Elsevier

Enzyme and Microbial Technology

Volume 92, October 2016, Pages 76-85
Enzyme and Microbial Technology

A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst

https://doi.org/10.1016/j.enzmictec.2016.06.018Get rights and content

Highlights

  • Surfactant-, NaCl-, urea-, and protease-tolerant mannanase was purified and characterized.

  • MnCSB39 had the lowest molecular mass of ∼30 kDa from Bacillus sp.

  • Optimal pH and temperature for enzymatic activity were 7.5 and 70 °C respectively.

  • The Km and Vmax values were 0.0819 mg/mL and 1099 ± 1.0 Umg−1, respectively.

  • Thermodynamics also support its hydrolytic efficiency, feasibility and spontaneity.

Abstract

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14 U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30 kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70 °C. The enzyme showed ˃55% activity at 5.0–15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37 °C for 60 min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3 M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85 kJmol−1 with a Kcat of 142.58 × 104 s−1. MnCSB39 had Km and Vmax values of 0.082 mg/mL and 1099 ± 1.0 Umg−1, respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q10, ΔGE-S, and ΔGE-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44 U mannanase from solid state fermentation of 1 g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst.

Introduction

Hemicelluloses are the second most abundant heteropolymer in nature. They are classified as mannans, xylans, arabinogalactans, or arabinans depending on their sugar backbone composition. Mannans are constructed from the simple sugar mannose as plant polysaccharides. These include mannan, glucomannan, galactomannan, and galacto- glucomannan, which consist of a β-1,4-linked linear backbone of mannose residues that carry other carbohydrates or acid substitutions. There are three enzymes that participate in the complete decomposition and conversion of mannan, namely endo-1,4-β-mannanase, exo-1,4-β-mannanase, and β-mannosidase [1], [2]. Nowadays, mannan and its degradation products (mannooligosaccharides) have been attracting attention of researchers in the food and pharmaceuticals industries because these oligosaccharides and poly-oligosaccharides exhibit various beneficial effects on human health [2].

Mannan and heteromannans are the major part of the hemicellulose fractions of plant cell walls. Mannan endo-1,4-β-mannosidase or 1,4-β-d-mannan mannanohydrolase (EC 3.2.1.78) catalyzes the random hydrolysis of the β-1,4 mannosidic linkages of mannans, glucomannan, galactomannan, and galactoglucomannan to yield mannooligosaccharides [3]. The synergistic action of endo-1,4-β-mannanases, β-mannosidases, β-glucosidases, α-galactosidases, and acetyl mannanesterases are required for the complete hydrolysis of softwood mannans. These enzymes belong to glycosyl hydrolase (GH) families 5 and 26 according to the carbohydrate active enzymes database (http://www.cazy.org) [4]. There are various applications of mannanases in pharmaceuticals and industrial processes, such as bio-bleaching of soft-wood pulps in the paper and pulp industries, improving the quality of food and feed, reducing the viscosity of coffee extracts, oil drilling, and as hydrolytic agents in detergents, slime control agents, and fish feed additives, etc. [5], [6].

The production of β-mannanases by microorganisms is more feasible due to its low cost, high production rate, and easily controlled conditions. β-1,4-mannanase has been isolated from a wide range of microorganisms, including Bacillus subtilis B36 [7], Bacillus subtilis NM-39 [8], Bacillus amyloliquefaciens CS47 [9], Bacillus sp. N16-5 [10], Bacillus sp. MG-33 [11], Paenibacillus illinoisensis ZY-08 [12], Bispora sp. MEY-1 [13], Aspergillus niger [14], Vibrio sp. Strain MA-138 [15], Sclerotium rolfsii [16], Cellulosimicrobium sp. strain HY-13 [17], Streptomyces lividans 66 [18], Streptomyces thermolilacinus [19], and Streptomyces sp. S27 [20]. Microbial mannanases are mainly extracellular and inducible in nature [5].

Different parameters influence the production of mannanase. The nutritional and physicochemical factors such as incubation time, temperature, pH, carbon and nitrogen sources, inorganic salts, agitation and dissolved oxygen concentration, affect the production of mannanase [2]. To overcome the problem of low yield of mannanase and high production costs for industrial application, we screened the predominant microorganism from fermented food (kimchi) in order to find a new strain that would produce mannanase with high activity. In our study, we selected the strain that can produce the extracellular enzyme to degrade the galactomannan (locust bean gum). This work describes the isolation of Bacillus sp. CSB39 from kimchi and purification of novel mannanase. Further, the biochemical and thermodynamic characterizations of the enzyme were performed to determine the potential of the enzyme for bio-industrial applications.

Section snippets

Materials

Locust bean gum (galactomannan) and mannose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thin layer chromatography (TLC) silica gel plates were purchased from Merck (Darmstadt, Germany). Mannobiose and mannotriose were purchased from Megazyme (Ireland). Sepharose CL-6B was purchased from Amersham Bioscience (Uppsala, Sweden). DEAE Sepharose Fast Flow was purchased from GE Healthcare Bio-Science AB (Uppsala, Sweden). All the reagents used were of analytical grade.

Isolation and two-stage screening of bacterial strain for mannanase production

Seventy-eight samples

Screening of bacterial strain for mannanase production

The presence of clear lytic zones around the colonies on the mannan agar plates indicates that mannan was utilized and degraded by a bacterial extracellular enzyme, which is present in mannan agar plate. Out of a total 78 isolates, only 21 formed clear lytic zones on mannan agar plates after incubation at 37 °C for 30 h, where the control had no effect (no lytic zones). The 21 strains were further cultured in galactomannan medium at 37 °C with shaking at 120 rpm for 60 h. As mention above, among the

Conclusion

We isolated and identified Bacillus sp. CSB39 from popular traditional Korean food kimchi. It was cultured in galactomannan media and purified to homogeneity using a two-step chromatographic separation technique. We found MnCSB39 to have a tolerance for surfactants-, NaCl-, urea-, and protease. MnCSB39 works optimally at a temperature of 70 °C and a pH of 7.5, retains its activity at a broad pH range (pH 4.6–11), and has a high thermostability. The kinetic and thermodynamic parameters of MnCSB39

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (NRF-2015R1A2A1A15056120, NRF-2015R1D1A1A 010 59 483) and Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (115073-2).

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