scholarly journals The effect of different substrates on chitinase activity from Bacillus sp. WS4F

2021 ◽  
Vol 924 (1) ◽  
pp. 012034
Author(s):  
P M Astuti ◽  
S Setyahadi ◽  
A K Wardani ◽  
A Sutrisno
Keyword(s):  
Ammonium Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pathogenic Fungi ◽  
Chitinase Activity ◽  
Plant Diseases ◽  
Partial Purification ◽  
Bacillus Sp ◽  
Ammonium Sulfate Precipitation ◽  
Shrimp Shells

Abstract One of the roles of chitinase is as an antifungal which is widely used as a biocontrol agent for plant diseases caused by pathogenic fungi. Bacillus sp. WS4F has chitinase activity which can inhibit the growth of Ganoderma boninense, a fungus that attacks oil palm and causes basal stem rot (BSR). This study aims to investigate the effect of different substrates on the activity of the chitinase from Bacillus sp. WS4F. Two kinds of substrates i.e. chitin flakes and shrimp shells were used in this study. Enzyme activity of chitinase was analyzed after partial purification of enzyme was performed using ammonium sulfate precipitation followed by dialysis. The highest activity of chitinase was achieved by the substrate using shrimp shells. The ammonium sulfate precipitation (60-80% saturation) 0.0949 U/mL for activity enzyme and 0.2639 mg/mL for protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 64.389 kDa.

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

2013 ◽  
Vol 10 (2) ◽  
pp. 29
Author(s):  
Normah Ismail ◽  
Nur' Ain Mohamad Kharoe
Keyword(s):  
Molecular Weight ◽  
Protein Content ◽  
Ammonium Sulfate ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Protease Activity ◽  
Protein Concentration ◽  
Temperature Stability ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage. 

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Characterization of a Host-Specific Protein Toxin (Ptr ToxB) from Pyrenophora tritici-repentis

1999 ◽  
Vol 12 (8) ◽  
pp. 728-732 ◽  
Author(s):  
Stephen E. Strelkov ◽  
Lakhdar Lamari ◽  
G. Murray Ballance
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Protein ◽  
Tan Spot ◽  
Partial Purification ◽  
Toxic Activity ◽  
Pyrenophora Tritici Repentis ◽  
Protein Toxin ◽  
Cation Exchange Column

Pyrenophora tritici-repentis, the causal agent of tan spot of wheat, differentially induces tan necrosis and/or chlorosis in wheat. A chlorosis-inducing, host-specific toxin, termed Ptr ToxB (formerly Ptr chlorosis toxin), was purified from the culture filtrates of a race 5 isolate of P. tritici-repentis. Partial purification was performed by 25 to 80% ammonium sulfate precipitation and passage through a carboxy-methyl-Sephadex C-25 cation exchange column. Final purification was performed by fast performance liquid chromatography, with a Mono S HR 5/5 cation exchanger, followed by size fractionation on a Superose 12 HR 10/30 column. The toxin was shown to be proteinaceous in nature, and purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of Ptr ToxB was determined to be 6.61 kDa. The amino acid composition and partial N terminus amino acid sequence of the toxin were also obtained. Ptr ToxB was found to be heat stable, maintaining full toxic activity after 1 h at 55°C. Infiltration of toxin concentrations as low as 14 nM produced chlorosis on susceptible cultivars.

scholarly journals Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp. Strain O-7 and Its Role in the Chitinolytic System

2002 ◽  
Vol 184 (7) ◽  
pp. 1865-1872 ◽  
Author(s):  
Katsushiro Miyamoto ◽  
Eiji Nukui ◽  
Hiroyuki Itoh ◽  
Takaji Sato ◽  
Takeshi Kobayashi ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Signal Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chitinase Activity ◽  
Maximum Activity ◽  
Sequencing Analysis ◽  
Gene Encoding ◽  
Calculated Molecular Mass ◽  
Chitin Binding Domain

ABSTRACT Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to α-chitin and β-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.

scholarly journals Biological Control of Fusarium Wilt of Cucumber by Chitinolytic Bacteria

