Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K-cat) of the enzyme was determined using colloidal chitin (49.71 +/- 1.15 S-1) and crystalline beta-chitin (17.20 +/- 0.83 S-1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5-9.0) and temperature (4-70 degrees C) with a peak activity at pH 5.5 and 55 degrees C. However, enzyme activity was found to be stable up to 45 degrees C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.