Abstract
Bacillus thuringiensis subsp. tenebrionis DSM‐2803 has been studied extensively and spore/crystal mixtures of this strain are used widely in commercial products to control coleopteran pests. The endochitinase chiA Btt gene of B. thuringiensis subsp. tenebrionis DSM‐2803 was cloned and expressed in Escherichia coli. The recombinant 6x‐histidine tagged protein (rChiA Btt, ~74 kDa), was purified by a HiTrap Ni affinity column. The Km of rChiA Btt was 0.847 μmol L−1 and its optimal activity occurred at pH 7 and ~40°C. Most divalent cations reduced endochitinase activity but only Hg+2 abolished activity of the enzyme. We report for the first time the characterization of a chitinase synthesized by B. thuringiensis subsp. tenebrionis DSM‐2803, and show that the purified rChiA74 Btt reduced the radial growth and increased the hyphal density of Colletotrichium gloeosporioides, the etiological agent of “anthracnose” in plants.
Highlights
IntroductionChitin is characteristically found in the exoskeleton of arthropods (insects, crustaceans) and in the cell wall of fungi and algae
Chitin, a β-1-4-linked polymer of N-acetylglucosamine (GlcNAc), is the second most abundant structural polysaccharide present in nature, eclipsed only by cellulose.Chitin is characteristically found in the exoskeleton of arthropods and in the cell wall of fungi and algae
This indicated that the main chitinolytic activity of B. thuringiensis subsp. tenebrionis DSM-2803 is an endochitinase, which is comparable to those of B. thuringiensis subsp. kurstaki HD73 and B. thuringiensis subsp. kurstaki HD1, as they showed activities of ~250 mU mL−1, 250 mU mL−1 and 270 mU mL−1, respectively (Fig. 1A), similar to the activity previously reported for B. thuringiensis subsp. kurstaki HD73 (Barboza-C orona et al 2009)
Summary
Chitin is characteristically found in the exoskeleton of arthropods (insects, crustaceans) and in the cell wall of fungi and algae. Bacteria do not synthesize chitin, but many species secrete chitinolytic enzymes that hydrolyze the polymer to release units of GlcNAc for use as carbon. Bacillus thuringiensis is a well-k nown bacterium that is widely used as an insect larvicide owing to the toxic effect of its proteinaceous Cry (crystal) and Cyt (cytolytic) toxins. In comparison to Cry and Cyt toxins, chitinases of B. thuringiensis have been less studied, but more recently have gained attention mainly because these enzymes have potential applied value to control phytopathogenic fungi (Driss et al 2005; Morales de la Vega et al 2006)
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