1999 ◽  
Vol 89 (1) ◽  
pp. 92-99 ◽  
Author(s):  
Pushpinder Paul Singh ◽  
Yong Chul Shin ◽  
Chang Seuk Park ◽  
Young Ryun Chung
Keyword(s):  
Fusarium Wilt ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrolytic Enzymes ◽  
Partial Purification ◽  
Streptomyces Sp ◽  
Chitinolytic Bacteria ◽  
Potting Medium ◽  
Paenibacillus Sp ◽  
Molecular Masses

Two chitinolytic bacterial strains, Paenibacillus sp. 300 and Streptomyces sp. 385, suppressed Fusarium wilt of cucumber (Cucumis sativus) caused by Fusarium oxysporum f. sp. cucumerinum in nonsterile, soilless potting medium. A mixture of the two strains in a ratio of 1:1 or 4:1 gave significantly (P < 0.05) better control of the disease than each of the strains used individually or than mixtures in other ratios. Several formulations were tested, and a zeolite-based, chitosan-amended formulation (ZAC) provided the best protection against the disease. Dose-response studies indicated that the threshold dose of 6 g of formulation per kilogram of potting medium was required for significant (P < 0.001) suppression of the disease. This dose was optimum for maintaining high rhizosphere population densities of chitinolytic bacteria (log 8.1 to log 9.3 CFU/g dry weight of potting medium), which were required for the control of Fusarium wilt. The ZAC formulation was suppressive when added to pathogen-infested medium 15 days before planting cucumber seeds. The formulation also provided good control when stored for 6 months at room temperature or at 4°C. Chitinase and β-1,3-glucanase enzymes were produced when the strains were grown in the presence of colloidal chitin as the sole carbon source. Partial purification of the chitinases, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and activity staining, revealed the presence of five bands with molecular masses of 65, 62, 59, 55, and 52 kDa in the case of Paenibacillus sp. 300; and three bands with molecular masses of 52, 38, and 33 kDa in the case of Streptomyces sp. 385. Incubation of cell walls of F. oxysporum f. sp. cucumerinum with partially purified enzyme fractions led to the release of N-acetyl-D-glucosamine (NAGA). NAGA content was considerably greater when pooled enzyme fractions (64 to 67) from Paenibacillus sp. were used, because they contained high β-1,3-glucanase activity in addition to chitinase activity. Suppression of Fusarium wilt of cucumber by a combination of these two bacteria may involve the action of these hydrolytic enzymes.

Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus

1983 ◽  
Vol 29 (11) ◽  
pp. 1526-1531 ◽  
Author(s):  
Susan E. Jensen ◽  
Donald W. S. Westlake ◽  
Saul Wolfe
Keyword(s):  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Isopenicillin N

Epimerase activity, which converts isopenicillin N to penicillin N, has been partially purified from cell-free extracts of Streptomyces clavuligerus. No stimulating cofactors of this activity were found, and neither EDTA nor anaerobic incubation caused significant inhibition of activity. Although pyridoxal phosphate did not stimulate epimerase activity, the presence of this cofactor was necessary for the stabilization of enzymic activity during the purification process. Epimerase activity was purified 35.5-fold by a combination of salt precipitation, gel filtration, and ion exchange chromatography. Gel filtration indicated that the epimerase has a molecular weight of 60 000 and sodium dodecyl sulphate – polyacrylamide gel electrophoresis of the 35.5-fold purified epimerase showed a major protein band running near that location. Pyridoxal phosphate antagonists did not uniformly inhibit epimerase activity, but the inhibitory effect of hydroxylamine could be partially reversed by pyridoxal phosphate.

scholarly journals The membrane attack mechanism of complement: the three polypeptide chain structure of the eigth component (C8).

1976 ◽  
Vol 143 (5) ◽  
pp. 1131-1139 ◽  
Author(s):  
W P Klob ◽  
H J Müller-Eberhard
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Ammonium Sulfate ◽  
Gel Electrophoresis ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chain Structure ◽  
Ammonium Sulfate Precipitation ◽  
Ion Exchange Column ◽  
Polypeptide Chains

The purification of human C8 in milligram quantities from outdated human serum was achieved by ammonium sulfate precipitation (37.5-50% saturation) and ion exchange column chromatography employing CM-32 cellulose and QAE-Sephadex. The yield of C8 activity ranged from 2-9%, and the average purification was 1,700-fold. Fully reduced C8 was shown by SDS polyacrylamide gel electrophoresis to have three polypeptide chains which were present in equimolor ratios: alpha, 77,000 daltons; beta, 63,000 daltons; and gamma, 13,700 daltons. C8 denaturation by SDS and urea in the absence of reducing agents revealed two noncovalently linked subunits: alpha-gamma, 99,000 daltons, and beta, 75,000 daltons.

