A rapid and sensitive assay for chitinase using tritiated chitin

Radioactive chitin, prepared by acetylation of chitosan with tritiated acetic anhydride, was used as substrate in a rapid and extremely sensitive assay for chitinase. The procedure is based on the insolubility of chitin and the solubility in water of the reaction product, diacetylchitobiose. The course of the chitinase reaction is nonlinear, a result that cannot be attributed to an artifact of the method, to inhibition by product, or to instability of the enzyme. Some evidence points to structural heterogeneity of the substrate as a cause for this behavior. Reacetylated chitosan was also used as an adsorbent in the purification of chitinase with better results than with the previously used colloidal chitin.     

Purification and characterization of extracellular chitinase from Aeromonas schubertii

A locally isolated stain Aeromonas schubertii was cultured and induced by powdered chitin for the production of chitinases. Extracellular proteins were purified by ammonium sulfate precipitation, dialysis to remove salts, and then preparative isoelectric focusing (IEF) to yield several chitinases. The purified enzymes were analyzed by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) with and without glycol chitin and were found to be SDS-resistant. The chitinase present in the highest abundance was the one with an estimated molecular weight of 75 kDa. The Michaelis constant and turnover number were determined to be 0.29 mM and 1 s⁻¹, respectively, for this enzyme using colloidal chitin azure as the substrate. However, the ethanol treatment of this enzyme could significantly increase its chitinolytic activity. Other chitinases obtained in the same IEF fraction were determined to have molecular weights of ca. 30, 38, and 110 kDa. Since the proteins with highest chitinase activity were collected from IEF fraction tube with pH value of 4.8, those chitinase were believed to be acidic. An activity assay method using colloidal chitin azure as the substrate was recommended since it possessed a broader range of linearity in comparison with conventional reducing sugar equivalent method.     

Preparation of fermentation-processed chitin and its application in chitinase affinity adsorption

A fermentation approach utilizing Paenibacillus sp. to process chitin was developed. The chitin obtained from this process is called fermentation-processed chitin (FPC), and it was further investigated with chitinase affinity adsorption studies together with three other adsorbents, i.e. crab shell chitin, colloid chitin, and enzyme-processed chitin. The results showed that FPC had the highest chitinase adsorption capacity. Under 15 °C and pH 5.0, FPC exhibited an optimal chitinase adsorption capacity of 85.9 U/g, which was 61.9% higher than that of the colloidal chitin. With 0.02 M acetic acid as the eluent, a purification-fold of 10.3 with 97% chitinase recovery was obtained. The results of surface morphology studies indicated that the FPC surface was modified to a fiber-like structure with deep pores. In comparison with the surface morphology of enzyme-processed chitin and colloidal chitin, it is inferred that the enhanced adsorption capacity of FPC for chitinase is attributed to both the effects of chitinase hydrolysis and the bacterial modification.     

Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation.

Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharide-degrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (A1, A2, B1, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pI 4.7 (A1); Mr 69,000, pI 4.5 (A2); Mr 38,000, pI 6.6 (B1); Mr 38,000, pI 5.9 (B2); Mr 39,000, pI 8.5 (C); and Mr 52,000, pI 5.2 (D). Among these chitinases, A1 and A2 had the highest colloidal-chitin-hydrolyzing activities. Chitinase A1 showed a strong affinity to insoluble substrate chitin. Purified chitinase A1 released predominantly chitobiose [(GlcNAc)2] and a trace amount of N-acetylglucosamine (GlcNAc) from colloidal chitin. N-terminal amino acid sequence analysis of chitinases A1 and A2 indicated that chitinase A2 was generated from chitinase A1, presumably by proteolytic removal of a C-terminal portion of chitinase A1. Since chitinase A2 did not have the ability to bind to chitin, the importance of the C-terminal region of chitinase A1 to the strong affinity of chitinase A1 to substrate chitin was suggested. Strong affinity of the chitinase seemed to be required for complete degradation of insoluble substrate chitin. From these results, it was concluded that chitinase A1 is the key enzyme in the chitinase system of this bacterium.     

