Extracellular Chitinase Activity Research Articles

Enhancement of resistance against fungal pathogens in peanut (Arachis hypogaea L.) cultivar L14 by heterologous expression of gene encoding chitinase 42 kDa from Trichoderma asperellum SH16

This study aims to extracellularly express the 42 kDa chitinase from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of a tissue-specific Asy promoter and the driving of the signal peptide of the rice amylase 3D gene. The 42 kDa chitinase genes were employed in this study, which included one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42–1 and syncodChi42–2) optimized for codon usage for plant expression. From 102 42 kDa chitinase transgenic peanut lines, seven elite lines were discovered, among them two for syncodChi42–1, three for syncodChi42–2, and two for Chi42. The study also revealed chitinase from two synthetic genes had higher expression levels than chitinase from the wild-type gene, with the highest specific activities of approximately 901 U/mg and 1124 U/mg, respectively, compared to 739 U/mg. All seven elite transgenic peanut lines exhibited strong extracellular chitinase activity, and some of them were able to fight the soil-borne pathogenic fungus Sclerotium rolfsii in both in vitro and in vivo assays. The significant chitinase activity of seven elite transgenic peanut lines, particularly five lines carrying one of two synthetic genes, suggests that transgenic crops might be used to prevent phytopathogenic fungi.

The transcription factor Ron1 is required for chitin metabolism, asexual development and pathogenicity in Beauveria bassiana, an entomopathogenic fungus

Ndt80-like transcription factor Ron1 is best known for its essential role in the regulation of N-acetylglucosamine (GlcNAc) catabolism. Ron1 was again found to be essential for sensing GlcNAc in Beauveria bassiana. Importantly, our study revealed that Ron1 is involved in the metabolic processes of chitin and asexual development. To further investigate the novel functions of Ron1 in B. bassiana, extracellular chitinase activity in the ΔRon1 mutant was found to decrease by 84.73% compared with wild type. The deletion of Ron1 made it difficult for the fungus to accumulate intracellular GlcNAc. Furthermore, transcriptomic analysis revealed that Ron1 exerted a significant effect on global transcription and positively regulated genes encoding chitin metabolism in respond to chitin nutrition. Yeast one-hybrid assay confirmed that Ron1 could bind to specific cis-acting elements in the promoters of chitinase and hexokinase. In addition, ΔRon1 displayed an impaired chitin component of the cell wall, with a chitin synthetase (ChsVII) predicted to function downstream of Ron1. Finally, the virulence of ΔRon1 mutant was significantly reduced in the Galleria mellonella insect model through cuticle infection or cuticle bypassing infection. These data functionally characterize Ron1 in B. bassiana and expand our understanding of how the transcription factor Ron1 works in pathogens.

Haemagglutinin And Chitinase Activities Of Virulent Aeromonas Hydrophila Islolated From Cyprinus Carpio

In the present study the Hemeagglutination and Chitinolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila were examined. The present study showed infectivity experiments, incomplete creation of extracellular products and chitinase activity of the A. hydrophila. In haemagglutination assay in the present experiment, bacterial agglutination takes place in 1:1, 1:2, 1:4, 1:8,1:16,1:32 dilutions in the E.S-2 treated O blood groups and in 1:1,1:4 where as the ES -1and ES-3 failed to agglutinate. Generally, whole cells showed a wider range of enzymic activities than ECP. The results showed that extra cellular product chitin could be a promising source for pathogenic factor in microorganisms.

Haemagglutinin And Chitinase Activities Of Virulent Aeromonas Hydrophila Islolated From Cyprinus Carpio

In the present study the Hemeagglutination and Chitinolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila were examined. The present study showed infectivity experiments, incomplete creation of extracellular products and chitinase activity of the A. hydrophila. In haemagglutination assay in the present experiment, bacterial agglutination takes place in 1:1, 1:2, 1:4, 1:8,1:16,1:32 dilutions in the E.S-2 treated O blood groups and in 1:1,1:4 where as the ES -1and ES-3 failed to agglutinate. Generally, whole cells showed a wider range of enzymic activities than ECP. The results showed that extra cellular product chitin could be a promising source for pathogenic factor in microorganisms.

