Rational protein engineering of alpha-L-arabinofuranosidase from Aspergillus niger for improved catalytic hydrolysis efficiency on kenaf hemicellulose

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 alpha-L-arabinofuranosidase (AnabfA) 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 AnabfA 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.

Automated summary

Through rational protein engineering, the catalytic efficiency of Aspergillus niger alpha-L-arabinofuranosidase towards pre-treated kenaf hemicellulose hydrolysis was improved, with the variant E449D/W453Y showing the highest hydrolysis of hemicellulose by producing up to 62% reducing sugar. Substituting amino acids at the substrate binding site reduces steric hindrance, allowing easier penetration of insoluble substrate to complement the enzyme's active site.