scholarly journals Lipolytic Potential ofAspergillus japonicusLAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Lívia Tereza Andrade Souza ◽  
Jamil S. Oliveira ◽  
Vera L. dos Santos ◽  
Wiliam C. B. Regis ◽  
Marcelo M. Santoro ◽  
...  
Keyword(s):  
Room Temperature ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Partial Purification ◽  
Extracellular Lipase ◽  
Inhibitory Effects ◽  
Ammonium Sulphate Precipitation ◽  
Lipase Enzyme ◽  
Alkaline Conditions ◽  
Triton X

Lipolytic potential ofAspergillus japonicusLAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu2+, Ni2+, and Al3+showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. TheKMandVmaxvalues of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

Rat lingual lipase: partial purification, hydrolytic properties, and comparison with pancreatic lipase

1984 ◽  
Vol 247 (4) ◽  
pp. G385-G393 ◽  
Author(s):  
I. M. Roberts ◽  
R. K. Montgomery ◽  
M. C. Carey
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Pancreatic Lipase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Dodecyl Sulfate ◽  
Triton X

We have partially purified lingual lipase from the serous glands of rat tongue. With a combination of Triton X-100 extraction or Triton X-114 phase-separation techniques, Bio-Bead SM-2 treatment, dialysis, and gel filtration on Sephadex G-200 or Sephacryl S-300, we obtained a sparingly soluble lipid-free protein demonstrating hydrolytic activity against triglycerides and negligible phospholipase or cholesteryl esterase activities. Compared with homogenate, specific activities of the enzyme were enriched 3- to 5-fold prior to gel filtration and 10-fold after gel filtration. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions (6 M guanidine X HCl or 0.1% sodium dodecyl sulfate) revealed one major glycoprotein band with Mr approximately 50,000. Gel filtration of the active enzyme in 0.1% Triton X-100 gave an Mr approximately 270,000-300,000, suggesting extensive self-aggregation. With both tributyrin and triolein, the pH optimum of the purified enzyme was 4.0 and activity extended from pH 2.0 to 8.0. In contrast to purified human pancreatic lipase, lingual lipase hydrolyzed triglyceride emulsions and mixed micelles stabilized with both short-chain (dihexanoyl) and long-chain (egg) lecithin and were inhibited only slightly (18-25%) by micellar concentrations of two common bile salts, taurodeoxycholate and taurocholate. Our results suggest that the hydrolysis of dietary fat by lingual lipase may extend from the pharynx through the esophagus and stomach and into the upper small intestine.

scholarly journals Partial purification and characterization of collagen galactosyltransferase from chick embryos

1976 ◽  
Vol 155 (1) ◽  
pp. 145-153 ◽  
Author(s):  
L Risteli ◽  
R Myllyä ◽  
K I Kivirikko
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Helical Conformation ◽  
Tissue Homogenate ◽  
Partial Purification ◽  
Molecular Weights ◽  
Soluble Collagen ◽  
Insoluble Collagen ◽  
Chick Embryo Extract ◽  
Triton X

Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.

scholarly journals Purification of a protein from Bacillus thuringiensis toxic to larvae of lepidoptera

1968 ◽  
Vol 106 (2) ◽  
pp. 445-454 ◽  
Author(s):  
K. E. Cooksey
Keyword(s):  
Bacillus Thuringiensis ◽  
Gel Filtration ◽  
Pieris Brassicae ◽  
Digestion Product ◽  
Toxic Protein ◽  
Protein Toxin ◽  
Ammonium Sulphate Precipitation ◽  
Alkaline Conditions ◽  
Crystal Toxin

The protein toxin of the parasporal body or crystal of Bacillus thuringiensis (Mattés isolate) has been purified severalfold by a combination of Sephadex G-200 gel filtration and ammonium sulphate precipitation. It has been shown that the use of highly alkaline conditions for dissolution of the crystals does not lead to serious artifacts. The crystal toxin has been shown to be quantitatively related to the crystal antigen. It is possible that there is a second distinct toxin present in the crystal and this too can be detected by its antigenic reaction. Purified toxic protein has been hydrolysed in vitro by regurgitated Pieris brassicae gut enzymes, chymotrypsin, trypsin and subtilisin. In each case the digest contained a product that was still antigenic, had mol.wt. about 40000 and was toxic to P. brassicae larvae. Smaller toxic molecules (mol.wt. approx. 10000) that did not react as antigens were also produced by proteolysis. It is possible that these smaller molecules were hydrolytic products of the larger digestion product.

scholarly journals New Lipase for Biodiesel Production: Partial Purification and Characterization of LipSB 25-4

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Aysel Ugur ◽  
Nurdan Sarac ◽  
Rukiye Boran ◽  
Berk Ayaz ◽  
Ozgur Ceylan ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Nitrogen Sources ◽  
Biodiesel Production ◽  
Partial Purification ◽  
Period Effect ◽  
Carbon And Nitrogen ◽  
Ammonium Sulphate Precipitation ◽  
Thermophilic Conditions