Diversity of culturable bacteria endowed with antifungal metabolites biosynthetic characteristics associated with tea rhizosphere soil of Assam, India

BackgroundRhizosphere soil is a crucial niche for the diverse beneficial microbial communities in plant-microbe interactions. This study explores the antagonistic potential and diversity of the rhizosphere soil bacteria from commercial tea estates of Assam, India which comes under the Indo-Burma mega-biodiversity hotspot. Rhizosphere soil samples were collected from six different tea estates to isolate the bacteria. The bacterial isolates were subjected to evaluate for the antagonistic activity against fungal pathogens. The potential isolates were investigated for chitinase production and the presence of chitinase gene. The bacterial genetic diversity was studied by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and BOX-PCR fingerprinting.ResultsA total of 217 rhizobacteria were isolated from tea rhizosphere soil, out of which 50 isolates exhibited the potential antagonistic activity against fungal pathogens. Among them, 12 isolates showed extracellular chitinase activity and the presence of chitinase genes. The chitinase genes were sequenced and the analysis of the sequences was performed by using PDB protein databank at the amino acid level. It showed the presence of ChiA and ChiA74 gene in the 6 most potent isolates which are involved in the hydrolysis of chitin. These isolates also exhibited antagonistic activity against all tested fungal pathogens. The diversity of 50 antagonistic bacterial isolates were analyzed through ARDRA and BOX-PCR fingerprinting. Diversity analysis and molecular identification of the rhizosphere isolates revealed that these antagonistic isolates predominantly belonged to the genus Bacillus followed by Enterobacter, Serratia, Lysinibacillus, Pseudomonas, and Burkholderia.ConclusionThe present study establishes that rhizobacteria isolated from the poorly explored tea rhizosphere soil could be a rich reservoir for the investigation of potential antagonistic bacterial candidates for sustainable agricultural and industrial applications.

Colonization of Beauveria bassiana 08F04 in root-zone soil and its biocontrol of cereal cyst nematode (Heterodera filipjevi).

Cereal cyst nematodes cause serious yield losses of wheat in Hunaghuai winter wheat growing region in China. Beauveria bassiana 08F04 isolated from the surface of cysts is a promising biological control agent for cereal cyst nematodes. As the colonization capacity is a crucial criteria to assess biocontrol effectiveness for a microbial agent candidate, we aimed to label B. bassiana 08F04 for efficient monitoring of colonization in the soil. The binary pCAM-gfp plasmid containing sgfp and hph was integrated into B. bassiana 08F04 using the Agrobacterium tumefaciens-mediated transformation. The transformation caused a significant change in mycelial and conidial yields, and in extracellular chitinase activity in some transformants. The cultural filtrates of some transformants also decreased acetylcholinesterase activity and the survival of Heterodera filipjevi second-stage juveniles relative to the wild-type strain. One transformant (G10) had a growth rate and biocontrol efficacy similar to the wild-type strain, so it was used for a pilot study of B. bassiana colonization conducted over 13 weeks. Real-time PCR results and CFU counts revealed that the population of G10 increased quickly over the first 3 weeks, then decreased slowly over the following 4 weeks before stabilizing. In addition, the application of wild-type B. bassiana 08F04 and transformant G10 significantly reduced the number of H. filipjevi females in roots by 64.4% and 60.2%, respectively. The results of this study have practical applications for ecological, biological and functional studies of B. bassiana 08F04 and for bionematicide registration.

Overexpression of Chitinase A Gene from Serratia marcescens in Bacillus subtilis and Characterization of Enhanced Chitinolytic Activity

Abstract Chitinase enzymes possess various usages in agriculture, biotechnology and medicine due to their chitin degrading property. Thus, efficient production of chitinase enzymes with desired properties has importance for its use. In this study, chitinase A (chiA) gene from Serratia marcescens Bn10 was cloned and heterologously overexpressed using pHT43 vector in Bacillus subtilis 168. The recombinant chitinase was characterized in terms of temperature, pH, and various effectors. The extracellular chitinase activity in recombinant B. subtilis was found 2.15-fold higher than the parental strain after 2 h of IPTG induction. Optimum temperature and pH for the extracellular chitinase activity in the recombinant B. subtilis were determined as 60 oC and pH 9.0, respectively. NaCl, Ca2+, Mn2+, Cu2+, Zn2+, sodium dodecyl sulfate (SDS), Tween-20, and ethanol increased the chitinase activity whereas Mg2+ caused an inhibition. The most notable increment on the chitinase activity was provided by Zn2+ (3.2 folds) and then by SDS (2.9 folds). The chitinase, overproduced by the recombinant B. subtilis 168 heterologously expressing chiA, was determined to have optimum activity at high temperature and alkaline conditions as well as various effectors increase its activity. The extracellular chitinase of recombinant B. subtilis might be a promising source for agricultural, biotechnological and medical applications.

