Biochemical Characterization of a GH46 Chitosanase Provides Insights into the Novel Digestion Specificity

Abstract

Endo-chitosanases (EC 3.2.1.132) are generally considered to selectively release functional chito-oligosaccharides (COSs) with degrees of polymerization (DPs) ≥ 2. Although numerous endo-chitosanases have been characterized, the digestion specificity of endo-chitosanases needs to be further explored. In this study, a GH46 endo-chitosanase OUC-CsnPa was cloned, expressed, and characterized from Paenibacillus sp. 1–18. The digestion pattern analysis indicated that OUC-CsnPa could produce monosaccharides from chitotetraose [(GlcN)4], the smallest recognized substrate, in a random endo-acting manner. Especially, the enzyme specificities during chitosan digestion including the regulation of product abundance through a transglycosylation reaction were also evaluated. It was hypothesized that an insertion region in OUC-CsnPa may form a strong force to be involved in stabilizing (GlcN)4 at its negative subsite for efficient hydrolysis. This is the first comprehensive report to reveal the digestion specificity and subsite specificity of monosaccharide production by endo-chitosanases. Overall, OUC-CsnPa described here highlights the previously unknown digestion properties of the endo-acting chitosanases and provides a unique example of possible structure–function relationships.

Rational Redesign of Chitosanase to Enhance Thermostability and Catalytic Activity to Produce Chitooligosaccharides with a Relatively High Degree of Polymerization

“…It may be due to the transglycosylation reaction of Csn46 at high temperature; similarly, Paenibacillus sp. 1–18 chitosanase (GH46 family) was used to hydrolyze chitosan, initially producing a mixture of COS, then converting DP 2–4 COS into DP > 4 COS by transglycosylation with a higher enzyme addition or after a long reaction time . Therefore, it appears that although the hydrolytic activity of the Csn46 wild type is lost at 70 °C, its transglycosylation activity is at least partially retained and produces high-DP COSs.…”

Chitosanase Production from the Liquid Fermentation of Squid Pens Waste by Paenibacillus elgii

“…1794 [ 50 ], and P. fukuinensis [ 51 ]) and the GH 46 family ( P. dendritiformis [ 49 ] and Paenibacillus sp. 1–18 [ 34 ]). The GH 46 chitosanases are usually smaller, with lower molecular weights than those of GH 8 (around 30 kDa) [ 16 ], such as Bacillus sp.…”

“…Nevertheless, subsequent to a comprehensive comparative analysis of 16S ribosomal RNA sequences, the aforementioned bacteria were reclassified as a unique genus in 1993 [29]. Several species in the genus of Paenibacillus have been reported to secrete different chitosanases [8,9,[30][31][32][33][34]. Kim et al (2004) introduced Paenibacillus elgii as a novel species of the genus Paenibacillus [35].…”

Structure-Assisted Design of Chitosanase Product Specificity for the Production of High-Degree Polymerization Chitooligosaccharides

“…It was noticed that Ca 2+ , Mg 2+ , Mn 2+ , Ba 2+ , K + , and Na 2 EDTA could activate OUC-CsnA4, and Mg 2+ had the most significant effect, which was consistent with the previous studies. 24,42,43 The presence of Fe 2+ , Zn 2+ , and Na + ions showed a negligible impact on the activity of OUC-CsnA4. In contrast, Fe 3+ and Cu 2+ ions, along with SDS, significantly inhibited its function, with SDS and Fe 3+ leading to a reduction in activity by over 90%.…”

“…According to the corresponding standard curves, the concentration and the relative proportion change of main products were calculated based on the peak areas. 24 2.8. Molecular Docking of Chitosanase OUC-CsnA4 and Mutants.…”