Enzymatic hydrolysis of low temperature alkali pretreated wheat straw using immobilized ß-xylanase nanoparticles.

A low temperature alkali (LTA) pretreatment method was used to treat wheat straw. In order to obtain good results, different factors like temperature, incubation time, NaOH concentration and solid to liquid ratio for the pretreatment process were optimized. Wheat straw is a potential biomass for the production of monomeric sugars. The objective of the current study was to observe the saccharification (%) of wheat straw with immobilized magnetic nanoparticles (MNPs). For this purpose, immobilized MNPs of purified ß-xylanase enzyme was used for hydrolysis of pretreated wheat straw. Wheat straw was pretreated using the LTA method and analyzed by SEM analysis. After completion of the saccharification process, saccharification% was calculated by using a DNS method. Scanning electron micrographs revealed that the hemicellulose, cellulose and lignin were partially removed and changes in the cell wall structure of the wheat straw had caused it to become deformed, increasing the specific surface area, so more fibers of the wheat straw were exposed to the immobilized ß-xylanase enzyme after alkali pretreatment. The maximum saccharification potential of wheat straw was about 20.61% obtained after pretreatment with optimized conditions of 6% NaOH, 1/10 S/L, 30 °C and 72 hours. Our results indicate the reusability of the ß-xylanase enzyme immobilized magnetic nanoparticles and showed a 15% residual activity after the 11th cycle. HPLC analysis of the enzyme-hydrolyzed filtrate also revealed the presence of sugars like xylose, arabinose, xylobiose, xylotriose and xylotetrose. The time duration of the pretreatment has an important effect on thermal energy consumption for the low-temperature alkali method.

Enzymatic hydrolysis of lignocellulosic biomass using a novel, thermotolerant recombinant xylosidase enzyme from Clostridium clariflavum: a potential addition for biofuel industry.

The present study describes the cloning, expression, purification and characterization of the xylosidase gene (1650 bp) from a thermophilic bacterium Clostridium clariflavum into E. coli BL21 (DE3) using the expression vector pET-21a(+) for utilization in biofuel production. The recombinant xylosidase enzyme was purified to homogeneity by heat treatment and immobilized metal ion affinity chromatography. SDS-PAGE determined that the molecular weight of purified xylosidase was 60 kDa. This purified recombinant xylosidase showed its maximum activity at a temperature of 37 °C and pH 6.0. The purified recombinant xylosidase enzyme remains stable up to 90 °C for 4 h and retained 54.6% relative activity as compared to the control. The presence of metal ions such as Ca2+ and Mg2+ showed a positive impact on xylosidase enzyme activity whereas Cu2+ and Hg2+ inhibit its activity. Organic solvents did not considerably affect the stability of the purified xylosidase enzyme while DMSO and SDS cause the inhibition of enzyme activity. Pretreatment experiments were run in triplicate for 72 h at 30 °C using 10% NaOH. Saccharification experiment was performed by using 1% substrate (pretreated plant biomass) in citrate phosphate buffer of pH 6.5 loaded with 150 U mL-1 of purified recombinant xylosidase enzyme along with ampicillin (10 µg mL-1). Subsequent incubation was carried out at 50 °C and 100 rpm in a shaking incubator for 24 h. Saccharification potential of the recombinant xylosidase enzyme was calculated against both pretreated and untreated sugarcane bagasse and wheat straw as 9.63% and 8.91% respectively. All these characteristics of the recombinant thermotolerant xylosidase enzyme recommended it as a potential candidate for biofuel industry.

Heterologous expression, molecular studies and biochemical characterization of a novel alkaline esterase gene from Bacillus thuringiensis for detergent industry.

Present study was aimed to clone and express the esterase encoding gene from Bacillus thuringiensis in E. coli BL21. Purification of recombinant esterase enzyme was achieved up to 48.6 purification folds by ion exchange chromatography with specific activity of 126.36 U mg-1. Molecular weight of esterase enzyme was 29 kDa as measured by SDS-PAGE. Purified esterase enzyme showed stability up to 90% at 90 °C and remained stable in a wide pH range (8-11). Molecular docking strengthens the experimental results by showing the higher binding energy with p-NP-butyrate. Enzyme activity was found to be reduced by EDTA but enhanced in the presence of other metal ions. Enzyme activity was reduced with 1% SDS, PMSF, and urea but organic solvents did not show considerable impact on it even at higher concentrations. Purified recombinant esterase was also found to be compatible with commercial laundry detergents and showed very good stability (up to 90%). All these properties proved the esterase enzyme from B. thuringensis a significant addition in detergent industry.

