Chitinolytic enzymes from<Emphasis Type="Italic">Clostridium aminovalericum</Emphasis>: Activity screening and purification

A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.     

Production of N-acetyl-β-d-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme

A bacterium, GJ-18, having strong chitinolytic activity was isolated from coastal soil. The isolated strain was identified as Aeromonas sp. by morphological and biochemical properties along with 16S rRNA gene sequence. The crude chitinolytic activity of culture supernatants was maximal on the 5th day of culture. Below 45°C, chitin was effectively hydrolyzed to N-acetyl--d-glucosamine (GlcNAc) by Aeromonas sp. GJ-18 crude enzymes, but hydrolysis decreased markedly above 50°C. The optimum pH for enzyme activity was 5.0. TLC and HPLC analysis revealed that, below 45°C, the major reaction product was GlcNAc with a small amount of (GlcNAc)₂ and (GlcNAc)₃, whereas above 50°C the major product was (GlcNAc)₂. When swollen chitin (100 mg) was incubated with crude enzyme preparations (10 U) at 40°C, chitin was hydrolyzed to 83.0 and 94.9% yield of GlcNAc within 5 and 9 days, respectively.     

Breakdown of chitin by Cytophaga johnsonii

Summary Decomposition of chitin by Cytophaga johnsonii was investigated. Unlike other chitinolytic bacteria, some strains of C. johnsonii did not liberate chitinase extracellularly; instead the cells of such strains had need for close contact with the chitin particles in order to hydrolyze them. The cell-free extract of one of these strains, viz., C. johnsonii C35 did show presence of chitinase. The remaining strains liberated an extracellular chitinase and a chitobiase. The partially purified chitinase from the culture filtrates of C. johnsonii C31 was most active at pH 6.3–6.5 and at 40°C.Metabolism of N-acetylglucosamine, a product of chitin hydrolysis, by C. johnsonii C35 and C31 was investigated. The nature of some of the products formed and the steps involved in their transformations by the resting cells and the cell-free extracts suggested that N-acetylglucosamine first gets deacetylated to glucosamine before it is further oxidized to glucosaminic acid by these strains. Both strains were also able to dehydrogenate gluconate to 2-ketogluconate, the former being in all probability the product of deamination of glucosaminic acid.     

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus chitonophagus</Emphasis>

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80°C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.     

Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli

Summary A DNA fragment encoding for 598 amino acids of chitinase protein from Bacillus circulans No. 4.1 was subcloned into pQE-30 expression vector and transformed into Escherichia coli M15 (pREP4). The molecular weight of the expressed protein was approximately 66 kDa. Enzymatic activity of the recombinant protein was assayed after purification using affinity chromatography on a nickel chelating resin. The enzyme hydrolyzed N-acetylchitooligosaccharides mainly to N-acetylchitobiose, and was active toward chitin, carboxymethyl-chitin, colloidal chitin, glycol chitin and 4-methylumbelliferyl-β-d-N, N′-diacetylchitobiose. The pH and temperature optima of the chitinase enzyme were 7.0 and 45 °C, respectively. This enzyme was stable in the pH range of 5.0–9.0 and at temperatures up to 50 °C. In addition, when cleaved by a proteolytic enzyme, the 20-kDa product could retain high chitinolytic activity.     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Chitinolytic activity of the anaerobic rumen fungus Piromyces communis OTS1

The anaerobic rumen fungus, Piromyces communis OTS1, was isolated from a fistulated goat. Its chitinolytic activity in the supernatant of media containing different types of chitin was studied. The fungus grew well in our basal medium, with and without colloidal chitin and chitin powder. N-Acetyl--glucosaminidase activity was not detected in any of the culture media. Chitinase activity, however, was detected in the basal medium with and without colloidal chitin and chitin powder. The extracellular chitinase concentrated from the fungal culture's supernatant by ammonium sulfate (80% saturation) showed highest activity at 40°C and at pH 5.5. In the other cell fractions of P. communis OTS1, N-acetyl--glucosaminidase was not detected, but chitinase activity was detected by 4-methylumbelliferyl reagents. Thus, it was found that the rumen fungus P. communis OTS1 has chitinase activity. Chitinases from the extracellular, cytosolic, and the microsomal fractiòns were mainly of the endo type of chitinase activity.     

