Chitin-containing Medium Research Articles

Гидролиз и окисление трудноразлагаемых субстратов микробными изолятами термальных источников

Traditional energy sources pollute the environment. Microbial fuel cells are an alternative energy source that can reduce the environmental burden. Microbial fuel cells also remove recalcitrant wastes from wastewater. This research featured the enzymatic potential of microbial isolates obtained from the Abakan Arzhan thermal spring.
 The study involved isolates of the genera Geobacter, Thermomonas, and Rhodopseudomonas. The keratinolytic analysis was in line with State Standard R 55987-2014. The chitinolytic activity was determined by injecting a bacterial suspension on Petri dishes with a chitin-containing medium. The lipolytic analysis involved cultivating the isolates in Stern’s glycerol fuchsin broth. The xylan hydrolysis depended on the reducing sugars. The cellulase activity was measured according to the standard method recom mended by the International Union of Pure and Applied Chemistry (IUPAC). The catalase potential was evaluated by the gasometric method on 1% gasoline media. The optimal parameters of consortium cultivation were determined by the voltage generated.
 The Geobacter isolate had the maximal keratinolytic activity while the Thermomonas isolate demonstrated the maximal protein hydrolysis (80.1 ± 1.5%). Both Geobacter and Rhodopseudomonas showed good lytic activity against chitin with the lysis zone of ≥ 3 mm. The Geobacter isolate demonstrated as many as 350 units of xylanase activity and 365 units of cellulase activity; Ther momonas had 350 units of xylanase activity and 360 units of cellulase activity; Rhodopseudomonas showed 310 units of xylanase activity and 304 units of cellulase activity. The maximal catalase properties belonged to Geobacter (1.40 units) and Thermomonas (1.38 units). The maximal energy generation by bacterial consortia occurred at pH 8 and 45°C after 48 h of cultivation. 
 In this research, isolates of the genera Geobacter, Thermomonas, and Rhodopseudomonas from the Abakan Arzhan thermal spring were able to remove recalcitrant components, thus demonstrating good prospects for biological treatment of industrial wastewater.

Morphological and structural characterization of chitin as a substrate for the screening, production, and molecular characterization of chitinase by Bacillus velezensis.

The processing of shellfishery industrial wastes is gaining much interest in recent times due to the presence of valuable components. Chitin is one of the valuable components and is insoluble in most common solvents including water. In this study, a novel gram-positive bacterial strain capable of solubilizing chitin was screened from a prawn shell dumping yard. The chitinolytic activity of the isolated strain was observed through the zone of hydrolysis plate assay. The hyper-producing isolate was identified as Bacillus velezensis through the 16S rRNA sequencing technique. The structural and morphological characterization of raw and colloidal chitin preparation was carried out using FTIR, XRD, and SEM analysis. The residual protein and mineral content, degree of polymerization, and degree of acetylation were reported for both raw and colloidal chitin preparations. There was a linear increase in the chitinase activity with an increase in the colloidal chitin concentration. The maximum activity of chitinase was observed as 38.98 U/mL for the initial colloidal chitin concentration of 1.5%. Supplement of additional carbon sources, viz., glucose and maltose, did not improve the production of chitinase and resulted in a diauxic growth pattern. The maximum chitinase activity was observed to be 33.10 and 30.28 U/mL in the colloidal chitin-containing medium with and without glucose as a secondary carbon source, respectively. Interestingly, the addition of complex nitrogen sources has increased the production of chitinase. A 1.95- and 2.14-fold increase in the enzyme activity was observed with peptone and yeast extract, respectively. The chitinase was confirmed using SDS-PAGE, native PAGE, and zymograms. The optimum pH and temperature for chitinase enzyme activity were found to be 7.0 and 44°C, respectively.

A Broad-Specificity Chitinase from Penicillium oxalicum k10 Exhibits Antifungal Activity and Biodegradation Properties of Chitin.

Penicillium oxalicum k10 isolated from soil revealed the hydrolyzing ability of shrimp chitin and antifungal activity against Sclerotinia sclerotiorum. The k10 chitinase was produced from a powder chitin-containing medium and purified by ammonium sulfate precipitation and column chromatography. The purified chitinase showed maximal activity toward colloidal chitin at pH 5 and 40 °C. The enzymatic activity was enhanced by potassium and zinc, and it was inhibited by silver, iron, and copper. The chitinase could convert colloidal chitin to N-acetylglucosamine (GlcNAc), (GlcNAc)2, and (GlcNAc)3, showing that this enzyme had endocleavage and exocleavage activities. In addition, the chitinase prevented the mycelial growth of the phytopathogenic fungi S. sclerotiorum and Mucor circinelloides. These results indicate that k10 is a potential candidate for producing chitinase that could be useful for generating chitooligosaccharides from chitinous waste and functions as a fungicide.

