Cloning, Sequences, and Characterization of Two Chitinase Genes from the Antarctic Arthrobacter sp. Strain TAD20: Isolation and Partial Characterization of the Enzymes

Abstract

ABSTRACT Arthrobacter sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell. Arthrobacter sp. strain TAD20 secretes two major chitinases, ChiA and ChiB ( Ar ChiA and Ar ChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in Escherichia coli . DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of Ar ChiA (881 amino acids [aa]) and Ar ChiB (578 aa). Ar ChiA and Ar ChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain of Ar ChiA exhibits 55% identity with a chitodextrinase from Vibrio furnissii . The Ar ChiB catalytic domain exhibits 33% identity with chitinase A of Bacillus circulans . The Ar ChiA chitin-binding domains are homologous to the chitin-binding domain of Ar ChiB. Ar ChiA and Ar ChiB were purified to homogeneity from the native Arthrobacter strain and partially characterized. Thermal unfolding of Ar ChiA, Ar ChiB, and chitinase A of Serratia marcescens was studied using differential scanning calorimetry. Ar ChiA and Ar ChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.