Modulation of a Loop Region in the Substrate Binding Pocket Affects the Degree of Polymerization of Bacillus subtilis Chitosanase Products

Abstract

The hydrolytic products of chitosanase from Streptomyces avermitilis (SaCsn46A) were found to be aminoglucose and chitobiose, whereas those of chitosanase from Bacillus subtilis (BsCsn46A) were chitobiose and chitotriose. Therefore, the sequence alignment between SaCsn46A and BsCsn46A was conducted, revealing that the structure of BsCsn46A possesses an extra loop region (194N-200T) at the substrate binding pocket. To clarify the impact of this loop on hydrolytic properties, three mutants, SC, TJN, and TJA, were constructed. Eventually, the experimental results indicated that SC changed the ratio of chitobiose to chitotriose hydrolyzed by chitosanase from 1:1 into 2:3, while TJA resulted in a ratio of 15:7. This experiment combined molecular research to unveil a crucial loop within the substrate binding pocket of chitosanase. It also provides an effective strategy for mutagenesis and a foundation for altering hydrolysate composition and further applications in engineering chitosanase.

Combining Flexible Region Design and Automatic Design to Enhance the Thermal Stability and Catalytic Efficiency of Leucine Dehydrogenase

“…The average RG value for the M2 mutant was 2.12 nm, slightly lower than the 2.14 nm of Es LeuDH, further demonstrating its enhanced structural stability. The SASA value reflects the extent of a protein’s surface contact with the solvent; a smaller SASA value indicates a more compact protein structure . The average SASA value for the M2 mutant was 182.23 nm 2 , lower than the 183.82 nm 2 of Es LeuDH (Figure C), indicating a more compact structure resulting from the mutation.…”