Abstract
Thermotoga maritima (Tma) contains genes encoding various hyperthermophilic enzymes with great potential for industrial applications. The gene TM1752 in Tma genome has been annotated as cellulase gene encoding protein Cel5B. In this work, the gene TM1752 was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Interestingly, the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxy methyl cellulose, which is the highest among thermophilic mannanases. However, the putative enzyme did not show sequence homology with any of the previously reported mannanases; therefore, the enzyme Cel5B was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B. Man/Cel5B exhibited maximum activity at 85°C and pH 5.5. This enzyme retained more than 50% activity after 5 h of incubation at 85°C, and retained up to 80% activity after incubated for 1 h at pH 5–8. The Km and Vmax of Man/Cel5B were observed to be 4.5 mg/mL galactomannan and 769 U/mg, respectively. Thin layer chromatography depicted that locust bean gum could be efficiently degraded to mannobiose, mannotriose, and mannooligosaccharides by Man/Cel5B. These characteristics suggest that Man/Cel5B has attractive applications for future food, feed, and biofuel industries.
Highlights
Mannan is a complex biopolymer, composed of mannose residues that are linked together by β-1,4 linkages named as pure mannan, or the polysaccharide may be a mixture of β-1,4-Dmannose and β-1,4-D-glucose units termed as glucomannan
Β-mannanase plays an essential role in the degradation of lignocellulosic biomass as β-mannan is the principal component of hemicellulose in soft woods. β-mannanase has been employed for the degradation of mannan in the cell wall of palm kernel cake for ethanol or biobutanol production (Cerveró et al, 2010; Shukor et al, 2016)
It can degrade mannan-rich agricultural crop residues, such as locust bean gum (LBG) and guar gum into mannooligosaccharides (MOS) which have numerous health benefits (Katrolia et al, 2013). β-mannanase is extensively used in the paper/pulp, food, and feed industries, in poultry feeds and prebiotic food supplements to decrease the immunogenic effect of mannan polymers, clarify fruit juices, and extract of oil from copra and detergent (Kaira et al, 2016; Singh et al, 2019)
Summary
Mannan is a complex biopolymer, composed of mannose residues that are linked together by β-1,4 linkages named as pure mannan, or the polysaccharide may be a mixture of β-1,4-Dmannose and β-1,4-D-glucose units termed as glucomannan. Β-mannanase has been employed for the degradation of mannan in the cell wall of palm kernel cake for ethanol or biobutanol production (Cerveró et al, 2010; Shukor et al, 2016) It can degrade mannan-rich agricultural crop residues, such as locust bean gum (LBG) and guar gum into mannooligosaccharides (MOS) which have numerous health benefits (Katrolia et al, 2013). Β-mannanase is extensively used in the paper/pulp, food, and feed industries, in poultry feeds and prebiotic food supplements to decrease the immunogenic effect of mannan polymers, clarify fruit juices, and extract of oil from copra and detergent (Kaira et al, 2016; Singh et al, 2019) In these industries, thermostable enzymes are preferentially used (Dhawan et al, 2016; Li and Nie, 2016) due to their robustness and enhanced rate of hydrolysis (Katsimpouras et al, 2016; Niu et al, 2017). Thermozymes exhibit low fluid viscosity, and high saccharification and production yield (Sarmiento et al, 2015), thereby improving the cost-effectiveness of the bioprocess (Arora et al, 2015)
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