Carnitine palmitoyltransferases in pea leaf chloroplasts: partial purification, location, and properties

2000 ◽  
Vol 78 (3) ◽  
pp. 328-335 ◽  
Author(s):  
Christine Masterson ◽  
Clifford Wood
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Carnitine Palmitoyltransferase ◽  
Chloroplast Envelope ◽  
Partial Purification ◽  
Western Blots ◽  
Chain Acyl ◽  
Long Chain ◽  
Inner Envelope

Carnitine palmitoyltransferase (EC 2.3.1.21), an enzyme that catalyses the reversible transfer of activated long-chain acyl groups between CoASH and L-carnitine, has been confirmed in pea leaf chloroplasts. This enzyme is bound to the chloroplast inner envelope membrane and has two isoforms, one bound to the outside (cytosol side) of the inner envelope and one bound to the inside (stromal side) of the inner envelope. Malonyl CoA inhibited the activity of the outer carnitine palmitoyltransferase, while stimulating the activity of the inner isoform and may be a regulator of these enzymes in vivo. Carnitine palmitoyltransferase was solubilized from the chloroplast envelope by detergent treatment and the two isoforms separated by Q-Sepharose anion exchange chromatography. Both proteins were immunochemically observed by probing Western blots of sodium dodecyl sulfate - polyacrylamide gel electrophoresis gels using an anti-beef heart mitochondrial carnitine palmitoyltransferase polyclonal antibody. The monomeric molecular mass of the protein recognized by this antibody was approximately 20 kDa. This 20-kDa protein also bound3H-carnitine. Both isoforms had broad acyl CoA substrate specificities, but showed increased activity with desaturated long-chain acyl CoAs, exhibiting a preference for linolenoyl CoA. A role for carnitine palmitoyltransferase in the shuttling of fatty acids across the chloroplast envelope is suggested.Key words: Pisum sativum, chloroplasts, carnitine palmitoyltransferase, fatty acid metabolism, eukaryotic pathway, membrane transport.

scholarly journals Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria

1984 ◽  
Vol 224 (1) ◽  
pp. 171-179 ◽  
Author(s):  
I R Cottingham ◽  
A L Moore
Keyword(s):  
Nadh Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Properties ◽  
Nadh Dehydrogenases ◽  
Arum Maculatum ◽  
A Minor ◽  
Considerable Loss

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.

scholarly journals Partial purification and characterization of a pyruvate dehydrogenase-complex-inactivating enzyme from rat liver

1978 ◽  
Vol 169 (2) ◽  
pp. 321-328 ◽  
Author(s):  
A Lynen ◽  
E Sedlaczek ◽  
O H Wieland
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Potassium Phosphate ◽  
Partial Purification ◽  
Acid Extraction ◽  
High Dilutions ◽  
Dehydrogenase Complex

An enzyme inactivating the pyruvate dehydrogenase complex (inactivase) was purified about 8000-fold from rat liver by differential centrifugation, acid extraction of a lysosomerich 25000 g pellet, acetone fractionation, and adsorption on calcium phosphate gel. By exclusion chromatography on Sephadex G-100 a molecular weight of 21 000 was estimated. The purified enzyme was most stable at pH 5.8 in potassium phosphate buffer, and at pH 4.5 in McIlvaine buffer. At high dilutions the enzyme was very labile and was remarkably stabilized by high salt concentrations. Enzyme activity is inhibited by native rat blood serum, iodoacetamide and leupeptin, but not by phenylmethanesulphonyl fluoride, suggesting that it belongs to the class of thiol proteinases. Among various enzymes tested, only 2-oxoglutarate dehydrogenase was attacked by the inactivase to a similar extent to the pyruvate dehydrogenase complex. Studies on the inactivation mechanism indicate that although the overall reaction is completely lost after treatment with inactivase, each individual step of the multienzyme complex retains full catalytic activity. As judged from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the transacetylase subunit appears to be degraded into several smaller fractions.

Sign in / Sign up

Export Citation Format

Share Document