First evidence of cryo-milling enhancing enzymatic production of chitooligosaccharides from chitin biomass

Abstract

Cryo-milling offers an energy-efficient approach to process α-chitin, the most prevalent and structurally robust chitin form. Unlike ball-milling, cryo-milling operates at low temperatures (<123 K), preventing heat-induced charring and loss of chitooligosaccharide. This study investigated the effects of various cryo-milling conditions on α-chitin, resulting in reduced crystalline plane intensity, increased surface area, and smaller particle size without causing substrate breakdown or functional group loss. Additionally, the study investigated cryo-milled α-chitin's enzymatic hydrolysis for the first time. Using Paenibacillus sp. LS1's chitin-active-secretome, cryo-milled α-chitin yielded twice as much GlcNAc compared to unmilled α-chitin. Hydrolysis with recombinant Chi5 from Paenibacillus sp. LS1 increased (GlcNAc)₂ and GlcNAc yields by twofold and fourfold, respectively, while recombinant StrChiA from Streptomyces sp. UH6 led to an elevenfold and twofold increase, respectively. Overall, cryo-milling emerges as a gentle yet effective alternative to ball-milling, significantly enhancing enzymatic saccharification of α-chitin by increasing surface area, thereby optimizing chitooligosaccharide production.