Chitin-degrading Activity Research Articles

Xanthomonas chitinilytica sp. nov., a novel chitinolytic bacterium isolated from a microbial fermentation bed material.

A Gram-stain-negative bacterium, H13-6T, was isolated from a microbial fermentation bed material collected from a pig farm located in Yan'an, Shaanxi, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain H13-6T was affiliated with the genus Xanthomonas and showed highest similarity to strain Xanthomonas maliensis M97T (98.38%), Xanthomonas prunicola CFBP 8353T (98.26%) and Xanthomonas oryzae ATCC 35933T (98.11%). The pairwise ortho Average Nucleotide Identity values and the digital DNA-DNA hybridization values between strain H13-6T and the other Xanthomonas species were all below their respective cut-offs. Two genes encoding for chitinase were found and the strain showed a strong chitin-degrading activity. The major fatty acids were Iso-C15:0 (55.9%), Antesio-C15:0 (7.4%) and Iso-C11:0 (5.5%) and the major polar lipids were diphosphatidylglycerol, phosphatidyglycerol and phosphatidylethanolamine. Based on the phenotypic properties and phylogenetic distinctiveness, Xanthomonas chitinilytica was proposed as a novel species of the genus Xanthomonas, with strain H13-6T (= CGMCC 1.61317T = NBRC 115641T) as type strain.

Multi-enzyme Machinery for Chitin Degradation in the Chitinolytic Bacterium Chitiniphilus shinanonensis SAY3T.

The chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T was examined to characterize its chitin-degrading enzymes in view of its potential to convert biomass chitin into useful saccharides. A survey of the whole-genome sequence revealed 49 putative genes encoding polypeptides that are thought to be related to chitin degradation. Based on an analysis of the relative quantity of each transcript and an assay for chitin-degrading activity of recombinant proteins, a chitin degradation system driven by 19 chitinolytic enzymes was proposed. These include sixteen endo-type chitinases, two N-acetylglucosaminidases, and one lipopolysaccharide monooxygenase that catalyzes the oxidative cleavage of glycosidic bonds. Among the 16 chitinases, ChiL was characterized by its remarkable transglycosylation activity. Of the two N-acetylglucosaminidases (ChiI and ChiT), ChiI was the major enzyme, corresponding to > 98% of the total cellular activity. Surprisingly, a chiI-disrupted mutant was still able to grow on medium with powdered chitin or GlcNAc dimer. However, its growth rate was slightly lower compared to that of the wild-type SAY3. This multi-enzyme machinery composed of various types of chitinolytic enzymes may support SAY3 to efficiently utilize native chitin as a carbon and energy source and may play a role in developing an enzymatic process to decompose and utilize abundant chitin at the industrial scale.

Chitin utilization by marine picocyanobacteria and the evolution of a planktonic lifestyle

Marine picocyanobacteria Prochlorococcus and Synechococcus, the most abundant photosynthetic cells in the oceans, are generally thought to have a primarily single-celled and free-living lifestyle. However, while studying the ability of picocyanobacteria to supplement photosynthetic carbon fixation with the use of exogenous organic carbon, we found the widespread occurrence of genes for breaking down chitin, an abundant source of organic carbon that exists primarily as particles. We show that cells that encode a chitin degradation pathway display chitin degradation activity, attach to chitin particles, and show enhanced growth under low light conditions when exposed to chitosan, a partially deacetylated soluble form of chitin. Marine chitin is largely derived from arthropods, which underwent major diversifications 520 to 535 Mya, close to when marine picocyanobacteria are inferred to have appeared in the ocean. Phylogenetic analyses confirm that the chitin utilization trait was acquired at the root of marine picocyanobacteria. Together this leads us to postulate that attachment to chitin particles allowed benthic cyanobacteria to emulate their mat-based lifestyle in the water column, initiating their expansion into the open ocean, seeding the rise of modern marine ecosystems. Subsequently, transitioning to a constitutive planktonic life without chitin associations led to cellular and genomic streamlining along a major early branch within Prochlorococcus. Our work highlights how the emergence of associations between organisms from different trophic levels, and their coevolution, creates opportunities for colonizing new environments. In this view, the rise of ecological complexity and the expansion of the biosphere are deeply intertwined processes.

