Nachweis von Clostridium botulinum Typ A, B, E und F mittels real-time-PCR

Clostridium botulinum is a Gram-positive, anaerobic, spore-performing bacterium with the ability to produce under certain conditions a protein with a characteristical neurotoxicity. Intoxications with C. botulinum toxin belong to the rare occuring food-poisonings; the mortality, however, is very high. C. botulinum produce seven different toxin types (type A to G), human intoxications are currently described to caused by toxin type A, B, E and F. C. botulinum is a strictly anaerobic growing bacterium, so the risk for the consumer’s health is mainly due to non-commercially produced food cans. A special form of botulism is the „infant botulism“. In contrast to the botulism of adults, where the disease is caused through toxin-containing food, spores of C. botulinum can sporulate and produce toxin in the intestines of an infant. The source of infant botulism can be honey, because it contains as a natural product C. botulinum spores. Because of the difficult and time-consuming cultural detection of C. botulinum, PCR methods to screen for the toxin genes A, B, E and F, which are relevant in the human medicine, have been used increasingly during the last years. In this presentation two real-time-PCR assays for C. botulinum, which can be applied in the routine laboratory, will be shown.

---

Zusammenfassung: Clostridium botulinum z?hlt zu den anaeroben sporenbildenden Bakterien, die unter bestimmten Bedingungen in der Lage sind, sich in Lebensmitteln zu vermehren und ein Protein mit charakteristischer Neurotoxizit?t zu bilden. Intoxikationen mit Clostridium botulinum-Toxin geh?ren zu den seltenen lebensmittelassoziierten Intoxikationen; die Mortalit?t bei einer Erkrankung ist allerdings sehr hoch. C. botulinum produziert sieben unterschiedliche Toxintypen (Typ A-G), wobei für menschliche Erkrankungsf?lle bisher die Toxintypen A, B, E und F beschrieben sind. Da es sich bei den genannten Keimen um strikt anaerob wachsende Bakterien handelt, stellen vor allem nicht kommerziell hergestellte Konserven, wie z. B. Kesselkonserven, ein Risiko für den Verbraucher dar. Als besondere Form des Botulismus wird der so genannte „S?uglingsbotulismus“ beschrieben. Im Gegensatz zur der Erkrankung, die bei Erwachsenen auftritt und die durch die Aufnahme des bereits toxinhaltigen Lebensmittels verursacht wird, k?nnen Sporen von C. botulinum im Darm von S?uglingen auskeimen und dort Toxine bilden. Ursache für den S?uglingsbotulismus ist h?ufig Honig, der als Naturprodukt C. botulinum-Sporen enthalten kann. Da der kulturelle Nachweis von C. botulinum aufwendig und eine endgültige Differenzierung schwierig ist, wird im Bereich der Routinediagnostik seit einigen Jahren verst?rkt mit PCR-Nachweisverfahren gearbeitet, die ein schnelles Screening auf das Vorhandensein der vier in der Humanmedizin relevanten Toxingene A, B, E und F erm?glichen. In dieser Arbeit werden zwei real-time-PCR-Systeme vorgestellt, die im Bereich der Routinediagnostik einsetzbar sind.

---

Eingegangen: 19. Januar 2007     

Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.     

Identification and characterization of a chitinase of Stenotrophomonas maltophilia, a bacterium that is antagonistic towards fungal phytopathogens

A rhizosphere strain of Stenotrophomonas maltophilia strain MUJ that is strongly antagonistic towards fungal phytopathogens secretes to the culture medium a single form of active chitinolytic enzyme belonging to family 18 of glycosyl hydrolases. The chitinase was purified by a two-stage procedure embracing fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme determined by SDS-PAGE was approximately 52 kDa. The enzyme demonstrated highest activity at 45°C and pH 6.8. The enzymatic protein showed considerable thermal stability during 2 h incubation at 45°C. The activity of the enzyme was strongly inhibited in the presence of Hg²⁺ and Cu²⁺. By applying mass spectrometry analysis, the peptides derived from the purified chitinase were assigned to amino acid sequences of the type ChiA chitinases synthesized by Stenotrophomonas bacteria. The purified enzyme inhibited the growth of fungal phytopathogens belonging to the genera Fusarium, Rhizoctonia and Alternaria.     