Characterisation and antifungal activity of extracellular chitinase from a biocontrol fungus, Trichoderma asperellum PQ34

ABSTRACTTrichoderma species were known as biological control agents against phytopathogenic fungi because they produce a variety of chitinases. Chitinases are hydrolytic enzymes that break down glycosidic bonds in chitin, a major component of the cell walls of fungi. The present study shows that extracellular chitinase activity reached a maximum value of approximately 22 U/mL after 96 h of T. asperellum PQ34 strain culture. The optimal temperature and pH of enzyme are 40°C and 7, respectively, whereas the thermal and pH stability range from 25°C to 50°C and 4 to 10, respectively. Chitinase at 60 U/mL inhibited nearly completely in vitro growth of Colletotrichum sp. (about 95%) and Sclerotium rolfsii (about 97%). In peanut plants, 20 U/mL of chitinase significantly reduced the incidence of S. rolfsii infection compared to controls. The fungal infection incidence of seeds before germination and 30 days after germination was only 2.22% and 2.38%, while the control was 13.33% and 17.95%. Besides, chitinase from T. asperellum PQ34 can also prevent anthracnose that is caused by Colletotrichum sp. on both mango and chilli fruits up to 72 h after enzyme pre-treatment at 40 U/mL. In mango and chilli fruits infected with anthracnose, 40 U/mL dose of chitinase inhibited the growth of fungi after 96 h of treatment, the diameter of lesion was only 0.88 cm for mango and 1.45 cm for chilli, while the control was 1.67 cm and 2.85 cm, respectively.

Phylogeny, chitinase activity, and pathogenicity of Beauveria, Metarhizium and Lecanicillium species against cowpea aphid, Aphis craccivora Koch

Cowpea aphid, Aphis craccivora Koch is a serious insect pest of cowpea distributed in almost all states of India and its management is essential to increase the quality and quantity of cowpea yield. This study sought to characterize the indigenous isolates of fungal entomopathogens, Beauveria bassiana, Metarhizium pingshaense, M. guizhouense, and Lecanicillium muscarium, using molecular phylogeny and evaluate their pathogenicity against A. craccivora under greenhouse conditions and compare their extracellular chitinase activity. Thirty isolates of fungal entomopathogens were collected from semi-natural and agricultural habitats. Molecular phylogeny of these isolates using partial nuclear sequences, Bloc and MzIGS3, and mitochondrial sequence, nad1, resolved four species. Screening of the isolates against A. craccivora using a single concentration of 1 × 106 conidia/ml revealed that all isolates were pathogenic, with mortality of aphids ranging from 16.67 to 100%. Bioassays of the highly pathogenic isolates of B. bassiana, M. pingshaense, and L. muscarium against A. craccivora revealed low LC50 value of L. muscarium isolate GKVK 03_11 (1.4 × 105 conidia/ml) under greenhouse conditions. Comparison of the extracellular chitinase activity of these isolates detected high chitinase activity of GKVK 03_11 isolate (0.621 U/ml). Study results suggest the biocontrol potential of indigenous L. muscarium isolate for the management of A. craccivora and the possible role of chitinase enzyme in the virulence of fungal entomopathogens.

Genetic diversity of Beauveria bassiana in semi natural and agricultural habitats and its biocontrol potential against cowpea aphid, Aphis craccivora Koch.