Chitinolytic properties of Bacillus pabuli K1

The chitinolytic properties of Bacillus pabuli K1 isolated from mouldy grain was studied. Chitinase activity was measured as the release of p -nitrophenol from p -nitrophenyl-N, N'-diacetylchitobiose. Influences of substrate concentration and different environmental variables on growth and chitinase activity were determined. The optimum environmental conditions for chitinase production were: 30°C, initial pH 8, initial oxygen 10% and a_w > 0.99. Chitinase production was induced when B. pabuli K1 was grown on colloidal chitin. The smallest chito-oligosaccharide able to induce chitinase production was N, N'-diacetylchitobiose, (GlcNAc)₂. Production was also induced by (GlcNAc)₃ and (GlcNAc)₄. When the bacterium was grown on glucose or N -acetylglucosamine, no chitinases were formed. The highest chitinase production observed was obtained with colloidal chitin as substrate. The production of chitinases by B. pabuli K1 growing on chitin was repressed by high levels (0.6%) of glucose. The production was also repressed by 0.6% starch, laminarin and β-glucan from barley and by glycerol. The addition of pectin and carboxymethyl cellulose increased chitinase production.     

Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101

A chitinase encoding gene from Bacillus sp. DAU101 was cloned in Escherichia coli. The nucleotide sequencing revealed a single open reading frame containing 1781 bp and encoding 597 amino acids with 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram. The chitinase was composed of three domains: a catalytic domain, a fibronectin III domain, and a chitin binding domain. The chitinase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 7.5 and 60 degrees C, respectively. The metal ions, Zn(2+), Cu(2+), and Hg(2+), were strongly inhibited chitinase activity. However, chitinase activity was increased 1.4-fold by Co(2+). Chisb could hydrolyze GlcNAc(2) to N-acetylglucosamine and was produced GlcNAc(2), when chitin derivatives were used as the substrate. This indicated that Chisb was a bifunctional enzyme, N-acetylglucosaminase and chitobiosidase. The enzyme could not hydrolyze glycol chitin, glycol chitosan, or CMC, but hydrolyzed colloidal chitin and soluble chitosan.     

Glycogen in Methanolobus and Methanococcus

Abstract Ultraviolet light and nitrosoguanidine were used to mutagenize a red pigmented culture of Serratia marcescens , strain EB415, which produced chitinase. After mutagenesis, a stable, non-pigmented mutant designated BL40 was isolated which produced larger colonies and zones of clearing on solid medium containing colloidal chitin.
In liquid medium with colloidal chitin as the sole carbon source both strains grew similarly but BL40 produced 160 units/ml of chitinase compared with 60 units/ml for EB 415, an increase of 167%. When chitin concentration was increased in the medium, chitinase production also increased. Chitinase appeared to be extracellular, since the supernatant from washed, sonicated cells for both strains showed no detectable amount of chitinolytic activity.     

兼具凝集素活性的芥菜几丁质酶BjCHI1

利用毕赤酵母表达系统表达芥菜几丁质酶基因BjCHI1及其两个衍生基因BjCHI2和BjCHI3,获得相应的蛋白质。经FPLC纯化后,测定了3种蛋白质的几丁质酶活性,发现它们均能降解CM-chitin-RBV和胶状几丁质。以CM-chitin-RBV为底物时的Km值分别为0.799mg/mL、0.544mg/mL和0.793mg/mL,差别甚微。而以胶状几丁质为底物时的Km值分别为0.281mg/mL、0.388mg/mL和1.643mg/mL,表现一定的差别,说明几丁质结合域影响了酶对不溶性底物的亲和力。3种蛋白中,只有BjCHI1在33μg/mL以上浓度具有凝集素活性,而BjCHI2和BjCHI3的浓度即使高达800μg/mL也无凝集素活性,表明2个几丁质结合域是BjCHI1具有凝集素活性的必需条件,这是植物中发现的第一个兼有几丁质酶和凝集素活性的蛋白质。      

Effects of carbon and nitrogen sources on the induction and repression of chitinase enzyme from<Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> isolates

Metarhizium anisopliae, an entomopathogenic hyphomycete, is being used effectively in Integrated Pest Management (IPM) system. Foliar application of these fungi is quite satisfactory as it invades its host by adhering to insect cuticles and formation of penetration structures called appresoria, which produces various extracellular enzymes, including chitinase that causes the insect cuticle breaching. The induction and repression mechanism of chitinase activity is not entirely understood and activity of this enzyme is different in response to different carbon and nitrogen sources. This report illustrates the effect of two carbon sources viz. colloidal chitin and dextrose and a nitrogen source, yeast extract on the chitinase production of fourteenM. Anisopliae isolates. The chitinase activity varied among the isolates and the different media used. A high enzymatic activity was observed in the medium containing an extra nitrogen source (yeast extract) followed by the medium containing colloidal chitin as a sole source of carbon and nitrogen. The exochitinase activity and the chitinase activity gel were also studied for the isolates showing high chitinase enzyme production. An array of chitinase isozymes were observed on chitinase activity gel with a common 14.3 kDa enzyme for all the isolates.     

