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1.
Braga FR Araújo JV Araujo JM Tavela Ade O Ferreira SR Freitas Soares FE Benjamin Ldos A Frassy LN 《Experimental parasitology》2011,(4):460-463
The continued maintenance of nematophagous fungi predatory activity under laboratory conditions is one of the basic requirements for a successful biological control. The purpose of this study was to evaluate the influence of time on the preservation of the fungus Duddingtonia flagrans (AC001 and CG722) stored in silica-gel for 7 years and their subsequent predatory activity on cyathostomin L3 larvae in 2% water-agar medium (2% WA). Samples of the isolates AC001 and CG722, originating from vials containing grains of silica-gel sterilized and stored for 7 years, were used. After obtaining fungal conidia, the predation test was conducted over 7 days on the surface of 9.0 cm Petri dishes filled with 2% WA. In the treated groups each Petri dish contained 500 cyathostomin L3 and conidia of fungal isolates in 2% WA. In the control group (without fungi) the plates contained 500 L3 in 2% WA. The experimental results showed that isolated AC001 and CG722 were efficient in preying on cyathostomin L3 (p < 0.01) compared to control (without fungus). However, no difference was observed (p > 0.01) in the predatory activity of the fungal isolates tested. Comparing the groups, there was a significant reductions of cyathostomin L3 (p < 0.01) of 88.6% and 78.4% on average recovered from the groups treated with the isolates AC001 and CG722, respectively, after 7 days. The results of this test showed that the fungus D. flagrans (AC001 and CG722) stored in silica-gel for at least 7 years maintained its predatory activity on cyathostomin L3. 相似文献
2.
A.O. Tavela J.V. Araújo F.R. Braga J.M. Araujo L.Q. Magalhães W.F. Silveira 《Biocontrol Science and Technology》2012,22(5):607-610
In vitro effects of nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34) and Pochonia chlamydosporia (VC1) were evaluated against eggs and third-stage infective larvae (L3) of horse cyathostomin (Nematoda: Strongylidae). The following percentage reductions compared with the control group were observed after a 20-day exposure period: AC001, 61.6%; NF34, 66.1%; VC1, 73.2%; group AC001 + VC1, 86.8%; NF34 + VC1, 77.3%; AC001 + NF34, 92.4%. The results showed that the fungal isolates (VC1, AC001 and NF34), acting alone or in conjunction, were efficient in controlling horse cyathostomin under in vitro conditions. 相似文献
3.
F.R. Braga J.V. Araújo F.E.F. Soares J.M. Araujo S.R. Ferreira L.N. Frassy 《Biocontrol Science and Technology》2011,21(11):1313-1320
The production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae from sheep were studied. D. flagrans was grown in liquid medium with glucose, casein, bibasic potassium phosphate (K2HPO4), magnesium sulfate (MgSO4), zinc sulfate (ZnSO4), ferrous sulfate (FeSO4), and copper sulfate (CuSO4). The proteolytic activity was measured within varied pHs and temperatures. To determine the thermostability, the crude extract was incubated at 28°C for 72 h. To study the effect of different chemical compounds on the activity of the crude extract, the samples were incubated in solutions containing (10 mM): calcium chloride (CaCl2), copper II sulfate (CuSO4), zinc sulfate (ZnSO4), magnesium sulfate (MgSO4), inhibitor phenylmethylsulfonyl fluoride (PMSF), and 0.5% SDS. Results showed that the highest activity obtained (79.23 U/mL) was at pH 9.0, while the optimum temperature was 60°C (119.6 U/mL). The thermostability analysis demonstrated that after 72 h the activity was maintained or increased. It was found that the CuSO4, ZnSO4, and PMSF strongly inhibited the proteolytic activity. Moreover, the MgSO4 and SDS, caused a weak inhibition of the proteolytic activity. There was a significant (P<0.01) reduction in number of treated L3 when compared to control (94.2%). The results suggest that the crude extract produced by D. flagrans (AC001) in liquid medium exerted larvicidal activity on trichostrongilid L3 and therefore may contribute to a large-scale industrial production. 相似文献
4.
Fabio Ribeiro Braga Filippe Elias de Freitas Soares José Humberto de Queiroz Juliana Milani Araujo Lorendane Millena de Carvalho Ingrid Ney Kramer de Mello 《Biocontrol Science and Technology》2013,23(11):1336-1341
The influence of casein and pH on the activity of the nematophagous fungus Duddingtonia flagrans (AC001) on trichostrongylide larvae was evaluated. A ‘positive influence’ was observed contributing to the reduction of 63% in the average number of recovered L3 in the media supplemented with casein and pH 7.0. 相似文献
5.
