Insect Chitinase Research Articles

Identification and characterization of a novel chitinase-like gene cluster ( AgCht5) possibly derived from tandem duplications in the African malaria mosquito, Anopheles gambiae

Insect chitinase 5 (Cht5), a well-characterized enzyme found in the molting fluid and/or integument, is classified as a group I chitinase and is usually encoded by a single gene. In this study, a Cht5 gene cluster consisting of five different chitinase-like genes ( AgCht5- 1, AgCht5- 2, AgCht5- 3, AgCht5- 4 and AgCht5- 5) was identified by a bioinformatics search of the genome of Anopheles gambiae. The gene models were confirmed by cloning and sequencing of the corresponding cDNAs and gene expression profiles during insect development were determined. All of these genes are found in a single cluster on chromosome 2R. Their open reading frames (ORF) range from 1227 to 1713 bp capable of encoding putative proteins ranging in size from 409 to 571 amino acids. The identities of their cDNA sequences range from 52 to 66%, and the identities of their deduced amino acid sequences range from 38 to 53%. There are four introns for AgCht5- 1, two for AgCht5- 2 and AgCht5- 3, only one for AgCht5- 4, but none for AgCht5- 5 in the genome. All five chitinase-like proteins possess a catalytic domain with all of the conserved sequence motifs, but only AgCht5-1 has a chitin-binding domain. Phylogenetic analysis of these deduced proteins along with those from other insect species suggests that AgCht5-1 is orthologous to the Cht5 proteins identified in other insect species. The differences in expression patterns of these genes at different developmental stages further support that these genes may have distinct functions. Additional searching of the genomes of two other mosquito species led to the discovery of four Cht5-like genes in Aedes aegypti and three in Culex quinquefasciatus. Thus, the presence of a Cht5 gene cluster appears to be unique to mosquito species and these genes may have resulted from gene tandem duplications.

Insect chitinase and chitinase-like proteins

Insect chitinases belong to family 18 glycosylhydrolases that hydrolyze chitin by an endo-type of cleavage while retaining the anomeric beta-(1-->4) configuration of products. There are multiple genes encoding chitinases and chitinase-like proteins in all insect species studied using bioinformatics searches. These chitinases differ in size, domain organization, physical, chemical and enzymatic properties, and in patterns of their expression during development. There are also differences in tissue specificity of expression. Based on a phylogenetic analysis, insect chitinases and chitinase-like proteins have been classified into several different groups. Results of RNA interference experiments demonstrate that at least some of these chitinases belonging to different groups serve non-redundant functions and are essential for insect survival, molting or development. Chitinases have been utilized for biological control of insect pests on transgenic plants either alone or in combination with other insecticidal proteins. Specific chitinases may prove to be useful as biocontrol agents and/or as vaccines.

Functional specialization among insect chitinase family genes revealed by RNA interference

The biological functions of individual members of the large family of chitinase-like proteins from the red flour beetle, Tribolium castaneum (Tc), were examined by using gene-specific RNAi. One chitinase, TcCHT5, was found to be required for pupal-adult molting only. A lethal phenotype was observed when the transcript level of TcCHT5 was down-regulated by injection of TcCHT5-specific dsRNA into larvae. The larvae had metamorphosed into pupae and then to pharate adults but did not complete adult eclosion. Specific knockdown of transcripts for another chitinase, TcCHT10, which has multiple catalytic domains, prevented embryo hatch, larval molting, pupation, and adult metamorphosis, indicating a vital role for TcCHT10 during each of these processes. A third chitinase-like protein, TcCHT7, was required for abdominal contraction and wing/elytra extension immediately after pupation but was dispensable for larval-larval molting, pupation, and adult eclosion. The wing/elytra abnormalities found in TcCHT7-silenced pupae were also manifest in the ensuing adults. A fourth chitinase-like protein, TcIDGF4, exhibited no chitinolytic activity but contributed to adult eclosion. No phenotypic effects were observed after knockdown of transcripts for several other chitinase-like proteins, including imaginal disk growth factor IDGF2. These data indicate functional specialization among insect chitinase family genes, primarily during the molting process, and provide a biological rationale for the presence of a large assortment of chitinase-like proteins.

Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum

Insect chitinase (CHT) family proteins are encoded by as many as 16 genes depending upon the species of interest. We have classified these proteins in three species into five different groups based on amino acid sequence similarities (Zhu et al., companion paper). The functions of most of the individual proteins of this family during growth and development are largely unknown. To help determine their enzymatic properties and physiological roles, we expressed representative members belonging to this protein family from Drosophila melanogaster (Dm) and Tribolium castaneum (Tc), and characterized their kinetic and carbohydrate-binding properties. Seven proteins, including DmCHT 4, 5, 9 and DmDS47 from Drosophila, and TcCHT5, TcIDGF2 and TcIDGF4 from Tribolium, belonging to groups I, IV or V of the chitinase-like family were expressed in a baculovirus-insect cell line expression system, purified and characterized. Their enzymatic and chitin-binding properties were compared to those of the well-characterized chitinase, MsCHT535, from Manduca sexta (Ms). All of these proteins, except those belonging to group V that are related to imaginal disc growth factors (IDGFs), exhibited chitinolytic activity against the long polymeric substrate, CM-Chitin-RBV, and/or the short oligomeric substrate, MU-(GlcNAc)3. TcCHT5, DmCHT5 and MsCHT535, which are members of group I chitinases, cleaved both polymeric and oligomeric substrates. Their enzymatic properties, including pH optima, kinetic parameters, and susceptibility to substrate inhibition by chitooligosaccharides, were similar. Two group IV chitinases, DmCHT4 and DmCHT9, also were characterized. DmCHT4 had one optimum pH of 6 towards the polymeric substrate and no detectable chitinolytic activity towards an oligosaccharide substrate. DmCHT9 had high activity from pH 4 to 8 towards the polymeric substrate and exhibited low activity towards the oligosaccharide substrate. The group V proteins, TcIDGF2 and TcIDGF4, contain all of the catalytically critical residues within conserved region II of family 18 chitinases but neither exhibited chitinolytic activity. Another group V protein, DmDS47, which lacks the critical glutamate residue in region II and the C-terminal CBD, also exhibited no chitinolytic activity. However, all three of the group V proteins bound to chitin tightly. A comparison of the amino acid sequences and homology model structures of group V proteins with enzymatically active members of the chitinase family indicated that the presence of additional loops of amino acids within the (βα)8-barrel structure of these proteins interferes with productive substrate binding and/or catalysis.

Effect ofBombyx moriChitinase against Japanese Pine Sawyer (Monochamus alternatus) Adults as a Biopesticide

Bombyx mori chitinase (Bm-CHI), with a molecular mass of 75 kDa, was investigated on the possibility that it can serve as a biocontrol agent against the adult Japanese pine sawyer (JPS), Monochamus alternatus (Coleoptera: Cerambycidae). Oral ingestion of purified chitinase at concentrations of 3 microM (11.25 microg/50 microl) and 0.3 micoM (1.125 microg/50 microl) caused high mortality in JPS, a significant decrease in bark consumption, and, only in high concentration, a slight reduction of body weight. Fluorescence assays indicated that peritrophic membrane (PM) chitin is degraded by the action of orally ingested Bm-CHI at 3 microM concentration only. Scanning electron micrographs clearly indicated that the beetles that ingested Bm-CHI of the same high concentration had their PM perforated and disrupted, but ultrastructural studies showed that the ingested chitinase did not affect the midgut epithelium. These findings open up the possibility of using insect chitinase as a biopesticidal enzyme. It should have agronomic potential for insect control.

Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linn�) and in vitro

With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid population's growth. Artificial diets were used to provide active (1, 10, 100 and 500 microg ml(-1)) and inactive (500 microg ml(-1)) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day population's doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.