Increasing Chitinase Activity of Serratia marcescens PT-6 through Optimization of Medium Composition

Chitin hydrolysate is one of the value added product derived from shrimp shell waste. Production of chitin hydrolysate using biological process offers an environmental friendly method compared to chemical process. Serratia marcescens PT-6, a gram negative chitinolytic bacterium isolated from shrimp pond sediment, shows good activity in hydrolyzing chitin. This study aimed to improve the chitinase activity of S. marcescens PT-6 culture by optimizing the component of chitin-containing medium (additional nitrogen source, additional carbon source, and colloidal chitin). The optimization of chitinase by S. marcescens PT-6 culture was done using one variable at a time method. The sequence of the research were to optimize 1) the type of additional carbon source (glucose, lactose, sucrose, and starch), 2) the type of additional nitrogen source (yeast extract, peptone, ammonium sulphate, and ammonium chloride), 3) the concentration of colloidal chitin (0.5; 1; 1.5; 2; and 2.5%), and 4) the concentration of the additional carbon and nitrogen source. The culture of S. marcescens PT-6 was incubated in colloidal chitin medium at 30 oC and chitinase activity from culture supernatant was analyzed. The results showed that starch gave the highest chitinase activity compare to other carbon source, meanwhile yeast extract was chosen as the best nitrogen source among others. The combination of 1.5% colloidal chitin with 0.5% starch and 0.1% yeast extract in medium increased the chitinase activity of S. marcescens PT-6 to 0.021 U/ml. These results indicated that an appropriate medium composition could increase the chitinase activity produced by S. marcescens PT-6 culture.

Identification of the glucosamine kinase in the chitinolytic pathway of Thermococcus kodakarensis

Although the chitinolytic pathway of the hyperthermophilic archaeon Thermococcus kodakarensis is well-studied, the genome does not contain genes homologous to previously identified glucosamine kinase genes. As some ADP-dependent glucokinases in the order Thermococcales exhibit phosphorylation activities for both glucose and glucosamine invitro, the homolog in T.kodakarensis, encoded by TK1110, was selected as a candidate for the missing glucosamine kinase gene. The purified, recombinant TK1110 enzyme exhibited phosphorylation activities for not only glucose but also glucosamine and N-acetylglucosamine. Kinetic analysis indicated that activity towards glucosamine was as significant as that towards glucose. In order to determine the physiological role of TK1110 in the chitinolytic pathway of T.kodakarensis, a gene disruption strain of TK1110 was constructed. When grown in chitin-containing medium, the TK1110 disruption resulted in almost complete impairment in chitin degradation, and a complete loss of chitin-dependent H2 production. As H2 production is tightly linked to cell growth in T.kodakarensis, the present results strongly suggest that TK1110 functions as the glucosamine kinase responsible for the chitin degradation in T.kodakarensis.

Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase

Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and ChiB) and a multi-modular lytic polysaccharide monooxygenase (LmLPMO10). These enzymes have been related to virulence and their role in chitin metabolism is poorly understood. It is thus of interest to functionally characterize the individual enzymes in order to shed light on their roles in vivo. Our results demonstrate that L. monocytogenes has a fully functional chitinolytic system. Both chitinases show substrate degradation rates similar to those of the nonprocessive endo-chitinase SmChiC from Serratia marcescens. Compared to the S. marcescens LPMO chitin-binding protein CBP21, LmLPMO10 shows a similar rate but different product profiles depending on the substrate. In LPMO-chitinase synergy experiments, CBP21 is able to boost the activity of both ChiA and ChiB more than LmLPMO10. Product analysis of the synergy assays revealed that the chitinases were unable to efficiently hydrolyse the LPMO products (chitooligosaccharide aldonic acids) with a degree of polymerization below four (ChiA and SmChiC) or three (ChiB). Gene transcription and protein expression analysis showed that LmLPMO10 is neither highly transcribed, nor abundantly secreted during the growth of L. monocytogenes in a chitin-containing medium. The chitinases on the other hand are both abundantly secreted in the presence of chitin. Although LmLPMO10 is shown to promote chitin degradation in tandem with the chitinases in vitro, the secretome and transcription data question whether this is the primary role of LmLPMO10 in vivo.