Fabio Ribeiro Braga Jackson Victor Araújo Filippe Elias Freitas Soares Juliana Milani Araujo Alexandre de Oliveira Tavela Lorendane Millena de Carvalho 《Biocontrol Science and Technology》2013,23(5):584-594
The present work aimed to evaluate the production and the characterisation of a chitinase from nematophagous fungus Duddingtonia flagrans (AC001) and observe the interaction of this fungus on engorged females of Amblyomma cajennense under laboratory conditions. In assay A, the engorged females of A. cajennense were separated and immersed for 5 seconds in a fungal suspension of 106 conidia/ml of the fungus D. flagrans and placed in Petri dishes, in the dark. In assay B, wheat bran supplemented with 1% chitin and liquid minimal medium was used [K2HPO4 (5.0 g/l), MgSO4 (0.10 g/l), ZnSO4 (0.0050 g/l), FeSO4 (0.001 g/l) e CuSO4 (0.50 mg/l)], as a substrate for chitinase production. To demonstrate the presence of chitinase in the crude extract obtained after the enzymatic extraction, a purification process was developed using a specific adsorption technique. The results from assay A demonstrated the interaction of the D. flagrans conidia produced from chitin-agar on engorged females of A. cajennense. In the assay B, D. flagrans produced a chitinase successfully, with a high value for enzyme activity. The molecular mass of semi-purified enzyme was estimated at approximately 34 kDa. It was concluded that the fungus produced a chitinase and has some entomopathogenic activity, as demonstrated here for the first time; however, it is strongly suggested that further studies are needed to elucidate the molecular mechanism of infection of target organisms by this fungus. 相似文献
6.
Paulo Wbiratan Lopes da Costa Felipe Boniedj Ventura Alvares Roberto Alves Bezerra Wlysse Ferreira Sarmento Francisca Flávia da Silva Jossiara Abrante Rodrigues 《Biocontrol Science and Technology》2019,29(11):1106-1117
ABSTRACTThe objective of this study was to evaluate the effectiveness of pelleted formulations of Duddingtonia flagrans and Monacrosporium thaumasium sodium alginate matrix stored for two and five years, by refrigeration of 2–8°C, on the predation of nematode infective larvae after passage of the gastrointestinal tract of asinines. Asinines were divided into seven groups, each group containing eight animals, in which each animal received a single dose of 100?g of pellets (containing 20?g of fungal mycelia) along with commercial feed to facilitate ingestion: GI – received D. flagrans pellets stored for five years; GII- received pellets of D. flagrans stored for two years; GIII – received newly produced D. flagrans pellets; GIV – received pellets of M. thaumasium stored for five years; GV – received pellets of M. thaumasium stored for two years; GVI – received pellets of newly-stocked M. thaumasium; and Control – received pellets without nematophagous fungi. It was observed that after passage of the pellets containing D. flangras (AC001) and M. thaumasium (NF34) by the gastrointestinal tract of the asinines, regardless of pellet storage time in assays A (Petri dishes) and B (coprocultures), there was a significant larval reduction (p?<?0.01) up to 72?h. It was concluded that the use of sodium alginate matrix pellets containing D. flagrans and M. thaumasium stored for two and five years were effective on the predation of infective nematode larvae after passage of the gastrointestinal tract from asinines. 相似文献
7.
Fabio Ribeiro Braga Jackson Victor Araújo Filippe Elias de Freitas Soares Juliana Milani Araujo Sebastião Rodrigo Ferreira José Humberto de Queiroz 《Biocontrol Science and Technology》2012,22(5):559-565
The objective of the present work was to study the conditions of predation of Duddingtonia flagrans conidia versus Panagrellus sp using response surface methodology. Conidia of D. flagrans (AC001) isolate were transferred into water-agar (WA) culture media at different pHs and different concentrations defined according to Central Composite Design (CCD). Five different concentrations of D. flagrans conidia: (1292, 500, 1000, 1500 and 1707) were used. For 2%WA media were used the following pHs were used: 6.29, 6.5, 7.0, 7.5 and 7.71. The response of the design corresponded to the number of larvae (transformed to square root scale) observed at the end of the experiment (10 days). At the tenth day, the non predated larvae were recovered in the Petri dishes. The results showed that 2%WA media at pH 7.0 contributed to improve the predatory activity of conidia of D. flagrans, and therefore this tool may be used in future studies under laboratory and natural conditions. 相似文献
8.
