Department of Biochemistry and Biotechnology, University of Gujrat, Hafiz Hayat Campus, Gujrat, Pakistan.
PLoS One. 2024 May 15;19(5):e0298716. doi: 10.1371/journal.pone.0298716. eCollection 2024.
The purpose of current research work was to investigate the effect of mutagenesis on endoglucanase B activity of indigenous strain of Aspergillus niger and its heterologous expression studies in the pET28a+ vector. The physical and chemical mutagens were employed to incorporate mutations in A. niger. For determination of mutations, mRNA was isolated followed by cDNA synthesis and cellulase gene was amplified, purified and sequenced both from native and mutant A. niger. On comparison of gene sequences, it was observed that 5 nucleotide base pairs have been replaced in the mutant cellulase. The mutant recombinant enzyme showed 4.5 times higher activity (428.5 µmol/mL/min) as compared to activity of native enzyme (94 µmol/mL/min). The mutant gene was further investigated using Phyre2 and I-Tesser tools which exhibited 71% structural homology with Endoglucanase B of Thermoascus aurantiacus. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible surface area (SASA), radius of gyration (Rg) and hydrogen bonds analysis were carried at 35°C and 50°C to explore the integrity of structure of recombinant mutant endoglucanase B which corresponded to its optimal temperature. Hydrogen bonds analysis showed more stability of recombinant mutant endoglucanase B as compared to native enzyme. Both native and mutant endoglucanase B genes were expressed in pET 28a+ and purified with nickel affinity chromatography. Theoretical masses determined through ExPaSy Protparam were found 38.7 and 38.5 kDa for native and mutant enzymes, respectively. The optimal pH and temperature values for the mutant were 5.0 and 50°C while for native these were found 4.0 and 35°C, respectively. On reacting with carboxy methyl cellulose (CMC) as substrate, the mutant enzyme exhibited less Km (0.452 mg/mL) and more Vmax (50.25 µmol/ml/min) as compared to native having 0.534 mg/mL as Km and 38.76 µmol/ml/min as Vmax. Among metal ions, Mg2+ showed maximum inducing effect (200%) on cellulase activity at 50 mM concentration followed by Ca2+ (140%) at 100 mM concentration. Hence, expression of a recombinant mutant cellulase from A. niger significantly enhanced its cellulytic potential which could be employed for further industrial applications at pilot scale.
本研究旨在探讨诱变对本土黑曲霉内切葡聚糖酶活性的影响及其在 pET28a+载体中的异源表达。采用物理和化学诱变剂在黑曲霉中引入突变。为了确定突变，从天然和突变的黑曲霉中分离 mRNA，然后进行 cDNA 合成和纤维素酶基因的扩增、纯化和测序。在比较基因序列时，观察到突变体纤维素酶中有 5 个碱基对被取代。与天然酶（94µmol/mL/min）相比，突变重组酶的活性提高了 4.5 倍（428.5µmol/mL/min）。进一步利用 Phyre2 和 I-Tesser 工具对突变基因进行研究，结果显示突变体与嗜热橙色高温菌的内切葡聚糖酶 B 具有 71%的结构同源性。在 35°C 和 50°C 下进行均方根偏差（RMSD）、均方根波动（RMSF）、溶剂可及表面积（SASA）、回转半径（Rg）和氢键分析，以研究重组突变内切葡聚糖酶 B 结构的完整性，这与它的最适温度相对应。氢键分析表明，与天然酶相比，重组突变内切葡聚糖酶 B 具有更高的稳定性。天然和突变的内切葡聚糖酶 B 基因均在 pET28a+中表达，并通过镍亲和层析进行纯化。通过 ExPaSy Protparam 确定的理论分子量分别为 38.7 和 38.5 kDa。突变体的最适 pH 和温度值分别为 5.0 和 50°C，而天然酶的最适 pH 和温度值分别为 4.0 和 35°C。在以羧甲基纤维素（CMC）为底物反应时，突变酶的 Km 值（0.452mg/mL）较低，Vmax 值（50.25µmol/ml/min）较高，而天然酶的 Km 值为 0.534mg/mL，Vmax 值为 38.76µmol/ml/min。在金属离子中，Mg2+在 50mM 浓度下对纤维素酶活性的诱导作用最大（200%），其次是 100mM 浓度下的 Ca2+（140%）。因此，黑曲霉重组突变体纤维素酶的表达显著提高了其纤维素酶活力，可在中试规模进一步用于工业应用。
