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Abstract：
BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate.
RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71.
CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.
RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71.
CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.
摘要：
背景：在这项研究中，我们分离出一种产纤维素酶的细菌,解淀粉芽孢杆菌菌株elh，从米皮。我们采用了两种优化方法来提高纤维素酶的产量。首先,我们采用了一次单变量(OVAT)方法来评估单个物理和化学参数的影响.随后，我们采用响应面方法（RSM）来研究这些因素之间的相互作用。我们使用克隆载体大肠杆菌DH5α异源表达纤维素酶编码基因。此外，我们进行了硅分子对接分析，以分析纤维素酶和羧甲基纤维素作为底物之间的相互作用。
结果：当在特定培养基中培养时，细菌分离物eh1表现出0.141±0.077U/ml的初始纤维素酶活性，即碱性液体介质（BLM），以米皮为基质。通过16SrRNA测序鉴定该菌株为解淀粉芽孢杆菌菌株elh1，在GenBank中分配了登录号OR920278。发现最佳孵育时间为发酵72h。尿素被确定为最合适的氮源，和葡萄糖作为最佳糖，导致产量增加到5.04±0.120U/ml。使用响应面方法（RSM）进行统计优化，纤维素酶的峰值活性达到14.04±0.42U/ml。该过程包括利用Plackett-Burman设计的初始筛选和利用BOX-Behnken设计的进一步改进。负责纤维素酶生产的基因,egl,在大肠杆菌DH5α中有效克隆和表达。转化的细胞表现出22.3±0.24U/ml的纤维素酶活性。egl基因序列保存在GenBank中,登录号为PP194445。计算机分子对接揭示了羧甲基纤维素的两个羟基与CMCase的结合袋内的Glu169的残基结合。这种相互作用形成两个氢键，亲和力得分为-5.71。
结论:与未优化的培养条件相比,培养条件的优化显著提高了纤维素酶的产量。此外,egl基因的异源表达表明，纤维素酶的重组形式具有活性，并且有效的表达系统可以有助于提高酶的产量。
结果：当在特定培养基中培养时，细菌分离物eh1表现出0.141±0.077U/ml的初始纤维素酶活性，即碱性液体介质（BLM），以米皮为基质。通过16SrRNA测序鉴定该菌株为解淀粉芽孢杆菌菌株elh1，在GenBank中分配了登录号OR920278。发现最佳孵育时间为发酵72h。尿素被确定为最合适的氮源，和葡萄糖作为最佳糖，导致产量增加到5.04±0.120U/ml。使用响应面方法（RSM）进行统计优化，纤维素酶的峰值活性达到14.04±0.42U/ml。该过程包括利用Plackett-Burman设计的初始筛选和利用BOX-Behnken设计的进一步改进。负责纤维素酶生产的基因,egl,在大肠杆菌DH5α中有效克隆和表达。转化的细胞表现出22.3±0.24U/ml的纤维素酶活性。egl基因序列保存在GenBank中,登录号为PP194445。计算机分子对接揭示了羧甲基纤维素的两个羟基与CMCase的结合袋内的Glu169的残基结合。这种相互作用形成两个氢键，亲和力得分为-5.71。
结论:与未优化的培养条件相比,培养条件的优化显著提高了纤维素酶的产量。此外,egl基因的异源表达表明，纤维素酶的重组形式具有活性，并且有效的表达系统可以有助于提高酶的产量。