Effective utilization of magnetic nano-coupled cloned ß-xylanase in saccharification process.

The ß-xylanase gene (DCE06_04615) with 1041 bp cloned from Thermotoga naphthophila was expressed into E. coli BL21 DE3. The cloned ß-xylanase was covalently bound to iron oxide magnetic nanoparticles coated with silica utilizing carbodiimide. The size of the immobilized MNPs (50 nm) and their binding with ß-xylanase were characterized by Fourier-transform electron microscopy (FTIR) (a change in shift particularly from C-O to C-N) and transmission electron microscopy (TEM) (spherical in shape and 50 nm in diameter). The results showed that enzyme activity (4.5 ± 0.23 U per mL), thermo-stability (90 °C after 4 hours, residual activity of enzyme calculated as 29.89% ± 0.72), pH stability (91% ± 1.91 at pH 7), metal ion stability (57% ± 1.08 increase with Ca2+), reusability (13 times) and storage stability (96 days storage at 4 °C) of the immobilized ß-xylanase was effective and superior. The immobilized ß-xylanase exhibited maximal enzyme activity at pH 7 and 90 °C. Repeated enzyme assay and saccharification of pretreated rice straw showed that the MNP-enzyme complex exhibited 56% ± 0.76 and 11% ± 0.56 residual activity after 8 times and 13 times repeated usage. The MNP-enzyme complex showed 17.32% and 15.52% saccharification percentage after 1st and 8th time usage respectively. Immobilized ß-xylanase exhibited 96% residual activity on 96 days' storage at 4 °C that showed excellent stability.

Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry.

The present study describes the cloning of the cellobiohydrolase gene from a thermophilic bacterium Clostridium clariflavum and its expression in Escherichia coli BL21(DE3) utilizing the expression vector pET-21a(+). The optimization of various parameters (pH, temperature, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration, time of induction) was carried out to obtain the maximum enzyme activity (2.78 ± 0.145 U ml-1) of recombinant enzyme. The maximum expression of recombinant cellobiohydrolase was obtained at pH 6.0 and 70 °C respectively. Enzyme purification was performed by heat treatment and immobilized metal anionic chromatography. The specific activity of the purified enzyme was 57.4 U mg-1 with 35.17% recovery and 3.90 purification fold. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of cellobiohydrolase was 78 kDa. Among metal ions, Ca2+ showed a positive impact on the cellobiohydrolase enzyme with increased activity by 115%. Recombinant purified cellobiohydrolase enzyme remained stable and exhibited 77% and 63% residual activity in comparison to control in the presence of n-butanol and after incubation at 80 °C for 1 h, respectively. Our results indicate that our purified recombinant cellobiohydrolase can be used in the biofuel industry.

Correction: Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry.

[This corrects the article DOI: 10.1039/D1RA00545F.].

Cloning, Purification, and Characterization of Recombinant Thermostable ß-Xylanase Tnap_0700 from Thermotoga naphthophila.

The gene of a ß-xylanase (Tnap_0700) was cloned from a hyperthermophilic Thermotoga naphthophila strain ATCC BAA-489 and expressed in Escherichia coli BL21 (DE3) via pET-21a (+) as an expression vector. The growth steps were upgraded for highest ß-xylanase expression via several factors, for example, IPTG concentration, time of induction, pH, and temperature. The pH and temperature optima for the extreme expression of ß-xylanase were 7.0 pH and 37 °C, correspondingly. Recombinant enzyme purified by heat treatment process, then later by immobilized metal ion affinity chromatography. Molecular mass of the purified ß-xylanase was 38 kDa observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at room temperature for 30 days. It exhibited high stability over wide series of temperature 50-90 °C and pH 4.0-9.0 upon the addition of 1 mM Ca+2 and reduced in the existence of Cu+2 and EDTA. The addition of about 10-30% different organic solvents have no considerable effect on enzyme. However, SDSF and urea acting as an inhibitor leads to decrease in the enzyme activity. The ß-xylanase enzyme was active to hydrolyze xylan from beechwood forming xylose. Thermostable ß-xylanase causes the breakdown of complex carbohydrates into monosaccharide components. This thermostable ß-xylanase revealed remarkable properties, which make it an encouraging candidate for various industrial applications especially in the alteration of renewable biomaterials into ethanol production, and biofuels from lignocellulosics has acknowledged much devotion subsequently in the last decade.