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<p>Xylanase is widely used in poultry feed to improve nutrient digestibility; however, its industrial application is limited by thermal instability and high production cost. In this study, wheat bran was used as a low-cost substrate for xylanase production by <i>Bacillus subtilis</i> KT004404. Fermentation parameters were optimized using Response Surface Methodology, yielding 113 U mL⁻¹ xylanase, representing a 1.85-fold increase over unoptimized conditions. The enzyme was partially purified by ammonium sulfate precipitation, achieving 2.3-fold purification with 54.58% yield while 5.7-fold purification with specific activity of 120 U mg⁻¹ after gel filtration chromatography. SDS–PAGE analysis indicated a molecular mass of ~ 22&#xa0;kDa. The enzyme showed stability across pH 4.0–8.0 and 30–70&#xa0;°C. Immobilization on bentonite improved enzyme stability, with &gt; 90% activity retention at elevated temperatures and 65–72% residual activity after 60 days of storage. Under simulated poultry gastrointestinal conditions, immobilized xylanase activity was better compared to free enzyme and pronounced improvements were observed in the acidic regions, where activity increased substantially in the proventriculus (15.6 vs. 3.2 U mL⁻¹) and gizzard (26.4 vs. 7.8 U mL⁻¹), indicating strong protection against low-pH inactivation. This study integrates statistical optimization (RSM-CCD), low-cost bentonite-based enzyme immobilization, and simulated gastrointestinal (GIT) validation using an agro-residue substrate. Unlike previous reports that focus on isolated aspects; this work provides an integrated process-level approach for improving enzyme, stability, and potential applicability in feed systems.</p> Graphical Abstract <p></p>

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Sustainable production and immobilization of thermostable xylanase from Bacillus subtilis KT004404 using wheat bran as an agro-industrial substrate

  • 期刊论文

  • 发表日期：

    2026年7月7日

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  • Aqsa Javaid,
  • Gerald C. Shurson,
  • Khunsa Saeed,
  • Beenish Asrar

摘要

Xylanase is widely used in poultry feed to improve nutrient digestibility; however, its industrial application is limited by thermal instability and high production cost. In this study, wheat bran was used as a low-cost substrate for xylanase production by Bacillus subtilis KT004404. Fermentation parameters were optimized using Response Surface Methodology, yielding 113 U mL⁻¹ xylanase, representing a 1.85-fold increase over unoptimized conditions. The enzyme was partially purified by ammonium sulfate precipitation, achieving 2.3-fold purification with 54.58% yield while 5.7-fold purification with specific activity of 120 U mg⁻¹ after gel filtration chromatography. SDS–PAGE analysis indicated a molecular mass of ~ 22 kDa. The enzyme showed stability across pH 4.0–8.0 and 30–70 °C. Immobilization on bentonite improved enzyme stability, with > 90% activity retention at elevated temperatures and 65–72% residual activity after 60 days of storage. Under simulated poultry gastrointestinal conditions, immobilized xylanase activity was better compared to free enzyme and pronounced improvements were observed in the acidic regions, where activity increased substantially in the proventriculus (15.6 vs. 3.2 U mL⁻¹) and gizzard (26.4 vs. 7.8 U mL⁻¹), indicating strong protection against low-pH inactivation. This study integrates statistical optimization (RSM-CCD), low-cost bentonite-based enzyme immobilization, and simulated gastrointestinal (GIT) validation using an agro-residue substrate. Unlike previous reports that focus on isolated aspects; this work provides an integrated process-level approach for improving enzyme, stability, and potential applicability in feed systems.

Graphical Abstract