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A new halotolerant xylanase from Aspergillus clavatus expressed in Escherichia coli with catalytic efficiency improved by site-directed mutagenesis

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Abstract

Daily agro-industrial waste, primarily cellulose, lignin, and hemicellulose, poses a significant environmental challenge. Harnessing lignocellulolytic enzymes, particularly endo-1,4-β-xylanases, for efficient saccharification is a cost-effective strategy, transforming biomass into high-value products. This study focuses on the cloning, expression, site-directed mutagenesis, purification, three-dimensional modeling, and characterization of the recombinant endo-1,4-β-xylanase (XlnA) from Aspergillus clavatus in Escherichia coli. This work includes evaluation of the stability at varied NaCl concentrations, determining kinetic constants, and presenting the heterologous expression of XlnAΔ36 using pET22b(+). The expression led to purified enzymes with robust stability across diverse pH levels, exceptional thermostability at 50 °C, and 96–100% relative stability after 24 h in 3.0 M NaCl. Three-dimensional modeling reveals a GH11 architecture with catalytic residues Glu 132 and 22. XlnAΔ36 demonstrates outstanding kinetic parameters compared to other endo-1,4-β-xylanases, indicating its potential for industrial enzymatic cocktails, enhancing saccharification. Moreover, its ability to yield high-value compounds, such as sugars, suggests a promising and ecologically positive alternative for the food and biotechnology industries.

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Acknowledgements

The authors thank Shivansh Mahajan for assistance in the gene cloning and expression and Mauricio de Oliveira for technical assistance.

Funding

This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/Brasil-financing Code 001, Programa de Doutorado Sanduíche no Exterior (PDSE/CAPES no. 88881.186934/2018-01), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)—Process numbers 2010/52322-3, 2018/07522-6, and 2014/50884-5, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process 563260/2010-6), and the National Institute of Science and Technology of the Bioethanol (465319/2014-9). T.M.P. was recipient of PDSE/CAPES Fellowship; R.C.L. was recipient of CAPES Fellowship; T.B.O. was recipient of FAPESP Fellowship (process 2017/09000-4); and M.L.T.M.P. is Research Fellow of CNPq (process 301963/2017–7).

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T.M.P, R.C.L, M.J.M, and M.L.T.M.P conceived and designed the experiments. T.M.P performed the experiments. T.M.P, T.B.O, M.J.M, and M.L.T.M.P analyzed the data. T.M.P, R.C.L, T.B.O, M.J.M, and M.L.T.M.P wrote the paper. All authors have read and approved the final version of this manuscript.

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Correspondence to Thiago M. Pasin.

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Pasin, T.M., Lucas, R.C., de Oliveira, T.B. et al. A new halotolerant xylanase from Aspergillus clavatus expressed in Escherichia coli with catalytic efficiency improved by site-directed mutagenesis. 3 Biotech 14, 178 (2024). https://doi.org/10.1007/s13205-024-04021-7

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