Integument

Characterization of a recombinant insect chitinase and a baculovirus chitinase

T. Shinoda 1 , J. Kobayashi 2 , M. Matsui 1 & Y. Chinzei 3

1 National Research Inst. of Vegetables, Ornamental Plants and Tea, Ano, Mie, 514-2392, Japan; 2 Faculty of Engineering, Mie Univ. Tsu, Mie, 514, Japan; 3 Dept. of Medical Zoology, School of Medicine, Mie Univ. Tsu, Mie, 514, Japan

Insect chitinase is an attractive target for selective insect regulators and also is indicated to be a good source for insect-resistant plants and efficient entomopathogens via genetic-engineering. It is thus important to gain more knowledge on this enzyme. We have isolated a chitinase cDNA (Slchi) from the common cutworm, Spodoptera litura, and characterized its recombinant protein. The Slchi cDNA encodes 552 amino-acid residues (aa) including a 19 aa putative signal peptide, with the calculated molecular mass of the putative mature protein 60,152 Da. The predicted amino acid sequence is highly similar to other insect and prawn chitinases. A major transcript of Slchi about 2.8 kb was detected in the epidermis only during molting suggesting that its digestive role of old cuticle at this period. To evaluate the chitinolytic activity of Slchi protein, we produced a recombinant Slchi protein using a Hyphantria cunea NPV (HycuNPV)-based protein expression system. The genome of HycuNPV also encodes a chitinase gene (chiA), which is similar to AcMNPV chiA, but shares only 15% amino acid residues with Slchi. To distinguish the chitinase activity derived from Slchi and chiA, we construct a recombinant HycuNPV, which expresses Slchi under the polyhedrin promoter and whose viral chitinase gene was knocked out. The recombinant Slchi protein produced from this virus exhibited high chitinolytic activity against 4MU-(GlcNAc)3 > 4MU-(GlcNAc)2 > 4MU-(GlcNAc)4, in this order. The viral chitinase also showed comparable chitinolytic activity, but in a different order, 4MU-( GlcNAc)2 > 4MU-(GlcNAc)3 = 4MU-(GlcNAc)4. No enzymatic activity was found against 4MU-(GlcNAc)1 in either chitinase. Interestingly, the Slchi protein was secreted rapidly into the culture medium from the infected cells, whereas the viral chitinase was retained predominantly in the infected cells. The differences in substrate specificity and the secretion properties suggest that the Slchi introduced into a baculovirus may enhance its insecticidal activity. Moreover, the recombinant Slchi protein can be used for screening of chitinase inhibitors and a supplement for entomopathogens.

Index terms: Spodoptera litura, Hyphantria cunea, NPV, chitinase, recombinant protein.

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