Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells

Abstract

With the aid of a signal sequence of a chitinase from Streptomyces lividans, a recombinant chitin deacetylase, whose gene originated from a Deuteromycete, Colletotrichum lindemuthianum, was produced in the culture medium of Escherichia coli cells, existing as a highly active form without the signal peptide. During the production of the recombinant chitin deacetylase, both a slight increase in the value of OD 600 nm in the culture medium and a drastic decrease in viable cell number were observed. When penta-N-acetyl-chitopentaose was used as the substrate, the recombinant chitin deacetylase had comparable kinetic parameters to those of the original enzyme from the fungus. The addition of a C-terminal six histidine sequence to the recombinant enzyme caused a slight decrease in the k cat value, and the further addition of a 12 amino acid sequence at its N-terminus caused a further decrease in the value. This production system allowed us to easily produce in the culture media the recombinant chitin deacetylases possessing as good properties as the original enzyme, without any disruption steps of the E. coli cells.z 1999 Federation of European Biochemical Societies.

A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation

“…A. nidulans CDA was obtained by cytoplasmic expression in E. coli, leading to the formation of inclusion bodies from which the active enzyme had to be refolded (38). CDA2 from S. cerevisiae was obtained as an active enzyme in the periplasmic space of E. coli (37), while ClCDA was secreted from the bacterial cells into the medium with the help of a signal sequence from Streptomyces lividans (36).…”

“…As the PgtCDA gene has a signal peptide (which was removed for expression in E. coli), the native enzyme is expected be secreted into the medium. Most extracellular fungal CDAs, such as ClCDA ATCC 56676, ClCDA DSM 16344, ClCDA UPS9, A. nidulans CDA, and S. cerevisiae CDA2p, have been reported to have an optimum pH ranging between pH 7 and 12 and an optimum temperature in the range of 50°C to 60°C (13,26,31,33,36,70,71). Chitin deacetylases are metalloenzymes requiring divalent cations, such as Zn 2ϩ , in their active site (25), and the addition of metal ions, such as Co 2ϩ in Mortierella sp.…”

Structure and Mechanism of Chitin Deacetylase from the Fungal Pathogen Colletotrichum lindemuthianum,

“…It has also been demonstrated that S. cereVisiae CDAdeficient strains show reduced ascospore wall rigidity and increased susceptibility to lytic enzymes (26). Recombinant ClCDA has previously been expressed in Escherichia coli (27) and recently in Pichia pastoris (28). ClCDA is a representative member of the CE-4 family and shares a number of sequence motifs, covering several conserved histidine and aspartic acid residues ( Figure 1B).…”

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells

“…CODs from the soil bacterium Rhizobium melitoti (John et al 1993) and the archaeon Thermococcus kodakaraensis KOD1 (Tanaka et al 2003) have been cloned and overproduced in transformed E. coli cells. It has been confirmed that chitin deacetylase from the mold Colletorichum lindemuthianum can be produced in the culture medium of transformed E. coli cells with the aid of a chitinase signal peptide from Streptomyces lividans (Tokuyasu et al 1999). These recombinant enzymes catalyze the hydrolysis of the acetamide bond on the non-reducing GlcNAc residue of (GlcNAc) 2 to produce GlcN-GlcNAc.…”