N-acetylglucosaminidase (chitobiase) from Serratia marcescens: Gene sequence, and protein production and purification in Escherichia coli

The chitobiase (Chb) encoding gene (chb) from Serratia marcescens (Sm) has been cloned, sequenced and expressed in Escherichia coli (Ec). Sequencing has revealed an open reading frame encoding a protein of 885 amino acids (aa). Ec cells harbouring plasmids containing chb can produce enzymatically active Sm Chb protein which is secreted into the periplasm. An efficient purification scheme using cation-exchange chromatography is presented. This yields about 3 mg of >95% pure Sm Chb per litre of Ec culture. The deduced aa sequence is 27-aa longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the cell membrane. Comparison with the other members of the family 20 of glycosyl hydrolases revealed that Chb has a conserved central region which aligns with almost all members of this family. According to the crystal structure of Sm Chb, this region comprises the catalytic domain of Chb which has an alpha/beta barrel fold.