Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the causal agent of the witches' broom disease of cacao. A multivariate statistical approach was employed to evaluate the effect of several variables, including carbon and nitrogen sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities. Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by glucose. Chitinase secretion was increased by yeast extract alone or in combination with other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin. The best cultivation times for non-secreted and secreted chitinase activities were 30 and 20 d, respectively. However, chitinase activity was always higher in the mycelium than in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely, higher mycelial growth was observed when the activity of the non-secreted chitinase was at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase activity by chitin decreased the mycelium growth. These results suggest that the culture medium, by the induction or repression of chitinases, affected the hyphal growth. Thus, as an essential component of M. perniciosa growth, chitinases may be a potential target for strategies to control disease.     

Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles

A serine protease and an enzyme preparation consisting of six chitinases, previously semi-purified from a liquid culture of Paecilomyces lilacinus strain 251, were applied to Meloidogyne javanica eggs to study the effect of the enzymes on eggshell structures. Transmission electron microscopic studies revealed that the protease and chitinases drastically altered the eggshell structures when applied individually or in combination. In the protease-treated eggs, the lipid layer disappeared and the chitin layer was thinner than in the control. The eggs treated with chitinases displayed large vacuoles in the chitin layer, and the vitelline layer was split and had lost its integrity. The major changes in the eggshell structures occurred by the combined effect of P. lilacinus protease and chitinases. The lipid layer was destroyed; the chitin layer hydrolyzed and the vitelline layer had lost integrity. The effect of P. lilacinus protease and chitinase enzymes on the hatching of M. javanica juveniles was also compared with a commercially available bacterial chitinase. The P. lilacinus protease and chitinase enzymes, either individually or in combination, reduced hatching of M. javanica juveniles whereas a commercial bacterial chitinase had an enhancing effect. Some juveniles hatched when the eggs were exposed to a fungal protease and chitinase mixture. We also established that P. lilacinus chitinases retained their activity in the presence of endogenous protease activity.     

Production of chitinolytic enzymes from a novel species ofAeromonas

A bacterial strain secreting potent chitinolytic activity was isolated from shrimp-pond water by enrichment culture using colloidal crab-shell chitin as the major carbon source. The isolated bacterium, designated asAeromonas sp No. 16 exhibited a rod-like morphology with a polar flagellum. Under optimal culture conditions in 500-ml shaker flasks, it produced a chitinolytic activity of 1.4 U ml^–1. A slightly higher enzymatic activity of 1.5 U ml^–1 was obtained when cultivation was carried out in a 5-liter jar fermentor using a medium containing crystalline chitin as the carbon source. The secretion of the enzyme(s) was stimulated by several organic nitrogenous supplements. Most carbon sources tested (glucose, maltose, N-acetylglucosamine, etc) enhanced cell growth, but they slightly inhibited enzyme secretion. Glucosamine (0.5% w/v) severely inhibited cell growth (16% of the control), but it did not significantly affect enzyme secretion. The production of chitinolytic enzymes was pH sensitive and was enhanced by increasing the concentration of colloidal chitin to 1.5%. The observed chitinolytic activity could be attributed to the presence of -N-acetylglucosaminidase and chitinase. Chitinase was purified by ammonium sulfate fractionation and preparative gel electrophoresis to three major bands on SDS-PAGE. An in-gel enzymatic activity assay indicated that all three bands possessed chitinase activity. Analysis of the enzymatic products indicated that the purified enzyme(s) hydrolyzed colloidal chitin predominantly to N,N-diacetyl-chitobiose and, to a much lesser extent, the mono-, tri, and tetramer of N-acetylglucosamine, suggesting that they are mainly endochitinases.     

Effective production of N-acetyl-β-D-glucosamine bySerratia marcescens using chitinaceous waste

The strain ofSerratia marcescens QM B1466 produces selectively large amount of chitinolytic enzymes (about 1mg/L medium). Enzymatic hydrolysis of chitin to N-acetyl-β-D-glucosamine (NAG) was performed with a system consisting of two hydrolases (chitinase and chitobiase) produced by optimization of a microbial host consuming chitin particles. For the development of Large-scale biological process for the production of NAG from chitinaceous waste, the selection and optimization of a microbial host, particle size of chitin and pretreatment of chitin source were investigated. Also, the effect of crab/shrimp chitin sources and initial induction time using chitin as a sole carbon source on chitinase/chitobiase production and NAG production were examined. Crab-shell chitin(1.5%) treated by dilute acid and, ball-milled with a nominal diameter less than 250m gave the highest chitinase activity over a 7 days culture. Crude chitinase/chitobiase solution obtained in a 10 L fed-batch fermentation showed a maximum activities of 23.6 U/mL and 5.1 U/mL, respectively with a feeding time of 3 hrs, near pH 8.5 at 30°C.     

Selection of chitinolytic strains of Bacillus thuringiensis

Three selected strains of Bacillus thuringiensis native to Mexico produced endochitinases, chitobiosidases, and N-acetyl--glucosaminidases in a medium containing colloidal chitin as a main carbon source. Two types of chitinases were clearly identified: endochitinases and chitobiosidases. Chromosomal location of a chitinase gene in B. thuringiensis LBIT-82 was resolved.     