Information on the biology and ecology of Beauveria bassiana in different habitats could provide essential knowledge in their development as biocontrol agents of insect pests. In this study, phylogenetic and genotypic information was used to evaluate the genetic diversity of B. bassiana within semi natural and agricultural habitats in Karnataka State of South India and assessed their extracellular chitinase activity and pathogenicity against cowpea aphid, Aphis craccivora. Multilocus phylogeny and microsatellite genotyping of B. bassiana conjointly resolved three phylogenetic species, Bb_1, Bb_2, and Bb_3, in semi natural and agricultural habitats. None of the three phylogenetic species of B. bassiana were associated with crop plants in agroecosystem or insect hosts in semi natural habitat. All the three phylogenetic species were detected with four genotypes each. All isolates of B. bassiana were pathogenic to A. craccivora in greenhouse bioassays. Isolate GKVK 01_13 caused a significantly high mortality of aphids and detected with an increased level of chitinase activity. The study results suggest that application of indigenous virulent strain of B. bassiana could provide effective control of native insect pest A. craccivora.

Assessment of bioactivity of endophytic actinomycetes from some medicinal plants

Out of 70 isolates of actinomycetes obtained from medicinal plants, 21 displayed antagonistic activity against Alternaria alternata, Fusarium oxysporum and Rhizoctonia solani. All the isolates were screened for hydrolytic enzymes production. Among these, 47 isolates produced amylase, 25 protease and 16 were having ability to produce chitinase. The extracellular chitinase activity of two selected isolates AR3 and O9 was evaluated (0.083 and 0.080 U/ml) with 0.6% colloidal chitin, which was found better as compared to 1% colloidal chitin. Scanning electron microscopy revealed rupture of F. oxysporum mycelial cell wall at the area of interaction with O9. The endophytic actinomycete isolate O9 was identified as Streptomyces rochei strain KMB-1 by 16S rDNA sequencing. This isolate was found to be promising in terms of seed germination and Fusarium wilt control in muskmelon under green house conditions.

Direct Regulation of Extracellular Chitinase Production by the Transcription Factor LeClp in Lysobacter enzymogenes OH11

Lysobacter enzymogenes is a gram-negative bacterial biological control agent that produces abundant extracellular enzymes capable of degrading the cell walls of fungal pathogens. In strain OH11, an isolate from China, the global regulator LeClp controls the production of extracellular chitinase by regulating the transcription of the chitinase-encoding gene chiA. Using a combination of bioinformatic, genetic, and biochemical methods, we show that LeClp regulates chiA transcription by directly binding to the chiA promoter region. Although LeClp appears to be important in this role, it is not the sole regulator of chiA transcription. Furthermore, the sequence analysis of putative LeClp binding sites indicated that the LeClp homolog could be involved in the regulation of extracellular chitinase production in diverse Lysobacter spp. by a mechanism similar to that in L. enzymogenes. Our findings present new insights into the molecular mechanism of LeClp in controlling extracellular chitinase activity, providing a fundamental road to elucidate how LeClp regulates the production of other extracellular lytic enzymes in L. enzymogenes.

Hfq regulates antibacterial antibiotic biosynthesis and extracellular lytic‐enzyme production inLysobacter enzymogenes OH11

Lysobacter enzymogenes is an important biocontrol agent with the ability to produce a variety of lytic enzymes and novel antibiotics. Little is known about their regulatory mechanisms. Understanding these will be helpful for improving biocontrol of crop diseases and potential medical application. In the present study, we generated an hfq (encoding a putative ribonucleic acid chaperone) deletion mutant, and then utilized a new genomic marker-free method to construct an hfq-complemented strain. We showed for the first time that Hfq played a pleiotropic role in regulating the antibacterial antibiotic biosynthesis and extracellular lytic enzyme activity in L. enzymogenes. Mutation of hfq significantly increased the yield of WAP-8294A2 (an antibacterial antibiotic) as well as the transcription of its key biosynthetic gene, waps1. However, inactivation of hfq almost abolished the extracellular chitinase activity and remarkably decreased the activity of both extracellular protease and cellulase in L. enzymogenes. We further showed that the regulation of hfq in extracellular chitinase production was in part through the impairment of the secretion of chitinase A. Collectively, our results reveal the regulatory roles of hfq in antibiotic metabolite and extracellular lytic enzymes in the underexplored genus of Lysobacter.

