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). Se quencing has revealed an open reading frame encoding a protein of 885 amino acids (aa). Ec cells harbouring plasmids containing chb can prod uce enzymatically active Sm Chb protein which is secreted into the per iplasm. An efficient purification scheme using cation-exchange chromat ography is presented. This yields about 3 mg of >95% pure Sm Chb per l itre of Ec culture. The deduced aa sequence is 27-aa longer at the N t erminus than that determined by sequencing of the purified protein, su ggesting that a leader sequence is removed during transport of the enz yme across the cell membrane. Comparison with the other members of the family 20 of glycosyl hydrolases revealed that Chb has a conserved ce ntral region which aligns with almost all members of this family. Acco rding to the crystal structure of Sm Chb, this region comprises the ca talytic domain of Chb which has an alpha/beta barrel fold.