on LBG agar (0.5% LBG and 1.8% agar) by individually spotting an aliquot of the culture supernatant (5 µL). The plates were incubated at 37 °C for 3 days. After 3 days, the plates were flooded with an aqueous solution of 0.25% Congo red (Nacalai, Kyoto, Japan) for 30 min to visualize the hydrolysis zones. The plates were then washed twice with 1 M NaCl for 15 min and 0.5% acetic acid to check the LBG degradation more clearly.

Molecular identification of the ID04-0555 strain
Molecular identification of the ID04-0555 strain was conducted based on the 16S rRNA gene as established by Lisdiyanti et al. (2010). The 16S rRNA gene was amplified via polymerase chain reaction (PCR) technique using a pair of 9F and 1510R primers (Additional file 1: Table S1) (Burggraf et al. 1992). The sequence was confirmed via ABI 3130 DNA sequencer (Applied Biosystems, Foster City, CA, USA) and then compared with others available in the GenBank/DDBJ/EMBL database using multiple sequence alignment (ClustalW).

Molecular cloning of a β-mannanase gene from the ID04-0555 strain
The ID04-0555 strain was cultured on inorganic salt starch agar plates. A single colony was cultured in TSB medium (17 g/L pancreatic digest of casein, 3.0 g/L enzymatic digest of soya bean contain papain, 2.5 g/L glucose, 5.0 g/L sodium chloride, and 2.5 g/L di-potassium hydrogen phosphate, Oxoid, Hampshire, England) for genomic DNA extraction. Genomic DNA was extracted using the method established by Kieser et al. (2000). A detailed method for cloning the β-mannanase gene appears in Additional file 1.

Expression and purification of recombinant β-mannanase in S. lividans 1326
Streptomyces lividans 1326/pUC702-pro-ManKs-(His) 6 (the recombinant β-mannanase expression strain) was inoculated into a test tube containing 5 mL of TSB medium (Becton, Dickinson and Company, Sparks, MD, USA) supplemented with 5 μg/mL of thiostrepton (EMD chemicals, San Diego, CA, USA), followed by cultivation at 28 °C for 2 days. Then, 1 mL of the seed culture was transferred into a baffled 500 mL shaking flask containing 100 mL of modified TSB medium with 30 g/L glucose (Nacalai, Kyoto, Japan) as a carbon source, 15 g/L tryptone (Nacalai, Kyoto, Japan) as a nitrogen source, and 5 μg/mL of thiostrepton. Cultivation was performed at 28 °C for 3 days.
Recombinant His-tagged β-mannanase was purified using Ni Sepharose ™ excel (GE Healthcare, Uppsala, Sweden) according to the manufacturer's instructions. Homogeneity and molecular mass of the purified β-mannanase were evaluted via 12% SDS polyacrylamide gel electrophoresis. Visualization of the protein bands was accomplished by staining with Coomassie Brilliant Blue G-250 (Nacalai, Kyoto, Japan). For western blotting, proteins were electroblotted onto Immobilon-P transfer membrane (Merck Millipore, Cork, Ireland) from SDS polyacrylamide gel and the His-tagged β-mannanase was allowed to react with Anti-His-tag HRP-DirecT (KDX, Aichi, Japan). The purified β-mannanase protein was electroblotted onto the transfer membrane, stained by Ponceau S (Nacalai, Kyoto, Japan), and analyzed using a peptide sequencer (Procise 492-HT Protein Sequencer, Applied Biosystems). Protein concentration was determined via Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard.

Enzyme assays
Standard β-mannanase activity was assayed via the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959). The standard reaction was conducted at 45 °C after exactly 15 min in 0.5 mL of a reaction mixture that contained appropriately diluted recombinant enzyme, 0.5% (w/v) LBG, and 50 mM MES buffer (pH 6.5). The amount of reducing sugars liberated in the enzyme reaction was assayed by mixing 0.5 mL of the DNS solution. The mixture was heated at 100 °C for 15 min and cooled on ice. The absorbance of the sample was measured at 540 nm. One unit of β-mannanase activity is defined as the amount of enzyme that liberates 1 µmol of reducing sugar per minute under a given set of experimental conditions. This experiment was repeated three times.