황토로부터 분리한 Bacillus licheniformis의 항진균 chitinase 생산과 효소 특성

In this study, we isolated two novel chitinase producing bacterial strains from yellow loess samples collected from Jullanamdo province. The chitinase producing bacteria were isolated based on the zone size of clearance in the chitin agar plates. Both of them were gram positive, rod (<TEX>$2{\sim}3{\times}0.3{\sim}0.4{\mu}m$</TEX>), spore-forming, and motility positive. They were facultative anaerobic, catalase positive and hydrolyzed starch, gelatin, and casein. From the 16s rRNA gene sequence analysis, the isolates were labeled as Bacillus licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02. The isolates showed higher extracellular chitinase activities than B. licheniformis ATCC 14580 as a control. The optimum temperature and pH for chitinase production were <TEX>$40^{\circ}C$</TEX> and pH 7.0, respectively. Response Surface Methodology (RSM) was used to optimize the culture medium for efficient production of the chitinase. Under this optimal condition, 1.5 times higher chitinase activity of B. licheniformis KYLS-CU02 was obtained. Extracellular chitinases of the two isolates were purified through ammonium sulfate precipitation and anion-exchange DEAE-cellulose column chromatography. The specific activities of purified chitinase from B. licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02 were 7.65 and 5.21 U/mg protein, respectively. The molecular weights of the two purified chitinases were 59 kDa. Further, the purified chitinase of B. licheniformis KYLS-CU01 showed high antifungal activity against Fusarium sp.. In conclusion, these two bacterial isolates can be used as a biopesticide to control pathogenic fungi.

The parasitic and lethal effects of Trichoderma longibrachiatum against Heterodera avenae

Heterodera avenae is a devastating plant pathogen that causes significant yield losses in many crops, but there is a lack of scientific information whether this pathogen can be controlled effectively using biocontrol agents. Here we determined the parasitic and lethal effects of Trichoderma longibrachiatum against H. avenae and the possible mechanism involved in this action. Both in vitro and greenhouse experiments were conducted. In vitro, T. longibrachiatum at the concentrations of 1.5×104 to 1.5×108spores per ml had a strong parasitic and lethal effect on the cysts of H. avenae, with the concentration of 1.5×108spores per ml having >90% parasitism 18days after treatments. In greenhouse, T. longibrachiatum inoculation decreased H. avenae infection in wheat (Triticum aestivum) significantly. Observations with microscopes revealed that after mutual recognition with cysts, the spore of T. longibrachiatum germinated with a large number of hyphae, and reproduced rapidly on the surface of cysts. Meanwhile, the cysts surface became uneven, with some cysts producing vacuoles, and the others splitting. Finally the cysts were dissolved by the metabolite of T. longibrachiatum. Chitinase activity increased in the culture filtrates of T. longibrachiatum and reached the maximum 4days after inoculation in the medium supplemented with colloidal chitin (1.02U/minperml) and nematode cysts (0.78U/minperml). The parasitism and inhibition of cysts through the increased extracellular chitinase activity serves as the main mechanism with which T. longibrachiatum against H. avenae. In conclusion, T. longibrachiatum has a great potential to be used as a biocontrol agent against H. avenae.

Enzymatic and genetic characterization of the DasD protein possessingN-acetyl-β-d-glucosaminidase activity inStreptomyces coelicolorA3(2)

The dasD gene is located just downstream of the dasABC gene cluster, encoding components of an ABC transporter for uptake of a chitin-degradation product N,N'-diacetylchitobiose [(GlcNAc)(2) ] in Streptomyces coelicolor A3(2). To clarify the roles of the DasD protein in the degradation and assimilation of chitin, we obtained and characterized a recombinant DasD protein and a dasD-null mutant of S.coelicolor A3(2). The recombinant DasD protein produced in Escherichia coli showed N-acetyl-β-d-glucosaminidase (GlcNAcase) activity and its optimum temperature and pH were 40°C and 7, respectively. dasD transcription was strongly induced in the presence of chitin, weakly by chitosan, but not by cellulose or xylan in S.coelicolor A3(2). Immuno-blot analysis demonstrated that DasD is a cytoplasmic protein. The dasD-null mutant exhibited cellular GlcNAcase activity which was comparable with that of the parent strain M145. DasD, thus, did not seem to be a major GlcNAcase. Induced extracellular chitinase activity in the dasD-null mutant was, interestingly, higher than M145, in the presence of colloidal chitin or (GlcNAc)(2) . In contrast to M145, (GlcNAc)(2) temporally accumulated in the culture supernatant of the dasD-null mutant in the presence of colloidal chitin.

