A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I–IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I β-1,3-glucanase against Fusarium solani germlings.     

Isolation and Characterization of a Barley Chitinase Genomic Clone—Expression in Powdery Mildew Infected Barley

Chitinases (E.C.3.2.1.14) are thought to play an important role in the defense of plants against fungal invasion. By screening a barley genomic library with a previously identified chitinase eDNA clone (clone 10), a genomic clone was isolated and characterized by DNA sequencing of the chitinase coding region and flanking sequences. This clone contains an open reading frame capable of coding for a 34 kD chitinase. Comparison of the amino acid sequence of the encoded protein with other barley chitinases suggests that the genomic clone encodes chitinase T, which has been characterized extensively by protein sequencing. Treatment of barley leaves and aleurone protoplasts with N-acetyl glucosamine oligomers which act as elicitors in other plants, did not lead to the elevation of the levels of the chitinases. However, infection of barley seedlings with the powdery mildew fungus, Erysiphe graminis, resulted in the induction of several isoforms of chitinase. The level and number of chitinase isozymes was correlated with the severity of infection. The infection-related chitinases found in barley leaves are different from those found in seeds.     

Entamoeba invadens: Cloning and molecular characterization of chitinases

Entamoeba histolytica, the causative agent of amebiasis infects through its cyst form and this transmission may be blocked using encystation specific protein as drug target. In this study, we have characterized the enzyme chitinase which express specifically during encystation. The reptilian parasite Entamoeba invadens, used as a model for encystation study contain three chitinases. We report the molecular cloning, over-expression and biochemical characterization of all three E. invadens chitinase. Cloned chitinases were over-expressed in bacterial system and purified by affinity chromatography. Their enzymatic profiles and substrate cleaving patterns were characterized. All of them showed binding affinity towards insoluble chitin though two of them lack the chitin binding domain. All the chitinases cleaved and released dimmers from the insoluble substrate and act as an exochitinase. Homology modeling was also done to understand the substrate binding and cleavage pattern.     

Garlic (Allium sativum) chitinases: characterization and molecular cloning

Leaves and bulbs of garlic ( Allium sativum L.) contain a chitinase which can be separated into three different isoforms with similar molecular structure and N- terminal amino acid sequence. SDS-PAGE of the alkylated chitinase revealed two distinct polypeptides of 32 and 33 kDa. Induction studies of the chitinase in leaves of garlic plants indicated that not only treatment with ethephon or salicylate and wounding but also a temperature shock strongly increased the enzyme level.
cDNA libraries constructed from poly(A)-rich RNA isolated from young garlic shoots and bulbs were screened for chitinase clones using the cDNA clone CCH4 encoding a basic potato chitinase as a probe. Two different cDNA clones (designated CHITAS 1 and CHITAS 2)of ca 1 000 bp were isolated and their sequences analyzed. The amino acid sequences deduced from both cDNA clones were homologous though not identical to the N-terminal sequences of the mature chitinases. Although both clones encode highly homologous chitinases their sequences definitely differ in that they have different signal peptides and one of them contains a glycine-rich domain. The garlic chitinases are apparently translated from an mRNA of 1200 nucleotides which encodes a proprotein of approximately 32 or 33 kDa for CHITAS 1 and CHITAS 2, respectively. Co-translational removal of the signal peptide will result in a 30 (for CHITAS 1) or 31 kDa (for CHITAS 2) protein with an isoelectric point of 4. 94 (for CHITAS 1) or 6. 12 (for CHITAS 2). Garlic chitinases are encoded by a small gene family as shown by Southern blot analysis of genomic DNA isolated from garlic.
The garlic chitinases show a high degree of sequence homology to the previously isolated chitinases from dicotyledonous as well as monocotyledonous species, indicating that these proteins have been conserved from an evolutionary point of view.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Cloning and expression analysis of Chitinase genes from Populus canadensis

Plant chitinases play a key role in conferring resistance to environmental stresses, including attack by fungal pathogens. In the present study, we employed rapid amplification of cDNA ends (RACE) to identify five chitinase genes in Populus canadensis Moench. Sequence alignment revealed that these genes belong to five subfamilies of chitinase genes. The full-length cDNAs of these genes ranged in size from 991 to 1358 bp and encoded proteins with mol wts from 29.5 to 40.3 kD. Five genes were grouped into three major clades based on amino acid sequences of encoded proteins. Exon-intron gene structure and protein domain analysis further supported the designation. A three-dimensional structure comparison showed the high similarity between five P. canadensis chitinases and three well-studied chitinases from other species. The expression levels of all five genes were up-regulated during Populus infection with the pathogenic fungus Marssonina brunnea, and four of them were highly induced by salt and drought stresses. Furthermore, such factors as elicitors, wounding, and low temperature also elevated the expression of these chitinase genes to varying extents. We postulated that these chitinase genes may be involved in pathways of the defense against fungal infection and function in response to various abiotic stresses.     

