Mechanoenzymatic Breakdown of Chitinous Material to N‐Acetylglucosamine: The Benefits of a Solventless Environment

Abstract

Chitin is not only the most abundant nitrogen‐containing biopolymer on the planet, but also a renewable feedstock that is often treated as a waste. Current chemical methods to break down chitin typically employ harsh conditions, large volumes of solvent, and generate a mixture of products. Although enzymatic methods have been reported, they require a harsh chemical pretreatment of the chitinous substrate and rely on dilute solution conditions that are remote from the natural environment of microbial chitinase enzymes, which typically consists of surfaces exposed to air and moisture. We report an innovative and efficient mechanoenzymatic method to hydrolyze chitin to the N‐acetylglucosamine monomer by using chitinases under the recently developed reactive aging (RAging) methodology, based on repeating cycles of brief ball‐milling followed by aging, in the absence of bulk solvent. Our results demonstrate that the activity of chitinases increases several times by switching from traditional solution‐based conditions of enzymatic catalysis to solventless RAging, which operates on moist solid substrates. Importantly, RAging is also highly efficient for the production of N‐acetylglucosamine directly from shrimp and crab shell biomass without any other processing except for a gentle wash with aqueous acetic acid.

Mechanoenzymatic Transformations in the Absence of Bulk Water: A More Natural Way of Using Enzymes

“…focused on chitinase activity, it also noted that the commercial enzyme preparation used for chitin hydrolysis contained, in addition to chitinases, α‐amylases. During the RAging reactions, these α‐amylases were found to hydrolyze the cryoprotectant present in the commercial chitinase preparation to glucose . This fortuitous observation strongly suggests that amylases could also be compatible with RAging reaction conditions.…”

“…The biomass was washed with diluteaqueousa cetic acid priortohydrolysis. [149] on wheat, [156] corn, [157] sago, [152,155] and rice [158,159] starch by bioextrusion.…”

“…B) Comparison of the yield and composition of the hydrolysis products from ten cycles of RAging, for commercial practical‐grade chitin (CC), biomass from shrimp (AcShrimp) and crab shells (AcCrab). The biomass was washed with dilute aqueous acetic acid prior to hydrolysis …”

“…A recent study by Therien et al . demonstrated that chitinases are not only several fold more efficient in the absence of bulk water, but are also more selective and generate a highly concentrated GlcNAc product.…”

“…[146] Use of chitinolytic enzymes,o rc hitinases, [147] in typical aqueous suspensions provides am ore environment-friendly depolymerization route, but is slow and still requires harsh pre-treatment of the biomass. [148] Ar ecent study by Therien et al [149] demonstrated that chitinases are not only severalf old more efficient in the absence of bulk water,b ut are also more selective and generate ah ighly concentrated GlcNAcp roduct. Under RAging conditions (h = 1.7 mLmg À1 ), only 0.7 wt %o fc hitinases from ac ommonplace terrestrial fungus A. niger was necessary to reach > 30 %h ydrolysis of chitin, of which 92 %w as GlcNAc.…”