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.     

Effective production of chitinase and chitosanase byStreptomyces griseus HUT 6037 using colloidal chitin and various degrees of deacetylation of chitosan

The advantages of the organismStreptomyces griseus HUT 6037 is that the chitinase and chitosanase using chitinaceouse substrate are capable of hydrolyzing both amorphous and crystalline chitin and chitosan. We attempted to investigate the optimization of induction protocol for high-level production and secretion of chitosanase and the influence of chitin and partially deacetylated chitosan sources (75–99% deactylation). The maximum specific activity of chitinase has been found at 5 days cultivation with the 48 hours induction time using colloidal chitin as a carbon source. To investigate characteristic of chitosan activity according to substrate, we used chitosan with various degree of deacetylation as a carbon source and found that this strain accumulates chitosanase in the culture medium using chitosanaceous substrates rather than chitinaceous substrates. The highest chitosanase activity was also presented on 4 days with 99% deacetylated chitosan. The partially 53% deacetylated chitosan can secrete both chitinase and chitosanase which was defined as a soluble chitosan. The specific activities of chitinase and chitosanase were 0.89 at 3 days and 1.33 U/mg protein at 5 days, respectively. It indicate that chitosanase obtained fromS. griseus HUT 6037 can hydrolyze GlcNAc-GlcN and GlcN-GlcN linkages by exo-splitting manner. This activity increased with increasing degree of deacetylation of chitosan. It is the first attempt to investigate the effects of chitosanase on various degrees of deacetylations of chitosan byS. griseus HUT 6037. The highest specific activity of chitosanase was obtained with 99% deacetylated chitosan.     

Solid-state cultivation of Bacillus thuringiensis R 176 with shrimp shells and rice straw as a substrate for chitinase production

In this study, shrimp shell powder, prepared by treating shrimp-processing waste by boiling and crushing, was used as a substrate for isolation of chitinase-producing microorganism. These organisms may have an important economic role in the biological control of rice and other fungal pathogens. Two hundred strains of bacteria with the ability to degrade chitin from shrimp shell waste were isolated from paddy soil, and of these, 40 strains showed chitinase activity in a solid state cultivation. One of the most potent isolates (strain R 176) was identified as Bacillus thuringiensis. Identification was carried out using morphological and biochemical properties along with 16S rRNA sequence analysis. This strain was able to produce high levels of extracellular chitinase in solid media containing shrimp shells as sole carbon source [1.36 U/g initial dry substrate (IDS)], which was 0.36-fold higher than the productivity in a liquid culture with colloidal chitin. The effects of medium composition and physical parameters on chitinase production by this organism were studied. The optimal medium contained shrimp shell mixed with rice straw in 1:1 ratio added with ball-milled chitin 0.5 % (w/v) and ammonium sulfate 0.5 % (w/v). The highest enzyme production (3.86 U/g IDS) by B. thuringiensis R 176 was obtained at pH 7, 37 °C after 14 days growth. With respect to the high amount of chitinase production by this strain in a simple medium, this strain could be a suitable candidate for the production of chitinase from chitinous solid substrates, and further investigations into its structure and characteristics are merited.     

Expression patterns of chitinase produced from Paenibacillus chitinolyticus with different two culture media

To investigate the expression patterns of chitinase isozymes on native-PAGE and SDS-PAGE gels Paenibacillus chitinolyticus MP-306 was cultured on culture media with and without chitin substrate. P. chitinolyticus MP-306 had a strong chitinolytic activity on colloidal chitin medium. Chitinase isozymes of MP-306 were expressed as six bands (CN1-CN6) on native-PAGE gels and thirteen bands (CS1-CS13) on SDS-PAGE gels after incubation in chitin medium. Three bands (CN1, CN2, and CN3) of chitinase isozymes of MP-306 on native-PAGE gels were expressed as nine bands (CS1, CS2, CS3, CS4, CS5, CS6, CS8, CS10, and CS13) of chitinase isozymes on SDS-PAGE gels. Three bands (CN4, CN5, and CN6) of chitinase isozymes of MP-306 were strongly inhibited by metal ions on native-PAGE and SDS-PAGE gels.     

