Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study

1992 ◽  
Vol 38 (4) ◽  
pp. 331-338 ◽  
Author(s):  
R. Balasubramanian ◽  
M. S. Manocha
Keyword(s):  
Chitinase Activity ◽  
Lysozyme Activity ◽  
Acid Protease ◽  
Glucanase Activity ◽  
Chromatographic Techniques ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Choanephora Cucurbitarum ◽  
Membrane Bound ◽  
Temperature Optima

Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS–PAGE the chitinase from both fungi migrated as a single band of Mr 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS–PAGE showed two bands for each fungus with Mr of 69.5 and 55 kDa in C. cucurbitarum and Mr 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N′-diacetylchitobiose, and N,N′,N′ ′-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but β-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar. Key words: chitinase, membrane-bound chitinase, cytosolic chitinase, Choanephora cucurbitarum, Phascolomyces articulosus.

scholarly journals Laundry Detergent Compatibility of Papain Like Protease Purified From Piper Betel Leaves

2018 ◽  
Vol 7 (3.3) ◽  
pp. 132
Author(s):  
A Mousami Shankar ◽  
Dr G.V.D. Sirisha ◽  
Dr K. Vijaya Rachel
Keyword(s):  
Laundry Detergent ◽  
Deae Cellulose ◽  
Maximum Activity ◽  
Native Page ◽  
Chromatographic Techniques ◽  
Detergent Compatibility ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
The Stability ◽  
Better Than

Enzymes have wide applications in detergent industry from early 1900’s. Mostly, clothes are soiled by protein based grime. Most of the detergents have either amylase / protease. Various sources were scrutinized for potent protease activity and Betel leaves were selected, the enzyme was then isolated, purified to homogeneity by ammonium sulphate precipitation, DEAE-Cellulose and gel permeation chromatographic techniques. The enzyme was monomeric in nature with a molecular mass of 38kDa as determined by native PAGE and SDS-PAGE. The enzyme shows maximum activity at 60oC and pH 4.0. The Km and Vmax of the enzyme were 4x10-3M and 54µmol/min/mg respectively. The enzyme was categorically inhibited by PCMB and iodo-acetamide suggesting it to have papain like nature. The stability of the enzyme is assessed over the stretch of alkaline pH and temperature. This evaluation validates the stability of the enzyme and its use in detergent formulations. It was evident that after adding the enzyme preparation the stains (tea, chocolate, blood) were removed much better than that of the controls, which affirms that papain like enzyme from betel leaves, enhances detergent activity.  

In vitro regulation of chitinase and chitin synthase activity of two mucoraceous hosts of a mycoparasite

1988 ◽  
Vol 34 (10) ◽  
pp. 1116-1121 ◽  
Author(s):  
M. S. Manocha ◽  
R. Balasubramanian
Keyword(s):  
Host Species ◽  
Neutral Type ◽  
Chitinase Activity ◽  
Chitin Synthase ◽  
Acid Proteinase ◽  
Neutral Proteinase ◽  
Choanephora Cucurbitarum ◽  
Membrane Bound

Effects of partially purified preparations of proteinases extracted from Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts of Piptocephalis virginiana, respectively, were studied in vitro on the activities of chitinase and chitin synthase of the same hosts. Both chitinase and chitin synthase are membrane bound, zymogenic, and activated by partial proteolysis. Host proteinases of acid and neutral type stimulated chitinase activity, but only the acid proteinase stimulated chitin synthase activity of the two hosts. Neutral proteinase of C. cucurbitarum inhibited its chitin synthase, whereas that of Ph. articulosus increased its chitin synthase activity. Partially purified preparations of neutral proteinase from the mycoparasite, Piptocephalis virginiana, however, enhanced chitinase and suppressed chitin synthase activity of both the hosts alike. The role of host proteinases in regulating the activity of chitinase and chitin synthase of the two host species is suggested.

scholarly journals Purification and Characterization of Peroxidase From Anthracnose Disease Infected Papaya (Carica papaya L.)

