Unraveling the dynamic mechanism of chitooligosaccharide biosynthesis through rational engineering of chitosanase product specificity

Abstract

Enzymatic preparation of high value-added chitooligosaccharides still faces challenges including uncontrollable product polymerization degrees and insufficient catalytic specificity of enzymes. In this study, three mutants, namely C41A, I17A/T47A, and S155A/T200A, were constructed via rational enzyme design, and the control mechanism of product specificity through differential conformational dynamics was systematically studied. Specifically, the C41A mutant located in the catalytic pocket modified the flexibility of the key loop structure, thus enhancing the selective cleavage toward chitosan. The proportion of chitoheptaose in the product reached 4.5%, which was 11.25-fold higher than that of the parental enzyme Csn46-Mut4. The I17A/T47A double mutant at the substrate channel entrance triggered rigid contraction of the channel entrance, restricting the entry of long-chain substrates and simplifying the hydrogen bond network. Consequently, the proportion of chitopentaose was improved to 22.5%, representing an 11.8-fold improvement compared with the parental enzyme. In contrast, the S155A/T200A double mutant formed a binding pocket compatible with larger long-chain substrates, enabling the biosynthesis of chitooctaose with a proportion of 0.88%, which represented a crucial breakthrough from zero detection. Molecular dynamics simulations and intermolecular interaction analyses confirmed that all three superior mutants followed the regulatory mechanism of “conformational dynamics-product specificity”. The T_m values of the mutants were increased by 0.16⁓2.30 °C relative to Csn46-Mut4. On the premise of maintaining enzyme activity, this work established a novel and efficient method for the precise preparation of chitooligosaccharides with specific high polymerization degrees, providing a solid foundation for technological innovation in this field and further promoting the application and development of high-polymer chitooligosaccharides in biomedicine and other related fields.

Graphical abstract

Keywords

Chitooligosaccharides / Chitosanase / Rational design / Molecular dynamics simulation

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Funding

The Ministry of Science and Technology of China(2025YFA0921202)

RIGHTS & PERMISSIONS

Jiangnan University