Cellulase from <i>Halomonas elongata</i> for biofuel application: enzymatic characterization and inhibition tolerance investigation
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Halophilic bacteria are promising candidates for biofuel production because of their efficient cellulose degradation. Their cellulases exhibit high activity, even in the presence of inhibitors and under extreme conditions, making them ideal for biorefinery applications. In this study, we isolated a strain of <i>Halomonas elongata</i> (Kadal6) from decomposed cotton cloth on a Rameshwaram seashore. Morphological, biochemical, and 16S rRNA analyses revealed that Kadal6 was 99.93% similar to the cellulase-producing strain, <i>H. elongata</i> MH25661. The tolerance of the cellulase to inhibitors was assessed through molecular docking with a cellulase model of MH25661 generated by I-TASSER and experimentally using response surface methodology (RSM) with Kadal6. A molecular docking study indicated a high inhibition constant for ethanol, hydroxymethylfurfural (HMF), and furfural. Cellulase from <i>H. elongata</i> Kadal6 (CellHe) showed a maximum inhibition rate of 44.27% at 55 °C, 15% ethanol, and 6.5 g/L furfural and HMF. The enzyme retained 50% of its activity in the presence of these inhibitors, and remained unaffected at 1 g/L furfural and HMF, although inhibition occurred at 3 g/L. <i>H. elongata</i> cellulase demonstrated significant tolerance to inhibition both in vitro (RSM) and in silico, indicating its potential for biorefinery applications in harsh environments. <i>Halomonas elongata</i> strain kadal6, isolated from partially decomposed cotton cloth at the seashore of Rameshwaram, shows a strong capacity for the breakdown of cellulose into glucose molecules.The resistance of the isolated strain to inhibition was assessed using response surface methodology and molecular docking analysis.In silico analysis revealed that CellHe possesses favorable industrial traits for biofuel production and showed tolerance to ethanol, HMF, and furfural, which are crucial for efficient cellulose hydrolysis.Cellulase was predicted to have a maximum inhibition rate of 44.27%, that is, cellulase remained approximately 50% active after exposure to inhibitory compounds under harsh conditions. <i>Halomonas elongata</i> strain kadal6, isolated from partially decomposed cotton cloth at the seashore of Rameshwaram, shows a strong capacity for the breakdown of cellulose into glucose molecules. The resistance of the isolated strain to inhibition was assessed using response surface methodology and molecular docking analysis. In silico analysis revealed that CellHe possesses favorable industrial traits for biofuel production and showed tolerance to ethanol, HMF, and furfural, which are crucial for efficient cellulose hydrolysis. Cellulase was predicted to have a maximum inhibition rate of 44.27%, that is, cellulase remained approximately 50% active after exposure to inhibitory compounds under harsh conditions.
嗜盐细菌（Halophilic bacteria）因其高效的纤维素降解能力，成为生物燃料生产极具潜力的候选者。其产生的纤维素酶（cellulase）即使在存在抑制剂及极端条件下仍表现出高活性，使其成为生物炼制应用的理想选择。本研究从拉梅什沃拉姆（Rameshwaram）海岸的腐烂棉布中分离出一株<i>长盐单胞菌</i>（<i>Halomonas elongata</i>，Kadal6株）。形态学、生化及16S rRNA分析表明，Kadal6株与产纤维素酶的<i>H. elongata</i> MH25661株相似度达99.93%。通过I-TASSER生成的MH25661株纤维素酶模型进行分子对接，以及利用Kadal6株通过响应面法（response surface methodology, RSM）开展实验，评估了该纤维素酶对抑制剂的耐受性。分子对接研究显示，该酶对乙醇、羟甲基糠醛（hydroxymethylfurfural, HMF）及糠醛具有较高的抑制常数。来自<i>H. elongata</i> Kadal6株的纤维素酶（CellHe）在55℃、15%乙醇及6.5 g/L糠醛与HMF条件下，最大抑制率为44.27%。该酶在这些抑制剂存在下仍保留50%的活性，且在1 g/L糠醛与HMF条件下不受影响，但在3 g/L时出现抑制。<i>长盐单胞菌</i>（<i>Halomonas elongata</i>）的纤维素酶在体外（in vitro，RSM实验）和计算机模拟（in silico）中均表现出显著的抑制耐受性，表明其在恶劣环境下生物炼制应用的潜力。 <i>长盐单胞菌</i>（<i>Halomonas elongata</i>）Kadal6株从拉梅什沃拉姆海岸的部分腐烂棉布中分离得到，具有将纤维素分解为葡萄糖分子的强大能力。通过响应面法和分子对接分析评估了该分离株的抑制抗性。计算机模拟分析显示，CellHe具有适合生物燃料生产的良好工业特性，并对乙醇、HMF和糠醛表现出耐受性——这些特性对高效纤维素水解至关重要。纤维素酶的最大抑制率预测为44.27%，即在恶劣条件下暴露于抑制化合物后，纤维素酶仍保留约50%的活性。 <i>长盐单胞菌</i>（<i>Halomonas elongata</i>）Kadal6株从拉梅什沃拉姆海岸的部分腐烂棉布中分离得到，具有将纤维素分解为葡萄糖分子的强大能力。通过响应面法和分子对接分析评估了该分离株的抑制抗性。计算机模拟分析显示，CellHe具有适合生物燃料生产的良好工业特性，并对乙醇、HMF和糠醛表现出耐受性——这些特性对高效纤维素水解至关重要。纤维素酶的最大抑制率预测为44.27%，即在恶劣条件下暴露于抑制化合物后，纤维素酶仍保留约50%的活性。
