Recombinant Expression of Chitinase CtChi70 in Komagataella phaffii for Sustainable N, N'-Diacetyl-Chitobiose Production From Shrimp Shells.

The rapid expansion of the global seafood processing industry has generated substantial quantities of shrimp shells and other chitin-rich waste. Recombinant chitinases provide a promising strategy for converting this biomass into high-value chitin oligosaccharides. In this study, we systematically optimized the secretory expression of the GH18 exochitinase CtChi70 in Komagataella phaffii (formerly Pichia pastoris) and examined its applicability for the enzymatic conversion of shrimp shell-derived chitin into N,N'-diacetyl-chitobiose ((GlcNAc)2). Through signal peptide optimization, gene copy number amplification, and HAC1 overexpression, extracellular chitinase activity in shake-flask cultures reached 181 U/L. Scale-up using high-cell-density fed-batch fermentation in a 7-L bioreactor further increased activity to 2151 U/L. A mild chitin extraction process was established using citric acid demineralization and low-concentration NaOH deproteinization, yielding a suitable substrate for enzymatic hydrolysis. The resulting chitin was successfully converted into high-purity (GlcNAc)2 within 24 h. Collectively, this work establishes an efficient expression strategy for CtChi70 production and demonstrates an integrated, environmentally sustainable approach for shrimp shell valorization.