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1.
Purification and characterization of Streptomyces albidoflavus antifungal components 总被引:1,自引:0,他引:1
M. Swiontek Brzezinska U. Jankiewicz A. Burkowska 《Applied Biochemistry and Microbiology》2013,49(5):451-457
Chitinolytic strain Streptomyces albidoflavus was isolated from soil of the central region of Poland. Its identification was based on analysis of 16S rRNA gene sequence. The colloidal chitin was revealed as the finest substrate for the production of chitinases by S. albidoflavus. The enzyme catalyzed the hydrolysis of the disaccharide 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotriose most efficiently and was, therefore, classified as an endochitinase. The chitinase of S. albidoflavus was purified by applying the two-step procedure: fractionation with ammonium sulphate and chitin affinity chromatography. The molecular weight of the purified enzyme determined by SDS-PAGE was approximately 50 kDa. The enzyme was characterised as thermostable during 180 min of preincubation at the temperature of 35°C and 40°C. The activity of the enzyme was strongly inhibited in the presence of Hg2+ and Mn2+ ions, SDS but stabilized by Ca2+ and Mg2+ ions. Both purified and crude chitinases from S. albidoflavus inhibited the development of fungal phytopathogens. Purified chitinase inhibited the growth of Alternaria alternata, Fusarium culmorum, Fusarium oxysporum and Botrytis cinerea. Additionally, the crude chitinase inhibited the growth of Fusarium solani. 相似文献
2.
Aspergillus niger LOCK 62 produces an antifungal chitinase. Different sources of chitin in the medium were used to test the production of the chitinase. Chitinase production was most effective when colloidal chitin and shrimp shell were used as substrates. The optimum incubation period for chitinase production by Aspergillus niger LOCK 62 was 6?days. The chitinase was purified from the culture medium by fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme was 43?kDa. The highest activity was obtained at 40?°C for both crude and purified enzymes. The crude chitinase activity was stable during 180?min incubation at 40?°C, but purified chitinase lost about 25?% of its activity under these conditions. Optimal pH for chitinase activity was pH 6–6.5. The activity of crude and purified enzyme was stabilized by Mg2+ and Ca2+ ions, but inhibited by Hg2+ and Pb2+ ions. Chitinase isolated from Aspergillus niger LOCK 62 inhibited the growth of the fungal phytopathogens: Fusarium culmorum, Fusarium solani and Rhizoctonia solani. The growth of Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum was not affected. 相似文献
3.
C. S. Anuratha Jenq-Kuen Huang Arunabala Pingali S. Muthukrishnan 《Journal of plant biochemistry and biotechnology.》1992,1(1):5-10
A 35 kD chitinase has been purified to apparent homogeneity from extracts of rice bran of cv New Bonnet by ammonium sulfate fractionation, chitin affinity chromatography, cation exchange chromatography on carboxymethyl cellulose and gel filtration. The purified enzyme has an isoelectric point of 8.8. The enzyme inhibited the growth of Rhizoctonia solani (the sheath blight pathogen), Trichoderma viride, T. harzianum, Fusarium graminaerum and F. culmorum in vitro. A cDNA clone for chitinase was isolated from a developing rice seed cDNA library by probing with a barley chitinase cDNA probe. The nucleotide sequence of this 654 bp clone was determined, it contains an open reading frame of 519 nucleotides. The protein product encoded by this clone is homologous to chitinases from tobacco, bean and barley. Southern blot analysis of rice genomic DNA with this probe revealed that chitinases are encoded by a small multi-gene family in the rice genome. 相似文献
4.
Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase
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Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips. 相似文献
5.
M. Swiontek Brzezinska U. Jankiewicz K. Lisiecki 《Applied Biochemistry and Microbiology》2013,49(2):154-159
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. 相似文献
6.