The lipolytic activities of 300 Streptomyces isolates were determined in Tributyrin and Rhodamine-B Agar. Lipase activities were also measured with p-nitrophenyl palmitate (p-NPP) as a substrate. The strain of Streptomyces bambergiensis OC 25-4 used in this study was selected among 300 strains of Streptomyces from MUCC as the best lipase producer. The incubation conditions were optimized and the inoculum amount, incubation period, effect of carbon and nitrogen sources, and rates of MgSO4 and CaCO3 were investigated. LipSB 25-4 (the lipase produced by S. bambergiensis OC 25-4 strain) was partially purified with ammonium sulphate precipitation, dialysis, and gel filtration chromatography 2.73-fold and with 92.12 U/mg specific activity. The optimal pH and temperature for LipSB 25-4 were determined as 8.0 and 50°C, respectively. The lipase has high stability in all pH and temperature values used in this study. While LipSB 25-4 was slightly activated in the presence of β-mercaptoethanol, it was slightly reduced by PMSF. The enzyme conserved approximately 75% of its activity at the end of 60 h, in the presence of methanol and ethanol. Since LipSB 25-4 displays high activity in the thermophilic conditions and stability in the presence of organic solvents, this lipase can catalyse the biodiesel production from olive oil by the transesterification reactions.

scholarly journals Partial Purification and Characterisation of Alcohol Dehydrogenase from Acetobacter aceti Isolated from Palm Wine

2016 ◽  
Vol 8 (2) ◽  
pp. 232-236
Author(s):  
Donatus Chimaobi ONAH ◽  
Sabinus Oscar O. EZE ◽  
Patrick Emeka ABA
Keyword(s):  
Alcohol Dehydrogenase ◽  
Alcoholic Beverage ◽  
Gel Filtration ◽  
Effective Concentration ◽  
Partial Purification ◽  
Acetobacter Aceti ◽  
Palm Wine ◽  
Effects Of Ph ◽  
Polyethelene Glycol ◽  
Triton X

Palm wine is a very important alcoholic beverage whose consumption is limited because it spoils easily. The study was designed to isolate Acetobacter aceti from palm wine, then extract, purify and characterize alcohol dehydrogenase (AD) from the A. aceti. Muller Hilton agar was used as medium for the growth of A. aceti for 48 h. The cells were harvested and subjected to ultrasonication using 500 watt ultrasonicator. Enzyme assay was carried out in both the supernatant and pellet. The enzyme was precipitated by polyethelene glycol 6000 while gel filtration was used for purifying the enzyme. The effects of pH, temperature and substrate concentration on AD were evaluated. The isolated A. aceti was gram negative, rod shaped, catalase positive, oxidase negative and was able to oxidize acetic acid to CO2 and H2O. Triton X-100 (0.3%) was the most effective concentration in solubilizing the protein (AD), while 15% polyethelene glycol 6000 was the most effective concentration for the precipitation of AD. An optimal pH of 5 was obtained with an optimal temperature of 50 °C. The most appropriate to solubilize and precipitate AD were 0.3% triton X-100 and 15% polyethelene glycol 6000 respectively, while AD activity was reduced under acidic pH, as well as for low and high temperatures.

scholarly journals Vacuolar H+-ATPase of adrenal secretory granules. Rapid partial purification and reconstitution into proteoliposomes

1990 ◽  
Vol 271 (1) ◽  
pp. 127-131 ◽  
Author(s):  
J R Perez-Castiñeira ◽  
D K Apps
Keyword(s):  
Atp Hydrolysis ◽  
Secretory Granules ◽  
Gel Filtration ◽  
Partial Purification ◽  
Weak Base ◽  
Chromaffin Granule ◽  
Octyl Glucoside ◽  
Rapid Purification ◽  
Adrenal Chromaffin ◽  
Triton X

A procedure has been developed for the rapid purification and reconstitution into phospholipid vesicles of the proton-translocating ATPase of bovine adrenal chromaffin-granule membranes. It involves fractionation of the membranes with Triton X-114, resolubilization of the ATPase with n-octyl glucoside, addition of purified lipids and removal of detergent by gel filtration. The entire process can be completed within 2 h. H+ translocation was detected by the ATP-dependent quenching of the fluorescence of a permeant weak base. The effect of varying the lipid composition of the vesicles on ATP hydrolysis and H+ translocation by the reconstituted enzyme was examined. ATPase activity was maximally increased about 4-fold by added lipid, but was relatively insensitive to its composition, whereas vesicle acidification was absolutely dependent on the addition of phospholipids and cholesterol.

scholarly journals Production process and characterization of extra cellular li-pids from bacterial strain from oil industries waste

