Efficient production of D-glucosamine by diacetylchitobiose deacetylase catalyzed deacetylation of N-acetyl-D-glucosamine

Abstract

Objective: D-Glucosamine (GlcN) is an important amino sugar with various applications in medicine, food & beverages, nutritional supplements, and dairy products. This study aimed to produce GlcN from N-acetyl-D-glucosamine (GlcNAc) with an efficient deacetylase, and apply different strategies to enhance GlcN production.

Results: We screened a series of deacetylases that involved in the deacetylation of GlcNAc to form GlcN. A diacetylchitobiose deacetylase (TKDac) from Thermococcus kodakarensis exhibited high-efficient deacetylation activity for GlcNAc, yet mostly in the form of inclusion bodies. The soluble expression of TKDac was improved by a co-expressing molecular chaperone (groEL) and TKDac, and insertion of rare codon ATA encoding isoleucine. As such, the recombinant strain TKEL4 was constructed to express TKDac, and 48 g/L GlcN was achieved by TKDac-catalyzed deacetylation. To overcome the inhibition of byproduct (acetate), immobilized TKDac was carried out to produce GlcN from GlcNAc. The immobilized TKDac was conveniently re-used for several batches (above 8) with a 90% conversion rate.

Conclusions: TKDac from T. kodakarensis was found to be an efficient deacetylase to produce GlcN. Co-expression of molecular chaperone and target protein, and insertion of rare codons were effective to improve the soluble expression of TKDac. The immobilized TKDac represents a promising method for future GlcN production.

Keywords: D-Glucosamine; Deacetylase; Deacetylation; Immobilization; Molecular chaperone; Rare codon.