Sheba M. J. Ignatius Jenq K. Huang Renu Khanna Chopra S. Muthukrishnan 《Journal of plant biochemistry and biotechnology.》1994,3(2):91-95
Chitinases (E.C.3.2.1.14) are thought to play an important role in the defense of plants against fungal invasion. By screening a barley genomic library with a previously identified chitinase eDNA clone (clone 10), a genomic clone was isolated and characterized by DNA sequencing of the chitinase coding region and flanking sequences. This clone contains an open reading frame capable of coding for a 34 kD chitinase. Comparison of the amino acid sequence of the encoded protein with other barley chitinases suggests that the genomic clone encodes chitinase T, which has been characterized extensively by protein sequencing. Treatment of barley leaves and aleurone protoplasts with N-acetyl glucosamine oligomers which act as elicitors in other plants, did not lead to the elevation of the levels of the chitinases. However, infection of barley seedlings with the powdery mildew fungus, Erysiphe graminis, resulted in the induction of several isoforms of chitinase. The level and number of chitinase isozymes was correlated with the severity of infection. The infection-related chitinases found in barley leaves are different from those found in seeds. 相似文献
7.
Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the Vmax of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions. 相似文献
8.
Karin Hjort Ilaria Presti Annelie Elväng Flavia Marinelli Sara Sjöling 《Applied microbiology and biotechnology》2014,98(6):2819-2828
Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F175DGIDIDWE183, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes. 相似文献
9.
Oshima H Miyazaki R Ohe Y Hayashi H Kawamura K Kikuyama S 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2002,132(2):381-388
An approximately 60-kDa protein with chitinase activity was purified from the pancreas of the toad Bufo japonicus. Its specific activity was 4.5 times higher than that of a commercial bacterial chitinase in fragmenting crab shell chitin, and its optimal pH was approximately 6.0. A cDNA clone encoding a protein consisting of 488 amino acid residues, including part of the peptide sequence determined from the isolated protein, was obtained from a toad pancreas cDNA library. The deduced amino acid sequence indicated that the protein contained regions with high homology to those present in chitinases from different species, with the amino acid residues for the chitinase activity and the chitin-binding ability being completely conserved. We designate the protein as toad pancreatic chitinase (tPCase). Northern blot analysis revealed the mRNA of this enzyme to be expressed exclusively in the pancreas. Toad PCase is the first amphibian chitinase to be identified as well as the first pancreatic chitinase identified in a vertebrate. 相似文献
10.
Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens 总被引:32,自引:0,他引:32
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Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N-terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen. 相似文献
11.
Chitinase in bean leaves: induction by ethylene,purification, properties,and possible function 总被引:6,自引:0,他引:6
Ethylene induced an endochitinase in primary leaves of Phaseolus vulgaris L. The enzyme formed chitobiose and higher chitin oligosaccharides from insoluble, colloidal or regenerated chitin. Less than 5% of the total chitinolytic activity was detected in an exochitinase assay proposed by Abeles et al. (1970, Plant Physiol. 47, 129–134) for ethylene-induced chitinase. In ethylene-treated plants, chitinase activity started to increase after a lag of 6 h and was induced 30 fold within 24 h. Exogenously supplied ethylene at 1 nl ml?1 was sufficient for half-maximal induction, and enhancement of the endogenous ethylene formation also enhanced chitinase activity. Cycloheximide prevented the induction. Among various hydrolases tested, only chitinase and, to a lesser extent, β-1,3-glucanase were induced by ethylene. Induction of chitinase by ethylene occurred in many different plant species. Ethylene-induced chitinase was purified by affinity chromatography on a column of regenerated chitin. Its apparent molecular weight obtained by sodium dodecyl sulfate-gel electrophoresis was 30,000; the molecular weight determined from filtration through Sephadex G-75 was 22,000. The purified enzyme attacked chitin in isolated cell walls of Fusarium solani. It also acted as a lysozyme when incubated with Micrococcus lysodeikticus. It is concluded that ethylene-induced chitinase functions as a defense enzyme against fungal and bacterial invaders. 相似文献
12.
《Enzyme and microbial technology》1986,8(10):588-592
Differences in polysaccharide composition of various fungal cell walls were indicated by their susceptibility to enzymatic digestion. This information was used to optimize the enzymatic extraction of intracellular enzymes or the preparation of fungal protoplasts in high yield. Bacterial glucanase and chitinase specially purified were used for this study. Mycelium of Aspergillus niger grown on uric acid was treated with mixtures of glucanase and chitinase. Cell wall breakdown products were analysed and the ratio of chitin to glucan was estimated to be 1:1.4. A. niger protoplast formation was optimized using this information. When the mixture of chitinase to glucanase was 1:1.4, similar to the fungal cell wall composition, a 95% yield of protoplasts was obtained after 30 min and their mean size was 7 μm. However, a ratio of 1.5 to 1 (chitinase to glucanase) was needed for the maximum extraction of uricase. Yield was 10.5 μ g−1cells after 1.5 h incubation at 28°C. Glucanase alone resulted in a maximum yield of 1.9 μ g−1while chitinase alone yielded 6.0 μ g−1under the same conditions. 相似文献
13.
