Strains, materials, and chemicals
The actinomycetes were obtained from Biotechnology Culture Collection (BTCC), Indonesian Institute of Sciences (LIPI). Streptomyces lividans 1326 (NBRC 15675) was purchased from National Institute of Technology and Evaluation (NITE, Chiba, Japan). Escherichia coli JM109 (Takara, Shiga, Japan) was used as the host strain for DNA manipulation. Locust bean gum (LBG) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ivory nut and konjac glucomannan were purchased from Megazyme (Wicklow, Ireland). Porang potato was purchased from PT Ambico (Surabaya, East Java, Indonesia). Suweg potato was purchased from a traditional market in East Java, Indonesia. Copra cake and palm kernel cake were purchased from a traditional market in Lampung, Sumatra, Indonesia. Mannose (M1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mannobiose (M2), mannotriose (M3), mannotetraose (M4), mannopentaose (M5), and mannohexaose (M6) were purchased from Megazyme (Wicklow, Ireland).
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.
The hydrolysis of mannan substrates by the culture supernatant from ID04-0555 strain (LBG as a carbon source) was analyzed via TLC. Hydrolysis of 0.4% (w/v) LBG, 0.2% (w/v) ivory nut, 0.2% (w/v) konjac glucomannan, or 0.3% (w/v) porang was performed containing 50 mM MES buffer (pH 6.5) and 1.0 U/mL of β-mannanase. Reaction mixtures were incubated for 0, 1, 2, 3, and 4 h in a shaker incubator at 30 °C. Reaction mixtures were heated at 100 °C for 5 min to terminate the reaction at various reaction times (0, 1, 2, 3, and 4 h). Reaction mixtures were spotted on TLC Silica gel 60F254, 20–20 cm (EMD/Merck, Darmstadt, Germany) and developed with a mixture of n-Butanol/Acetic Acid/water (2:1.1:1, v/v/v). Spots were stained using DAP that contained diphenylamine, aniline, acetone, and phosphoric acid (Merck KGaA, Darmstadt, Germany), and subsequently heated at 120 °C for 15 min. Mannose (M1), mannobiose (M2), mannotriose (M3), mannotetraose (M4), mannopentaose (M5), and mannohexaose (M6) were used as standards.
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.
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 CaCl2, CuCl2, CoCl2, NaCl, KCl, MgCl2, MnCl2, and ZnCl2, 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, GenBank database under the accession number KY576672) has been deposited in the GenBank database under the accession number LC012037.