Improvement of cross-linking and stability on cross-linked enzyme aggregate (CLEA)-xylanase by protein surface engineering

Mohd Khairul Hakimi Abdul Wahab, Hesham Ali El-Enshasy, Farah Diba Abu Bakar, Abdul Munir Abd. Murad, Jamaliah Md Jahim, Rosli Md Illias

Research output: Contribution to journalArticle

Abstract

Cross-linked enzyme aggregate (CLEA) is a well-known enzyme immobilization technique that is efficient and cost-effective. In this study, we proposed a combination of the surface modification of an enzyme and CLEA to increase the number of free amino groups. Site-directed mutagenesis was applied to selected residues of xylanase from Aspergillus fumigatus RT-1 and the xylanase was subsequently cross-linked using glutaraldehyde. Surface analysis of the xylanase revealed that 9 residues were exposed to the environment and only 3 were lysines. Thus, four additional lysines were substituted for residues opposite of the catalytic region. After optimizing the CLEA parameters, a stable cross-linked mutant xylanase with the addition of BSA (mXyn-CLEA-BSA) was obtained where the enzyme was 1.09-fold, 1.35-fold and 1.77-fold more stable than the cross-linked recombinant xylanase with the addition of BSA (rXyn-CLEA-BSA), without the addition of BSA (rXyn-CLEA) and free enzyme (rXyn), respectively. In terms of reusability, rXyn-CLEA can be used up to 5 cycles, rXyn-CLEA-BSA and mXyn-CLEA up to 7 cycles and mXyn-CLEA-BSA up to 8 cycles until the total activity is lost. The increase in stability and reusability using this approach provides a promising biocatalyst that can be further utilized in the production of prebiotics in the biomass industry.

Original languageEnglish
JournalProcess Biochemistry
DOIs
Publication statusAccepted/In press - 1 Jan 2019

Fingerprint

Protein Engineering
Membrane Proteins
Enzymes
Proteins
Reusability
Lysine
Enzyme immobilization
Mutagenesis
Biocatalysts
Prebiotics
Aspergillus
Surface analysis
Aspergillus fumigatus
Glutaral
Site-Directed Mutagenesis
Surface treatment

Keywords

  • Cross-linked enzyme aggregate
  • Enzyme immobilization
  • Surface modification
  • Xylanase

ASJC Scopus subject areas

  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Improvement of cross-linking and stability on cross-linked enzyme aggregate (CLEA)-xylanase by protein surface engineering. / Abdul Wahab, Mohd Khairul Hakimi; El-Enshasy, Hesham Ali; Abu Bakar, Farah Diba; Abd. Murad, Abdul Munir; Md Jahim, Jamaliah; Illias, Rosli Md.

In: Process Biochemistry, 01.01.2019.

Research output: Contribution to journalArticle

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AU - Abdul Wahab, Mohd Khairul Hakimi

AU - El-Enshasy, Hesham Ali

AU - Abu Bakar, Farah Diba

AU - Abd. Murad, Abdul Munir

AU - Md Jahim, Jamaliah

AU - Illias, Rosli Md

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N2 - Cross-linked enzyme aggregate (CLEA) is a well-known enzyme immobilization technique that is efficient and cost-effective. In this study, we proposed a combination of the surface modification of an enzyme and CLEA to increase the number of free amino groups. Site-directed mutagenesis was applied to selected residues of xylanase from Aspergillus fumigatus RT-1 and the xylanase was subsequently cross-linked using glutaraldehyde. Surface analysis of the xylanase revealed that 9 residues were exposed to the environment and only 3 were lysines. Thus, four additional lysines were substituted for residues opposite of the catalytic region. After optimizing the CLEA parameters, a stable cross-linked mutant xylanase with the addition of BSA (mXyn-CLEA-BSA) was obtained where the enzyme was 1.09-fold, 1.35-fold and 1.77-fold more stable than the cross-linked recombinant xylanase with the addition of BSA (rXyn-CLEA-BSA), without the addition of BSA (rXyn-CLEA) and free enzyme (rXyn), respectively. In terms of reusability, rXyn-CLEA can be used up to 5 cycles, rXyn-CLEA-BSA and mXyn-CLEA up to 7 cycles and mXyn-CLEA-BSA up to 8 cycles until the total activity is lost. The increase in stability and reusability using this approach provides a promising biocatalyst that can be further utilized in the production of prebiotics in the biomass industry.

AB - Cross-linked enzyme aggregate (CLEA) is a well-known enzyme immobilization technique that is efficient and cost-effective. In this study, we proposed a combination of the surface modification of an enzyme and CLEA to increase the number of free amino groups. Site-directed mutagenesis was applied to selected residues of xylanase from Aspergillus fumigatus RT-1 and the xylanase was subsequently cross-linked using glutaraldehyde. Surface analysis of the xylanase revealed that 9 residues were exposed to the environment and only 3 were lysines. Thus, four additional lysines were substituted for residues opposite of the catalytic region. After optimizing the CLEA parameters, a stable cross-linked mutant xylanase with the addition of BSA (mXyn-CLEA-BSA) was obtained where the enzyme was 1.09-fold, 1.35-fold and 1.77-fold more stable than the cross-linked recombinant xylanase with the addition of BSA (rXyn-CLEA-BSA), without the addition of BSA (rXyn-CLEA) and free enzyme (rXyn), respectively. In terms of reusability, rXyn-CLEA can be used up to 5 cycles, rXyn-CLEA-BSA and mXyn-CLEA up to 7 cycles and mXyn-CLEA-BSA up to 8 cycles until the total activity is lost. The increase in stability and reusability using this approach provides a promising biocatalyst that can be further utilized in the production of prebiotics in the biomass industry.

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