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.

Methodological aspects of assessing chitin utilization by soil microorganisms

A computational method for estimating specific activity of chitin decomposition by microorganisms is proposed. Spectrophotometric and gas chromatographic methods have been used to determine the rates of chitinase production, biomass accumulation, and carbon dioxide emission by pure cultures of microorganisms grown on a chitin-containing medium. Among dominants of the chitinolytic community of chernozem (Trichoderma viride, Stretomyces albolongus, Alcaligenes, and Arthrobacter), the highest chitinolytic activity is characteristic of prokaryotes. In brown desert-steppe soil, the main destructor prokaryotes are actinomycetes (S. roseolilacinus). The biomass of the fungus T. viride growth on the chitin-containing medium markedly exceeds that of prokaryotes, but the specific activity of respiration and chitinase production in actinomycetes S. roseolilacinus and S. albolongus is an order of magnitude higher than in T. viride.

Extracellular hydrolases of strain Bacillus sp. 739 and their involvement in the lysis of micromycete cell walls

The mycolytic bacterial strain Bacillus sp. 739 produces extracellular enzymes which degrade in vitro the cell walls of a number of phytopathogenic and saprophytic fungi. When Bacillus sp. 739 was cultivated with Bipolaris sorokiniana, a cereal root-rot pathogen, the fungus degradation process correlated with the levels of the beta-1,3-glucanase and protease activity. The comparative characteristic of Bacillus sp. 739 enzymatic preparations showed that efficient hydrolysis of the fungus cell walls was the result of the action of the complex of enzymes produced by the strain when grown on chitin-containing media. Among the enzymes of this complex, chitinases and beta-1,3-glucanases hydrolyzed most actively the disintegrated cell walls of B. sorokiniana. However, only beta-1,3-glucanases were able to degrade the cell walls of native fungal mycelium in the absence of other hydrolases, which is indicative of their key role in the mycolytic activity of Bacillus sp. 739.

Optimization of cultivation conditions for the production of chitinase from Paenibacillus sp CHE-N1

A bacterial strain capable of secreting high levels of chitinase was isolated from chitin-enriched soil and identified as Paenibacillus sp. CHE-N1. This strain, which shows high potential as an antagonist against fungi, was grown in a chitin-containing medium to evaluate its ability to produce chitinase. The statistical design method was used to determine the optimal chitinase-producing conditions. Factors, including carbon sources and nitrogen sources in the medium compositions, initial pH, and the temperature of fermentation, were investigated. The obtained optimal conditions were: CSCP 55.4g/L, peptone 1.8g/L, and (NH4)2SO4 1.6g/L, with an optimal initial pH and temperature of 8.5 and 34.3℃, respectively. Under these conditions, the optimal volumetric enzyme activity could reach 8.1U/mL, about a 4-fold increase as compared to results under basic conditions.

Regulation of N-acetyl-β- d-glucosaminidase produced by Trichoderma harzianum: evidence that cAMP controls its expression

Trichoderma harzianum is a filamentous fungus reported to be a producer of extracellular N-acetyl-β- d-glucosaminidase (NAGase) when grown in chitin-containing medium. An approximately 64-kDa protein with NAGase activity was purified by gel filtration and ion exchange chromatography. The involvement of cyclic AMP (cAMP) in the synthesis of NAGase from T. harzianum in chitin-containing medium was also investigated. Molecules that increase the intracellular levels of cAMP, including caffeine, aluminium tetrafluoride and dinitrophenol, were used. Western blot analysis showed that NAGase synthesis was repressed by increasing the levels of intracellular cAMP. Using specific nag primers in a reverse transcription-polymerase chain reaction-based approach, NAGase synthesis was shown to be regulated at the level of gene transcription.

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a β-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract–peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS–PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10 μg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.

Transformants of Metarhizium anisopliae sf. anisopliae overexpressing chitinase from Metarhizium anisopliae sf. acridum show early induction of native chitinase but are not altered in pathogenicity to Manduca sexta.

