The laminarinase system of Streptomyces sp., ATCC 11238

Summary Extracellular proteins from Streptomyces sp. ATCC 11238 grown on fungal mycelia and chitin as C- and N-sources were concentrated by ultrafiltration and acetone precipitation. The crude preparation containing chitin and laminarin degrading enzymes was fractionated by repeated gel filtrations. Three different types of -1,3-glucanases were found. Besides oligomeric breakdown products laminaritriose is the main product of laminarin hydrolysis by one endo--1,3-glucanase. A second laminarin degrading (exo-splitting) enzyme yields predominantly laminaribiose. Another exo--1,3-glucanase liberates glucose but no, oligosaccharides from the nonreducing end of laminarin.     

Localization and release mechanism of cellulases in Trichoderma reesei QM 9414

Summary A significant increase in the extracellular yield of -glucosidase was observed when Trichoderma reesei QM 9414 was cultivated on a cellulose medium containing chitin. Measurement of enzyme activities in the various fractions of the mycelium revealed that endoglucanase was truly extracellular while -glucosidase was cell wall bound. Treatment of Trichoderma mycelium with cell wall degrading enzymes (produced from Trichoderma) led to a release of -glucosidase from the mycelium. Apparently chitin, in the presence of cellulose, induces the synthesis of chitinase and other cell wall lytic enzymes which promote release of the intramural -glucosidase into the medium.     

A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa,the causal agent of witches' broom disease of cocoa

A Trichoderma harzianum isolate (1051), which was able to antagonize in field the phytopathogen Crinipellis perniciosa, the causal agent of witches' broom disease of cocoa, produces several hydrolytic enzymes. A chitinase, with molecular mass of about 37 kDA, which was secreted by the Trichoderma in the culture medium containing chitin, was partially purified by gel filtration followed by hydrophobic chromatography. The optimal pH and temperature for chitin hydrolysis by the partially purified enzyme were 4.0 and 37 °C, respectively. Chitobiose, laminarin, cellulosic substrates including aryl-glucosides, xylan, starch and -galactomannan were not hydrolysed by the enzyme. Remarkably, the partially purified enzyme drastically affected the cell wall of the phytopathogen C. perniciosa in vitro.     

Two simple and rapid methods for the detection of polymer-degrading enzymes on high-resolution, alkaline, cold, in situ-native (HiRACIN)-PAGE and high-resolution, in situ-inhibited native (HiRISIN)-PAGE

Two sensitive, high-resolution and exceedingly versatile methods for the detection of isoenzymes of polymer-degrading enzymes on high-resolution, alkaline, cold, in situ-native (HiRACIN)-PAGE and high-resolution in situ-inhibited, native (HiRISIN)-PAGE are described. Extracellular crude extracts containing xylanases and carboxymethylcellulases from Scopulariopsis sp. and glucoamylases from Aspergillus niger were subjected to non-denaturing PAGE containing substrates in the resolving gel. In case of HiRACIN-PAGE, the enzymes were prevented from degrading their respective substrates during run by carrying out electrophoresis at 4°C and the pH of running and resolving gel buffer systems were increased from 8.5 to 10.6. In case of HiRISIN-PAGE, adding competitive inhibitor of the enzyme, cellobiose, in the resolving gel prevents the degradation of polymer during the run. These techniques were successfully applied, for the first time, to visualize four, three and four sharp and distinct bands of xylanases, glucoamylases and CMCases, respectively.     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.     

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.     

Purification and characterization of two chitinases from Streptomyces albovinaceus S-22

Two chitinases, A and B, were purified from the culture supernatant of Streptomyces albovinaceus S-22 by ammonium sulphate fraction (80%) and Sephadex G-200 gel filtration. Both enzymes had molecular weights estimated to be 43 and 45kDa by SDS polyacrylamide gel electrophoresis. The enzymes were active at 40°C and pH 5.6 after 120min, and stable at temperatures below 40°C in the absence of chitin. The purified enzyme had potential for cell wall lysis of fungal pathogenesis tested.     

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.     