Andrew C. Chen Richard T. Mayer John R. DeLoach 《Archives of biochemistry and biophysics》1982,216(1):314-321
Chitinase that appears as a single band by electrophoresis was purified from stable fly pupae. The chitinase has no cation requirements for activity, and a broad pH optimum around 5. The molecular weight of the chitinase, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 48,000, and the isoelectric point is 4.85. Kinetic properties were determined using acetylated chitosan. The Km is 33 mm and V is 1.21 μmol/min/mg protein. The insect growth regulator diflubenzuron had no effect on chitinase activity. 相似文献
14.
Patricia E. B. Verwer Marian T. ten Kate Franco H. Falcone Shaun Morroll Henri A. Verbrugh Irma A. J. M. Bakker-Woudenberg Wendy W. J. van de Sande 《PloS one》2013,8(10)
Objectives
Caspofungin, currently used as salvage therapy for invasive pulmonary aspergillosis (IPA), strangely only causes morphological changes in fungal growth in vitro but does not inhibit the growth. In vivo it has good efficacy. Therefore the question arises how this in vivo activity is reached. Caspofungin is known to increase the amount of chitin in the fungal cell wall. Mammals produce two chitinases, chitotriosidase and AMCase, which can hydrolyse chitin. We hypothesized that the mammalian chitinases play a role in the in vivo efficacy of caspofungin.Methods
In order to determine the role of chitotriosidase and AMCase in IPA, both chitinases were measured in rats which did or did not receive caspofungin treatment. In order to understand the role of each chitinase in the breakdown of the caspofungin-exposed cells, we also exposed caspofungin treated fungi to recombinant enzymes in vitro.Results
IPA in immunocompromised rats caused a dramatic increase in chitinase activity. This increase in chitinase activity was still noted when rats were treated with caspofungin. In vitro, it was demonstrated that the action of both chitinases were needed to lyse the fungal cell wall upon caspofungin exposure.Conclusion
Caspofungin seemed to alter the cell wall in such a way that the two chitinases, when combined, could lyse the fungal cell wall and assisted in clearing the fungal pathogen. We also found that both chitinases combined had a direct effect on the fungus in vitro. 相似文献15.
Tzu-Wen Liang Tung-Yen Hsieh San-Lang Wang 《Bioprocess and biosystems engineering》2014,37(6):1201-1209
A thermostable chitinase was purified by chitin affinity from the culture supernatant of Bacillus cereus TKU028 with shrimp head powder (SHP) as the sole carbon/nitrogen source. TKU028 chitinase was purified using a one-step affinity adsorbent system, and the molecular mass of TKU028 chitinase (approximately 40 kDa) was then determined using SDS-PAGE. The enzyme was stable for 60 min at temperatures below 60 °C and stable over a broad pH range of 4–9 for 60 min. In addition, the temporal changes of a bacterial community in mangrove river sediment of the Tamsui River with added SHP were also analysed by PCR–denaturing gradient gel electrophoresis to investigate the effects of B. cereus TKU028 on the degradation of SHP. The 6-week incubation sample of SHP and B. cereus TKU028-amended mangrove river sediment displayed the highest amount of biomass, reducing sugar and total sugar, and some variance of bacterial community composition existed in the soils. 相似文献
16.
17.
Regulation of Protein Synthesis in Plant Embryo by Protein Phosphorylation : I. Purification and Characterization of a cAMP-Independent Protein Kinase and Its Endogenous Substrate 总被引:1,自引:1,他引:0
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A cyclic AMP-independent protein kinase, which strongly inhibits in vitro protein synthesis, was purified to homogeneity from barley embryo by affinity and ion exchange chromatography. The Mr of the purified enzyme is 95,000 with two nonidentical subunits of Mr 58,000 and 39,000. The enzyme activity is not stimulated by cAMP, cGMP, or calmodulin. The endogenous phosphate acceptor of this kinase is a protein of Mr 52,000, was isolated by purified protein kinase immobilized Sepharose column. Using antibodies raised against this protein kinase, the levels of the enzyme during embryogenesis and germination are determined. An inverse relationship has been observed between protein kinase level and rate of protein synthesis. 相似文献
18.