Specificity and Affinity of Natural Product Cyclopentapeptide Inhibitors against A. fumigatus, Human, and Bacterial Chitinases

Family 18 chitinases play key roles in organisms ranging from bacteria to man. There is a need for specific, potent inhibitors to probe the function of these chitinases in different organisms. Such molecules could also provide leads for the development of chemotherapeuticals with fungicidal, insecticidal, or anti-inflammatory potential. Recently, two natural product peptides, argifin and argadin, have been characterized, which structurally mimic chitinase-chitooligosaccharide interactions and inhibit a bacterial chitinase in the nM-mM range. Here, we show that these inhibitors also act on human and Aspergillus fumigatus chitinases. The structures of these enzymes in complex with argifin and argadin, together with mutagenesis, fluorescence, and enzymology, reveal that subtle changes in the binding site dramatically affect affinity and selectivity. The data show that it may be possible to develop specific chitinase inhibitors based on the argifin/argadin scaffolds.

Cloning, expression and functional characterisation of chitinase from larvae of tomato moth ( Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase

Chitinases are vital to moulting in insects, and may also affect gut physiology through their involvement in peritrophic membrane turnover. A cDNA encoding chitinase was cloned from larvae of tomato moth ( Lacanobia oleracea), a Lepidopteran pest of crops. The predicted protein contains 553 amino acid residues, with a signal peptide of 20 a.a. Sequence comparison showed 75–80% identity with other Lepidopteran chitinases. L. oleracea chitinase was produced as a functional recombinant enzyme in the yeast Pichia pastoris. A fusion protein containing chitinase joined to the N-terminus of snowdrop lectin (GNA) was also produced, to determine whether GNA could deliver chitinase to the haemolymph of Lepidopteran larvae after oral ingestion. The purified recombinant proteins exhibited similar levels of chitinase activity in vitro. Both proteins were highly toxic to L. oleracea larvae on injection, causing 100% mortality at low dose (2.5 μg/g insect). Injection of chitinase prior to the moult resulted in decreased cuticle thickness. The recombinant proteins caused chronic effects when fed, causing reductions in larval growth and food consumption by up to 60%. The oral toxicity of chitinase was not increased by attaching GNA in the fusion protein, due to degradation in the larval gut, preventing GNA acting as a “carrier”.

Introduction and constitutive expression of a tobacco hornworm (Manduca sexta) chitinase gene in soybean

Embryogenic soybean [Glycine max (L.) Merrill] cultures were transformed with a Manduca sexta chitinase (msc) gene using microprojectile bombardment. A 1.7 kb DNA fragment encoding a tobacco hornworm chitinase was cloned into the rice transformation vector pGL2, under the control of the maize ubiquitin promoter and linked to the hpt gene as a selectable marker. After bombardment, hygromycin-resistant tissues were isolated and cultured to give rise to clones of transgenic material. Four hygromycin-resistant clones were converted into plants. Two clones were positive for the msc gene via polymerase chain reaction (PCR) and Southern blot analysis. The integration inheritance, and expression of transgenes were confirmed by molecular analysis of transgenic soybean plants. Progeny analysis showed that the introduced genes were inherited and segregated in a 3:1 Mendelian fashion. DNA blot experiments and progeny inheritance analysis indicated that the plants contained several copies of the msc gene and that the insertion occurred at a single locus. Northern blotting analysis confirmed the expression of the transgenes. Western blot analysis of transgenic plants and their progeny revealed the presence of a protein with a molecular weight of 48kDa that reacted with the Manduca sexta antibody. Progeny from the chitinase-positive plants were tested for their resistance to the soybean cyst nematode. Plants expressing the insect chitinase did not manifest enhanced resistance to the soybean cyst nematode.

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a β-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract–peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS–PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10 μg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.

A potent insect chitinase inhibitor of fungal origin.

A water-soluble polysaccharide was isolated from the culture filtrate of a fungal strain, Sphaeropsis sp. TNPT116-Cz, as a novel insect chitinase inhibitor. It was purified to chromatographic homogeneity by ethanol precipitation, anion-exchange and gel filtration chromatography. Its molecular weight was estimated to be 16 kDa by gel filtration HPLC. Monosaccharide analysis showed that it contained glucose, galactose, N-acetylglucosamine and a deoxysugar. This polysaccharide showed potent and specific inhibitory activity against Spodoptera litura chitinase with an IC50 value of 28 nM.

