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Background <p>The efficient degradation of lignocellulosic biomass is essential for sustainable bioprocessing industries, including biofuel production and agro-waste valorization. This study screened bacterial isolates from black soils in Jharkhand, India, to identify strains producing cellulolytic and xylanolytic enzymes with industrial potential. <i>Bacillus subtilis</i> SSMK413 was selected for its coproduction of cellulase and xylanase, and the enzymes were purified and characterized for biochemical and kinetic properties. Additionally, their application in lignocellulosic biomass saccharification was evaluated to assess industrial relevance.</p> Results <p>Among the screened bacterial isolates, <i>Bacillus subtilis</i> strain SSMK413, isolated from the Baulia region of Jharkhand (24.131955°N, 85.546774°E), exhibited robust coproduction of cellulase and xylanase. Primary screening on carboxymethyl cellulose (CMC) and xylan agar plates confirmed its enzymatic activities, with molecular identification via 16&#xa0;S rRNA gene sequencing verifying its taxonomic classification. Optimal enzyme production was achieved after 48&#xa0;h of incubation. Enzyme purification was performed using ammonium sulfate precipitation, dialysis, and Sephadex G-100 gel filtration chromatography, resulting in purification folds of 14.3 for cellulase and 11.5 for xylanase. Biochemical characterization indicated optimal activities at 40–50&#xa0;°C, with pH optima of 5.0 for cellulase and 6.0 for xylanase. Enzyme activities were significantly enhanced by Mn²⁺, Mg²⁺, and Fe³⁺ ions, whereas Cs⁺ and EDTA acted as inhibitors. Kinetic studies revealed a lower Km for cellulase (0.192&#xa0;mg mL<sup>− 1</sup>) compared to xylanase (2.615&#xa0;mg mL<sup>− 1</sup>), indicating higher substrate affinity for cellulase. SDS-PAGE and zymogram analyses determined molecular masses of 55–65&#xa0;kDa for cellulase and 240–270&#xa0;kDa for xylanase, suggesting a monomeric structure for cellulase and a multimeric complex for xylanase.</p> Conclusions <p><i>B. subtilis</i> (SSMK413) is a promising candidate for industrial enzyme production due to its efficient coproduction of cellulase and xylanase with favorable biochemical properties. The microbial diversity from black soils in Jharkhand represents a valuable genetic resource for future biotechnological exploration. Further studies should focus on enzyme immobilization and genetic enhancement to improve catalytic efficiency and operational stability.</p>

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Exploring black soil microbiota for industrial enzymes: cellulase and xylanase characterization from Bacillus subtilis SSMK413

  • 期刊论文

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  • 发表日期：

    2025年12月18日

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  • Shweta Shah,
  • Bairagi C. Mallick,
  • Bhagwati Devi,
  • Manoj Kumar

摘要

Background

The efficient degradation of lignocellulosic biomass is essential for sustainable bioprocessing industries, including biofuel production and agro-waste valorization. This study screened bacterial isolates from black soils in Jharkhand, India, to identify strains producing cellulolytic and xylanolytic enzymes with industrial potential. Bacillus subtilis SSMK413 was selected for its coproduction of cellulase and xylanase, and the enzymes were purified and characterized for biochemical and kinetic properties. Additionally, their application in lignocellulosic biomass saccharification was evaluated to assess industrial relevance.

Results

Among the screened bacterial isolates, Bacillus subtilis strain SSMK413, isolated from the Baulia region of Jharkhand (24.131955°N, 85.546774°E), exhibited robust coproduction of cellulase and xylanase. Primary screening on carboxymethyl cellulose (CMC) and xylan agar plates confirmed its enzymatic activities, with molecular identification via 16 S rRNA gene sequencing verifying its taxonomic classification. Optimal enzyme production was achieved after 48 h of incubation. Enzyme purification was performed using ammonium sulfate precipitation, dialysis, and Sephadex G-100 gel filtration chromatography, resulting in purification folds of 14.3 for cellulase and 11.5 for xylanase. Biochemical characterization indicated optimal activities at 40–50 °C, with pH optima of 5.0 for cellulase and 6.0 for xylanase. Enzyme activities were significantly enhanced by Mn²⁺, Mg²⁺, and Fe³⁺ ions, whereas Cs⁺ and EDTA acted as inhibitors. Kinetic studies revealed a lower Km for cellulase (0.192 mg mL− 1) compared to xylanase (2.615 mg mL− 1), indicating higher substrate affinity for cellulase. SDS-PAGE and zymogram analyses determined molecular masses of 55–65 kDa for cellulase and 240–270 kDa for xylanase, suggesting a monomeric structure for cellulase and a multimeric complex for xylanase.

Conclusions

B. subtilis (SSMK413) is a promising candidate for industrial enzyme production due to its efficient coproduction of cellulase and xylanase with favorable biochemical properties. The microbial diversity from black soils in Jharkhand represents a valuable genetic resource for future biotechnological exploration. Further studies should focus on enzyme immobilization and genetic enhancement to improve catalytic efficiency and operational stability.