Hinokitiol inhibits Aspergillus fumigatus by interfering with the cell membrane and cell wall.

Hinokitiol (β-thujaplicin) is an important component of the essential oil extracted from Chamaecyparis obtuse, which prevents the decay and decomposition of temple and shrine buildings in Japan. Hinokiol has been shown to have a detrimental effect on various fungi such as Candida albicans and saprophytic fungi. However how hinokitiol works against Aspergillus fumigatus (A. fumigatus) has not been claimed. This study aims to investigate the adverse effects of hinokitiol on the disruption of the cell wall and cell membrane of A. fumigatus and to explore possible potential mechanisms or pathways. According to our results, hinokitiol negatively altered mycelium morphology, growth density, and cell plasma composition content. When incubated with human corneal epithelial cells (HCECs), hinokitiol saw a safe effect with concentrations below 12 μg/ml. Hinokitiol was shown to increase the cell membrane's permeability by decreasing the cell membrane's ergosterol content. The integrity of the cell wall was disrupted, as well as a significant increase in chitin degradation and chitinase activity. As determined by RNA-seq results, subsequent analysis, and qRT-PCR, altered transcript levels of cell walls and cell membranes-related genes (such as eglC) illustrated how hinokitiol affected the genetic profile of A. fumigatus. With this study, we recommend hinokitiol as an effective anti-A. fumigatus agent by reducing the amounts of key components in the cell wall and membrane by preventing production and accelerating breakdown.

Irreversible evolutionary loss of chitin-degrading ability in the chitinase-like protein Ym1 under positive selection in rodents.

Ym1 (chitinase-like 3, Chil3) expressed in mice is a nonenzymatic chitinase-like protein, which shows 67% identity with mouse acidic chitinase (Chia). Similar to Chia, Ym1 is overexpressed in asthma and parasitic infections in mouse lungs. Due to the lack of chitin-degrading activity, the biomedical role of Ym1 under these pathophysiological conditions remains to be determined. In this study, we investigated what region and amino acid changes in Ym1 resulted in the loss of enzymatic activity. Replacing two amino acids at the catalytic motif to obtain a Chia-like sequence (N136D and Q140E; MT-Ym1) did not activate the protein. We conducted a comparative study of Ym1 and Chia. We found that three protein segments-(i) the catalytic motif residues, (ii) exons 6 and 7, and (iii) exon 10-are responsible for chitinase activity loss in Ym1. We show that replacing each of these three segments in Chia that are also involved in substrate recognition and binding by the Ym1 sequence can fully abolish the enzymatic activity. In addition, we show that there have been extensive gene duplication events at the Ym1 locus specific to the rodent lineages. Consistent with this result, Ym1 orthologs from the rodent genome were under positive selection when analyzed through the CODEML program. These data suggest that numerous amino acid substitutions in the regions involved in the chitin recognition, binding, and degradation ability of the ancestor Ym1 molecule lead to the irreversible inactivation of the protein.

Development of Fermented Shrimp Shell Product with Hypoglycemic and Hypolipidemic Effects on Diabetic Rats.

In 2020, approximately 9.3 billion tons of crustaceans were consumed, and 45–48% of shrimp shell (SS) by-products were discarded as waste. In this study, the SS of Litopenaeus vannamei was fermented by Lactobacillus plantarum LV33204, Stenotrophomonas maltophilia LV2122 (strong proteolytic activity), and Aeromonas dhakensis LV1111 (chitin-degrading activity), and the optimal fermentation conditions of liquid-fermented SS was established. Contents of total peptide, astaxanthin, and total phenolic content of the fermented SS were significantly higher than that of unfermented SS. In the presence of fermented SS, glucose uptake and insulin resistance of TNF-α-stimulated FL83B hepatocytes were markedly improved. Furthermore, daily oral supplement of fermented SS to streptozotocin (STZ)/nicotinamide (NA)-induced diabetic rats for 7 weeks significantly reduced plasma glucose and insulin resistance. Meanwhile, ingestion of fermented SS might enhance hepatic catabolism of glucose by increasing hexokinase and glucose-6-phosphate dehydrogenase activity and decreasing glucose-6-phosphatase activity. In addition, the fermented SS downregulated plasma total cholesterol (TG), triglycerides (TCs), low-density lipoprotein cholesterol (LDL-C), liver TG, and TC and lipid peroxidation levels in diabetic rats. In conclusion, a biorefinery process for waste SS was established through mixed strain fermentation. The in vitro and in vivo data reveal that the fermented SS is a promising functional food for the management of diabetic hyperglycemia and hyperlipidemia.