Induction of chitinases and of translatable mRNA for these enzymes in melon plants infected with Collectotrichum lagenarium

In melon plants infected with Colletotrichum langenarium, there is a strong increase in the activity of chitinase, an enzyme with a potential defensive function against pathogens. In order to investigate the molecular mechanisms involved in the regulation of chitinase gene expression, antisera have been raised against two purified chitinases (I and II) from infected melon plants. Changes induced by infection in the rate of synthesis of chitinases were determined using direct immunoprecipitation of enzymes labelled in vivo with [³⁵S]methionine. A large but transient increase in the rate of chitinase synthesis occurred 5 days after inoculation. The in vitro synthesis of chitinases was then studied in healthy and infected melon plants. Poly(A)RNA was fractionated by sucrose gradient density centrifugation, and translated in a rabbit reticulocyte lysate. The transition products were then separated by fast protein liquid chromatography (FPLC) and further analysed by SDS-polyacrylamide gel electrophoresis. The identification of in vitro synthesized chitinases was performed by immunoprecipitation. The obtained results indicated the presence of chitinases in in vitro translation products of mRNA from infected, but not from healthy melon seedlings. It is concluded therefore, that infection of melon seedlings by a pathogen caused an increase in the translatable mRNAs for host chitinases.     

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.     

Purification and characterization of Streptomyces albidoflavus antifungal components

Chitinolytic strain Streptomyces albidoflavus was isolated from soil of the central region of Poland. Its identification was based on analysis of 16S rRNA gene sequence. The colloidal chitin was revealed as the finest substrate for the production of chitinases by S. albidoflavus. The enzyme catalyzed the hydrolysis of the disaccharide 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotriose most efficiently and was, therefore, classified as an endochitinase. The chitinase of S. albidoflavus was purified by applying the two-step procedure: fractionation with ammonium sulphate and chitin affinity chromatography. The molecular weight of the purified enzyme determined by SDS-PAGE was approximately 50 kDa. The enzyme was characterised as thermostable during 180 min of preincubation at the temperature of 35°C and 40°C. The activity of the enzyme was strongly inhibited in the presence of Hg²⁺ and Mn²⁺ ions, SDS but stabilized by Ca²⁺ and Mg²⁺ ions. Both purified and crude chitinases from S. albidoflavus inhibited the development of fungal phytopathogens. Purified chitinase inhibited the growth of Alternaria alternata, Fusarium culmorum, Fusarium oxysporum and Botrytis cinerea. Additionally, the crude chitinase inhibited the growth of Fusarium solani.     

Purification and characterization of a 54 kDa chitinase from Bombyx mori

The 54 kDa protein that was suggested to be processed from the 65 kDa and 88 kDa chitinases of Bombyx mori [Koga et al., Insect Biochem. Mol. Biol. 27, 757–767 (1997)] was purified and proved to be a third chitinase (EC 3.2.1.14). This chitinase was purified from the fifth larval instar of B. mori by chromatography on DEAE-Cellulofine A–500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650(M) columns. The apparent molecular mass was confirmed to be 54 kDa by SDS–PAGE. Its optimum pH was 6.0 toward a short substrate, N-acetylchitopentaose (GlcNAc₅), while in its reaction with a longer substrate, glycolchitin, the enzyme showed a wide pH-range between 4.0 and 10. Kinetic parameters for the chitinase could be obtained in the hydrolysis of glycolchitin but not in that of N-acetylchitooligosaccharides (GlcNAc_n, n=2–6) because of substrate inhibition. The chitinase hydrolyzed N-acetylchitooligosaccharides except for dimer as follows: trimer to monomer plus dimer, tetramer to two molecules of dimer, pentamer to dimer plus trimer, and hexamer to dimer plus tetramer as well as two molecules of trimer. These results suggest that the 54 kDa chitinase is an endo-type hydrolase and preferred the longer-chain N-acetylchitooligosaccharides. Moreover, the anomeric forms of N-acetylchitooligosaccharides were analyzed in the reaction with the 54-kDa chitinase. It was revealed that this enzyme cleaves the substrate to produce the β anomeric product. With respect to inhibition of the 54 kDa chitinase, it was specifically inhibited by allosamidin in a competitive way with K_i values depending on the pH of the reaction mixture (K_i=0.013−0.746 μM). Comparing the properties and kinetic behavior of this chitinase with those of the 88 and 65 kDa chitinases from B. mori, regarding the specific activity of the three enzymes, the 65-kDa chitinase was 2.15 and 2.8 times more active than the 88 and 54-kDa chitinases, respectively. However, in the overall reaction of glycolchitin (k_cat/K_m), the 88-kDa enzyme was 4 and 40 times more active than the 65-kDa and the 54-kDa enzymes, respectively. Concerning the affinity (1/K_m) to glycolchitin, the 88 kDa chitinase affinity (at pH 6.5) was 5.8 times higher than that of the 65 kDa chitinase (at pH 5.5) and 4.0 times higher than that of the 54 kDa chitinase (at pH 6.0). These kinetic results suggest that B. mori chitinases are processed during ecdysis from the larger chitinase to smaller ones that leads to changes in their kinetic properties such as K_m, k_cat and k_cat/K_m successively.     

Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.     

Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (M_r) of 102069 Da, similar in size to its M_r of about 100 000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3′-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.     

Purification, characterization, and molecular cloning of a chitinase from the seeds of Benincasa hispida

A chitinase was purified from the seeds of Benincasa hispida, a medicinal plant also called white gourd, and a member of the Cucurbitaceae family. Purification was done by using a procedure consisting of only two fractionation steps: an acid denaturation step followed by ion-exchange chromatography. The sequence of the N-terminal forty amino acid residues was analyzed and the sequence indicated that the enzyme is a class III chitinase. The enzyme, which is a basic chitinase, is one of at least five chitinases detected in the seed extract of B. hispida. Like other class III chitinases, this enzyme also has lysozyme activity. A genomic clone of the gene encoding the enzyme was isolated and sequenced. The gene has the potential to encode a protein of 301 amino acid residues. The deduced amino acid sequence of the protein, as expected from the N-terminal amino acid sequence, shares high degrees of similarity with other class III chitinases.     

Identification, characterization and functional analysis of a GH-18 chitinase from Streptomyces roseolus

A 40 kDa chitinase from Streptomyces roseolus DH was purified to homogeneity from culture medium. The N-terminal sequence was TPPPAKAVKLGYFTNWGVYG, which was highly homologous to the glycoside hydrolase (GH) 18 conserved domain of Streptomyces chitinases and included the two crucial Trp and Tyr sites. The purified enzyme showed maximal activity at 60 °C, pH 6.0 and exhibited good thermal and pH stabilities. The enzyme displayed strict substrate specificity on colloidal or glycol chitin, but not on chitosan derivatives. It was activated by Mg²⁺, Ba²⁺ and Ca²⁺, and inhibited by Cu²⁺, Co²⁺, Mn²⁺, whereas Zn²⁺ and ethylenediamine tetraacetic acid showed little inhibitory effects. Morphological changes observed by scanning electron microscopy revealed the occurrence of regular pores on the surface with the progress of enzymatic chitinolysis. Additionally, this GH-18 chitinase had a marked inhibitory effect on fungal hyphal extensions. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.     

Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco

Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.     

Isolation of a new fungi and wound-induced chitinase class in corms of Crocus sativus

In plants, various chitinases have been identified and categorized into several groups based on the analysis of their sequences and domains. We have isolated SafchiA, a novel class of chitinase from saffron (Crocus sativus L.). The cDNA encoding SafchiA is mainly expressed in roots and corms, and its expression is induced by elicitor treatment, methyl jasmonate, wounding, and by the fungi Fusarium oxysporum, Beauveria and Phoma sp., suggesting a defence role of the protein. Furthermore, in vitro assays with the recombinant native protein showed chitinolytic, and antifungal activity. The deduced protein shares high similarity with chitinases belonging to family 19 of glycosyl-hydrolases, although some changes in the enzyme active site are present. To explore the properties of SafchiA we have expressed recombinant SafchiA in Escherichia coli and generated four different mutants affected in residues involved in the catalytic activity. One glutamic acid essential for family 19 chitinases activity is not present in C. sativus chitinase suggesting that only one acidic residue is necessary for the enzyme activity, in a similar manner as family 18 glycosyl-hydrolases.     

Isolation and sequence of a novel amphibian pancreatic chitinase

An approximately 60-kDa protein with chitinase activity was purified from the pancreas of the toad Bufo japonicus. Its specific activity was 4.5 times higher than that of a commercial bacterial chitinase in fragmenting crab shell chitin, and its optimal pH was approximately 6.0. A cDNA clone encoding a protein consisting of 488 amino acid residues, including part of the peptide sequence determined from the isolated protein, was obtained from a toad pancreas cDNA library. The deduced amino acid sequence indicated that the protein contained regions with high homology to those present in chitinases from different species, with the amino acid residues for the chitinase activity and the chitin-binding ability being completely conserved. We designate the protein as toad pancreatic chitinase (tPCase). Northern blot analysis revealed the mRNA of this enzyme to be expressed exclusively in the pancreas. Toad PCase is the first amphibian chitinase to be identified as well as the first pancreatic chitinase identified in a vertebrate.