High NaCl concentrations induce the resistance to thermal denaturation of an extremely halotolerant (salt-activated) ß-mannanase from Bacillus velezensis H1.

ß-mannanase catalyzes the hydrolysis of mannans ß-1,4-mannosidic linkages to produce industrially relevant oligosaccharides. These enzymes have numerous important applications in the detergent, food, and feed industries, particularly those that are resistant to harsh environmental conditions such as salts and heat. While, moderately salt-tolerant ß-mannanases are already reported, existence of a high halotolerant ß-mannanase is still elusive. This study aims to report the first purification and characterization of ManH1, an extremely halotolerant ß-mannanase from the halotolerant B. velezensis strain H1. Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis revealed a single major peak with a molecular mass of 37.8 kDa demonstrating its purity. The purified enzyme showed a good thermostability as no activity was lost after a 48 h incubation under optimal conditions of 50 °C and pH 5.5. The enzyme's salt activation nature was revealed when its maximum activity was obtained in the presence of 4 M NaCl, it doubled compared to the no-salt condition. Moreover, NaCl strengthens its resistance to thermal denaturation, as its melting temperature (Tm) increased steadily with increasing NaCl concentrations reaching 75.5 °C in the presence of 2.5 M NaCl. The Km and Vmax values were 5.63 mg/mL and 333.33 µmol/min/mL, respectively, using carob galactomannan (CG) as a substrate. The enzyme showed a significant ability to produce manno-oligosaccharides (MOS) from lignocellulosic biomass releasing 13 mg/mL of reducing sugars from olive mill wastes (OMW) after 24 h incubation. The results revealed that this enzyme may have significant commercial values for agro-waste treatment, and other potential applications.

Box-Behnken design optimization of xylanase and cellulase production by Aspergillus fumigatus on Stipa tenacissima biomass.

Optimization of xylanase and cellulase production by a newly isolated Aspergillus fumigatus strain grown on Stipa tenacissima (alfa grass) biomass without pretreatment was carried out using a Box-Behnken design. First, the polysaccharides of dried and ground alfa grass were characterized using chemical methods (strong and diluted acid). The effect of substrate particle size on xylanase and carboxymethylcellulase (CMCase) production by the selected and identified strain was then investigated. Thereafter, experiments were statistically planned with a Box-Behnken design to optimize initial pH, cultivation temperature, moisture content, and incubation period using alfa as sole carbon source. The effect of these parameters on the two enzyme production was evaluated using the response surface method. Analysis of variance was also carried out, and production of the enzymes was expressed using a mathematical equation depending on the influencing factors. The effects of individual, interaction, and square terms on production of both enzymes were represented using the nonlinear regression equations with significant R2 and P-values. Xylanase and CMCase production levels were enhanced by 25% and 27%, respectively. Thus, this study demonstrated for the first time the potential of alfa as a raw material to produce enzymes without any pretreatment. A set of parameter combinations was found to be effective for the production of xylanase and CMCase by A. fumigatus in an alfa-based solid-state fermentation.

Production of a halotolerant endo-1,4-ß-glucanase by a newly isolated Bacillus velezensis H1 on olive mill wastes without pretreatment: purification and characterization of the enzyme.

Facing the critical issue of high production costs for cellulase, numerous studies have focused on improving the efficiency of cellulase production by potential cellulolytic microorganisms using agricultural wastes as substrates, extremophilic cellulases, in particular, are crucial in the biorefinery process because they can maintain activity under harsh environmental conditions. This study aims to investigate the ability of a potential carboxymethylcellulose-hydrolyzing bacterial strain H1, isolated from an Algerian saline soil and identified as Bacillus velezensis, to use untreated olive mill wastes as a substrate for the production of an endo-1,4-ß-glucanase. The enzyme was purified 44.9 fold using only two steps: ultrafiltration concentration and ion exchange chromatography, with final recovery of 80%. Its molecular mass was estimated to be 26 kDa by SDS-PAGE. Enzyme identification by LC-MS analysis showed 40% identity with an endo-1,3-1,4-ß-glucanase of GH-16 family. The highest enzymatic activity was significantly measured on barley ß-glucan (604.5 U/mL) followed by lichenan and carboxymethylcellulose as substrates, confirming that the studied enzyme is an endo-1,4-ß-glucanase. Optimal enzymatic activity was at pH 6.0-6.5 and at 60-65 °C. It was fairly thermotolerant, retaining 76.9% of the activity at 70 °C, and halotolerant, retaining 70% of its activity in the presence of 4 M NaCl. The enzyme had a Vmax of 625 U/min/mL and a high affinity with barley ß-glucan resulting a Km of 0.69 mg/mL. It also showed a significant ability to release cello-oligosaccharides. Based on such data, the H1 endo-1,4-ß-glucanase may have significant commercial values for industry, argo-waste treatment, and other biotechnological applications.