2014 ◽  
Vol 6 (2) ◽  
pp. 49-57 ◽  
Author(s):  
L Bari ◽  
P Hassan ◽  
N Absar ◽  
S Khatun ◽  
MI Hossain
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Potassium Cyanide ◽  
Ammonium Sulphate Precipitation ◽  
Reducing Conditions ◽  
Sds Page ◽  
Peroxidase Enzyme ◽  
Km Value ◽  
Temperature Optima

Peroxidase enzyme was isolated and purified from the pulp of disease infected ripen papaya of local variety by 90% ammonium sulphate precipitation, chromatography on DEAEcellulose followed by hydrophobic chromatography on Phenyl Sepharose CL-4B and the purifications achieved was about 7.2 fold with 2.5% recovery. The purified enzyme was homogeneous as judged by polyacrylamide slab gel electrophoresis. The purified enzyme had a Mr of about 55,000 and 50 000 as determined by gel filtration on Sephadex G-100 and SDS-PAGE, respectively. The molecular mass of the enzyme was found to be very similar under both reducing and non-reducing conditions indicating that the enzyme contains no subunit. The enzyme has the following characteristics: pH optima at 6.0, temperature optima around 38°C, enzyme activity was found to be strongly inhibited in the presence of potassium cyanide and Fe+2 while the activity was found to be remarkably increased in the presence of ammonium sulphate. The Km value for the peroxidase obtained with pyrogallol as substrate was 0.027 mM. DOI: http://dx.doi.org/10.3329/bjmb.v6i2.17643 Bangladesh J Med Biochem 2013; 6(2): 49-57

Antimicrobial activity of bacteriocin isolated and purified from rumen liquor collected from slaughtered goats

2015 ◽  
Vol 49 (6) ◽  
Author(s):  
S. Barathiraja ◽  
J. Thanislass ◽  
P. X. Antony ◽  
S. Venkatesaperumal
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Diffusion Method ◽  
Chromatographic Techniques ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Overlay Method

Bacteriocin like substance with antimicrobial activity was purified from freshly collected rumen liquor using 60% ammonium sulphate precipitation followed by ion exchange(SP-Sepharose) and gel filtration (Sephadex G25) chromatographic techniques. Purity of the product was checked on SDS-PAGE, having molecular weight of 6.5 kDa. Anti-microbial activity was demonstrated using <italic>Bacillus subtilis</italic> by gel overlay method and agar cut well diffusion method. Proteomic analysis confirmed the substance as bacteriocin. The purified sample was resistant to the action of protease. The substance was active at pH 4, 7 and 10. It was also active at autoclave temperature. The peptide purified was found to inhibit the growth of <italic>Staphylococcus aureus</italic> (MTCC87), <italic>Listeria monocytogenes</italic> (MTCC 657) and <italic>Pseudomonas aeurginosa</italic> (MTCC 424).

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

scholarly journals Evidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in Saccharomyces cerevisiae.

1995 ◽  
Vol 42 (2) ◽  
pp. 269-274 ◽  
Author(s):  
U Lenart ◽  
J Haplova ◽  
P Magdolen ◽  
V Farkas ◽  
G Palamarczyk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Deae Cellulose ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sds Page ◽  
Nonionic Detergent ◽  
Membrane Bound ◽  
Labeled Probe ◽  
Triton X ◽  
Cellulose Column

The membrane-bound sterolglucoside synthase from the yeast Saccharomyces cerevisiae has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with [di-125I]-5-[3-(p-azidosalicylamide)]allyl-UDPglucose, a photoactive analogue of UDP glucose, which is a substrate for this enzyme. Upon photolysis the 125I-labeled probe was shown to link covalently to the 66 kDa protein. The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Since photoinsertion of the probe to protein of 66 kDa correlates with the molecular mass of the protein visualized upon enzyme purification we postulate that the 66 kDa protein is involved in sterolglucoside synthesis in yeast.

scholarly journals Coal Depolymerising Activity and Haloperoxidase Activity of Mn Peroxidase fromFomes durissimusMTCC-1173

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Sunil Kumar Singh ◽  
Meera Yadav ◽  
Sudha Yadava ◽  
Kapil Deo Singh Yadav
Keyword(s):  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Sodium Lactate ◽  
Low Ph ◽  
Exchange Chromatography ◽  
Fungal Strain ◽  
Mn Peroxidase ◽  
Sds Page ◽  
Catalytic Rate ◽  
Temperature Optima