Insight Into Genes Involved in the Production of Extracellular Chitinase in a Biocontrol Bacterium Lysobacter enzymogenes C-3

The chitinase producing Lysobacter enzymogenes C-3 has previously been shown to suppress plant pathogens in vitro and in the field, but little is known of the regulation of chitinase production, or its role in antimicrobial activity and biocontrol. In this study, we isolated and characterized chitinase-defective mutants by screening the transposon mutants of L. enzymogenes C-3. These mutations disrupted genes involved in diverse functions: glucose-galactose transpoter (gluP), disulfide bond formation protein B (dsbB), Clp protease (clp), and polyamine synthase (speD). The chitinase production of the SpeD mutant was restored by the addition of exogenous spermidine or spermine to the bacterial cultures. The speD and clp mutants lost in vitro antifungal activities against plant fungal pathogens. However, the gluP and dsbB mutants showed similar antifungal activities to that of the wild-type. The growth of the mutants in nutrient rich conditions containing chitin was similar with that of the wild-type. However, growth of the speD and gluP mutants was defective in chitin minimal medium, but was observed no growth retardation in the clp and dsbB mutant on chitin minimal medium. In this study, we identified the four genes might be involved and play different role in the production of extracellular chitinase and antifungal activity in L. enzymogenes C-3.

Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, Mamestra brassicae.

Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinaseencoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21-392), linker region (AA 393-498), and C-terminal chitin-binding domain (AA 499-562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels.

Chitinase from Bacillus licheniformis DSM13: Expression in Lactobacillus plantarum WCFS1 and biochemical characterisation

The gene chi, coding for a GH18 chitinase from the Gram-positive bacterium Bacillus licheniformis DSM13 (ATCC 14580), was cloned into the inducible lactobacillal expression vectors pSIP403 and pSIP409, derived from the sakacin-P operon of Lactobacillus sakei, and expressed in the host strain Lactobacillus plantarum WCFS1. Both the complete chi gene including the original bacillal signal sequence as well as the mature chi gene were compared, however, no extracellular chitinase activity was detected with any of the constructs. The chitinase gene was expressed intracellularly as an active enzyme with these different systems, at levels of approximately 5 mg of recombinant protein per litre of cultivation medium. Results obtained for the two different expression vectors that only differ in the promoter sequence were well comparable. To further verify the suitability of this expression system, recombinant, His-tagged chitinase Chi was purified from cell extracts of L. plantarum and characterised. The monomeric 65-kDa enzyme can degrade both chitin and chitosan, and shows properties that are very similar to those reported for the native chitinase purified from other B. licheniformis isolates. It shows good thermostability (half lives of stability of 20 and 8.4 days at 37 and 50 °C, respectively), and good stability in the pH range of 5–10. The results presented lead the way to overproduction of chitinase in a food-grade system, which is of interest for the food and feed industry.

Functional expression and structural characterization of ORF cDNA encoding chitinase of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)

Chitin is an important component of the exoskeleton and peritrophic matrix in insects. Its bio-degradation is initiated by the endo-splitting chitinase. We cloned an ORF cDNA encoding chitinase from the last instar larva of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), into E. coli to confirm its functionality. Its amino acid sequence was compared with previously described lepidopteran chitinases. S. exigua chitinase expression enhanced cell growth approx. 1.5 fold in transformed E. coli than in the wild strain in a 1% colloidal chitin-containing medium with insufficient regular nutrients. Compared with the wild strain, the two intracellular chitin degradation derivatives, glucosamine and N-acetylglucosamine, increased approx. 5.8 and 1.5 fold, respectively, and extracellular chitinase activity in the transformed strain was about 1.6 fold higher. The ORF of S. exigua chitinase-encoding cDNA including stop codon was composed of 1674 bp nucleotides and the calculated molecular weight of the deduced 557 amino acid residues was about 62.6 kDa. The ORF consisted of an N-terminal leading signal peptide (AA 1–20), a catalytic domain (AA 21–392), a linker region (AA 393–493), and a C-terminal chitin-binding domain (AA 494–557) showing a typical molting fluid chitinase structure. Phylogenetic analysis determined that all 5 noctuid chitinases were grouped together, while two bombycid enzymes and one tortricid enzyme mapped together in one lineage. In the noctuid group, the sub-lineages reflected their taxonomic relationships at the Genus level.