The Insect Pathogen Photorhabdus luminescens Protects Plants from Phytopathogenic Fusarium graminearum via Chitin Degradation.

Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable and biological alternatives are urgently needed. The insect-pathogenic bacterium Photorhabdus luminescens, already used in combination with entomopathogenic nematodes (EPNs) as a biocontrol agent, is characterized by two different phenotypic cell forms, called primary (1°) and secondary (2°). The 1° cells are symbiotic with EPNs and are used for biocontrol, and the 2° cells are unable to undergo symbiosis with EPNs, remain in the soil after insect infection, and specifically interact with plant roots. A previous RNA sequencing (RNAseq) analysis showed that genes encoding the exochitinase Chi2A and chitin binding protein (CBP) are highly upregulated in 2° cells exposed to plant root exudates. Here, we investigate Chi2A and CBP functions and demonstrate that both are necessary for P. luminescens 2° cells to inhibit the growth of the phytopathogenic fungus Fusarium graminearum. We provide evidence that Chi2A digests chitin and thereby inhibits fungal growth. Furthermore, we show that 2° cells specifically colonize fungal hyphae as one of the first mechanisms to protect plants from fungal phytopathogens. Finally, soil pot bioassays proved plant protection from F. graminearum by 2° cells, where Chi2A and CPB were essential for this process. This work gives molecular insights into the new applicability of P. luminescens as a plant-growth-promoting and plant-protecting organism in agriculture. IMPORTANCE The enteric enterobacterium Photorhabdus luminescens is already being used as a bioinsecticide since it is highly pathogenic toward a broad range of insects. However, the bacteria exist in two phenotypically different cell types, called 1° and 2° cells. Whereas only 1° cells are symbiotic with their nematode partner to infect insects, 2° cells were shown to remain in the soil after an insect infection cycle. It was demonstrated that 2° cells specifically interact with plant roots. Here, we show that the bacteria are beneficial for the plants by protecting them from phytopathogenic fungi. Specific colonization of the fungus mycelium as well as chitin-degrading activity mediated by the chitin binding protein (CBP) and the chitinase Chi2A are essential for this process. Our data give evidence for the novel future applicability of P. luminescens as a plant-growth-promoting organism and biopesticide.

Symbiotic chitin degradation by a novel anaerobic thermophilic bacterium Hydrogenispora sp. UUS1-1 and the bacterium Tepidanaerobacter sp. GT38

Chitin is the second most abundant organic compound in nature. Although mesophilic bacteria degrade insoluble chitin, there is a paucity of data describing degradation of insoluble chitin by anaerobic thermophilic bacteria. In this report, we screened cow manure compost for new chitin degradation systems, and identified a chitinolytic bacterial community (CBC) that showed high chitin degradation activity under thermophilic conditions, i.e., 1% (w/v) chitin powder degraded completely within 7 days at 60 °C. Metagenomic analysis revealed that the CBC was dominated by two bacterial genera from Hydrogenispora, an uncultured taxonomic group, and Tepidanaerobacter. Hydrogenispora were abundant in the early-to-mid stages of culturing with chitin, whereas the population of Tepidanaerobacter increased during the later stages of culturing. Strains UUS1-1 and GT38, which were isolated as pure cultures using the roll-tube method with colloidal chitin, N-acetyl-d-glucosamine, and glucose as carbon sources, were found to be closely related to H. ethanolica and T. acetatoxydans, respectively. Strain UUS1−1 readily degraded chitin and is the first anaerobic thermophilic chitinolytic bacterium reported, whereas strain GT38 showed no chitinolytic activity. Based on phylogenetic analysis, UUS1−1 and GT38 should be classified as novel genera and species. Zymogram analysis revealed that UUS1−1 produces at least two chitinases with molecular weights of 150 and 40 kDa. A coculture of UUS1−1 and GT38 degraded crystalline chitin faster with lower accumulation of lactate compared with UUS1−1 alone, indicating that the strains maintained a symbiotic association through assimilation of organic acids in chitin degradation and that strain GT38 consumed end-products to reduce end-product inhibition and enhance the degradation of crystalline chitin.