Mode of action and antifungal properties of two cold-adapted chitinases

The mode of action of two chitinases from the Antarctic Arthrobacter sp. strain TAD20 on N-acetyl-chitooligomers and chitin polymers has been elucidated. Identification of the length of chitin oligomers following enzymatic hydrolysis was verified by using HPLC-based analysis. It was observed that the length of the oligomer is important for enzyme action. The enzymes cannot effectively hydrolyze chitin oligomers with a degree of polymerization lower than four. ArChiA is an endochitinase which hydrolyzes chitin substrates randomly, whereas ArChiB is an exochitinase which degrades chitin chains and N-acetyl-chitooligomers from the nonreducing end, releasing N-N-diacetyl-chitobiose. ArChiB (100 g/ml) inhibited spore germination and hyphal elongation of the phytopathogenic fungus Botrytis cinerea by 15% and 30%, respectively. A more pronounced effect was observed with ArChiA (100 g/ml) resulting in 70% inhibition of spore germination and 60% inhibition of germ tube elongation. A slight additive effect was observed, when the two enzymes were used in combination, only on the inhibition of germ tube elongation.Communicated by G. Antranikian     

Moult cycle and seasonal activities of chitinolytic enzymes in the integument and digestive tract of the Antarctic krill,Euphausia superba

Summary Chitinolytic activity was quantified in euphausiid integuments in relation to moulting. In Euphausia superba, shortly before moult the activity increased in chitinase and N-acetyl--D-glucosaminidase to pronounced maxima indicating the onset of massive resorption of cuticular material. Enzymatic activity of E. superba corresponded to values in Meganyctiphanes norvegica, a boreal euphausiid which was investigated for comparison, as well as in insecta. Antarctic krill from winter catches displayed activities comparable to summer material suggesting physiological preparation for moulting. Accordingly, moulting did not cease during winter. Both enzymes were also active in the digestive tract in summer as well as in winter krill: chitin containing food of phyto-and zooplankton origin is digestable. Seasonally stable activities did not point to changes in nutritional preference. In contrast to other crustacea, digestive enzyme activity was not reduced around moult, suggesting a high capacity to continuously utilize food sources including chitin. This property can be linked directly to the high energy need caused by the necessity of constant active swimming in both krill species.Supported by German Research Counsil (DFG), grant-nos. Ad 24/9 and Bu 548/1Dedicated to Professor Dr. G. Hempel on the occasion of his 60th birthday     

Electrophoretic Studies of Alkaline Proteinases from Strains of Aspergillus flavus Group

Entevobacter sp. G-1 which produces chitinolytic and chitosanolytic enzymes, was previously isolated in our laboratory. One major chitinase, designated ChiA, was purified 42.9-fold from a culture filtrate of Entevobacter sp. G-L To purify the chitinase, ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration on Sephadex G-100 column chromatography were used. The ChiA protein had a molecular weight of 60,000 estimated by SDS polyacrylamide gel electrophoresis and an isoelectric point of 6.6. The optimal pH and optimal temperature of ChiA against colloidal chitin were pH 7.0, and 40°C, respectively. The purified ChiA degraded colloidal chitin mainly to GlcNAc₂ with a small amount of GlcNAc₃ and GlcNAc₄. ChiA hydrolyzed flaked chitin, colloidal chitin, and ethylenglycol chitin, but did not hydrolyze carboxymethyl cellulose (CMC), nor >90% deacetylated flaked chitosan. The chitinase activity was 42% inhibited by 10mm EDTA, but was not inhibited by Ca²⁺ (<50 mm) or NaCl (<400 mm). The purified ChiA hydrolyzed colloidal chitin and chitin-related compounds in an endo splitting manner.     

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 °C, but N,N-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)₂] were selectively produced from chitin. At 45 °C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 °C (GlcNAc)₂ was the major product (86%) with a yield of 35% within 5 d.Revisions requested 29 September 2004; Revisions received 1 November 2004     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Genomic comparison of chitinolytic enzyme systems from terrestrial and aquatic bacteria