Nachweis und Charakterisierung von Clostridium perfringens mittels real-time-PCR

Clostridium perfringens is a Gram-positive, anaerobic bacterium. The strains are classified into five types (type A to E), depending on the ability to produce alpha-, beta-, epsiloon and iota-toxin, but food-poisonings are mostly caused by C. perfringens type A isolates. Ingestion of contaminated food is followed by gastrointestinal disease, when enzyme-resistant C. perfringens enterotoxins (CpE) are set free during sporulation. In most cases the bacterium has to grow up to more than 10⁶ cfu/g food to cause a gastrointestinal disease (BVET, 2005; BfR, 2005). Cultural methods, normally used for the detection of C. perfringens, are not able to differentiate isolates into the five different toxin types. This is the reason, why the detection of C. perfringens in food under the present legal regulations can only used as an indication for inadequate production hygiene or a toxin-infection with contaminated food as source. For the detection of the toxin genes to differentiate C. perfringens in five different toxin types, three duplex real-time-PCR assays for the routine diagnostic were developed and validated. The assays can be used for quick detection and easy classification of C. perfringens isolates and as a rapid screening-system in suspected cases of C. perfringens food-poisonings.

---

Zuszammenfassung:  Clostridium perfringens geh?rt zur Gruppe der gram-positiven, anaerob bzw. aerotolerant wachsenden Bakterien. Abh?ngig von der F?higkeit der einzelnen C. perfringens St?mme, alpha-, beta-, epsilon und iota-Toxin zu bilden, erfolgt eine Einteilung in die Typen A bis E, wobei C. perfringens Typ A für den überwiegenden Teil der lebensmittelassoziierten menschlichen Erkrankungen verantwortlich ist. Ursache solcher Toxin-Infektionen ist das erst im Darm im Rahmen der Sporulation der mit den Lebensmitteln aufgenommenen vegetativen Zellen gebildete Enterotoxin (CpE). In der Regel muss sich der Erreger im Lebensmittel auf mehr als 10⁶ KbE/g Lebensmittel vermehren, damit beim Konsumenten eine Erkrankung ausgel?st wird (BVET, 2005; BfR, 2005). Da die derzeit vorhandenen kulturellen Nachweisverfahren keine M?glichkeit bieten, die gewonnenen Isolate einem bestimmten Toxintyp zuzuordnen, kann der Nachweis von C. perfringens in einem Lebensmittel nach gültiger Rechtslage lediglich als ein Hinweis auf eine mangelhafte Produktions- oder Verarbeitungtechnologie oder auf das Vorliegen einer lebensmittelbedingten Toxin-Infektion gewertet werden. Zum Nachweis der Toxingene, die eine Einteilung von C. perfringens in die fünf verschiedene Toxintypen erm?glichen, wurden drei Duplex-real-time-PCR-Assays etabliert und die Einsatzm?glichkeiten in der Routinediagnostik getestet. Die Systeme sollen für einen schnellen Nachweis und eine rasche Differenzierung von C. perfringens sowie als Screening-System bei Verdacht auf eine lebensmittelbedingte Erkrankung genutzt werden.

---

Eingegangen: 30. Januar 2007     

Prevalence of Clostridium difficile in retailed meat in the Netherlands

Recent reports indicate that a large proportion of community-acquired Clostridium difficile infections (CA-CDI) are not linked to recent antibiotic therapy, older age, significant comorbidity or previous hospitalization. Possible community sources for CA-CDI include animals and food, and therefore a surveillance study on the prevalence of C. difficile in meat was performed. Samples of different meat species were collected from the retail trade and analyzed for the presence of C. difficile using a method that included selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping. Of 500 samples tested, 8 (1.6%) were positive for the presence of C. difficile: 1 from lamb (6.3%) and 7 from chicken meat (2.7%). The isolated strains belonged to PCR ribotypes different from those that are currently most frequently found in patients with CDI in the Netherlands, except for C. difficile PCR ribotype 001 which was found in one chicken meat sample. This observation suggests that other matrices than meat may serve as a source for CA-CDI.     

Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria

A new real-time PCR assay was developed targeted to the psychrotolerant spoilage bacteria, Clostridum estertheticum, a causative agent of 'blown-pack' spoilage of vacuum packaged meats during chilled storage. Further, a robust validation of the sensitivity and specificity in different meat processing related matrices was carried out. Results show that real-time PCR is a valid method for the detection of C. estertheticum spores as long as consideration is given to the matrix being tested and the sensitivity of detection required. For meat, hide, blood/drip and environmental swabs it was possible to detect low numbers of C. estertheticum spores (approx 3 spores per ml) by direct real-time PCR (without pre-enrichment of the samples). For faeces and soil matrices, a cold temperature enrichment step was required prior to DNA extraction and real-time PCR analysis to increase the ability to detect samples containing C. estertheticum spores; this was particularly important when the samples contained low numbers of spores (less than 3 spores per ml). For matrices with high levels of PCR inhibitors such as soil, it was necessary to dilute the extracted DNA sample 100 fold especially for detection of high levels of contamination (greater than 10(3)per ml) otherwise a pre-enrichment was required.     

Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE

The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 °C and showed a higher k_cat/K_m value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.     

cspB encodes a major cold shock protein in Clostridium botulinum ATCC 3502

The relative expression of three cold shock protein coding genes (cspA, cspB and cspC) of Clostridium botulinum ATCC 3502 was studied with quantitative RT-PCR analysis following a cold shock shift from 37 °C to 15 °C. A significant increase in the relative expression of all three genes was observed upon the temperature downshift. To validate these findings, single-gene insertional inactivation of cspA, cspB and cspC was undertaken with the ClosTron gene knock-out system. In growth experiments, mutations in cspB or cspC, but not cspA, resulted in a cold-sensitive phenotype. No growth of the cspB mutant was observed at 15 °C over a ten day period, whereas at 20 °C the growth rate was 70% lower than that of wild type strain. The growth rate of cspC mutant was 70% and 80% lower than the growth rate of the wild type strain at 15 °C and 20 °C, respectively. At 37 °C the growth of cspB mutant did not differ from, but the growth rate of cspC mutant was 30% lower than, that of the wild type strain. The cspA mutant grew somewhat faster than the wild type strain at all studied temperatures. Since the inactivation of cspB resulted in the most prominent defect in growth at low temperatures, we suggest that cspB encodes the major cold shock protein of C. botulinum ATCC 3502. Understanding the mechanisms behind cold tolerance of C. botulinum helps to evaluate the safety risks this foodborne pathogen poses in the modern food industry.     

Involvement of Clostridium gasigenes and C. algidicarnis in 'blown pack' spoilage of Brazilian vacuum-packed beef

The objectives of this study were to isolate psychrotrophic clostridia from Brazilian vacuum-packed beef cuts (spoiled or not) and to identify the isolates by using 16S rRNA gene sequencing. Anaerobic psychrotrophic microorganisms were also enumerated and samples were collected to verify the incidence of psychrotrophic clostridia in the abattoir environment. Vacuum-packed beef cuts (n = 8 grossly distended and n = 5 non-spoiled) and environmental samples were obtained from a beef packing plant located in the state of São Paulo, Brazil. Each sample was divided in three subsamples (exudate, beef surface and beef core) that were analyzed for vegetative forms, total spore-forming, and sulfide reducing spore-forming, both activated by alcohol and heat. Biochemical profiles of the isolates were obtained using API20A, with further identification using 16S rRNA gene sequencing. The growth temperature and the pH range were also assessed. Populations of psychrotrophic anaerobic vegetative microorganisms of up to 10¹⁰ CFU/(g, mL or 100 cm²) were found in ‘blown pack’ samples, while in non-spoiled samples populations of 10⁵ CFU/(g, CFU/mL or CFU/100cm²) was found. Overall, a higher population of total spores and sulfide reducing spores activated by heat in spoiled samples was found. Clostridium gasigenes (n = 10) and C. algidicarnis (n = 2) were identified using 16S rRNA gene sequencing. Among the ten C. gasigenes isolates, six were from spoiled samples (C1, C2 and C9), two were isolated from non-spoiled samples (C4 and C5) and two were isolated from the hide and the abattoir corridor/beef cut conveyor belt. C. algidicarnis was recovered from spoiled beef packs (C2). Although some samples (C3, C7, C10 and C14) presented signs of ‘blown pack’ spoilage, Clostridium was not recovered. C. algidicarnis (n = 1) and C. gasigenes (n = 9) isolates have shown a psychrotrophic behavior, grew in the range 6.2-8.2. This is the first report on the isolation of psychrotrophic Clostridium (C. gasigenes and C. algidicarnis) in Brazil. This study shows that psychrotrophic Clostridium may pose a risk for the stability of vacuum-packed beef produced in tropical countries during shelf-life and highlights the need of adopting control measures to reduce their incidence in abattoir and the occurrence of ‘blown pack’ spoilage.     