Effect of the C-terminal domain of Vibrio proteolyticus chitinase A on the chitinolytic activity in association with pH changes

Aims: To reveal the cause of the difference in activity of chitinase A from Vibrio proteolyticus and chitinase A from a strain of Vibrio carchariae (a junior synonym of Vibrio harveyi), we investigated the pH‐dependent activity of full‐length V. proteolyticus chitinase A and a truncated recombinant corresponding to the V. harveyi form of chitinase A. Methods and Results: After overexpression in Escherichia coli strain DH5α, the full‐length and truncated recombinant chitinases were purified by ammonium sulphate precipitation and anion exchange column chromatography. Chitinase activity was measured at various pH values using α‐crystal and colloidal chitins as the substrate. The pH‐dependent patterns of the relative specific activities for α‐crystal chitin differed between the full‐length and truncated recombinant chitinases, whereas those for colloidal chitin were similar to each other. Conclusion: The difference in the activity of V. proteolyticus chitinase A and V. harveyi chitinase A might be partly due to a change in the pH dependence of the chitinase activities against α‐crystal chitin, resulting from C‐terminal processing. Significance and Impact of Study: The present results are important findings for not only ecological studies on the genus Vibrio in association with survival strategies, but also phylogenetic studies.     

Impact of process parameters on chitinase production by an alkalophilic marine Beauveria bassiana in solid state fermentation

A chitinolytic fungus, Beauveria bassiana was isolated from marine sediment and significant process parameters influencing chitinase production in solid state fermentation using wheat bran were optimised. The organism was strongly alkalophilic and produced maximum chitinase at pH 9·20. The NaCl and colloidal chitin requirements varied with the type of moistening medium used. Vegetative (mycelial) inoculum was more suitable than conidial inoculum for obtaining maximal enzyme yield. The addition of phosphate and yeast extract resulted in enhancement of chitinase yield. After optimisation, the maximum enzyme yield was 246·6 units g⁻¹ initial dry substrate (U gIDS⁻¹). This is the first report of the production of chitinase from a marine fungus.     

Statistical optimization of medium components for production of extracellular chitinase by Basidiobolus ranarum: a novel biocontrol agent against plant pathogenic fungi

The influence of concentration of medium components such as colloidal chitin, lactose, malt extract, yeast extract, and peptone on the chitinase production from Basidiobolous ranarum at the flask level were studied by using statistical tool Central Composite Design (CCD) and analysed by Response Surface Methodology (RSM). The results revealed that colloidal chitin, malt extract and peptone had significant effect (P < 0.01) on the chitinase production at their individual levels. The polynomial equation of the model developed incorporates 3 linear, 3 quadratic and 5 interactive terms. Maximum chitinase production of 3.47 U ml(-1) was achieved with 1.5% colloidal chitin, 0.125% lactose, 0.025% malt extract and 0.075% peptone. After optimization, chitinase activity was increased by 7.71 fold. A second order polynomial equation was found to be useful for the development of efficient bioprocess for chitinase production. To screen the biotechnological potential of this enzyme, degradation of fungal mycelia by ammonium sulphate precipitate of the same was studied for several pathogenic fungi-in vitro which showed promising results particularly against Rhizoctonia solani and Fusarium solani. This study provides the first evidence showing the effectiveness of RSM for the development of a robust statistical model for the chitinase production by Basidiobolus and for its application in the biocontrol of phytopathogenic fungi. (? 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

Streptomyces lunalinharesii spores contain chitin on the outer sheath

Chitin from Streptomyces lunalinharesii spores, detected on its outermost surface layer, was isolated and characterized by chemical and spectroscopic methods, transmission electron microscopy and flow cytometry analysis. Gold–chitinase- and gold–lectin ( Lycopersicum esculentum agglutinin, LEA)-conjugated labels were used in microscopy experiments, whereas a fluorescence–lectin (LEA) conjugate was used in flow cytometry analysis. Chitin isolation consisted of several steps of hot alkali and nitrous acid treatment, and the final material was obtained in the colloidal form. The infrared and the ¹³C CP/MAS NMR spectra of Streptomyces sp. colloidal chitin and colloidal chitin obtained from commercial crab shell chitin were very similar. Incubation of the spores with gold-labeled lectin, or gold-labeled recombinant chitinase, showed the presence of gold particles around the spore surface, indicating the specific binding of the lectin or the recombinant chitinase with the chitin present on the outermost surface. Flow cytometry analysis, using the fluorescence–lectin conjugate, confirmed these results. According to scanning electron microscopy, S. lunalinharesii presented spore surface ornamentation belonging to the spiny group. This is the first detailed characterization of chitin on the spore's outermost layer from a Streptomyces species.     

