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bioRxiv · 10.1101/2024.01.31.578261

Heterologous expression and characterization of mutant cellulase from indigenous strain of Aspergillus niger

Zafar, M.·Ahmad, W.·Anwar, Z.·Mehmood Ashraf, N.·Ahmad, S.
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Abstract

The current study highlights 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 {micro}mol/mL/min) as compared to activity of native enzyme (94 {micro}mol/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 {degrees}C and 50 {degrees}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 {degrees}C while for native these were found 4.0 and 35 {degrees}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 {micro}mol/ml/min) as compared to native having 0.534 mg/mL as Km and 38.76 {micro}mol/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.

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Zafar, M., Ahmad, W., Anwar, Z., Mehmood Ashraf, N., Ahmad, S.. 2024-02-02. Heterologous expression and characterization of mutant cellulase from indigenous strain of Aspergillus niger. https://doi.org/10.1101/2024.01.31.578261

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