Chitin degradation ability is known for many aquatic and terrestrial bacterial species. However, differences in the composition of chitin resources between aquatic (mainly exoskeletons of crustaceans) and terrestrial (mainly fungal cell walls) habitats may have resulted in adaptation of chitinolytic enzyme systems to the prevalent resources. We screened publicly available terrestrial and aquatic chitinase‐containing bacterial genomes for possible differences in the composition of their chitinolytic enzyme systems. The results show significant differences between terrestrial and aquatic bacterial genomes in the modular composition of chitinases (i.e. presence of different types of carbohydrate binding modules). Terrestrial Actinobacteria appear to be best adapted to use a wide variety of chitin resources as they have the highest number of chitinase genes, the highest diversity of associated carbohydrate‐binding modules and the highest number of CBM33‐type lytic polysaccharide monooxygenases. A ctinobacteria do also have the highest fraction of genomes containing β‐1, 3‐glucanases, enzymes that may reinforce the potential for degrading fungal cell walls. The fraction of bacterial chitinase‐containing genomes encoding polyketide synthases was much higher for terrestrial bacteria than for aquatic ones supporting the idea that the combined production of antibiotics and cell‐wall degrading chitinases can be an important strategy in antagonistic interactions with fungi.     

Influence of manganese on morphology and cell wall composition of Aspergillus niger during citric acid fermentation

Morphology and cell wall composition of Aspergillus niger were studied under conditions of manganese sufficient or deficient cultivation in an otherwise citric acid producing medium. Omission of Mn²⁺ (less than 10^-7 M) from the nutrient medium of Aspergillus niger results in abnormal morphological development which is characterized by increased spore swelling, and squat, bulbeous hyphae. Fractionation and analysis of manganese deficient cell walls revealed increased chitin and reduced -glucan contents as well as reduction of galactose containing polymers, as compared to cell walls from manganese sufficient grown hyphae. Addition of copper induced the same effect as manganese deficiency, both on morphology and cell wall composition. Addition of cycloheximide also produced a very similar type of morphology with increased chitin and reduced -glucan contents of the cell wall but its effect on galactose was less pronounced.Dedicated to emer. Prof. Dr. J. Kisser on the occasion of his 80th birthday     

The effect of acquired microbial enzymes on assimilation efficiency in the common woodlouse,Tracheoniscus rathkei

Summary The digestive tract of the common woodlouse, Tracheoniscus rathkei Brandt (Isopoda: Oniscoidea), contains digestive enzymes active against -1,4-glucans, which are the chief storage polysaccharides of vascular plants, algae, fungi, and animals, and -1,3-glucans, which are present in algae and fungi. Digestive tract extracts also exhibit significant activity toward xylan and carboxymethyl-cellulose but negligible activity toward microcrystalline cellulose, substrates representative of the major structural polysaccharides of vascular plants. Low activity was detected toward pectin, and no activity was detected toward chitin. Activity toward xylan is due in part to microbial enzymes acquired from the leaf litter which was the isopod's normal food. Although ingested microbial xylanases are stable and active in the gut fluid, they do not make a quantitatively significant contribution to the isopod's ability to assimilate the hemicellulosic component of its diet. However, the assimilation of carbon from labeled plant fiber is enhanced in isopods which have acquired a cellulase by ingestion of leaf litter amended with a commercial preparation of the cellulase complex from the fungus, Penicillium funiculosum. This result demonstrates the potential contribution of acquired enzymes to the digestion of plant fiber in terrestrial detritivores. We urge caution, however, in assigning an important digestive function to ingested enzymes on the basis of evidence that only indicates that such enzymes are present in the gut fluid without additional evidence that their presence results in an enhancement of digestive efficiency.     

Enhancing the efficiency of the bioagent Bacillus subtilis JF419701 against soil-borne phytopathogens by increasing the productivity of fungal cell wall degrading enzymes

This study was devoted to increasing the production of fungal cell wall degrading enzymes by Bacillus subtilis JF419701 to enhance its efficiency in the biological control process. In dual culture, B. subtilis JF419701 showed the highest antagonistic effect of the 256 bacterial strains tested against six soil-borne pathogens, Alternaria alternata, Exserohilum rostratum, Fusarium oxysporum, Macrophomina phaseolina, Pythium ultimum and Rhizoctonia solani. The production potentiality of the enzymes α-1,3-glucanase, β-1,3-glucanase, chitinase and protease by B. subtilis JF419701 was studied in vitro. Results proved that the maximum production of enzymes by this bacterium was achieved after a two-day incubation period at a slightly alkaline pH (8). The addition of colloidal chitin or S-glucan to the growth media enhanced the production of all the enzymes except protease, which was stimulated by casein. This study therefore recommends that to obtain an efficient and strong bioagent culture of B. subtilis JF419701, it is necessary to grow this micro-organism on a specific medium containing either chitin or its derivatives at pH 8 for two days.     

