Engineering the substrate-binding domain of chitinase for enhanced bioconversion of crustacean shell waste in probiotic feed fermentation.

Background

Crustacean shell waste, generating 8 million tons annually from global aquaculture, represents an underutilized chitin-rich feedstock. However, the high crystallinity of native α-chitin currently prevents cost-effective enzymatic valorization, limiting its conversion into value-added products for feed applications.

Results

In this study, a chitinase (PbChi) from Paenibacillus barengoltzii was engineered by replacing its native chitin-binding domain to enhance its hydrolytic activity toward insoluble chitinous substrates and to improve its efficacy in solid state probiotic feed fermentation. The enzyme was expressed in Bacillus subtilis by both spore surface display and extracellular secretion, yielding active immobilized and free forms with optimal activity at 55-60 °C and pH 5.5. The native chitin-binding domain was replaced with a homologous B. subtilis chitin-binding domain (BsCBD), which increased chitinase activity by 2.16-fold relative to the wild-type and conferred hydrolytic activity toward powdered chitin and shrimp shell powder. Homology modeling, molecular docking and molecular dynamics simulation suggested that the engineered enzyme adopted a more expanded conformation and exhibited a 17% increase in solvent-accessible surface area, which may contribute to the improved hydrolysis of insoluble substrates observed experimentally. In a solid state co-fermentation system containing probiotics and shrimp shell powder, the engineered chitinase increased reducing sugar content to 18.61 ± 2.14% compared to 5.51% for the wild-type enzyme, at the same time as lowering feed pH to 5.1.

Conclusion

These results demonstrate that chitin-binding domain engineering in a GRAS (i.e. Generally Recognized As Safe) host not only enhanced chitinase activity (2.16-fold) and expanded substrate specificity toward insoluble shell substrates, but also increased reducing sugar yield by over 3-fold in probiotic solid state fermentation, providing a measurable and viable strategy for converting crustacean shell waste into value-added feed ingredients without chemical pretreatment. © 2026 Society of Chemical Industry.