Extracellular chitinase activity has been implicated in the pathogenesis of several fungal infections. Following induction with chitin, the insect pathogens Metarhizium anisopliae sf. acridum ARSEF strain 324 and Metarhizium anisopliae sf. anisopliae ARSEF strain 2575 secrete 44-kDa basic and acidic isoforms of endochitinase, respectively. The gene from strain 324 (Chit1) was cloned and inserted into the genome of strain 2575 under the control of Aspergillus regulatory elements such that transgenic 2575 (2575-Chit(+)) expressed CHIT1 in a noninducing medium (i.e., not containing chitin). Isoelectric focusing followed by a zymogram technique revealed that neither wild-type 2575 nor 2575-Chit(+) produced significant amounts of the native 2575 acidic chitinase in a noninducing medium. However, in a chitin-containing medium, 2575-Chit(+) produced the native chitinase earlier than strain 2575, soon after secretion of CHIT1. We hypothesize that this is due to the production of soluble inducers following chitin hydrolysis by CHIT1 and that M. anisopliae uses enzymes expressed at low levels to sense the nature of the polymeric nutrient present in the immediate environment. However, the chitinase overproducers did not show altered virulence to caterpillars (Manduca sexta) compared to the wild-type fungus, suggesting that wild-type levels of chitinase are not limiting for cuticle penetration.

Aquatic Actinomycete-Fungal Interactions and Their Effects on Organic Matter Decomposition: A Microcosm Study.

The role of fungi in the decomposition of organic matter in streams has been well examined, although the role of bacterial antagonists in such processes has gained little attention. To examine bacterial-fungal interactions, experiments involving pairwise combinations of four actinomycete isolates (A1+ and A2+ could remove chitin from chitin-containing media, and A1? and A2? could not) and two fungal isolates (F+ a true fungus, F? an oomycote) were conducted. For each bacterial-fungal combination, 250-ml microcosms were sampled at 8 day intervals for 32 days. Microbial biomass and organic matter, as well as the activities of five extracellular enzymes, were measured. Each experiment consisted of a control group and four treatment groups. Controls comprised sterilized stream water and macrophytes. The first treatment was inoculated with only actinomycetes (~103 cells ml-1), the second treatment was inoculated with only fungi (~102 cells ml-1), the third group was inoculated simultaneously with actinomycetes and fungi, and the fourth group was inoculated with actinomycetes 2 days after fungal establishment. For all combinations, the lowest rates of organic matter decomposition were expected in the controls, as a result of only physical degradation. In contrast, the greatest rates of organic matter decomposition were predicted in treatments inoculated with F+ 2 days prior to A1? or A2?. Greater than 50% of the organic matter was decomposed in each of the fungal treatments. Fungal-actinomycete interactions resulted in reduced fungal biomass relative to the fungal-only treatments. However, when inoculated 2 days apart, combinations of F? and actinomycetes resulted in enhanced rates of organic matter decomposition, as well as greater levels of extracellular enzyme activities. These results demonstrate that actinomycete-fungal interactions and their colonization dynamics affect the accumulation of biomass, extracellular enzyme activities, and rates of organic matter decomposition.

Antifungal activity of chitinases produced by some fluorescent pseudomonads against Colletotrichum falcatum Went causing red rot disease in sugarcane

Chitinase production and growth of certain fluorescent pseudomonads isolated from sugarcane rhizosphere on different subtrates were studied. When chitin was substituted for glycerol in King's B medium, 3 of the 4 strains showed enhanced bacterial multiplication. Bacterial cells grown on chitin-containing medium showed enhanced antifungal activity against Colletotrichum falcatum Went causing red rot disease in sugarcane. Chitinase production was significantly higher when chitin was amended to King's B medium. Higher chitinase production was also recorded when fluorescent pseudomonad strains were grown in the medium containing crab-shell chitin. Cell-free bacterial culture filtrate from chitin-containing medium significantly inhibited mycelial growth of the pathogen. These cell-free conditioned media contained 3 to 7 polypeptides. Western blot analysis revealed five isoforms of chitinase with molecular masses of 47, 36, 32, 20 and 18.5 kDa. A possible role of chitinases in red rot disease management is discussed.