Chitinolytic enzymes from the newly isolated <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> SBK1: study of the mosquitocidal activity

Aeromonas hydrophila SBK1 (GenBank accession no. HM802878.1), a potent chitinolytic bacterium, was isolated from a pool of 30 chitinolytic isolates. The isolate showed higher chitinolytic activity in respect to clear zone to colony size ratio of 2.15. Maximum production of chitinolytic enzymes, viz., β-N-acetyl-glucosaminidase and chitinase (specific activity 655.3 and 71.6 U mg⁻¹, respectively) by A. hydrophila SBK1 was observed in the synthetic media, containing (w/v)-colloidal chitin, 4.0%; peptone, 0.3%; phosphate, 0.3% (0.15% of each KH₂PO₄ and K₂HPO₄); NaCl, 0.25%; MgSO₄, 0.05%; KCl, 0.05%; pH 7.0 and at 35°C after 72 h of incubation. Both carbon-to-nitrogen (C/N) and carbon-to-phosphate (C/P) ratio of 13.33 were found optimum for chitinase production. Enzyme productivity increased about twofold in optimized culture condition in respect to its un-optimized state. The crude enzyme showed optimum activity against Culex quinquefasciatus larvae in native water at pH 7.0 and 35°C (LD₅₀ 0.60 U ml⁻¹ at 48 h). Therefore, the studied chitinases can be used as an effective mosquitocidal agent.     

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.     

Characterization of gastrointestinal chitinase in the lizard Sceloporus undulatus garmani (Reptilia: Phrynosomatidae)

Most studies on chitinase activity in lizards have been concerned with Palaearctic (European) and Laurasian (Middle Eastern and Asian) taxa. Several genera of Old World lizards, Anguis, Uromastix, Chamaeleo and Lacerta, have been shown to possess chitinolytic activity. To date, only one New World lizard, Anolis carolinensis, has been reported to exhibit chitinolytic activity. In the present study, chitinase activity was characterized in a second New World taxon, Sceloporus undulatus garmani, a New World, phrynosomatid lizard. Chitinolytic activity was measured by incubating tissue extracts with a radioactive chitin substrate, acetyl-[H³]chitin and determining acid soluble radioactivity as an estimate for chitin hydrolysis. Chitinolytic activity was present in stomach, small intestine and pancreas extracts, with the stomach and pancreas having the highest specific activities. Chitinolytic activity was higher at pH 4.5 than at pH 7.5. The stomach chitinase is immunologically similar to the gastric chitinase previously described for rainbow trout. Western blot analysis showed anti-chitinase cross-reactivity in the extracts of the stomach, but no cross-reactivity in the pancreatic or intestinal extracts, suggesting different isoforms of chitinase. There was no detected lysozyme activity (less than 0.01 mg/ml lysozyme) present in the extracts of the stomach, small intestine and pancreas. The localization of chitinolytic activity in S. u. garmani is in agreement with earlier reptilian reports on the distribution of chitinase.     

Characterization of chitinase from Streptomyces luridiscabiei U05 and its antagonist potential against fungal plant pathogens

Streptomyces luridiscabiei U05 was isolated from wheat rhizosphere. It produced chitinase, which showed in vitro antifungal properties. The crude enzyme inhibited the growth of Alternaria alternata, Fusarium oxysporum, F. solani, Botrytis cinerea, F. culmorum and Penicillium verrucosum. The chitinase enzyme of the molecular weight of 45 kDa was purified using affinity chromatography of chitin. Streptomyces luridiscabiei U05 produced different chitinolytic enzymes. The highest enzyme activity was observed with the use of 4‐MU‐(GlcNAc)_, which points to the presence of an β‐N‐acetylhexosaminidase. The optimum activity was obtained at 35–40°C and pH 7–8. The enzyme showed thermostability at 35–40°C during 240 min of preincubation and lost its activity at 50°C and 60°C in 60 min. The chitinase activity from S. luridiscabei U05 was strongly inhibited by Hg²⁺ and Pb²⁺ ions, and sodium dodecyl sulphate (SDS). The Ca²⁺, Cu²⁺ and Mg²⁺ ions stimulated the activity of the enzyme.     