Characterization of fructose 6 phosphate phosphoketolases purified from Bifidobacterium species

An extracellular chitin deacetylase activity has been purified to homogeneity from autolyzed cultures of Aspergillus nidulans. This enzyme is an acidic glycoprotein with a pI of 2.75 and a 28% (wt/wt) carbohydrate content. The apparent M _r of the enzyme estimated by SDS/PAGE and Superose 12 (f.p.l.c.) was around 27,000. The enzyme had an optimum pH at 7.0 and was stable in the pH range 4.0–7.5. Its optimum temperature of reaction was 50°C, and it was stable from 30° to 100°C after 1 h of preincubation. The enzyme hydrolyzed glycol chitin and oligomers of N-acetylgucosamine and to a lesser extent chitin, colloidal chitin, carboxymethylchitin, and an -1 3,1 6-N-acetylgalactosamine-galactan among other substances with amido groups, but the enzyme did not hydrolyze peptide bonds. The role of this enzyme could be deacetylation of chitin oligosaccharides during autolysis, after action of endochitinase on cell walls.     

Chitovibrin: a chitin-binding lectin fromVibrio parahemolyticus

A novel 134 kDa, calcium-independent chitin-binding lectin, chitovibrin, is secreted by the marine bacteriumVibrio parahemolyticus, inducible with chitin or chitin-oligomers. Chitovibrin shows no apparent enzymatic activity but exhibits a strong affinity for chitin and chito-oligomers >dp9. The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0–4m NaCl. Chitovibrin appears to be completely different from other reported Vibrio lectins and may function to bindV. parahemolyticus to chitin substrates, or to capture or sequester chito-oligomers. It may be a member of a large group of recently described proteins in Vibrios related to a complex chitinoclastic (chitinivorous) system.Abbreviations (GlcNAc)₂ N,N-diacetylchitobiose - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PTS phosphotransferase system     

Surface carbohydrates and cell wall structure of in vitro-induced uredospore infection structures ofUromyces viciae-fabae before and after treatment with enzymes and alkali

Summary Uredospores ofUromyces viciae-fabae differentiate to form germ tubes, appressoria, infection hyphae and haustorial mother cells on oil-containing collodion membranes. The cell walls of these infection structures were studied with the electron microscope and with FITC-labeled lectins before and after treatment with enzymes and inorganic solvents. Binding of the FITC-labeled lectins was measured with a microscope photometer. The enzymes pronase E, laminarinase, chitinase and lipase had different effects on each infection structure. Pronase treatment uncovered the chitin of germ tubes, appressoria and haustorial mother cells, but not of substomatal vesicles and infection hyphae. A mixture of - and -1,3-glucanase which also contained chitinase activity dissolved germ tubes and appressoria completely, but not infection pegs, substomatal vesicles, infection hyphae and haustorial mother cells. After treatment with laminarinase or lipase, an additional layer, which is especially obvious over the substomatal vesicle, infection hypha and haustorial mother cell, bound to LCA-FITC. In the wall of the haustorial mother cell, a ring, which surrounds the presumed infection peg, had strong affinity for WGA after protease and sodium hydroxide treatment. The infection structures have a fibrillar skeleton. The main constituent seems to be chitin. This skeleton is more dense or has a higher chitin content in the walls of appressoria and haustorial mother cells. The fibrils of the skeleton extend throughout the cell wall of the germ tube and appressorium. They are embedded within amorphous material of complex chemical composition (-1,3-glucan, -1,3-glucan, glycoprotein). The chitin of the infection peg, substomatal vesicle, infection hypha and haustorial mother cell is covered completely with this amorphous material. These results show, that each infection structure has distinct surface and wall characteristics. They may reflect the different tasks of the infection structures during host recognition and leaf penetration.Abbreviations AP appressorium - FITC fluorescein isothiocyanate - GT germ tube - HC haustorial mother cell - IH infection hypha - IP infection peg - LCA Lens culinaris agglutinin - n nucleus - neu neuramic acid - p pyranoside - R ring - s septum - SV substomatal vesicle - WGA wheat germ agglutinin