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A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (-7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (-3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20-30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Enzyme hydrolysis faces a bottleneck due to the recalcitrance of the lignocellulose biomass. The protein engineering of GH11 xylanase from Aspergillus fumigatus RT-1 was performed near the active site and at the N-terminal region to improve its catalytic efficiency towards pretreated kenaf (Hibiscus cannabinus) hydrolysis. Five mutants were constructed by combined approaches of error-prone PCR, site-saturation and site-directed mutagenesis. The double mutant c168 t/Q192H showed the most effective hydrolysis reaction with a 13.9-fold increase in catalytic efficiency, followed by mutants Y7L and c168 t/Q192 H/Y7L with a 1.6-fold increase, respectively. The enhanced catalytic efficiency evoked an increase in sugar yield of up to 28% from pretreated kenaf. In addition, mutant c168 t/Q192 H/Y7L improved the thermostability at higher temperature and acid stability. This finding shows that mutations at distances less than 15 Å from the active site and at putative secondary binding sites affect xylanase catalytic efficiency towards insoluble substrates hydrolysis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

• Mutation at substrate binding site improves hemicellulose catalytic efficiency. • Amino acid substitution reduces steric hindrance at the catalytic site. • Larger tunnel space enable complex substrate to access the active site. • Enzyme hydration increases the enzyme structure flexibility. • Flexibility enhances higher substrate diffusion and hydrolytic activity. Lignocellulosic biomass utilisation as an alternative to fossil resources was hampered due to its recalcitrant to enzymatic hydrolysis. Herein, rational protein engineering strategy has been carried out on Aspergillus niger α-L-arabinofuranosidase (An abf A) substrate binding pathway to improve its catalytic efficiency towards pre-treated kenaf (Hibiscus cannabinus) hemicellulose hydrolysis. A total of five variants (N246D, L371 V, E449D, W453Y and E449D/W453Y) were constructed based on An abf A sequence and structure information. Substitutions from bulky to smaller amino acids and hydrophobic to less hydrophobic residues were shown to improve the enzyme catalytic reaction towards insoluble substrate. Variant E449D/W453Y induces the highest hydrolysis of hemicellulose by producing up to 62 % reducing sugar. It is evident that substituting amino acids at the substrate binding site reduces steric hindrance, thus allowing insoluble substrate to easily penetrate and complement the enzyme's active site. [ABSTRACT FROM AUTHOR]