Assaying Arabidopsis lipase activity

2000 ◽  
Vol 28 (6) ◽  
pp. 773-775 ◽  
Author(s):  
F. Beisson ◽  
V. Arondel ◽  
R. Verger
Keyword(s):  
Lipase Activity ◽  
Crude Extract ◽  
Specific Activity ◽  
Colorimetric Assay ◽  
Fluorescence Assay ◽  
Plant Lipase ◽  
Parinaric Acid ◽  
Fluorescence Assays ◽  
Continuous Assay

A low lipase activity from a crude extract of Arabidopsis seedlings was assayed using three sensitive methods (radiolabelled triacylglycerols, commercial resorufin ester and triacylglycerols containing the naturally fluorescent parinaric acid as substrates). The specific activity of the extract was found to be similar using the three methods. However, the plant lipase activity measured using the radioactivity and the fluorescence assays could be abolished by heating the extract, contrary to the apparent activity measured using the commercial colorimetric assay. Unlike the radioactivity assay, the fluorescence assay can be monitored continuously. The parinaric acid-based method is therefore the only one to provide a sensitive, specific and continuous assay.

scholarly journals Antibacterial and Cytotoxicity Activities of Major Compounds from Tinospora cordifolia Willd. Growing on Mangifera indica L.

2019 ◽  
Vol 3 (4) ◽  
pp. 32-42
Author(s):  
Thongchai Taechowisan
Keyword(s):  
Antibacterial Activity ◽  
Cell Lines ◽  
Crude Extract ◽  
Hek293 Cell ◽  
Colorimetric Assay ◽  
Mangifera Indica ◽  
Tinospora Cordifolia ◽  
Cytotoxicity Activity ◽  
Stem Extract ◽  
Cytotoxicity Activities

Objective To investigate the major constituents of Tinosporacordifolia Willd. growing on Mangiferaindica, and to evaluate the efficacy of their antibacterial and cytotoxicity activities. Methods The ethanolic stem extract of T. cordifolia was subjected to silica gel 60 column chromatography, thin layer chromatography and medium pressure liquid chromatography for isolation of the major compounds. Identification of purified compounds was achieved by spectroscopic methods.. The crude extract and purified compounds were screened for their antibacterial and cytotoxicity properties using standard procedures. Results Two alkaloids were purified and identified as Magnoflorin (1) and Tembetarine (2). These compounds showed high antibacterial activity against Bacillus cereus and Staphylococcus aureus with both MIC (32-64 µg/ml) and MBC (128-256 µg/ml). The cytotoxicity activity of the purified compounds and crude extract was determined using MTT colorimetric assay against L929 and HEK293 cell lines. This showed weak cytotoxicity activity with IC50 values of 1162.24 to 2290.00 µg/ml and 1376.67 to 2585.06 µg/ml towards L929 and HEK293 cell lines, respectively. Conclusion The major compounds present in ethanolic stem extract of T. cordifolia growing on M. indica were extracted, purified and identified. This study suggests that these compounds exhibit great potential for antibacterial activity with weak cytotoxicity activity. They may be useful for their medicinal functions.

scholarly journals Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX

2018 ◽  
Vol 84 (16) ◽  
Author(s):  
Wei Zhou ◽  
Rui Huang ◽  
Zhiguang Zhu ◽  
Yi-Heng P. Job Zhang
Keyword(s):  
High Activity ◽  
Double Layer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Specific Activity ◽  
Colorimetric Assay ◽  
Petri Dish ◽  
Rapid Identification ◽  
Thermobifida Fusca ◽  
Content Type

ABSTRACT Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

A novel spectrophotometric assay for lipase activity utilizingcis-parinaric acid

Lipids ◽  
1989 ◽  
Vol 24 (6) ◽  
pp. 518-525 ◽  
Author(s):  
A. M. Rogel ◽  
W. L. Stone ◽  
F. O. Adebonojo
Keyword(s):  
Lipase Activity ◽  
Spectrophotometric Assay ◽  
Parinaric Acid

scholarly journals Determination the Optimum Conditions of the Activity and Stability of Lipase Extracted from Sunflower Germinated Seeds

2021 ◽  
pp. 431-440
Author(s):  
Alaa M. Dh. Al-Haidari ◽  
Ibrahim S. Alsaadawi ◽  
Saad Hussein Khudhair
Keyword(s):  
Lipase Activity ◽  
Specific Activity ◽  
Enzyme Reaction ◽  
Total Activity ◽  
Enzyme Concentration ◽  
Sunflower Seeds ◽  
Optimum Conditions ◽  
Inhibitory Effects ◽  
Mineral Salts ◽  
Lipase Enzyme