Mn peroxidase has been purified to homogeneity from the culture filtrate of a new fungal strainFomes durissimusMTCC-1173 using concentration by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The molecular mass of the purified enzyme has been found to be 42.0 kDa using SDS-PAGE analysis. The values using MnSO4and H2O2as the variable substrates in 50 mM lactic acid-sodium lactate buffer pH 4.5 at were 59 μM and 32 μM, respectively. The catalytic rate constants using MnSO4and H2O2were 22.4 s−1and 14.0 s−1, respectively, giving the values of 0.38 μM−1s−1and 0.44 μM−1s−1, respectively. The pH and temperature optima of the Mn peroxidase were 4 and , respectively. The purified MnP depolymerises humic acid in presence of H2O2. The purified Mn peroxidase exhibits haloperoxidase activity at low pH.

Tissue Distribution and Purification of Prophenoloxidase in Larvae of Asian Corn Borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

2004 ◽  
Vol 36 (5) ◽  
pp. 360-364 ◽  
Author(s):  
Cong-Jing Feng ◽  
Wen-Jun Fu
Keyword(s):  
Relative Molecular Mass ◽  
Ostrinia Furnacalis ◽  
Immunofluorescence Test ◽  
Corn Borer ◽  
Indirect Immunofluorescence Test ◽  
Ammonium Sulphate Precipitation ◽  
Asian Corn Borer ◽  
Sds Page ◽  
Lepidoptera Pyralidae ◽  
Blue Sepharose

Abstract Using ammonium sulphate precipitation, Blue-Sepharose CL-6B, Phenyl-Sepharose CL-4B, prophenoloxidase (PPO) was isolated and purified from hemolymph of Ostrinia furnacalis larvae. This zymogen was a heterodimer, and composed of two subunits with the relative molecular mass ranging from 66.2 kD to 97.4 kD determined by SDS-PAGE. Western blotting and indirect immunofluorescence test showed that PPO was present in integument, hemolymph plasma and cell membrane of granular hemocytes and oenocytoids of O. furnacalis larvae.

scholarly journals Characterization of the soluble, secreted form of urinary meprin

1996 ◽  
Vol 315 (2) ◽  
pp. 461-465 ◽  
Author(s):  
Robert J. BEYNON ◽  
Simon OLIVER ◽  
Duncan H. L. ROBERTSON
Keyword(s):  
Proteolytic Activity ◽  
Protein Band ◽  
Peptide Sequence ◽  
Soluble Form ◽  
Α Subunit ◽  
Alternative Processing ◽  
Sds Page ◽  
Processing Pathways ◽  
Membrane Bound

A soluble form of the kidney membrane metalloendopeptidase, meprin, is present in urine. Urinary meprin is expressed in BALB/C mice with the Mep-1a/a genotype (high meprin, expressing meprin-α and meprin-β) but not in BALB.K mice of the Mep-1b/b genotype (that only express meprin-β). Western blotting with antisera specific to the meprin-α and the meprin-β subunits established that the only form of meprin present in urine samples was derived from meprin-α. This form of meprin is partially active, and comprises at least three variants by non-reducing SDS/PAGE and by zymography and two protein bands on reducing SDS/PAGE. Sequencing of these two bands established that the N-terminus of the larger protein band begins with the pro-peptide sequence of the α-subunit (VSIKH..), whereas the smaller band possessed the mature meprin N-terminal sequence (NAMRDP..). Trypsin is able to remove the pro-peptide, with a concomitant activation in proteolytic activity. After deglycosylation, the size of the pro- and mature forms of urinary meprin are consistent with cleavage in the region of the X–I boundary. There is a pronounced sexual dimorphism in urinary meprin expression. Females secrete a slightly larger form, and its proteolytic activity is about 50% of that released by males. The urinary meprin is therefore a naturally occurring secreted form of this membrane-bound metalloendopeptidase and is more likely to be generated by alternative processing pathways than by specific release mechanisms.

Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus).

2008 ◽  
Vol 55 (4) ◽  
pp. 753-760 ◽  
Author(s):  
Władysław Polcyn
Keyword(s):  
Mass Spectrometry ◽  
Nitrate Reductase ◽  
Biochemical Properties ◽  
Validation Criteria ◽  
Sds Page ◽  
Mass Spectrometry Identification ◽  
Bradyrhizobium Sp ◽  
Rhizobial Species ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was undertaken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.

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