Cloning and expression of chitinase a from serratia marcescens for large-scale preparation of N,N-Diacetyl chitobiose

Yue Jin Wu, Chih Yu Cheng*, Yaw-Kuen Li

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

The gene of Serratia marcescens chitinase A(chiA) 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.

Original languageEnglish
Pages (from-to)688-695
Number of pages8
JournalJournal of the Chinese Chemical Society
Volume56
Issue number4
DOIs
StatePublished - 1 Jan 2009

Keywords

  • Chitinase A
  • Colloidal chitin
  • N,N-Diacetyl chitobiose
  • Serratia marcescens

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