Characterization of Chitinase Produced by the Alkaliphilic <Emphasis Type="Italic">Bacillus mannanilyticus</Emphasis> IB-OR17 B1 Strain

The paper reports on the isolation of an extracellular chitinase produced by the alkaliphilic Bacillus mannanilyticus IB-OR17 B1 strain grown in media containing crab shell and bee chitin at a pH of 8–11. The enzyme was 860-fold purified by ultrafiltration and chitin sorption. The molecular weight of the purified chitinase was shown by denaturing electrophoresis to be 56 kDa. The enzyme showed maximum activity at a pH of 7.5–8.0 and 65°C and was stable within a pH range of 3.5–10.5 and temperature range of 75–85°C. With colloidal chitin as substrate, the kinetic characteristics of the chitinase were determined as follows: K_M ~ 1.32 mg/mL and V_max ~ 5.05 μM min^–1. N-acetyl-D-glucosamine and its dimer were the main products of enzymatic chitin cleavage, while the trisaccharide was detected just in minor quantities. The chitinase actively hydrolyzed p-nitrophenyl-GlcNAc₂ according to the exo-mechanism of substrate hydrolysis characteristic of chitobiosidases.     

Chitin in the peritrophic membrane of Acarus siro (Acari: Acaridae) as a target for novel acaricides

The peritrophic membrane in Acarus siro L. (Acari: Acaridae) is produced by distinct cells located in the ventriculus. In this study, the chitin inside the peritrophic membrane was detected using wheat germ-lectin conjugated with colloidal gold (10 nm). The chitin fibrils of the peritrophic membrane were a target for chitin effectors, including 1) chitinase, which hydrolyzes chitin fibers inside the peritrophic membrane; 2) calcofluor, which binds to chitin and destroys the peritrophic membrane mesh structure; and 3) diflubenzuron, which inhibits chitin synthesis. In addition, soybean trypsin protease inhibitor (STI) and cocktails of chitinase/calcofluor, diflubenzuron/calcofluor and chitinase/STI were tested. These compounds were supplemented in diets and an increase of population initiated from 50 individuals was observed after 21 d of cultivation. Final A. siro densities on experimental and control diets were compared. The chitin in the peritrophic membrane was determined to be a suitable target for novel acaricidal compounds for suppressing the population growth of A. siro. The most effective compounds were calcofluor and diflubenzuron, whereas the suppressive effects of chitinase and STI were low. The failure of chitinase could be due to its degradation by endogenous proteases. The combination of chitinase and STI suppressed A. siro population growth more effectively than when they were tested in oral admission separately. The combinations of calcofluor/chitinase or calcofluor/difluorbenzuron showed no additive effects on final A. siro density. The presence of chitin in peritrophic membrane provides a target for novel acaricidal compounds, which disrupt peritrophic membrane structure. The suitability of chitin effectors and their practical application in the management of stored product mites is discussed.     

Biochemistry of insect differentiation: A system for studying the mechanism of chitinase activity in vitro

Results obtained with an in vitro system for the study of chitinase are described. The system involves soluble enzyme protein(s) and an insoluble substrate preparation. With insect molting fluid chitinase, it shows properties that parallel those observed during in vivo breakdown of cuticle during the molt. For example, molting fluid chitinase activity not previously exposed to chitin is stronly and specifically adsorbed to the substrate, in contrast to other enzymatic activities including hexosaminidase (chitobiase) present in molting fluid. This leads to partial purification of molting fluid chitinase activity reflected in increased specific activity of chitinase associated with the insoluble chitin substrate; we have previously reported increase of specific chitinase activity of (deproteinized) cuticle resulting from its incubation with molting fluid (M. L. Bade and A. Stinson, 1978, Biochem. Biophys. Res. Commun.84, 381–388). Soluble end product is generated rapidly and linearly with time by the in vitro system; the end product is assumed to be N-acetylglucosamine since the specific radioactivity of this compound is unchanged during the 10 min required for assay. Molting fluid chitinase activity may involve a number of polypeptides ranging in molecular weight from 145,000 to less than 20,000 daltons. The system described gives results consistent with a processive mechanism for molting fluid chitinase, i.e., data are given demonstrating that molting fluid chitinase continues to act on the same chitin particle(s) with which it initially associates rather than diffusing freely from substrate particle to substrate particle, and the product of its action appears to be a monosaccharide rather than a mixture of oligosaccharides. Processive behavior for chitinase would be predicted from the known structure, and the in vivo measured rate of breakdown, of cuticle chitin during the molt; the preliminary nature of this conclusion, based on what is so far known about the structure of the substrate used in the in vitro system, is briefly discussed.