The present study was conducted to determine the optimum conditions required for lipase enzyme activity extracted from germinated sunflower seeds, including temperature, pH, agitation, time of incubation, enzyme concentration, substrate type, and concentrations of mineral salts and EDTA. Optimum pH, temperature and time of incubation required for lipase stability were also determined. The results showede optimum lipase activity (3.251U/ml) wasund at 30 ̊C and pH 7 after 20 minutes of incubation when using 1 ml lipase enzyme with 0.02 ml of CaCl2 (10 mM) at 100 rpm of agitation and in the presence of olive oil as the substrate for enzyme reaction. EDTA appeared to have inhibitory effects, while Ca+2 and Mg+2 have stimulatory effects on lipase activity. The values of lipase activity, total activity, and specific activity measured under optimum conditions were increased by 36.99%, 36.95%, and 38.21% over control, respectively. The enzyme showed stability at a temperature ranged between 30 to 50 ˚C, pH between 7 to 8, and time of incubation between 10 to 40 minutes. These results suggest that lipase enzyme extracted from germinated sunflower seeds have stability that depends on pH, temperature, and incubation period, which enables it to be used in different industries.

scholarly journals Bromelain Activity of Waste Parts of Two Pineapple Varieties

2018 ◽  
Vol 2 ◽  
pp. 21-28 ◽  
Author(s):  
Edmund Ofosu Benefo ◽  
Isaac Williams Ofosu
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulphate ◽  
Crude Extract ◽  
Specific Activity ◽  
High Demand ◽  
Digestion Method ◽  
Waste Products ◽  
Food Industries ◽  
Ethanol Precipitation ◽  
Ammonium Sulphate Precipitation

Bromelain, a protease found in pineapples, is of high demand in the pharmaceutical, cosmetic and food industries. Along the pineapple processing chain, waste products such as peels, crowns, stems and cores result. These parts are usually discarded, though they contain significant amounts of the enzyme bromelain. This study sought to determine the bromelain activity of the crowns and peels of two pineapple varieties grown in Ghana;MD2andSugarloaf. The crude extract was obtained by homogenising the peels and crowns in a cold phosphate buffer and centrifuging at 3000 rpm for 15 min. Ethanol and ammonium sulphate precipitation were carried out on the extract between 30% – 80% precipitation levels. The enzyme activity was determined using the casein digestion method. Results showed that bromelain was precipitated mainly in the 30% – 60% precipitation range.Sugarloafcrowns yielded the highest enzyme activity of 20.82 U/ml and a specific activity of 194.58 U/mg at the 40% ammonium sulphate precipitation level. This was followed by theSugarloafpeels with an enzyme activity of 19.98 U/ml at 50% ethanol precipitation level. Ethanol precipitation resulted in fractions with lower bromelain activity. Enzyme activity was higher in theSugarloafvariety and also in the crowns of both varieties. The two pineapple varieties have significant levels of bromelain activity and could be exploited for commercialisation.

scholarly journals Lipolytic Activities of Bacteria and Fungi Isolated from Soil Samples

2021 ◽  
pp. 27-43
Author(s):  
Racheal Oluwayemisi Fashogbon ◽  
Bose Adebayo ◽  
Victoria Musa ◽  
Titilayo Femi-Ola
Keyword(s):  
Lipase Activity ◽  
Metal Ion ◽  
Specific Activity ◽  
Soil Samples ◽  
Bacillus Sp ◽  
State University ◽  
Sudan Black B ◽  
Fusarium Sp ◽  
Microbial Lipases ◽  
Bacteria And Fungi