Antifungal chitinases from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> SG2: preliminary report

A halotolerant bacterium capable of chitinase production was previously isolated from saline soils of Gavkhooni marsh, Iran. The strain was identified as Bacillus pumilus strain SG2. This strain secretes two chitinases into the medium in response to the presence of chitin in the medium. The two chitinases, ChiS and ChiL, were active on both polymeric as well as oligosaccharide substrates. In this study, these two extracellular chitinases were purified from the supernatant of the Bacillus culture by ammonium sulfate precipitation. The optimum pH and temperature for both of these chitinases were pH 6 and 37°C, respectively. According to the conserved domain analysis, ChiS and ChiL were categorized in family 18 of the glycosylhydrolases. Three essential conserved amino acid residues (Asp-194, Asp-196 and Glu-198) in ChiS and (Asp-228, Asp-230 and Glu-232) in ChiL were found within the active site. Antifungal activities of the two purified chitinases were assessed on Rhizoctonia solani, Verticillium sp., Nigrospora sp., Stemphyllium botryosum and Bipolaris sp. demonstrating inhibition of all the tested strains. However, these chitinases did not inhibit cell wall formation of the non-chitin-containing fungi (oomycetes) Phytophthora citricola and Phytophthora capsici. This is the first investigation demonstrating antifungal activity of chitinases purified from B. pumilus SG2.     

Production of chitinase and its optimization from a novel isolate Alcaligenes xylosoxydans: potential in antifungal biocontrol

A novel, highly chitinolytic strain of Alcaligenes xylosoxydans was isolated which showed potential for use as an antifungal biocontrol agent for the control of two fungal plant pathogens. It could degrade and utilize dead mycelia of Rhizoctonia bataticola and Fusarium sp. (fungal plant pathogens of Cajanus cajan). In vitro it could inhibit the growth of Fusarium sp. and R. bataticola. Chitin at 10–15 g/l was found to be good carbon and nitrogen source. Alcaligenes xylosoxydans showed optimum chitinase production at 72 h, pH optima at 8 and growth peak at 120 h. Yeast extract, arabinose, Tween 20 and several other surfactants enhanced chitinase production.     

Distribution of Chitinases in the Entomopathogen <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine in Protein Secretion

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.     

低温几丁质酶基因在乳酸克鲁维酵母中的重组表达及酶学性质表征

【目的】通过构建假交替单胞菌(Pseudoalteromonassp.DL-6)低温几丁质酶(chitinaseA,chi A;chitinase C,chi C)的重组乳酸克鲁维酵母菌株、纯化重组蛋白并对其进行酶学性质表征,为低温几丁质酶潜在工业化生产几丁寡糖奠定理论基础。【方法】人工合成密码子优化的几丁质酶基因,构建重组乳酸克鲁维酵母表达质粒(p KLAC1-chi A、p KLAC1-chi C)并用电脉冲法转化到乳酸克鲁维酵母中,实现低温几丁质酶的可溶表达。利用镍柱亲和层析纯化得到高纯度的重组几丁质酶。【结果】成功构建产低温几丁质酶的重组乳酸克鲁维酵母并纯化获得高纯度的重组几丁质酶。经SDS-PAGE分析在110 k Da与90 k Da附近出现符合预期大小的蛋白条带。铁氰化钾法测得Chi A和Chi C的酶活分别为51.45 U/mg与108.56 U/mg。最适反应温度分别为20°C和30°C,最适p H分别为8.0和9.0。在低于40°C,p H 8.0–12.0时,Chi A和Chi C重组酶较稳定。Chi A和Chi C对胶体几丁质以及粉状底物α-几丁质与β-几丁质具有明显的降解活性,且具有一定协同降解能力。【结论】首次实现假交替单胞菌来源的低温几丁质酶在乳酸克鲁维酵母中的重组表达、纯化、酶学性质及其降解产物分析,为其他低温几丁质酶的研究提供借鉴意义。     

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

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.     

Purification and partial characterization of two chitinases from the mycoparasitic fungus <Emphasis Type="Italic">Talaromyces flavus</Emphasis>

Chitinases were produced by Talaromyces flavus CGMCC 3.4301 when it was grown in the presence of chitin. Two chitinases from the culture filtrate of T. flavus were purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE–Sepharose and Phenyl–Sepharose hydrophobic interaction chromatography. By SDS–PAGE, the molecular weight of the two enzymes was estimated to be 41 and 32 kDa, respectively. The 41 kDa chitinase (CHIT41) had a 4.0 pH optimum; the 32 kDa chitinase (CHIT32) optimum activity was at pH 5.0. The optimum temperature for the two chitinase activities was 40 °C. The two chitinases had activity against cell wall of Verticillium dahliae, Sclerotinia sclerotiorum and Rhizoctonia solani, and inhibited spore germination and germ tube elongation of Alternaria alternata, Fusarium moniliforme, and Magnaporthe grisea.     