Isolation and Characterization of the Genes Encoding Basic and Acidic Chitinase in Arabidopsis thaliana 总被引:2,自引:1,他引:1
Plants synthesize a number of antimicrobial proteins in response to pathogen invasion and environmental stresses. These proteins include two classes of chitinases that have either basic or acidic isoelectric points and that are capable of degrading fungal cell wall chitin. We have cloned and determined the nucleotide sequence of the genes encoding the acidic and basic chitinases from Arabidopsis thaliana (L.) Heynh. Columbia wild type. Both chitinases are encoded by single copy genes that contain introns, a novel feature in chitinase genes. The basic chitinase has 73% amino acid sequence similarity to the basic chitinase from tobacco, and the acidic chitinase has 60% amino acid sequence similarity to the acidic chitinase from cucumber. Expression of the basic chitinase is organ-specific and age-dependent in Arabidopsis. A high constitutive level of expression was observed in roots with lower levels in leaves and flowering shoots. Exposure of plants to ethylene induced high levels of systemic expression of basic chitinase with expression increasing with plant age. Constitutive expression of basic chitinase was observed in roots of the ethylene insensitive mutant (etr) of Arabidopsis, demonstrating that root-specific expression is ethylene independent. Expression of the acidic chitinase gene was not observed in normal, untreated Arabidopsis plants or in plants treated with ethylene or salicylate. However, a transient expression assay indicated that the acidic chitinase promoter is active in Arabidopsis leaf tissue. 相似文献
19.
d-lactate dehydrogenase has been purified from horseshoe crab (Limulus polyphemus) skeletal muscle and the seaworm (Nereis virens). The purified Limulus dehydrogenase was shown to be a dimer, with a molecular weight of approximately 70 000. Sephadex gel filtration and equilibrium sedimentation yield molecular weights of about 80 000 and 70 000 respectively. Acid dissociation yields a molecular weight species of about 35 000. The native enzyme has an so20w of 3.95. Extrapolation of para-hydroxymercuribenzoate inhibition curves to 100% inhibition corresponds to two molecules of para-hydroxymercuribenzoate bound per molecule of enzyme. Studies on the stoichiometric binding of reduced coenzyme show two molecules bound per molecule of enzyme. The number of tryptic peptides has been found to be one-half that expected from the amino acid composition. The electrophoretic pattern of isoenzymic forms can be best interpreted as suggesting that the enzyme is dimeric. In vitro high salt, freeze-thaw hybridizations of the isolated Limulus muscle isoenzymes yield the electrophoretic pattern predicted by a dimeric structure.The physical properties ot Nereis lactate dehydrogenase have been found to be similar to those for the Limulus muscle lactate dehydrogenase. 相似文献
20.
Monomeric malate synthase from a thermophilic Bacillus. Molecular and kinetic characteristics 总被引:1,自引:0,他引:1
The molecular weight of malate synthase purified from a thermophilic Bacillus was determined to be 62,000 by sedimentation equilibrium methods, confirming the value obtained earlier by the gel filtration technique. This enzyme and its homologs from other bacteria, which are all monomeric proteins with molecular weights of approximately 60,000. therefore differ from the considerably larger and multimeric malate synthases from yeast, Neurospora crassa, and other eucaryotic microorganisms and plants. Amino acid analysis reveals the thermophile synthase to be relatively rich in glutamic acid and to have a higher content of arginine in comparison with the yeast enzyme. The Bacillus enzyme is an acidic protein with an isoelectric pH of 4.6 and has two sulfhydryl groups titratable with 5,5′-dithiobis(2-nitrobenzoic acid). Its parameters indicative of its overall hydrophobicity and of levels of helicity and turn, which were deduced from the amino acid composition, lie well within the range recorded for a number of mesophile and thermophile enzymes. However, the level of β-sheet structure is considerably lower than that calculated for the yeast synthase; this supports a trend recently observed for certain other thermophile proteins. The synthase isolated from the thermophilic Bacillus appears to be homogeneous by several criteria, although upon electrophoresis in the native state in polyacrylamide it yields two protein bands that are both enzymatically active. Several kinetic characteristics of this enzyme are also reported. 相似文献