This study was carried out at the Department of Microbiology, Microbiology Laboratory, Ado-Ekiti State University, Ekiti State, Nigeria between July, 2018 to March, 2019. Due to the diverse biotechnological importance of lipases as a biocatalytic enzyme, extracellular production of microbial lipases has to gain lots of interest. This study, therefore, focused on the physicochemical parameters of lipase producing microorganisms from different soil samples. Microorganisms were isolated from four different soil samples using Nutrient Agar (NA) and Potato Dextrose Agar (PDA). The isolates were identified and characterized. Production, an assay for Lipase enzymes, purification, the effect of pH, Temperature and metal ion was investigated. The isolates were culturally, morphologically and biochemically characterized. Two of the bacteria strains (Bacillus sp. and Staphylococcus sp.) and four fungi (Fusarium sp., Aspergillus fumigatus, Aspergillus niger, and Trichophyton sp.) isolates were able to produce lipid using Sudan Black B Fat staining techniques. Fusarium sp. isolated from dumpsite soil had the highest specific lipase activity (21.16 µmol/min/ml) while Bacillus sp. isolated from red oil spill soil had the highest lipase activity (0.59 µmol/min/mg). The specific activity of partially purified lipase for Fusarium sp. was 2.39 µmol/min/mg while Bacillus sp. had a specific activity of 2.46 µmol/min/mg. 30oC - 50oC, pH 7.0 to 9.0 and KCl2 (139.672%) supported the highest production of lipase by the Bacillus sp. and Fusarium sp. This study demonstrated that the Bacillus sp. produced a high amount of lipase activity followed by Fusarium sp. Extensive and persistent screening for new microorganisms and their lipolytic activities will help to provide faster ways to solve most environmental soil pollution.

High-resolution colorimetric detection of lipase activity based on enzyme-controlled reshaping of gold nanorods

2019 ◽  
Vol 11 (17) ◽  
pp. 2286-2291 ◽  
Author(s):  
Hao Zhang ◽  
Shengnan Wu ◽  
Linghua Zhang ◽  
Ling Jiang ◽  
Fengwei Huo ◽  
...  
Keyword(s):  
High Resolution ◽  
Gold Nanorods ◽  
Lipase Activity ◽  
Colorimetric Assay ◽  
Colorimetric Detection ◽  
Sensing Mechanism

The sensing mechanism of a high resolution colorimetric assay for the visual readout of lipase activity based on AuNR reshaping.

scholarly journals Effect of diabetes on acid and neutral triacylglycerol lipase and on lipoprotein lipase activities in isolated myocardial cells from rat heart

1986 ◽  
Vol 238 (1) ◽  
pp. 233-238 ◽  
Author(s):  
I Ramírez ◽  
D L Severson
Keyword(s):  
Lipoprotein Lipase ◽  
Lipase Activity ◽  
Specific Activity ◽  
Subcellular Fractions ◽  
Capillary Endothelium ◽  
Myocardial Cells ◽  
Protamine Sulphate ◽  
Triacylglycerol Lipase ◽  
Neutral Lipase ◽  
High Concentrations

A neutral triacylglycerol lipase activity that is separate and distinct from lipoprotein lipase (LPL) could be measured in homogenates of myocardial cells if protamine sulphate and high concentrations of albumin were included in the assay. This neutral lipase was predominantly particulate, with the highest relative specific activity in microsomal subcellular fractions. The induction of diabetes by the administration of streptozotocin to rats resulted in a decrease in LPL activity in myocyte homogenates and in particulate subcellular fractions, but the percentage of cellular LPL activity that was released during incubation of myocytes with heparin was normal. In contrast, neutral lipase activity was increased in diabetic myocyte homogenates and microsomal fractions. Acid triacylglycerol lipase activity was not changed in diabetic myocytes. The decrease in LPL in myocytes owing to diabetes may result in the decreased functional LPL activity at the capillary endothelium of the diabetic heart.

scholarly journals Specificity and localization of lipolytic activity in adult Drosophila melanogaster

1994 ◽  
Vol 304 (3) ◽  
pp. 775-779 ◽  
Author(s):  
G M Smith ◽  
K Rothwell ◽  
S L Wood ◽  
S J Yeaman ◽  
M Bownes
Keyword(s):  
Drosophila Melanogaster ◽  
Lipase Activity ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Accessory Gland ◽  
Null Mutant ◽  
Male Accessory Gland ◽  
Accessory Glands ◽  
Esterase 6 ◽  
Adult Drosophila

The triacylglycerol lipases present in adult Drosophila melanogaster have been investigated. Different lipase activities are present in various tissues in the fly. In particular, an abundant lipase activity is present in the male accessory gland. An esterase null mutant was used to confirm that the enzyme activity was due to a distinct lipase and not non-specific activity from esterase 6 which is also abundant in accessory glands. The properties of the accessory-gland lipase were investigated, and pH optima and substrate utilization suggest that it has some similarities to vertebrate bile-salt-stimulated lipase. Lipase activity is significantly reduced in males and increased in females shortly after mating. This finding suggests that lipase activity is transferred to the female and may be important in mating and reproduction in Drosophila.

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