Improvement of catalytic efficiency and thermal stability of l-asparaginase from Bacillus subtilis 168 through reducing the flexibility of the highly flexible loop at N-terminus

Abstract

Abstract Herein, the catalytic efficiency and thermal stability of l -Asparaginase (ASN, EC 3.5.1.1) from Bacillus subtilis 168 were enhanced by modifying its N-terminus highly flexible loop. A continuous non-conserved region (residues20–29) within the N-terminal loop of the ASN was identified and substituted with the structurally equivalent region of ASNs from Escherichia coli, Helicobacter pylori, Wolinella succinogenes, Pseudomonas aeruginosa, Erwinia chrysanthemi, and Pectobacterium carotovorum, yielding the ASN loop variants L1, L2, L3, L4, L5, and L6, respectively. In contrast to the wild-type enzyme, L6 exhibited the highest increase (2.1-fold) in specific activity. Molecular dynamics analysis suggested a negative correlation between the flexibility of the continuous non-conserved region and specific activity. Saturation mutagenesis and complex mutation on flexible residues Ala26 and Gly29 of L6 obtained a double variant L6-A26N/G29F with 3.44-fold and 7.76-fold increases in specific activity and half-life at 65 °C in comparison with wild-type enzyme, respectively. As suggested by structural analysis, the reduced flexibility of the continuous non-conserved region benefit the correct orientation of the active site Thr16 and closure of the substrate pocket. Our results demonstrated that reducing the flexibility of the highly flexible loop was an efficient method to enhance the catalytic efficiency and thermal stability of ASN.