Characterization of β-mannanase activity
To determine the optimal pH of β-mannanase activity, evaluations were conducted between pH 4.0 and 10 under standard assay conditions using the following buffers: 50 mM Acetate buffer (pH 4.0-5.0), MES buffer (pH 5.0-7.0), MOPS buffer (pH 7.0-8.5), and glycine-NaOH buffer (pH 8.5-10), respectively. The optimal temperature of activity was evaluated by incubating the enzyme samples with the substrate at temperatures ranging from 30 to 90 °C in 50 mM MES buffer (pH 6.5). Thermal stability of the activity was determined by incubating the enzyme in 50 mM MES buffer (pH 6.5) at 45, 50, and 55 °C for a maximum of 120 min. The effect of various metal ions and chemical reagent on the activity was determined by incubating the enzyme in 50 mM MES (pH 6.5) containing 0.5% (w/v) LBG in the presence of 1 mM of CaCl 2 , CuCl 2 , CoCl 2 , NaCl, KCl, MgCl 2 , MnCl 2 , and ZnCl 2 , and 5 mM of EDTA. These experiments were repeated three times.
Relative activity of β-mannanase against LBG, ivory nut, konjac glucomannan, and porang potato was determined by 0.5% (w/v) of each substrate in 50 mM MES buffer (pH 6.5) at 45 °C for 15 min. For determination of the kinetic parameters, concentrations of LBG were varied from 0.05 to 0.5% (w/v) and the activity of the recombinant β-mannanase was monitored in 50 mM MES buffer (pH 6.5) at 45 °C for 30 min. Michaelis-Menten parameters (V max and K m values) were estimated by the Hanes-Woolf plot. These experiments were repeated three times.
The hydrolysis products from mannan polymers by the recombinant β-mannanase were analyzed via TLC. Details of this method were shown earlier in materials and methods. Hydrolysis of mannan polymers was performed containing 50 mM MES buffer (pH 6.5) and 1.0 U/mL of the recombinant β-mannanase. Reaction mixtures were incubated for 0, 1, 2, 3, 4, 24, 48, and 72 h at 30 °C. Additionally, hydrolysis of 0.5% (w/v) M1-M6 was performed containing 50 mM MES buffer (pH 6.5) and 3.1 U/mL of the recombinant β-mannanase.

Nucleotide sequence accession number
The nucleotide sequence of the β-mannanase gene isolated from the ID04-0555 strain (BTCC B-806, Gen-Bank database under the accession number KY576672) has been deposited in the GenBank database under the accession number LC012037.

Screening using mannan polymers
At first, we screened 500 isolates to identify strains exhibiting high mannanase activity among the actinomycetes collected by biotechnology culture collection (BTCC) from Indonesian soils and leaf-litters. These strains were cultivated in the ISP2 medium using LBG as a carbon source, and qualitative analysis of hydrolysis activity was performed using Congo red dye (such as ID04-0555 strain shown in Fig. 1a). One strain, the ID04-0555 strain, was selected with the high activity among these isolates. We also confirmed that the hydrolysis activity of the ID04-0555 strain was induced by various sources of Indonesian mannan biomass, such as porang potato, copra cake, palm kernel cake, and suweg potato substrates. (Additional file 1: Figure S1).
To investigate hydrolytic patterns of the secreted mannan degrading enzymes from the ID04-0555 strain, reactions were measured based on TLC analysis using the culture supernatant. The culture supernatant from the ID04-0555 strain was able to hydrolyze mannan polymers into oligosaccharides (Fig. 1b-e). LBG was hydrolyzed into mannose and various types of oligosaccharides for 4 h, and trisaccharide was one of the major products. A similar degradation pattern was observed using ivory nut. The hydrolysis of glucomannan, such as the konjac glucomannan and porang potato, also produced various oligosaccharides with trisaccharides as main products.

Molecular identification of the ID04-0555 strain
The ID04-0555 strain was isolated in a soil sample collected from Cibinong, West Java, Indonesia. Phylogenetic analysis confirmed that this strain belongs to the family Streptomycetaceae and to the genus Kitasatospora. Moreover, neighbor-joining phylogenetic analysis based on a 16S rRNA gene sequence revealed that the strain belongs to the genus Kitasatospora and is closely related to Kitasatospora cineracea and Kitasatospora niigatensis (97% sequence similarity) (Fig. 2).

Cloning and sequence analysis of a β-mannanase gene from the ID04-0555 strain
We amplified a partial β-mannanase gene from the ID04-0555 strain via a PCR reaction with a set of primers designed by β-mannanase sequences of five Streptomyces strains from NCBI/NBRC. Then, an entire open reading frame of the β-mannanase sequence was identified from the partial gene sequence (for details of the method, see Additional file 1). We obtained 1435 nucleotides in the DNA sequence, which revealed an open reading frame (ORF) of 1302 nucleotides encoding a protein with 434 amino acids and a calculated molecular mass of 41.4 kDa (referred to as ManKs_4-555) (Fig. 3).
Alignment of the putative amino acid sequence showed that ManKs_4-555 was comprised of a putative N-terminal catalytic domain (from E51 to L288) and two cellulose binding modules (CBMs) at the C-terminus region (from A355 to V384 and from A398 to V427). The putative catalytic domain of ManKs_4-555 resembles the domain structure of a glycoside hydrolase family 5 (GH5). The carbohydrate-binding modules are similar to those of family 10 CBMs (Fig. 3).

