Rational Design of the Chitinase from <i>Paenibacillus Barengoltzii</i> for Efficient Production of Lacto- <i>N</i> -Triose II from Chitin Powder

Chitin, a major component of shellfish waste, is a promising resource for producing bioactive compounds. To enhance the hydrolysis efficiency of Chitinase, a combined engineering strategy was employed to optimize the Chitinase (m<i>Pb</i>Chi70) from <i>Paenibacillus barengoltzii</i>. Through computer-aided rational design and chitin-binding domain modification, an enhanced mutant, <i>m</i>-m<i>Pb</i>Chi70 (Trp163Phe/Asp199Glu/Lys688Pro), was developed with significantly improved catalytic performance. The mutant exhibited optimal activity at pH 6.0 and 50 °C. Its specific activity toward chitin powder increased by 24.1% compared to m<i>Pb</i>Chi70. During chitin powder hydrolysis, <i>m</i>-m<i>Pb</i>Chi70 exhibited a 1.8-fold increase in <i>N</i>-acetyl-chitobiose yield (39.1 mmol·L^-1) and a 67.4% reduction in <i>N</i>-acetyl-d-glucosamine accumulation. Furthermore, the mutant enabled efficient and sustainable production of lacto-<i>N</i>-triose II (LNT2) from chitin powder, achieving a yield of 15.0 g·L¹. This study not only presents an innovative strategy for Chitinase engineering but also offers a sustainable and cost-effective approach for converting chitin waste into high-value LNT2.