Alkaline Serine Protease Is an Exotoxin of Vibrio alginolyticus in Kuruma Prawn,Penaeus japonicus 总被引:1,自引:0,他引:1
An extracellular lethal toxin produced by Vibrio
alginolyticus strain Swy originally isolated from diseased kuruma prawn
(Penaeus japonicus) was partially purified by Fast Protein Liquid
Chromatography with hydrophobic interaction (Phenyl Sepharose High
Performance) chromatography and gel filtration columns. The toxin is an
alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF),
and showed maximal activity at pH 10, having a molecular weight of about 33
kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the
toxin was also completely inhibited by FeCl2 but partially
inhibited by CaCl2, CuCl2, CoCl2,
MnCl2, and ZnCl2, and not inhibited by ethylenediamine
tetraacetic acid (EDTA), ethylene glycol-bis(β-amino-ethyl ether)
N,N,N′,N′-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecyl
sulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both
the crude extracellular products (ECP) and the partially purified toxin are
lethal for kuruma prawn at LD50 values of 0.30 and 0.27 μg
protein/g body weight, respectively. The addition of PMSF completely
inhibited the lethal toxicity of both the ECP and the partially purified
toxin, indicating that this serine protease is a lethal factor produced by
the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic
protease produced by V. alginolyticus strain Swy.
Received: 12 April 1996 / Accepted: 31 July 1996 相似文献
9.
Extracellular proteases produced by Scytalidium thermophilum, grown on microcrystalline cellulose, were most active at pH 6.5–8 and 37–45 °C when incubated for 60 min. Highest protease activity was at day 3 where endoglucanase activity was low. Protease activity measurements with and without the protease inhibitors, p-chloromercuribenzoate, PMSF, antipain, E-64, EDTA and pepstatin A, suggest production of thiol-containing serine protease and serine proteases. Endoglucanase and Avicel-adsorbable endoglucanase activity in culture medium was not significantly affected by protease inhibitors. 相似文献
10.
F. R. Braga J. V. Araújo A. K. Campos J. M. Araújo R. O. Carvalho A. R. Silva A. O. Tavela 《World journal of microbiology & biotechnology》2008,24(8):1559-1564
This work evaluated the in vitro action of four isolates of the nematophagous fungi Duddingtonia flagrans (AC001), Monacrosporium sinense (SF53) and Pochonia chlamydosporia (VC1 and VC4) on eggs of Fasciola hepatica. The eggs were plated on 2% water-agar with the grown isolates and control without fungus. After 7, 14 and 21 days, the eggs
were removed and classified according to the following parameters: effect type 1, lytic effect with no morphological damage
to eggshells; type 2, lytic effect with morphological changes in eggshells and embryos; and type 3, lytic effect with morphological
changes in embryos and eggshells, with hyphal penetration and internal egg colonization. Pochonia chlamydosporia showed ovicidal activity on F. hepatica eggs in the studied intervals of the type-3 effect, of 12.8% (VC1) and 16.5% (VC4); 14.4% (VC1) and 18.7% (VC4), 20.1% (VC1)
and 21.5 % (VC4), over 7, 14 and 21 days respectively. No statistical difference was found (P > 0.01) among the isolates VC1 and VC4 for effects type 1, 2 and 3 during the studied intervals. Duddingtonia flagrans (AC001) and Monacrosporium sinense fungi only showed effect type 1, with no significant difference between them, with the following results: 60.1% (AC001) and
57.5% (SF53); 62.3% (AC001) and 62.0% (SF53); 66.5% (AC001) and 73.4% (SF53), over 7, 14 and 21 days respectively. Pochonia chlamydosporia fungi negatively influenced the in vitro F. hepatica viability. Therefore it can be considered as a potential biological control agent for this helminth. 相似文献
11.
Chellakere K. Manjunath Gwendolyn E. Goings Ernest Page 《The Journal of membrane biology》1985,85(2):159-168
Summary Gap junctions (GJ) isolated from rat hearts in presence of the protease inhibitor phenylmethylsulfonylfuoride (PMSF) contain a Mr 44,000 to 47.000 major polypeptide and have a urea-resistant layer of fuzz on their cytoplasmic surfaces, whereas junctions isolated without PMSF are proteolyzed to a Mr 29.500 polypeptide by a serine protease and have smooth cytoplasmic surfaces (C.K. Manjunath, G.E. Goings & E. PageAm. J. Physiol.
246:H865–H875, 1984). Rat liver GJ isolated with or without PMSF contain a Mr 28,000 polypeptide and have smooth cytoplasmic surfaces. Here we examine the origin, type and inhibitor sensitivity of the heart protease; why similar proteolysis is absent during isolation of rat liver gap junctions; and whether the Mr 44.000 to 47,000 cardiac GJ polypeptide is the precursor of the Mr 29,500 subunit. We show that the Mr 44,000 to 47,000 polypeptide corresponds to the unproteolyzed connexon subunit; that proteolysis of this polypeptide occurs predominantly during exposure to high ionic strength solution (0.6m KI) which releases serine protease from mast cell granules; that this protease is inhibitable with PMSF and (less completely) soybean trypsin inhibitor and chymostatin; and thatin vivo degranulation of mast cells by injecting rats with compound 48/80 fails to prevent breakdown of cardiac GJ during isolation. The results support the concept that GJ from rat heart and liver differ in protein composition. 相似文献
12.