Properties of catalytic, linker and chitin-binding domains of insect chitinase

Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in insect cells using baculovirus vectors. Three additional recombinant proteins composed of the catalytic domain fused with one or two insect or plant chitin-binding domains (CBDs) were also generated and characterized. The catalytic and chitin-binding activities are independent of each other because each activity is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide primarily through an effect on the K m for the former substrate. The linker region, which connects the two domains, facilitates secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region is extensively modified by O-glycosylation and the catalytic domain is moderately N-glycosylated. Immunological studies indicated that the linker region, along with elements of the CBD, is a major immunogenic epitope. The results support the hypothesis that the domain structure of insect chitinase evolved for efficient degradation of the insoluble polysaccharide to soluble oligosaccharides during the molting process.

Macromolecular Insect Chitinase Inhibitors Produced by Fungi: Screening and Partial Characterization

Culture broths of fungal strains were screened for novel insect chitinase inhibitors using the Spodoptera litura chitinase inhibitory assay. The culture filtrates of 5 strains showed potent and specific inhibitory activity against the insect chitinase. Partial characterization showed that the active compounds produced by these strains were water-soluble macromolecular compounds which had not been hitherto reported as chitinase inhibitors. These novel chitinase inhibitors are, therefore, expected to be potential agents for insect control.

A novel putative insect chitinase with multiple catalytic domains: hormonal regulation during metamorphosis.

We have used differential display to identify genes that are regulated by juvenile hormone in the epidermis of the beetle Tenebrio molitor. One of the genes encodes T. molitor chitinase 5 (TmChit5), a chitinase possessing an unusual structure. Sequence analysis of TmChit5 identified five 'chitinase units' of approx. 480 amino acids with similarity to chitinase family 18. These units are separated by less conserved regions containing putative PEST (rich in proline, glutamic acid, serine and threonine) sequences, putative chitin-binding domains and mucin domains. Northern-blot analysis identified a single transcript of approx. 9 kb, whose abundance correlated with that of 20-hydroxyecdysone during metamorphosis. Injection of pupae with 20-hydroxyecdysone alone, or in combination with cycloheximide, indicated that TmChit5 expression is directly induced by the hormone. Further experiments indicated that methoprene (a juvenile hormone analogue) indirectly induced TmChit5 mRNA expression. On the basis of the present results and previous studies, we propose a molecular mechanism for cuticle digestion during the moulting process.

Site-directed mutagenesis and functional analysis of an active site tryptophan of insect chitinase

Chitinase is an enzyme used by insects to degrade the structural polysaccharide, chitin, during the molting process. Tryptophan 145 (W145) of Manduca sexta (tobacco hornworm) chitinase is a highly conserved residue found within a second conserved region of family 18 chitinases. It is located between aspartate 144 (D144) and glutamate 146 (E146), which are putative catalytic residues. The role of the active site residue, W145, in M. sexta chitinase catalysis was investigated by site-directed mutagenesis. W145 was mutated to phenylalanine (F), tyrosine (Y), isoleucine (I), histidine (H), and glycine (G). Wild-type and mutant forms of M. sexta chitinases were expressed in a baculovirus-insect cell line system. The chitinases secreted into the medium were purified and characterized by analyzing their catalytic activity and substrate or inhibitor binding properties. The wild-type chitinase was most active in the alkaline pH range. Several of the mutations resulted in a narrowing of the range of pH over which the enzyme hydrolyzed the polymeric substrate, CM-Chitin-RBV, predominantly on the alkaline side of the pH optimum curve. The range was reduced by about 1 pH unit for W145I and W145Y and by about 2 units for W145H and W145F. The W145G mutation was inactive. Therefore, the hydrophobicity of W145 appears to be critical for maintaining an abnormal pK a of a catalytic residue, which extends the activity further into the alkaline range. All of the mutant enzymes bound to chitin, suggesting that W145 was not essential for binding to chitin. However, the small difference in K m’s of mutated enzymes compared to K m values of the wild-type chitinase towards both the oligomeric and polymeric substrates suggested that W145 is not essential for substrate binding but probably influences the ionization of a catalytically important group(s). The variations in k cat’s among the mutated enzymes and the IC 50 for the transition state inhibitor analog, allosamidin, indicate that W145 also influences formation of the transition state during catalysis.