Fusarium udum is resistant to the mycolytic activity of a biocontrol strain of Bacillus subtilis AF 1

In vitro interaction of the pigeonpea wilt pathogen Fusarium udum and a biocontrol strain of Bacillus subtilis AF 1 showed that the fungus forms chlamydospore-like structures and increases vacuolation, when both cultures are simultaneously inoculated into potato dextrose broth. If B. subtilis AF 1 was inoculated after 24 h of growth of F. udum, chlamydospores were not formed and regular conidiation was observed. The growth of F. udum was significantly affected in the presence of AF 1 in terms of dry weight. Extracellular proteins of B. subtilis AF 1 reduced the growth of F. udum in proportion to the concentration of the protein precipitate. Cell-free filtrates of B. subtilis AF 1 prepared from chitin-containing medium showed chitinase activity. The formation of chlamydospore-like structures and vacuolated portions in mycelium of F. udum in the presence of AF 1 suggests that F. udum has a mechanism to tolerate mycolytic activity and grows slowly with regular conidiation.

Fusarium udum is resistant to the mycolytic activity of a biocontrol strain of Bacillus subtilis AF 1

In vitro interaction of the pigeonpea wilt pathogen Fusarium udum and a biocontrol strain of Bacillus subtilis AF 1 showed that the fungus forms chlamydospore-like structures and increases vacuolation, when both cultures are simultaneously inoculated into potato dextrose broth. If B. subtilis AF 1 was inoculated after 24 h of growth of F. udum, chlamydospores were not formed and regular conidiation was observed. The growth of F. udum was significantly affected in the presence of AF 1 in terms of dry weight. Extracellular proteins of B. subtilis AF 1 reduced the growth of F. udum in proportion to the concentration of the protein precipitate. Cell-free filtrates of B. subtilis AF 1 prepared from chitin-containing medium showed chitinase activity. The formation of chlamydospore-like structures and vacuolated portions in mycelium of F. udum in the presence of AF 1 suggests that F. udum has a mechanism to tolerate mycolytic activity and grows slowly with regular conidiation.

Properties of the chitinase of the antifungal biocontrol agent Streptomyces lydicus WYEC108

An extracellular chitinase from culture filtrates of Streptomyces lydicus WYEC108, a broad spectrum antifungal biocontrol agent, was characterized and purified. Its role in the antifungal activity of this actinomycete was studied. Low constitutive levels of the enzyme were observed when cultures were grown with both simple and complex carbon substrates. The optimal temperature and substrate concentration for maximal chitinase production were 25–30°C and 0.4–0.8 g ml −1 chitin, respectively. High chitinase production was obtained when 1% colloidal chitin was present in the medium as a growth substrate. Activity was induced by n-acetylglucosamine or n,n′-diacetylchitobiose (GlcNac) 2 and repressed by glucose, xylose, arabinose, raffinose, and carboxymethyl cellulose. Strong catabolite repression of the chitinase was observed. Addition of pectin, laminarin, starch, or β-glucan to the chitin-containing medium, however, increased chitinase production. Probing the S. lydicus genomic DNA with the chiA gene from S. lividans has localized the gene to a 2.5 kb DNA fragment of genomic DNA. The chitinase appears to play a role in the antifungal activities of S. lydicus WYEC108. Production was greatly enhanced when cells were grown in a medium containing colloidal chitin supplemented with certain fungal cell wall preparations, in particular those from Pythium or Aphanomyces species. Crude fungal cell walls were lysed by partially purified chitinase. While S. lydicus also produces one or more antifungal antibiotics, its chitinase probably plays a significant role in the in vivo antifungal biocontrol activity of this rhizosphere-colonizing actinomycete.

Production of N-Acetylglucosamine Deacetylase by Vibrio cholerae Non-O1.

Vibrio cholerae non-O1 (1148 A) produced β-N-acetylglucosamini-dase and N-acetylglucosamine (GlcNAc) deacetylase intracellularly when grown in chitin or GlcNAc containing medium. It also secreted chitinase only in the chitin-containing medium. The partially purified GlcNAc deacetylase deacetylated GlcNAc but not chitin oligosaccharides, the dimer to hexamer of GlcNAc. We also detected the reaction product by capillary electrophoresis.

Endochitinase fromGliocladium virens: Isolation, Characterization, and Synergistic Antifungal Activity in Combination with Gliotoxin

The culture filtrate from the biocontrol agent Gliocladium virens strain 41 grown in chitin-containing medium was strongly inhibitory to mycelial growth of different plant-pathogenic fungi. The antibiotic gliotoxin was isolated from the culture liquid. The culture filtrate also contained different types of chitinolytic enzyme activities, including endochitinase, chitin 1,4β-chitobiosidase, and glucan N-acetyl-β-D-glucosaminidase, as well as glucan 1,3-β-glucosidase activity. An endochitinase was purified to homogeneity []