Premium
Cloning and Expression of Chitinase A from Serratia Marcescens for Large‐Scale Preparation of N,N ‐Diacetyl Chitobiose
Author(s) -
Wu YueJin,
Cheng ChihYu,
Li YawKuen
Publication year - 2009
Publication title -
journal of the chinese chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.329
H-Index - 45
eISSN - 2192-6549
pISSN - 0009-4536
DOI - 10.1002/jccs.200900103
Subject(s) - chitobiose , chemistry , chitinase , serratia marcescens , chromatography , escherichia coli , biochemistry , electrospray ionization , chitosanase , chitin , molecular mass , enzyme , mass spectrometry , chitosan , gene
The gene of Serratia marcescens chitinase A ( chi A) was cloned by PCR. The complete gene was constructed into a pRSET vector and expressed in Escherichia coli . The recombinant enzyme was purified to > 90% homogeneity by hydrophobic interaction chromatography followed by ion‐exchange separation. Measured with an electrospray‐ionization mass spectrometer, the molecular mass of the protein was 58,607 Da, consistent with a theoretical calculation of the deduced protein without the signal peptide. The recombinant enzyme was characterized and tested for the preparation of chitobiose. In general, the recombinant Chtinase A exhibited an exo‐type catalytic activity toward colloidal chitin and released both N ‐acetylglucosamine and N,N ‐diacetyl chitobiose as products. After extensive testing, we produced N,N ‐diacetyl chitobiose as the predominant product when the enzymatic reaction was performed in sodium acetate buffer at pH 5.5; under such conditions, an enzymatic process is established for the production of the disaccharide on a 100‐g scale.