A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin

Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multicatalytic enzymes contain several glycoside hydrolase domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd1381 from the actinobacterium Jonesia denitrificans, that uniquely combines two different polysaccharide-degrading activities. We found that Jd1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans, degraded both α- and β-chitin. Both the chitinase and the LPMO activities of Jd1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd1381 was comparable with or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens Importantly, comparison of the chitinolytic efficiency of Jd1381 with the efficiencies of combinations of truncated variants-JdLPMO10 and JdCBM5-Chi18 or JdLPMO10-CBM5 and JdChi18-indicated that optimal Jd1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion.

Chitin degradation potential and whole-genome sequence of Streptomyces diastaticus strain CS1801

The aim of this study was to evaluate the chitin degradation potential and whole-genome sequence of Streptomyces diastaticus strain CS1801, which had been screened out in our previous work. The results of fermentation revealed that CS1801 can convert the chitin derived from crab shells, colloidal chitin and N-acetylglucosamine to chitooligosaccharide. Additional genome-wide analysis of CS1801 was also performed to explore the genomic basis for chitin degradation. The results showed that CS1801 possesses a chromosome with 5,611,479 bp (73% GC) and a plasmid with 1,388,284 bp (73% GC). The CS1801 genome consists of 7584 protein-coding genes, 90 tRNA and 21 rRNA operons. In addition, the results of genomic CAZyme analysis indicated that CS1801 comprises 103 glycoside hydrolase family genes, which could regulate the glycoside hydrolases that contribute to chitin degradation. The whole-genome information of CS1801 could highlight the mechanism underlying the chitin degradation activity of CS1801, strongly indicating that CS1801 is characterized by a substantial number of genes encoding chitinases and the complete metabolic pathway of chitin, conferring CS1801 with promising potential applicability in chitooligosaccharide production.

Complete Genome of the Chitin-Degrading Bacterium, Paenibacillus xylanilyticus W4

Chitinases possess an extraordinary ability to directly hydrolyze highly insoluble chitin polymers to low-molecular-weight chito-oligomers, which possess particular biological functions, such as elicitor action and antitumor activity. A novel strain, Paenibacillus xylanilyticus W4, which was isolated from soil, showed strong chitin degradation activity. Here, we first reported the complete genome information of P. xylanilyticus. Paenibacillus xylanilyticus W4 contains a 5,532,141 bp single circular chromosome with 47.33% GC content. The genome contains 5,996 genes, including 39 rRNA- and 109 tRNA-coding genes. Phylogenetic analysis and Genome-to-Genome Distance revealed its taxonomic characterization into a separate family. Six glycoside hydrolase 18 (GH18) and 2 GH23 enzymes involved in chitin degradation. Although many of the chitinases were conserved in Paenibacillus, several GH18 chitinases share high similarity with Bacillus circulans. The genome information provided here could benefit for understanding the chitin-degrading properties of P. xylanilyticus as well as its potential application in biotechnological and pharmaceutical fields.

The Development of Macrophomina phaseolina (Fungus) Resistant and Glufosinate (Herbicide) Tolerant Transgenic Jute.