Characterization of Antifungal Chitinase from Marine Streptomyces sp. DA11 Associated with South China Sea Sponge Craniella Australiensis

The gene cloning, purification, properties, kinetics, and antifungal activity of chitinase from marine Streptomyces sp. DA11 associated with South China sponge Craniella australiensis were investigated. Alignment analysis of the amino acid sequence deduced from the cloned conserved 451 bp DNA sequence shows the chitinase belongs to ChiC type with 80% similarity to chitinase C precursor from Streptomyces peucetius. Through purification by 80% ammonium sulfate, affinity binding to chitin and diethylaminoethyl-cellulose anion-exchange chromatography, 6.15-fold total purification with a specific activity of 2.95 Umg⁻¹ was achieved. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular weight of approximately 34 kDa and antifungal activities were observed against Aspergillus niger and Candida albicans. The optimal pH, temperature, and salinity for chitinase activity were 8.0, 50°C, and 45 g‰ psu, respectively, which may contribute to special application of this marine microbe-derived chitinase compared with terrestrial chitinases. The chitinase activity was increased by Mn²⁺, Cu²⁺, and Mg²⁺, while strongly inhibited by Fe²⁺ and Ba²⁺. Meanwhile, SDS, ethyleneglycoltetraacetic acid, urea, and ethylenediaminetetraacetic acid were found to have significantly inhibitory effect on chitinase activity. With colloidal chitin as substrates instead of powder chitin, higher V _max (0.82 mg product/min·mg protein) and lower K _m (0.019 mg/ml) values were achieved. The sponge’s microbial symbiont with chitinase activity may contribute to chitin degradation and antifungal defense. To our knowledge, it was the first time to study sponge-associated microbial chitinase.     

Use of a promoterless lacZ gene insertion to investigate chitinase gene expression in the marine bacterium Pseudoalteromonas sp. strain S9.

Sequence data for genes encoding 16S rRNA indicated that the marine strain previously named Pseudomonas sp. strain S9 would be better identified as a Pseudoalteromonas sp. By use of transposon mutagenesis, a chitinase-negative mutant of S9 with a lacZ reporter gene insertion was isolated. Part of the interrupted gene was cloned and sequenced. The deduced amino acid sequence had homology to sequences of bacterial chitinases. Expression of the chitinase gene promoter was quantified by measuring the lacZ reporter gene product, beta-galactosidase, beta-Galactosidase production was induced 10-fold by N-acetylglucosamine and 3-fold by chitin in minimal medium. Repression of beta-galactosidase synthesis was observed in rich medium either with or without chitin but was not observed in minimal medium containing glucose. The chitinase gene promoter was induced by starvation and higher-than-ambient levels of carbon dioxide but not by cadmium ion, heat or cold shock, or UV exposure.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

Characterization of chitinases excreted by Bacillus cereus CH

Bacillus cereus CH was shown to excrete chitinases into the culture supernatant when cultivated in a medium containing 0.2% colloidal chitin, whereas the removal of colloidal chitin resulted in a low activity. After concentration of the culture supernatant by precipitation with ammonium sulfate, the induced chitinases were purified by sequential chromatography. Four different chitinases, A, B1, B2, and B3 with molecular masses of 35, 47, 58, and 64 kDa, respectively, were separated. All chitinases showed similarities in their kinetic parameters when observed with colloidal chitin, including an optimal pH of 5.0-7.5, and an optimal temperature between 50-60 degrees C. Chitinase A hydrolyzed glycol chitin and p-nitrophenyl-di-N-acetyl-beta-chitobioside at similar rates to that of colloidal chitin, whereas group B chitinases hydrolyzed both substrates in much lower rates. From analyses of the reaction products, it is most likely that chitinase A and all group B chitinases hydrolyze the substrates tested in an endo-fashion. However, group B chitinases were distinct from chitinase A in possessing high transglycosylation activity. From amino terminal sequencing, chitinases B1, B2, and B3 were shown to have almost identical sequences, which differed from that of chitinase A. The similarities in the reaction modes and amino terminal sequences among chitinases B1, B2, and B3 suggest that these chitinases may be derived from a presumptive precursor protein through C-terminal processing.     

A Paecilomyces fumosoroseus mutant over-producing chitinase displays enhanced virulence against Bemisia tabaci

A Paecilomyces fumosoroseus strain was mutagenized by u.v. Among 200 colonies, one mutant (M84), showed a large and stable chitin hydrolysis-halo. Glucose consumption and biomass production were similar for M84 and the parental strain. Chitinase was inducible by chitin and repressed by glucose in both strains but, when they were grown on minimal medium plus colloidal chitin as sole carbon source, the parental and M84 strains yielded 198 and 690 mol N-acetylglucosamine, respectively. This results indicate that the mutant strain synthesized a chitinase with a higher activity. Bioassays against Bemisia tabaci nymph, showed that M84 incited a 2-fold higher incidence of disease compared to the parental strain.