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

• Published in: Process biochemistry (1991) Vol. 86; pp. 40 - 49
• Main Authors: Abdul Wahab, Mohd Khairul Hakimi, El-Enshasy, Hesham Ali, Bakar, Farah Diba Abu, Murad, Abdul Munir Abdul, Jahim, Jamaliah Md, Illias, Rosli Md
• Format: Journal Article
• Language: English
• Published: Barking Elsevier Ltd 01.11.2019; Elsevier BV
• Subjects: Amino groups; Cross-linked enzyme aggregate; Crosslinking; Enzyme immobilization; Enzymes; Glutaraldehyde; Immobilization; Protein engineering; Residues; Site-directed mutagenesis; Surface analysis (chemical); Surface modification; Xylanase; Surface modification; Enzyme immobilization; Cross-linked enzyme aggregate; Xylanase
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2019.07.017

[Display omitted] •Surface analysis revealed rXyn contain low number of lysine on the surface.•Four lysines were substituted on the surface of rXyn.•Better structure rigidity and compactness when new lysines were substituted.•mXyn-CLEA-BSA possess higher stability than free Xyn and traditional CLEA-Xyn. 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.