Secreted expression of ManKs_4-555 in a heterologous host
The ManKs_4-555 β-mannanase gene, fused to a sequence encoding a histidine tag at the C-terminal, was ligated with the pUC702 vector under the control of the Streptoverticillium cinnamoneum phospholipase D promoter (Ogino et al. 2004). The constructed vector (referred to as pUC702-pro-ManKs-(His) 6 , Additional file 1: Table S1) was transformed into Streptomyces lividans 1326, which has been employed as a host strain for recombinant protein expression. The transformant strain was named S. lividans 1326/pUC702-pro-ManKs-(His) 6 (Additional file 1: Table S1).
After the secreted recombinant β-mannanase was expressed in the host strain, the culture supernatant was analyzed by SDS-PAGE and western blotting (Fig. 4a). A single protein band corresponding to the β-mannanase was observed, whereas no protein band was observed in the supernatant from either S. lividans 1326 wild-type strain or S. lividans 1326/pUC702-pro strain, which is a transformant harboring the pUC702-pro vector as a control ( Fig. 4a; Additional file 1: Table S1). Maximum β-mannanase activities of culture supernatants from S. lividans 1326/pUC702-pro-ManKs-(His) 6 , S. lividans 1326 wild-type, and S. lividans 1326/pUC702-pro strains reached 84, 0.39, and 0.41 U/mL, respectively, after cultivation at 28 °C for 72 h. These results indicate that recombinant β-mannanase was expressed in the S. lividans 1326/pUC702-pro-ManKs-(His) 6 strain. The maximum yield was 1.4 mg based on a 100 mL culture scale after purification. N-terminal amino acid sequence analysis (A-T-T-G-I-H-V-A-N-G) using a peptide sequencer indicates that the recombinant β-mannanase had a signal peptide from M1 to A37, and the sequence between A37 and A38 was cleaved during the secretory process.

Characterization of recombinant ManKs_4-555
The effect of pH on ManKs_4-555 activity was evaluated in 50 mM of various buffers at 45 °C for 15 min using LBG as a substrate. Maximum activity was observed at pH 6.5, and more than 80% maximum activity was detectable in the neutral pH region of 6.0-7.0 (Fig. 4b).
Specific activity and kinetic parameters of ManKs_4-555 were determined using LBG as a substrate. The specific activity of the ManKs_4-555 was 944 ± 54 U/mg under standard conditions (as mentioned in the "Materials and methods"). K m and V max values of 0.55 mg/mL and 1054 μmol/min/mg were determined using 0.05-0.5% LBG. The hydrolytic activity of the ManKs_4-555 was assayed against various mannan polymers as substrates. The ManKs_4-555 hydrolyzes LBG galactomannan, ivory , and glycine-NaOH buffer (pH 8.5-10, filled circle). ManKs_4-555 activity at pH 6.5 was taken to be 100%. c Temperature dependence of ManKs_4-555. Activity was measured at 30-90 °C using 50 mM MES buffer (pH 6.5) containing 0.5% (w/v) LBG. β-Mannanase activity at 65 °C was taken to be 100%. d Thermal stability of ManKs_4-555. Activity was measured at 45 °C (circle), 50 °C (square), and 55 °C (diamond) using 50 mM MES buffer (pH 6.5) for a maximum of 120 min. β-Mannanase activity at the starting point was taken to be 100% nut, konjac glucomannan and porang potato glucomannan with a relative hydrolysis of 100, 40, 90, and 65, respectively ( Table 2).

Hydrolysis of mannan polymers using recombinant ManKs_4-555
To investigate the hydrolytic pattern of mannan polymers, reactions with various mannan substrates were evaluated based on TLC method using recombinant ManKs_4-555, and results are shown in Fig. 5. ManKs_4-555 has the ability to hydrolyze some substrates such as LBG, ivory nut, konjac glucomannan, and porang potato for up to 72 h of reaction time. This β-mannanase produced various length oligosaccharides from LBG (Fig. 5a). On the other hand, it produced mannobiose, mannotriose, and mannotetraose from ivory nut as the main products (Fig. 5b). From glucomannan such as konjac glucomannan and porang potato, the β-mannanase produced oligosaccharides presumably derived from disaccharide, trisaccharide, and tetrasaccharide (Fig. 5c, d). We also analyzed the hydrolysis of mannose and oligosaccharides (from mannobiose to mannohexaose) using the ManKs_4-555 (Additional file 1: Figure S2). The substrates from mannose to mannotetraose were hardly hydrolyzed by the β-mannanase, whereas mannopentaose and mannohexaose were hydrolyzed into mannobiose, mannotriose, and mannotetraose (Additional file 1: Figure S2).