2016 ◽  
Vol 4 (1) ◽  
pp. 74
Author(s):  
Vadivel Balamurugan ◽  
Veluchamy Balakrishnan ◽  
Arjunan Sundaresan ◽  
Kodhandapani Vasanthi ◽  
Selvaraj Venkatesan
Keyword(s):  
Carbon Source ◽  
Oil Industry ◽  
Downstream Processing ◽  
Partial Purification ◽  
Photometric Method ◽  
Factors Affecting ◽  
Industry Waste ◽  
Ammonium Sulphate Precipitation ◽  
Lipase Enzyme

The Bacillus sp was isolated from oil industry waste. The isolated strain was screened for the production of lipase enzyme. The production was done by shake flask fermentation. After downstream processing, the partial purification was done my ammonium sulphate precipitation & dialysis and the assay was done by photometric method. The various factors affecting production of extra cellular lipase activity was assayed which include pH, different substrate, temperature and additives. Besides, production was made using different carbon source and crude medium. Result showed that pH 6 and 37°C is an optimum environmental parameter for the growth of the isolate. In addition, the sucrose was found to be better carbon source.

scholarly journals Partial Purification and Characterization of Endoxylanase from a fungus, Leohumicola incrustata

Bio-Research ◽  
2021 ◽  
Vol 19 (1) ◽  
pp. 1192-1201
Author(s):  
Olusegun Richard Adeoyo ◽  
Brett Ivan Pletschke ◽  
Joanna Felicity Dames
Keyword(s):  
Industrial Applications ◽  
Glycoside Hydrolases ◽  
Precipitation Method ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Incubation Periods ◽  
Rf Values ◽  
Layer Chromatography

Xylanases are glycoside hydrolases (GH) that degrade β-1,4-xylan, a linear polysaccharide found as hemicellulose in cell wall of plants. Endoxylanase (Endo-1,4-β-xylanase, EC 3.2.1.8) randomly catalyses xylan to produce varying short xylooligosaccharides (XOS). This study aimed to determine the characteristics of a partially purified endoxylanase from Leohumicola incrustata. Enzyme production was carried out using beechwood (BW) xylan, after which the cell-free crude filtrate was concentrated using the ammonium sulphate precipitation method. The hydrolysed products were analysed by thin-layer chromatography (TLC) and zymography. The result showed that the enzyme produced varying smaller-sized linear xylooligosaccharides with Rf values corresponding to those of xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose and other higher oligomers. The endoxylanase had a molecular mass of 72 kDa. The enzyme is stable in the presence of K+, Na+, Ca2+, Fe2+, Mg2+, Zn2+, Co2+, pH of 5.0 and temperature of 37oC. However, the activity gradually decreased after 60 min at 50oC and retained over 69% activity after 120 min, while at 60 and 70oC, the enzyme activity sharply decreased (pre-incubation periods). Endoxylanase from L. incrustata is comparable to those of other microorganisms and should be considered an attractive candidate for future industrial applications.

scholarly journals Extraction, Characterization and Partial Purification of L-Asparaginase from the Leaves of Arachis Hypogaea L.

2019 ◽  
Vol 16 (3) ◽  
pp. 681-691
Author(s):  
Niranjana. J Niranjana ◽  
Kandasamy Arun Gandhi ◽  
D. Sunmathi ◽  
P. Nanthavanan
Keyword(s):  
Metal Ions ◽  
Arachis Hypogaea ◽  
Specific Activity ◽  
Lymphoblastic Leukemia ◽  
Gel Filtration ◽  
Size Exclusion ◽  
Partial Purification ◽  
Crude Enzyme Extract ◽  
Ammonium Sulphate Precipitation ◽  
Arachis Hypogaea L

L-asparaginase has been a promising therapeutic agent in the treatment of acute lymphoblastic leukemia and lymphoma. In recent times, due to the side effects of commercially available bacterial L-asparaginase and its unavoidable importance, plants are being explored as the source of L-asparaginase. The enzyme L-asparaginase was partially purified from Arachis hypogaea L. The crude enzyme extract was subjected to different purification steps including ammonium sulphate precipitation, dialysis followed by separation on Sephadex G-100 gel filtration (size exclusion chromatography) to obtain partially pure form of L - asparaginase. The enzyme was partially purified to 118 folds and contained specific activity of 4686.86 U/mg with 9.85% yield. SDS-PAGE electrophoresis of the partially purified enzyme revealed that it was a single protein with molecular weight of 70 kDa. The study on physiochemical properties showed that L - asparaginase from Arachis hypogaea L. was potassium-dependent in nature, where its optimum pH of enzyme activity was found to be 8.0 and temperature as 40°/50°C with reaction time of 15 - 20 minutes. Also it was observed that the L-asparaginase activity increased with the presence of metal ions such as Na+, Mg++, making it an enzyme dependent on metal ions for its reaction. In addition to this, it was revealed that the enzyme was partially inhibited in presence of certain chelators. The specificity of L-asparaginase obtained from Arachis hypogaea L. with lack of urease activity and minimal glutaminase activity along with less cytotoxicity on human blood indicated it as an efficient chemotherapeutic agent that could be investigated further in future studies.

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