Partial purification and properties of extracellular chitinase produced by Acremonium obclavatum,an antagonist to the groundnut rust,Puccinia arachidis

An extracellular chitinase of Acremonium obclavatum was partially purified. It had an M _r of 45 kDa on SDS-PAGE, and was optimally active at pH 3 to 4 and 50°C. Hg and Mn (10 mm) inhibited activity. The chitinase hydrolysed colloidal chitin more rapidly than crude chitin or isolated A. obclavatum cell walls. The partially-purified enzyme inhibited uredospore germination and germ-tube growth of Puccinia arachidis.The authors are with the Centre for Advanced Study in Botany, University of Madras, Guindy campus, Madras 600 025, India     

Characterization and role of a metalloprotease induced by chitin in <Emphasis Type="Italic">Serratia</Emphasis> sp. KCK

A metalloprotease induced by chitin in a new chitinolytic bacterium Serratia sp. Strain KCK was purified and characterized. Compared with other Serratia enzymes, it exhibited a rather broad pH activity range (pH 5.0–8.0), and thermostability. The cognate ORF, mpr, was cloned and expressed. Its deduced amino acid sequence showed high similarity to those of bacterial zinc-binding metalloproteases and a well-conserved serralysin family motif. Pretreatment of chitin with the Mpr protein promoted chitin degradation by chitinase A, which suggests that Mpr participates in, and facilitates, chitin degradation by this microorganism.     

Production and purification of a chitinase from Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus

The chitinolytic properties of Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus, are reported. E. americana was shown to produce chitinolytic activity in the absence of chitin and in the presence of glucose and N-acetylglucosamine, indicating constitutive synthesis by these strains. A single 33-kDa protein with chitinolytic activity was purified to homogeneity from culture filtrates, by hydrophobic interaction chromatography using a phenyl-group substituted matrix. This enzyme, by virtue of differential activity against chromogenic chitooligosaccharides and against dye-labelled soluble carboxymethylated chitin (CM-chitin-RBV), was demonstrated to be an endochitinase. Our data suggest this 33-kDa chitinase appeared to be the only chitinolytic enzyme produced by E. americana, strains of which do not grow using chitin as a carbon source. The significance of these findings in the context of mushroom disease is discussed.     

Purification and characterization of chitinase from Alcaligenes xylosoxydans

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecularmass of chitinase was estimated to be 45 kDa and44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 °C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l^–1. Cu²⁺ and Na⁺ at 5 mM inhibited chitinase activity to 25% while Ca²⁺, Mg²⁺ and Ba²⁺ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Isolation of novel chitinolytic bacteria and production optimization of extracellular chitinase

Chitin is one of the most abundant biopolymers widely distributed in the marine and terrestrial environments. Chitinase enzyme has received increased attention due to its wide range of biotechnological applications, especially in agriculture for biocontrol of phytopathogenic fungi and harmful insects. In the present study, 58 bacterial isolates were screened for chitinolytic activity and on the basis of chitin hydrolysis zone 6 isolates were selected for chitinase production in broth media. Based on enzyme production, two most potent isolates identified as Aeromonas hydrophila HS4 and Aeromonas punctata HS6 were selected for further study. The effects of media composition and various fermentation conditions for optimization of chitinase production were studied. The maximum chitinase production was obtained at 37 °C and pH 8.0 after 24–48 h of incubation by HS4; and at 37 °C and pH 7 after 48 h incubation by HS6. Among the substrates colloidal chitin was the best for both the strains. Regarding carbon sources, starch (1%) was the best for both strains; while malt and yeast extract (1%) was found as the best nitrogen source for HS4 and HS6, respectively. Out of metal ions Mn²⁺ and Cu²⁺ enhanced enzyme production in the case of HS6. However, Co²⁺ was the most appropriate for HS4.     

An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product

An extracellular chitinase of Bacillus sp. WY22 was purified by 9.6-fold. It had a Mr of 35 kDa, an apparent K _m value for colloidal chitin of 3 mg ml^–1 and was optimally active at 37 °C and pH 5.5 over 1 h. The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin. It formed chitotriose as a major product from colloidal chitin and glycol chitin.     

Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis

Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 isolated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis. Chitinase activity was detected from the second day of cultivation, then increased gradually and reached maximum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the enzyme was strongly inhibited in the presence of Pb²⁺, Hg²⁺ and stabilized by the ions Mg²⁺. Purified chitinase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Penillium sp.     

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.