A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst
Graphical abstract
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).
References (44)
- et al.
Softwood hemicellulose-degrading enzymes from Aspergillus niger: purification and properties of a β-mannanase
J. Biotechnol.
(1998) - et al.
Cloning DNA sequencing, and expression of the β-1,4-mannanase gene from a marine bacterium, Vibrio sp. strain MA-138
J. Ferment. Bioeng.
(1997) - et al.
Hydrolysis of isolated coffee mannan and coffee extract by mannanases of Sclerotium rolfsii
J. Biotechnol.
(2000) - et al.
Cloning and characterization of a modular GH5 β-1,4-mannanase with high specific activity from the fibrolytic bacterium Cellulosimicrobium sp. strain HY-13
Bioresour. Technol.
(2011) - et al.
Characterization of calcium ion sensitive region for β-mannanase from Streptomyces thermolilacinus
Biochim. Biophys. Acta
(2011) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding
Anal. Biochem.
(1976)- et al.
High-level production, purification and characterization of a thermostable β-mannanase from the newly isolated Bacillus subtilis WY34
Carbohydr. Polym.
(2006) - et al.
Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32
Bioresour. Technol.
(2008) - et al.
Influence of water activity and temperature on xylanase biosynthesis in pilot-scale solid-state fermentation by Aspergillus sulphureus
Enzyme Microb. Technol.
(2003) Recent process developments in solid-state fermentation
Process Biochem.
(1992)
Thermostable enzymes
Biotechnol. Adv.
Acidic β-mannanase from Penicillium pinophilum C1: cloning, characterization and assessment of its potential for animal feed application
J. Biosci. Bioeng.
Kinetics and heat-inactivation mechanisms of d-amino acid oxidase
Process Biochem.
Thermozymes: identifying molecular determinants of protein structural and functional stability
Trends Biotechnol.
Cloning and strong expression of a Bacillus subtilis WL-3 mannanase gene in B. subtilis
J. Microbiol. Biotechnol.
An overview of mannan structure and mannan-degrading enzyme systems
Appl. Microbiol. Biotechnol.
Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain
Appl. Environ. Microbiol.
The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics
Nucleic Acids Res.
Microbial mannanases: an overview of production and applications
Crit. Rev. Biotechnol.
Mannanases: microbial sources, production, properties and potential biotechnological applications
Appl. Microbiol. Biotechnol.
A beta-mannanase from Bacillus subtilis B36: purification, properties, sequencing, gene cloning and expression in Escherichia coli
Z. Naturforsch. C
Purification and properties of mannanase from Bacillus subtilis
World J. Microbiol. Biotechnol.
Cited by (25)
Hyperproduction of a bacterial mannanase and its application for production of bioactive mannooligosaccharides from agro-waste
2023, Process BiochemistryCitation Excerpt :MAN-7 also showed high pH stability in the wide pH range of 5.0–8.0 as it retained > 65% activity after 12 h of preincubation (Fig. 3). Mannanases from most of the bacterial strains have been reported to be active in the neutral pH range [24,25]. Apart from few exceptions such as mannanases from Bacillus velezensis and Bacillus sp.
The production of β-mannanase from Kitasatospora sp. strain using submerged fermentation: Purification, characterization and its potential in mannooligosaccharides production
2020, Biocatalysis and Agricultural BiotechnologyCitation Excerpt :Whereas, hydrolysis of LBG by Penicillium occitanis produced M3 and M4 as a main products (Blibech et al., 2011), hydrolysis of LBG by Aspergillus terreus mainly produced M2-M6 (Soni et al., 2016) and hydrolysis of LBG by Bacillus sp. CSB39 just produced M2 (Regmi et al., 2016). Hydrolysis of palm kernel cake formed M2-M6, with M3 and M4 as the chief component (Fig. 7B).
Development and catalytic characterization of L-asparaginase nano-bioconjugates
2019, International Journal of Biological MacromoleculesThermo and alkali stable β-mannanase: Characterization and application for removal of food (mannans based) stain
2019, International Journal of Biological MacromoleculesCitation Excerpt :However, its wide applicability in a range of industries such as cosmetic, food, pharmaceutical, detergent, etc. where different forms of mannans are involved, makes it commercially desirable. In the current work, we purified and characterized an alkaline thermostable β-mannanase from newly isolated K. pneumoniae strain SS11 which produced extracellular mannanase [14] with high activity as desired for industrial applications. The predominant microbe was screened from landfill site and a strain was selected that could produce extracellular β-mannanase to degrade locust bean gum (LBG).
- 1
Both these authors contributed equally to this work.