Man/Cel5B, a Bifunctional Enzyme Having the Highest Mannanase Activity in the Hyperthermic Environment

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.

Valorization Potential of a Novel Bacterial Strain, Bacillus altitudinis RSP75, towards Lignocellulose Bioconversion: An Assessment of Symbiotic Bacteria from the Stored Grain Pest, Tribolium castaneum

“…Our observations are in congruence with those of Vettath and coworkers who stated that many genes (875 out of 3820 protein-coding genes) in B. altitudinis encode for carbohydrate and protein metabolism [66]. Since mannan constitutes an important component of prebiotics for animal husbandry and nutritional supplements [67], B. altitudinis RSP75 could prove to be a promising candidate for mannan oligosaccharide-based dietary supplements of dairy and feedstock. Bacillus altitudinis RSP75 also showed a growth pattern similar to B. megaterium S3, showing slow and extended growth on CMC media [68] due to a metabolic shift to use cellulose as a carbon source.…”

Heterologous expression and characterization of glycoside hydrolase with its potential applications in hyperthermic environment

“…70PC53 and thermoacidophile Alicyclobacillus acidocaldarius ATCC27009 ( Eckert et al, 2002a , Eckert et al, 2002b , Ng et al, 2009 ). The optimum temperature reported here is relatively lower than related glycosyl hydrolases as reported previously ( Chhabra et al, 2002 , Sadaqat et al, 2021 ). The enzyme partially reamained stable upto 70 °C and then activity declines as increase in temperature.…”

Modifying Thermostability and Reusability of Hyperthermophilic Mannanase by Immobilization on Glutaraldehyde Cross-Linked Chitosan Beads

“…β-1,4-mannanase (mannan mannohydrolase, EC 3.2.1.78) is an endo-acting hydrolase that randomly cleaves the β-1,4-mannosidic linkages in the main chain of mannans (linear mannan, galactomannan, glucomannan, and galactoglucomannan), therefore producing mannooligosaccharides (MOS) of various lengths [ 5 ]. β-mannanase has wide industrial applications as it is used for the production of partially hydrolyzed guar gum, MOS, fruit juice clarification, paper/pulp bio-bleaching, synthesis of detergents, amelioration of animal or poultry feed, and saccharification of biomass [ 6 , 7 ].…”

“…In the current study, hyperthermostable β-mannanase from T. maritima (Man/Cel5B); which was purified and characterized in a previous study [ 6 ]; was immobilized on glutaraldehyde cross-linked chitosan beads. Properties of the immobilized and free Man/Cel5B were compared.…”