Homology modeling of the insect chitinase catalytic domain–oligosaccharide complex and the role of a putative active site tryptophan in catalysis

Knowledge-based protein modeling and substrate docking experiments as well as structural and sequence comparisons were performed to identify potential active-site residues in chitinase, a molting enzyme from the tobacco hornworm, Munduca sexta. We report here the identification of an active-site amino acid residue, W145. Several mutated forms of the gene encoding this protein were generated by site-directed mutagenesis, expressed in a baculovirus-insect cell–line system, and the corresponding mutant proteins were purified and characterized for their catalytic and substrate-binding properties. W145, which is present in the presumptive catalytic site, was selected for mutation to phenylalanine (F) and glycine (G), and the resulting mutant enzymes were characterized to evaluate the mechanistic role of this residue. The wild-type and W145F mutant proteins exhibited similar hydrolytic activities towards a tri-GlcNAc oligosaccharide substrate, but the former was approximately twofold more active towards a polymeric chitin-modified substrate. The W145G mutant protein was inactive towards both substrates, although it still retained its ability to bind chitin. Therefore, W145 is required for optimal catalytic activity but is not essential for binding to chitin. Measurement of kinetic constants of the wild-type and mutant proteins suggests that W145 increases the affinity of the enzyme for the polymeric substrate and also extends the alkaline pH range in which the enzyme is active.

Insect resistance of transgenic tobacco expressing an insect chitinase gene.

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control.

Insect Chitinases: Molecular Biology and Potential Use as Biopesticides

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.

Synthesis and Biological Activity of Allosamidin and Allosamidin Analogues

The chitinase inhibitor/insect ecdysis inhibitor allosamidin and eight allosamidin analogues have been synthesised from simple carbohydrate starting materials. Allosamidin was assayed against Tineola bisselliella (Hummel) larvae and all nine compounds were examined for their effects on the development of larvae of Lucilia cuprina (Wiedemann). High larval mortality compared to controls resulted when T. bisselliella and L. cuprina larva were exposed to allosamidin. The (1→3) linked regioisomer, the dimeric analogue and the gluco-configurated dimeric analogue of allosamidin all showed high activity against L. cuprina larvae. The regioisomer, the (1→3) linked isomer and its dimeric analogue, as well as the monomer allosamizoline and its regioisomer, were inactive. These new in-vivo results are consistent with known in-vitro insect chitinase inhibition data, in that greatest larval mortality was exhibited by the best inhibitors.

Characterization of a 46 kda insect chitinase from transgenic tobacco

A 46 kDa Manduca sexta (tobacco hornworm) chitinase was isolated from leaves of transgenic tobacco plants containing a recombinant insect chitinase cDNA, characterized, and tested for insecticidal activity. The enzyme was purified by ammonium sulfate fractionation, Q-Sepharose anion-exchange chromatography and mono-S cation-exchange chromatography. Although the gene for the chitinase encoded the 85 kDa full-length chitinase as previously reported by Kramer et al. [Insect Biochem. Molec. Biol. 23, 691–701 (1993)], the enzyme is produced in tobacco as a 46 kDa protein that is approximately four-fold less active than the 85 kDa chitinase. The N-terminal amino acid sequence of the 46 kDa chitinase is identical to that of the 85 kDa chitinase. The former enzyme is not glycosylated, whereas the latter contains approximately 25% carbohydrate. The pH and temperature optima of the 46 kDa chitinaseare similar to those of the 85 kDa chitinase. The former enzyme is more basic than the latter. The 46 kDa chitinase likely consists of the N-terminal catalytic domain of the 85 kDa chitinase and lacks the C-terminal domain that contains several potential sites for glycosylation. The 46 kDa chitinase is expressed in a number of plant organs, including leaves, flowers, stems and roots. Enzyme levels are higher in leaves and flowers than in stems and roots, and leaves from the middle portion of the plant have more chitinase than leaves from the top and bottom portions. Little or no enzyme is secreted outside of the plant cells because it remains in the intracellular space, even though its transit sequence is processed. When fed at a 2% dietary level, the 46 kDa chitinase caused 100% larval mortality of the merchant grain beetle, Oryzaephilis mercator. The results of this study support the hypothesis that insect chitinase is a biopesticidal protein for insect pests feeding on insect chitinase gene-containing transgenic plants.