Discussion
Recently, the mannan degrading enzymes have gained interest for application in feed and food industries by the production of potentially health-promoting  mannooligosaccharides (MOS). Here, we cloned and characterized a β-mannanase from actinomycetes to have potential applications for mannan utilization, such as the production of MOS.
The activity of ManKs_4-555 was enhanced with Co 2+ and Mn 2+ ions (Table 1). The activity of some Streptomyces β-mannanases was reported to be enhanced with Mn 2+ ion (Shi et al. 2011;Montiel et al. 2002), whereas the activity of these β-mannanases was not reported to be enhanced with Co 2+ ion. Cellulosimicrobium sp. strain HY-13 β-mannanase, which has amino acid sequences corresponding to ManKs_4-555, showed a similar tendency toward enhanced activity with Co 2+ and Mn 2+ ions (Kim et al. 2011). In contrast, the activity of ManKs_4-555 was inhibited with Zn 2+ ion (Table 1). Several β-mannanases from Streptomyces strains (Kumagai et al. 2013) and Cellulosimicrobium sp. (Kim et al. 2011) had a similar tendency to inhibit activity with Zn 2+ ion. The ManKs_4-555 is presumably sensitive to metal ion, because the activity of the β-mannanase was inhibited with EDTA (Table 1).
Recombinant ManKs_4-555 produced oligosaccharides from mannan polymers, including various length oligosaccharides from LBG (Fig. 5a) and mannobiose, mannotriose, and mannotetraose mainly from ivory nut (Fig. 5b). ManKs_4-555 was different from the extracellular enzymes of the wild-type ID04-0555 strain in the production of oligosaccharides (trisaccharides as the main product from the enzymes of the ID04-0555 strain in Fig. 1b, c). Other mannan degradation enzymes might be also secreted from the ID04-0555 strain with the exception of the ManKs_4-555. In addition, the mannan degradation pattern for ManKs_4-555 was different from other previously reported Streptomyces and Cellulosimicrobium β-mannanases showing amino acid sequences corresponding to ManKs_4-555. For example, Streptomyces sp. S27 β-mannanase produced trisaccharide (22.14%) as the main product from LBG along with mannose to pentasaccharide (Shi et al. 2011), and S. thermolilacinus β-mannanase produced disaccharide and trisaccharide from LBG (Kumagai et al. 2011). The Cellulosimicrobium sp. strain HY-13 β-mannanase produced mannan polysaccharide to primarily pentasaccharide from LBG, but the β-1,4-mannanase produced M 4 (25.2%) and various length oligosaccharides from ivory nut mannan (Kim et al. 2011). Some β-mannanases other than actinomycetes were reported to produce mainly mannobiose and mannotriose from ivory nut mannan (Ademark et al. 1998;Sachslehner and Haltrich 1999;Stålbrand et al. 1993;Jiang et al. 2006). Several reported β-mannanases, such as Bacillus subtilis WY34 and Penicillium occitanis Pol6, were similar to ManKs_4-555 in the degradation  Montiel et al. (2002) pattern of copra mannan or ivory nut mannan (Jiang et al. 2006;Blibech et al. 2010). ManKs_4-555 showed the production of oligosaccharides from various mannan substrates, and this feature might be useful for the production of MOS based on mannan biomass. In conclusions, a Kitasatospora strain exhibiting high mannanase activity in the production of oligomannan was screened from actinomycetes strains isolated in Indonesia. A β-mannanase was then cloned from the strain and characterized. The amino acid sequence of the Kitasatospora β-mannanase showed a 58-71% similarity with the amino acid sequences of Streptomyces β-mannanases. Several characteristics, such as high hydrolysis activity and production of oligosaccharides from various mannan including raw mannan polymers, were found to be specific to the Kitasatospora β-mannanase, and these are thought to have important practical applications. Such properties suggest the potential utilization of ManKs_4-555 (or also the mother strain) for research in biodegradation of raw mannan biomass and prebiotics. We examined an actinomycete strain not yet previously reported for the cloning of β-mannanase. The investigation of different actinomycetes strains might be potentially useful for obtaining β-mannanases with varied enzymatic properties.