Characterization of a yam class IV chitinase produced by recombinant Pichia pastoris X-33.

A yam (Dioscorea opposita Thunb) class IV chitinase, whose genomic DNA was cloned by Mitsunaga et al. (2004), was produced by the recombinant Pichia pastoris X-33 in high yields such as 66 mg/L of culture medium. The chitinase was purified by column chromatography after Endoglycosidase H treatment and then characterized. It showed properties similar to the original chitinase E purified from the yam tuber reported by Arakane et al. (2000). This Pichia-produced chitinase also showed strong lytic activity against Fusarium oxysporum and Phytophthora nicotianae, wide pH and thermal stability, optimum activity at higher temperature such as 70 °C, and high substrate affinity, indicating that one can use this Pichia-produced yam chitinase as a bio-control agent.

Purification and characterization of a novel isozyme of chitinase from Bombyx mori.

75-kDa chitinase, which showed potential as a biocontrol agent against Japanese pine sawyer, was characterized after purification from the integument of the fifth instar larvae of Bombyx mori by chromatography on diethylaminoethyl (DEAE)-Toyoperal 650 (M), hydroxylapatite, and Fractogel EMD DEAE 650 (M) columns. The optimum pH was 6.0 toward N-acetylchitopentaose (GlcNAc5) and 10 toward glycolchitin. The optimum temperature was 60 degrees C toward GlcNAc5 and 25 degrees C toward glycolchitn. The enzyme was stable at pH 7-10 and below 40 degrees C. Kinetic analysis and reaction-pattern analysis using glycolchitin and N-acetylchitooligosacchraides as substrates indicated that 75-kDa chitinase is an endo- or random-type hydrolytic enzyme to produce the beta anomeric product and that it prefers the longer N-acetylchitooligosaccharides, suggesting, together with the N-terminal amino acid sequence, that the 75-kDa chitinase belongs to family 18 of glycosyl hydrolases.

Effect of Bombyx mori chitinase 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.

Alternative splicing of the primary transcript generates heterogeneity within the products of the gene for Bombyx mori chitinase.

The gene of chitinase in the silkworm, Bombyx mori, generates four mRNA products by alternative splicing. Nucleotide sequences of the entire gene for chitinase and respective cDNAs demonstrate that the pre-mRNA undergoes alternative splicing at both the 5' and 3' regions. At the 5' region, the pre-mRNA experienced differential splicing through two alternative 5'-intron consensus splicing sites. These products differ in the last amino acid of the signal peptide and the first amino acid of the mature N-terminal sequences: one with Cys(20)-Ala(21) and the other with Ser(20)-Asp(21). The product with Cys(20)-Ala(21) residues is one amino acid larger than the other with Ser(20)-Asp(21). At the 3' region the pre-mRNA of the chitinase gene undergoes alternative splicing in three different fashions. It is spliced either through retaining or excluding the upstream 121-bp direct repeat found at the 3' region of the coding sequences or through retaining or excluding of an insertion of 9 bp in a combinatorial manner. Retention or exclusion of the upstream 121-bp direct repeat results in a protein with a deduced amino acid sequence similar in size to the one retaining both direct repeats. However, exclusion of the insert of the 9 bp from the mRNA results in a protein with 22 extra amino acids. All of the mRNA products appear to be generated from a single gene as demonstrated by testing the 3' region of the genomic DNA and variant chitinase mRNA products. B. mori chitinase expression in the fifth instar larvae epidermal tissues appears to be developmentally regulated, but the phenomenon of alternative splicing of the pre-mRNA is not stage-dependent. Furthermore, the four mRNA products showed chitinase activity when expressed in Escherichia coli, which demonstrates the role of the alternative splicing process in generating multiple isoforms of the silkworm's chitinase.

Intracellular localization of a class IV chitinase from yam.

Genomic DNA encoding a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves in previous research (Biosci. Biotechnol. Biochem., 68, 1508-1517 (2004)). But this chitinase had an additional sequence composed of eight amino acids (a C-terminal extension) at the C-terminal, compared with class IV chitianses from other plants. In order to clarify the role of this C-terminal extension in cellular localization, plants and suspension-cultured cells of Nicotiana tabacum were transformed with either the cloned yam class IV chitinase gene carrying the C-terminal extension or its truncated gene by the Agrobacterium-mediated method, and then their localization was investigated. The results suggest that the C-terminal extension of yam class IV chitinase plays a role as a targeting signal for plant vacuoles. This is the first report presenting the existence of vacuolar type class IV chitinase.

Plant chitinase as a possible biocontrol agent for use instead of chemical fungicides.

We investigated whether a plant chitinase can be used as a biocontrol agent instead of chemical fungicides by spraying chitinase E (family 19; class IV) from a yam (Dioscorea opposita Thunb) alone or together with beta-1,3-glucanase directly onto the surface of a powdery mildew infecting strawberry berries and leaves. Results were observed by eye and with a scanning electron microscope. The powdery mildew infecting the strawberries was degraded, mainly by the chitinase, and the disease did not appear again for more than 2 weeks. These results indicated that this kind of plant chitinase might be safe and biodegradable biocontrol agent for use instead of conventional fungicides.

Molecular cloning of a genomic DNA encoding yam class IV chitinase.

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.

Kinetic analysis of a chitinase from red sea bream, Pagrus major.

Kinetic analysis was done on the 46-kDa chitinase (EC 3.2.1.14) purified from the stomach of red sea bream, Pagrus major, using glycolchitin and N-acetylchitooligosaccharides (GlcNAc(n), n=2-6) as substrates. High activity was observed at two pHs, such as 2.5 and 9.0, toward glycolchitin as seen in other insect chitinases, and also at both pH 2.5 and 5.0 even toward a short substrate, N-acetylchitopentasaccharide. Allosamidin competitively inhibited chitinase with Ki value of 0.0214 microM at pH 2.5 and 0.0024 microM at pH 9.0 in the reaction of glycolchitin. Substrate inhibition was observed in the reaction of N-acetylchitopentasaccharide. The anomeric forms of the products from N-acetylchitooligosaccharides were analyzed to be beta anomer by the high pressure liquid chromatography (HPLC) method. The data for both beta-anomer formation and allosamidin inhibition suggest that red sea bream chitinase belongs to family 18 of glycosyl hydrolases. This suggestion is also supported by the results for the N-terminal amino acid sequence.

Analysis of hydrolytic activity of a 65-kDa chitinase from the silkworm, Bombyx mori.

The hydrolytic reactions of Bombyx mori 65-kDa chitinase with the short substrates, N-acetyl-chitooligosaccharides, were analyzed by HPLC. Analysis of the hydrolyzed products showed that the newly produced oligosaccharides are all beta anomers, suggesting that, similar to other family 18 glycosyl hydrolases, the 65-kDa chitinase acts in the retaining mechanism. Furthermore, the enzyme cleaves the N-acetylchitooligosaccharides mainly at the linkage between the second and the third GlcNAc moieties from the non-reducing end, while the other sites were cleaved in smaller proportions. Moreover, the initial reaction rates of the enzyme with the longer N-acetylchitooligosaccharides were higher than those with shorter ones. These results suggest that the enzyme is an endo-cleaving type and more efficient on the longer substrates.