Inhibitory effects of polyphosphates on Clostridium perfringens growth, sporulation and spore outgrowth

This study evaluated the effects of various polyphosphates (SPP, STPP, SAPP and TSPP) on growth, sporulation and spore germination of Clostridium perfringens, and germination and outgrowth of C. perfrinegns spores in poultry meat. We have found that the requirements of polyP (0.8–1.0%) to inhibit C. perfringens bacterial growth were higher than those reported for other bacteria. Sub-lethal concentrations of polyP significantly (p<0.01) inhibited sporulation of C. perfringens by reducing sporulating cells (heat-resistant cells) 5–6 log₁₀. While C. perfringens spores were able to germinate in the presence of 1% STPP, their outgrowth was significantly (p<0.01) inhibited. Finally, a significant (p<0.01) reduction of survival of C. perfringens was observed when meat samples contaminated with a cocktail of spores of C. perfringens isolates carrying enterotoxin gene on the chromosome were treated with 1% STPP. Collectively, this study demonstrated the inhibitory effects of polyP on growth, sporulation and spore outgrowth of C. perfringens, and suggests that polyP can be used not only as an enhancer of the functional properties of meat products, but also as a promising C. perfringens antimicrobial agent.     

Occurrence of toxigenic Clostridium difficile in edible bivalve molluscs

Clostridium difficile is an anaerobic bacterium commonly considered to be responsible for antibiotic-associated gastrointestinal diseases, ranging from diarrhea of varying severity to pseudomembranous colitis. The aim of this study was to assess the occurrence of C. difficile in marine edible bivalve molluscs, which, as filter feeding organisms, are able to accumulate particles suspended in water, including microorganisms. Samples of Mytilus galloprovincialis, Tapes philippinarum, and Venus verrucosa were collected from mussel farms and fishmongers in the province of Naples (Southern Italy). C. difficile was found in 49% of the 53 samples investigated. Sixteen isolates were grouped in 12 known different PCR ribotypes (001, 002, 003, 010, 012, 014/020, 018, 045, 070, 078, 106, and 126), whereas 10 additional isolates were grouped in 8 new PCR riboprofiles. Two toxinotypes (0 and V) were found. Fifty eight percent of the isolates were toxigenic. These findings indicate that toxigenic C. difficile strains can be isolated in bivalve molluscs. Marine filter feeding organisms, therefore, may be considered as reservoir of toxigenic strains of C. difficile. The ingestion of raw or poorly cooked contaminated seafood and the high temperature resistance of the spore-forming C. difficile could represent an important source of exposure and pose human health concern.     

Characterization of a marine origin aerobic nitrifying-denitrifying bacterium

The bacterial strain F6 was isolated from a biological aerated filter that is used for purifying recirculating water in a marine aquaculture system and was identified as Marinobacter sp. based on the analysis of its 16S rRNA gene sequence. Strain F6 showed efficient aerobic denitrifying ability. One hundred percent of nitrates and 73.10% of nitrites were removed, and the total nitrogen (TN) removal rates reached 50.08% and 33.03% under a high nitrate and nitrite concentration in the medium, respectively. N(2)O and (15)N(2), as revealed by GC-MS and GC-IRMS, were the products of aerobic denitrification. Factors affecting the growth and aerobic denitrifying performance of strain F6 were investigated. The results showed that the optimum aerobic denitrification conditions for strain F6 were the presence of sodium succinate as a carbon source, a C/N ratio of 15, salinity ranging from 32-35?g/L of NaCl, incubation temperature of 30°C, an initial pH of 7.5, and rotation speed of 150?rpm [dissolved oxygen (DO) 6.75?mg/L]. In addition, strain F6 was confirmed to be a heterotrophic nitrifier through its NO(2)(-) generation and 25.96% TN removal when NH(4)(+) was used as the sole N source. Therefore, strain F6, the first reported member of genus Marinobacter with aerobic heterotrophic nitrifying-denitrifying ability, is an excellent candidate for facilitating simultaneous nitrification and denitrification (SND) in industry and aquaculture wastewater.     