The worldwide demand for natural bast fibers is met aptly by the long, golden and silky fibers of jute. This highest bast fiber producing crop is of great applicability and is extensively used in paper and textile industry. Macrophomina phaseolina (Tassi) Goid is a severely devastating necrotrophic fungal pathogen causing stem rot, root rot, and charcoal rot diseases in both the cultivated species of jute – Corchorus capsularis and Corchorus olitorius. Another major problem faced in jute cultivation is profuse weed infestation in the fields. Huge losses in quality fiber production is caused by this pathogenic fungi and cultivation cost increases as well due to weed management expenditure during cropping season. To solve these long persisting jute cultivation challenges, the chitinase (chi11) gene (to provide fungus resistance) and the bar gene (to provide herbicide tolerance) have been incorporated in C. capsularis JRC-321 via Agrobacterium transformation and analyzed up to T2 generation. Stable integration and expression of these two genes in the jute genome was confirmed upon extensive analyses. Transgenic plants showed higher chitinase expression and chitin degrading activity than non-transgenic control plants. Antifungal activity significantly increased in transgenic plants as confirmed by detached leaf and whole plant M. phaseolina bioassay. Herbicide tolerance was analyzed by growing transgenic plants in 10 mg/l glufosinate ammonium containing media and by spraying 0.25% (v/v) glufosinate herbicide Basta® on them. Assessment of residual phytotoxicity effects of Basta® on soil confirmed no negative impact on growth of indicator plants corn and cucumber. Transgenic jute plants were at par with non-transgenic (control) jute plants in all phenotypic aspects. Non-transgenic (control) jute plants suffered significant losses in fiber yield and quality due to M. phaseolina infection whereas the transgenic lines maintained the quality of fiber even after the infection.

Biological Potential of Chitinolytic Marine Bacteria.

Chitinolytic microorganisms secrete a range of chitin modifying enzymes, which can be exploited for production of chitin derived products or as fungal or pest control agents. Here, we explored the potential of 11 marine bacteria (Pseudoalteromonadaceae, Vibrionaceae) for chitin degradation using in silico and phenotypic assays. Of 10 chitinolytic strains, three strains, Photobacterium galatheae S2753, Pseudoalteromonas piscicida S2040 and S2724, produced large clearing zones on chitin plates. All strains were antifungal, but against different fungal targets. One strain, Pseudoalteromonas piscicida S2040, had a pronounced antifungal activity against all seven fungal strains. There was no correlation between the number of chitin modifying enzymes as found by genome mining and the chitin degrading activity as measured by size of clearing zones on chitin agar. Based on in silico and in vitro analyses, we cloned and expressed two ChiA-like chitinases from the two most potent candidates to exemplify the industrial potential.

Heterologous expression of the antifungal β-chitin binding protein CBP24 from bacillus thuringiensis and its synergistic action with bacterial chitinases.

The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to β-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of β-N-acetylglucosaminidase from Thermotoga maritima encoded by the Tm0809 gene.

β-N-acetylglucosaminidase (NagA) protein hs a chitin-degrading activity and chitin is one of the most abundant polymers in nature. NagA contains a family 3 glycoside (GH3)-type N-terminal domain and a unique C-terminal domain. The structurally uncharacterized C-terminal domain of NagA may be involved in substrate specificity. To provide a structural basis for the substrate specificity of NagA, structural analysis of NagA from Thermotoga maritima encoded by the Tm0809 gene was initiated. NagA from T. maritima has been overexpressed in Escherichia coli and crystallized at 296 K using ammonium sulfate as a precipitant. Crystals of T. maritima NagA diffracted to 3.80 Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 231.15, b = 133.62, c = 140.88 Å, β = 89.97°. The crystallization of selenomethionyl-substituted protein is in progress to solve the crystal structure of T. maritima NagA.

Guava (Psidium guajava L.) rhizosphere Glomus mosseae spores harbor actinomycetes with growth promoting and antifungal attributes

