Preparation of N-acetyl-D-glucosamine and N,N-diacetylchitobiose by enzymatic hydrolysis of squid pen chitin

Abstract

Squid pen chitin (β-chitin) was hydrolyzed by crude enzymes from two sources; fungal enzyme from Aspergillus fumigatus and bacterial enzyme from cloned Serratia sp. to selectively produced N-acetyl-D-glucosamine (GIcNAc) and N,N'-diacetylchitobiose ((GIcNAc)₂) respectively. The crude enzyme (4 U/1 g of chitin) from A. fumigatus hydrolyzed chitin (3% w/v) at pH 3, 40℃ and gave 72% HPLC yield of GIcNAc within 2 days. The hydrolysis of chitin (3% w/v) with the crude enzyme (1 U/1 g of chitin) from cloned bacteria Serratia sp. at pH 6, 37℃ for 6 day gave 43% and 2.6% HPLC yield of (GIcNAc)₂ and GIcNAc, respectively. The isolation of GIcNAc by ethanol precipitation from the highly concentrated solution of crude product followed by the decoloration with the activated charcoal gave pure GIcNAc with 64% isolated yield. The hydrolysis of chitin by bacterial enzyme 5 U/1 g chitin followed by isolation of (GIcNAc)₂ by activated charcoal column chromatography using stepwise-gradient elution of water up to 30% ethanol gave pure (GIcNAc)₂ in 40% yield.