Cloning and characterization of chitinase gene from hepatopancreas from black tiger shrimp Penaeus monodon

Abstract

Chitinase (E.C. 3.2.1.14) is an enzyme that catalyzes the degradation of chitin. Chitinase is useful enzyme fo wide applications in several industries. A computer program was used for searching the chitinase gene in the black tiger shrimp (Penaeus monodon) database (http://pmonodon.biotec.or.th). A total of 10,100 clones were searched for chitinase gene. Three clones, two clones from a hemocyte library and another clone from hepatopancreas library, were identified as a putative chitinase gene and reconfirmed by BLASTN program. The chitinase gene, ChiHP74, found in hepatopancreas cDNA library of black tiger shrimp P. monodon has been cloned. The ChiHP74 contains an open reading frame of 1995 bp. The cDNA encoded for a polypeptide chain of 665 amino acid residues with a predicted molecular weight of 74 kDa. Sequence analysis of the deduced amino acid sequence of chitinase comprised a family 18 catalytic domain (CatD), a tachycitin like domain, and two proline rich domains at the C-terminus. In an expression of this gene, bacterial expression system and yeast expression system were used to express ChiHP74. The result showed this chitinase could not be expressed by both systems. Because of no expression of full-length fragment, CatD was chosen to clone and express instead. It was successfully expressed by using PET-17b in E. coli strain BL21 (DE3) called Chi-CatD which showed an optimum pH in two ranges of 4 and 7. The optimum temperature of this enzyme was 60degrees Celsius. Chi-CatD hydrolyzed B-chitin the best and only dimmer [(GlcNAc)2] was detected.