A total of eight actinomycetes were isolated from Glomus mosseae spores predominant in the rhizosphere of guava (Psidium guajava L. cv Arka Mridula) orchards in Karnataka State, southern India. Five isolates were identified using RNA polymerase β-subunit gene primers as Streptomyces fradiae, S. avermitilis, S. cinnamonensis, Leifsonia poae and Streptomyces canus. All the isolates were tested for beneficial attributes like growth promotion, production of growth hormones like IAA and GA3, phosphate solubilization, siderophore production and antagonistic activity against pathogens including chitinase activity. S. canus recorded increase in guava height and plant dry matter (25.58%, 30.89%) over control followed by S. avermitilis (20.75%, 30%) and S. cinnamonensis (14.05%, 22%) and L. poae (11.09%, 20%) in 10 month-old P. guajava seedlings grown with actinomycetes cultures mixed in coco peat growth medium at 10−8cfug−1 substrate. All the isolates produced growth hormones at varying levels. S. canus produced 10.1μgml−1 of IAA and 12.0μgml−1 of GA3, the highest, compared to others. S. avermitilis, S. canus and L. poae exhibited higher activity of phosphate solubilization, siderophore production and chitin degrading activity out of the five isolates. All the isolates tested in dual culture possessed strong antifungal activity against Fusarium oxysporum and Alternaria solani. The findings from this current study clearly indicated the possibilities of using mycorrhizae associated actinomycetes as bioinoculant for growth promotion, nutrient mobilization and biocontrol agent in guava seedling production.

High expression levels of chitinase genes inStreptomyces coelicolorA3(2) grown in soil

Although Streptomyces species are major chitin-degraders in soil ecosystems, the expression of the diverse chitinase genes within Streptomyces coelicolor grown in soil has not been assessed. As a first step, the induction pattern of nine chitinase genes in S. coelicolor growing in autoclaved soil was compared with those in liquid cultures. The relative expression levels of nine chitinase genes were measured using real-time reverse transcription PCR. The expression of all chitinase genes was induced by chitin in both autoclaved soil and liquid cultures, but to different levels. The expression levels of five chitinase genes in autoclaved soil were significantly higher than those in the liquid cultures. In particular, a putative chitinase gene, chitinase H, showed the highest induction in autoclaved soil. The same induction pattern was confirmed in nonautoclaved soil, indicating that soil contains some factors affecting the expression of chitinase genes. The chiH gene product, ChiH, cloned in Streptomycetes lividans was secreted and exhibited chitin degradation activity that was stable within a wide range of acidic pHs. The disruption of dasR, a transcriptional regulator for the uptake of N-acetylglucosamine, abolished the expression of chiH, demonstrating that DasR is required for the regulation of ChiH expression.

Purification, Cloning, and Sequence Analysis of β-N-Acetylglucosaminidase from the Chitinolytic BacteriumAeromonas hydrophilaStrain SUWA-9

A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A beta-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-beta-D-glucosaminide and pNP-N-acetyl-beta-D-galactosaminide. A gene coding for the purified beta-N-acetylglucosaminidase was isolated. The ORF identified is 2661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial beta-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.

HETEROLOGOUS EXPRESSION OF A CHITINASE GENE FROM AEROMONAS CAVIAEIN PSEUDOMONAS FLUORESCENS

A transcriptional fusion for an Aeromonas caviae chitinase gene was constructed under the control of a constitutive promoter of the kanaraycin resistance gene (PKmR ). The construct was inserted into a medium copy number broad host range plasmid vector to yield recombinant plasmid pAM340, which harbored transcriptional fusion PKmR - chi. Another transcriptional fusion, Ptac-chi, in a recombinant plasmid pAM630, was conducted as comparison. Triparental mating of E. coli carrying the recombinant plasmids with Pseudomotws fluorescens 5100, a phyllosphere bacterium, was performed. Pseudomonas fluorescens 5100 exconjugants were examined for constitutive expression of chitinase employing a spectrophotometric assay; they showed stronger chitin degradation activity than Escherichia coli transformants. Using a fungal antagonism plate assay, this chitinolytic P. fluorescens, however, could not inhibit selected phytopathogenic fungi. Keywords: Aeromonas caviae/ chitinase gene/transcriptional fusion/PKm'V Vtac-chilPseudomonas fluorescens

Purification of chitinolytic protein from Rehmannia glutinosa showing N-terminal amino acid sequence similarity to thaumatin-like proteins.

We have purified a 21-kDa protein, designated as P1, from Rehmannia glutinosa to homogeneity by ammonium sulfate precipitation, anion exchange chromatography, hydrophobic interaction chromatography, and preparative native PAGE. The purified P1 had chitin degradation activity. The N-terminal amino acid sequence of P1 indicated that it is very similar to those of thaumatin and other reported thaumatin-like proteins.