Effect of a bacteriocin produced by Pediococcus acidilactici against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat

The inhibitory effect of a bacteriocin, produced by Pediococcus acidilactici, against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat surface, was evaluated by in situ assays. Samples were incubated with the bacteriocin and then with a culture of the pathogenic bacteria. The treatment with 500, 1000 or 5000 bacteriocin units/ml (BU/ml) reduced the counts of L. monocytogenes after storage at 15°C during 72h by 1, 2 or 3 log cycles and with 1000 or 5000 BU/ml after storage at 4°C during 21 days by 2.5 or 3.5 log cycles, respectively, compared to the control. With C. perfringens a bacteriostatic effect could be observed.     

Molecular cloning, identification and characterization of 2-methyl-3-hydroxypyridine-5-carboxylic-acid-dioxygenase-coding gene from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

The gene (mlr6788) of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099 has been identified as a gene coding for 2-methyl-3-hydroxypyridine-5-carboxylic acid dioxygenase (MHPCO), the seventh enzyme in degradation pathway I for pyridoxine, a free form of vitamin B(6). The gene was cloned and overexpressed in Escherichia coli cells co-transformed with chaperonin genes. The homogeneous recombinant enzyme showed similar enzymatic properties to the enzyme from Pseudomonas sp. MA-1. MHPCO was essential for the assimilation of pyridoxine in M. loti, but not for its growth in a nutrient-rich medium. From the infection experiment of a symbiotic plant Lotus japonicus with an M. loti mlr6788 gene disruptant, MHPCO was demonstrated to be dispensable for at least nodule formation on roots of seedlings in symbiosis.     

Streptococcus thermophilus 580 produces a bacteriocin potentially suitable for inhibition of Clostridium tyrobutyricum in hard cheese

A strain of Streptococcus thermophilus that inhibits Clostridium tyrobutyricum has been isolated from raw milk. The active compound produced disappears after a treatment with protease. However, unlike most bacteriocins, it is not thermoresistant, and the activity is completely lost after 1 h at 60 degrees C. Its inhibitory spectrum is limited to other thermophilic streptococci, Brochothrix, and sporulated gram-positive rods. So this bacteriocin could be different from those already described. This bacteriocin-producing strain could be used in thermophilic starter for hard cheese making because the bacteriocin is not active against thermophilic lactobacilli. It is produced in M17 medium during the decreasing temperature phase of the hard cheese-making process temperature cycle and is also produced in milk. Moreover, when Streptococcus thermophilus was cocultured with a Lactobacillus delbrueckii subsp. lactis starter strain, it seems to enhance the bacteriocin production. However the level of activity always decreases drastically during the stationary phase. But inhibition of Clostridium tyrobutyricum spores can be obtained in small-scale curds.     

Honey major protein characterization and its application to adulteration detection

The major proteins in honey have different molecular weights depending upon the honeybee species. To confirm the origin of major honey proteins, honey protein produced by Apis cerana or Apis mellifera were purified and analyzed by MALDI-TOF. Two major proteins were identified as a major royal jelly protein 1. Although two major proteins shared primary structure, they showed different molecular weights of 56 and 59 kDa, respectively. To discriminate the honeybee species producing honey using SDS–PAGE, artificial marker proteins, 56 and 59 kDa, were produced from Escherichia coli. Two artificial marker proteins were co-electrophoresed with honey samples and the difference in molecular weight was readily distinguished by SDS–PAGE. Therefore, the measurement of major proteins in honey is a useful method to discriminate the honey that produced from different honeybee species.     

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO₂, ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO₂ in comparison to the other strains of Dekkera. These strains were inoculated (10³–10⁴ cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 °C and 21 °C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10⁵ cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 °C while slower growth was observed at 15 °C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 °C compared to 21 °C.     

Expression of a gene encoding chitinase (pCA 8 ORF) from Aeromonas sp. no. 10S-24 in Escherichia coli and enzyme characterization

A gene encoding chitinase from Aeromonas sp. no. 10S-24 was expressed using pTrc99A in Escherichia coli JM 105 which yielded a 5-fold higher activity than when pUC19 was used. Three different truncated enzymes (SA-1, SA-2 and SA-3) were obtained after purification. Their isoelectric points were 7.0, 6.9, and 6.7, respectively. The enzymes showed two optimum pHs, 4.0 and 7.0, when incubated with ethylene glycol chitin as the substrate, and were stable over a wide pH range (3.0–9.0). The optimum temperature was 60°C and the enzymes were stable up to 50°C. The chitinases exhibited wide substrate specificities for chitin-related compounds.