Gene cloning, expression and characterization of chitinase from black tiger shrimp Penaeus monodon

Abstract

Chitinases are essential enzymes for crustaceans and animal alike for their molting and digestion of foods containing chitin. From the Penaeus monodon EST database, cDNA contigs and singletons for three chitinases, namely PmChi1, 2 and 3, were identified. The complete sequences of the mature PmChi1, 3 and partial PmChi2 were amplified and cloned. The reading frames of PmChi1 and 3 encoded mature proteins of 644 and 468 amino acids with calculated molecular weights of 72.4 and 51.9 kDa, respectively. The amino acid sequence comparison among the penaeid chitinases revealed very high homology around 90%. They were, thus, grouped together along with those of other crustaceans and insects into three groups separated from those of mammals. The PmChi1 and 3 were expressed mainly in hepatopancreas, whereas PmChi2 were in gill. Small amount of them could be expressed in other tissues. After molting, only the expression of PmChi2 was down-regulated while the expression of PmChi1 and 3 was relatively unchanged. The results suggested that the PmChi2 was likely involved in molting while the others might function in the digestion of chitinous foods. The recombinant PmChi1 (rPmChi1) over-produced from Escherichia coli had its optimal pH 5 but it was most stable at neutral pH. Interestingly, the optimal temperature was relatively high at 55 ºC. Nevertheless, it was stable at lower temperature below 40 ºC. The rPmChi1 preferentially hydrolyzed the more soluble substrates like partially N-acetylated chitin (PNAC) and colloidal chitin from shrimp shell as compared to the β-chitin from squid pen.