Alexander Ya. Strongin Lara S. Izotova Zakhar T. Abramov Lidia M. Ermakova Dmitrii I. Gorodetsky Valentin M. Stepanov 《Archives of microbiology》1978,119(3):287-293
While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by nonionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.Abbreviations ISP
intracellular serine protease
- ISP-A-Bsu A-50 and ISP-B-Bsu A-50
molecular forms A and B of B. subtilis A-50 intracellular serine protease, respectively
- SDS
sodium dodecyl sulfate
- PMSF
phenylmethyl sulfonylfluoride
- pNA
p-nitroanilide
- Buffer A
50 mM Tris-(hydroxymethyl)aminomethane-1 mM CaCl2 adjusted to pH 8.5 with HCl 相似文献
13.
A metagenomic cosmid library was constructed, in which the insert DNA was derived from the coastal sediment near Antarctic
China Zhongshan Station. One clone (ACPRO001) expressing protease activity was isolated from the library using milk agar plates.
Sequencing of the clone revealed a novel protease gene. The amino acid sequence comparison and phylogenetic analysis indicated
that it could be classified as a subtilisin-like serine protease, though the highly conserved residue Asp was replaced by
Ala. The ACPRO001 protease gene was expressed in pET-His and purified for characterization. The optimal temperature and pH
for the activity of the ACPRO001 protease were 60°C and pH 9.0, respectively. The enzyme retained about 73% of residual activity
after 2 h incubation at 50°C in the presence of Ca2+. The presence of Ca2+ increased the thermostability of ACPRO001 protease obviously. The enzymatic activity was inhibited by 1 mM phenylmethyl sulfonylfluoride
(PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. 相似文献
14.
Tooba Naz Shamsi Romana Parveen Priyankar Sen 《Preparative biochemistry & biotechnology》2017,47(5):513-519
The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20?mM tris-CaCl2 buffer (pH 8.2) with a flow rate of 0.5?mL min?1. The molecular weight and purity of ~23?kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697?U?mg?1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source. 相似文献
15.
Lecanicillium psalliotae produced an extracellular protease (Ver112) which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 32 kDa. The optimum activity of Ver112 was at pH 10 and 70 °C (over 5 min). The purified protease degraded a broad range of substrates including casein, gelatin, and nematode cuticle with 81% of a nematode (Panagrellus redivivus) being degraded after treating with Ver112 for 12 h. The protease was highly sensitive to PMSF (1 mM) indicating it to be a serine protease. The N-terminal amino acid residues of Ver112 shared a high degree of similarity with other cuticle-degrading proteases from nematophagous fungi which suggests a role in nematode infection. 相似文献
16.
In this paper it is described for the first time the capability of Myrothecium verrucaria to grow in submerged and solid state cultures using poultry feathers as the only substrate. The fungus produced a protease
with an unusual keratinolytic activity among plant pathogenic fungi. Its crude protease hydrolyzed keratinous substrates at
pH 9.0 and 40 °C in the following order: poultry feather keratin > sheep wool keratin > human nail keratin > human hair keratin.
Protease activity was highly sensitive to phenylmethyl sulphonyl fluoride (PMSF) indicating that the enzyme belonged to the
serine protease family. 相似文献
17.
Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic
Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar
substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative
molecular mass (M
r) of 28.7 kDa, whereas protease B, with a M
r of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1
U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate
for chymotrypsin-like serine proteases. However, the K
m values of these two proteases on SAAPF-pNa were higher than that for α-chymotrypsin, indicating a lower affinity of proteases
A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein,
and they stained poorly with the silver staining method. However, NH2-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost
total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and
TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease
family. Journal of Industrial Microbiology & Biotechnology (2001) 26, 387–393.
Received 05 November 2000/ Accepted in revised form 23 April 2001 相似文献
18.
An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography,
ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal
activity at pH 10 and with a temperature of 55°C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl
fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined
with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology
with protease from another B. pumilus strain.
Received: 17 April 2002 / Accepted: 24 May 2002 相似文献
19.
A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mm CaCl2. The enzyme was completely inactivated by PMSF, EDTA and -mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.R. Dhandapani and R. Vijayaragavan are with the Centre for Plant Molecular Biology & Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, India 相似文献
20.
《Bioscience, biotechnology, and biochemistry》2013,77(7):1166-1168
A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CyS03H7-Gly8, Val12-Glu13, Tyr16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or α2-macroglubulin. 相似文献