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

[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.