scholarly journals Zymography for Picogram Detection of Lipase and Esterase Activities

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1542
Author(s):  
Andre Mong Jie Ng ◽  
Hongfang Zhang ◽  
Giang Kien Truc Nguyen
Keyword(s):  
Lipolytic Activity ◽  
Industrial Applications ◽  
Buffering Capacity ◽  
Detection Sensitivity ◽  
Lipolytic Enzyme ◽  
Liquid Chromatography Mass Spectrometry ◽  
Lipolytic Enzymes ◽  
Detection And Identification ◽  
Detection Assay ◽  
Esterase Activities

Lipases and esterases are important catalysts with wide varieties of industrial applications. Although many methods have been established for detecting their activities, a simple and sensitive approach for picogram detection of lipolytic enzyme quantity is still highly desirable. Here we report a lipase detection assay which is 1000-fold more sensitive than previously reported methods. Our assay enables the detection of as low as 5 pg and 180 pg of lipolytic activity by direct spotting and zymography, respectively. Furthermore, we demonstrated that the detection sensitivity was adjustable by varying the buffering capacity, which allows for screening of both high and low abundance lipolytic enzymes. Coupled with liquid chromatography-mass spectrometry, our method provides a useful tool for sensitive detection and identification of lipolytic enzymes.

scholarly journals Metaproteomic Discovery and Characterization of a Novel Lipolytic Enzyme From an Indian Hot Spring

2021 ◽  
Vol 12 ◽  
Author(s):  
Dennis Sander ◽  
Yanfei Yu ◽  
Premankur Sukul ◽  
Sina Schäkermann ◽  
Julia E. Bandow ◽  
...  
Keyword(s):  
Chain Length ◽  
Prolonged Exposure ◽  
Hot Spring ◽  
Hydrolytic Activity ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Lipolytic Enzyme ◽  
Lipolytic Enzymes ◽  
Optimum Activity ◽  
Solvent Stable

Lipolytic enzymes are produced by animals, plants and microorganisms. With their chemo-, regio-, and enantio-specific characteristics, lipolytic enzymes are important biocatalysts useful in several industrial applications. They are widely used in the processing of fats and oils, detergents, food processing, paper and cosmetics production. In this work, we used a new functional metaproteomics approach to screen sediment samples of the Indian Bakreshwar hot spring for novel thermo- and solvent-stable lipolytic enzymes. We were able to identify an enzyme showing favorable characteristics. DS-007 showed high hydrolytic activity with substrates with shorter chain length (<C8) with the maximum activity observed against p-nitrophenyl butyrate (C4). For substrates with a chain length >C10, significantly less hydrolytic activity was observed. A preference for short chain acyl groups is characteristic for esterases, suggesting that DS-007 is an esterase. Consistent with the high temperature at its site of isolation, DS-007 showed a temperature optimum at 55°C and retained 80% activity even after prolonged exposure to temperatures as high as 60°C. The enzyme showed optimum activity at pH 9.5, with more than 50% of its optimum activity between pH 8.0 and pH 9.5. DS-007 also exhibited tolerance toward organic solvents at a concentration of 1% (v/v). One percent of methanol increased the activity of DS-007 by 40% in comparison to the optimum conditions without solvent. In the presence of 10% methanol, DMSO or isopropanol DS-007 still showed around 50% activity. This data indicates that DS-007 is a temperature- and solvent-stable thermophilic enzyme with reasonable activity even at lower temperatures as well as a catalyst that can be used at a broad range of pH values with an optimum in the alkaline range, showing the adaptation to the habitat’s temperature and alkaline pH.

scholarly journals Mycoplasma hyopneumoniae p65 Surface Lipoprotein Is a Lipolytic Enzyme with a Preference for Shorter-Chain Fatty Acids

2004 ◽  
Vol 186 (17) ◽  
pp. 5790-5798 ◽  
Author(s):  
Jono A. Schmidt ◽  
Glenn F. Browning ◽  
Philip F. Markham
Keyword(s):  
Fatty Acids ◽  
Signal Sequence ◽  
Lipolytic Activity ◽  
Mycoplasma Hyopneumoniae ◽  
Lipolytic Enzyme ◽  
Protein Cleavage ◽  
Lipolytic Enzymes ◽  
Hydrolysis Of ◽  
Chain Fatty Acids

ABSTRACT Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39°C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.

scholarly journals Studies on lipid digestion in the preruminant calf. The source of lipolytic activity in the abomasum

1976 ◽  
Vol 36 (3) ◽  
pp. 439-447 ◽  
Author(s):  
Joyce Toothill ◽  
S. Y. Thompson ◽  
J. D. Edwards-Webb
Keyword(s):  
Fatty Acids ◽  
Dietary Fat ◽  
Milk Fat ◽  
Lipolytic Activity ◽  
Lipolytic Enzyme ◽  
Lipid Digestion ◽  
Lipolytic Enzymes ◽  
Ph Values ◽  
Proteolytic Activities ◽  
Pouch Secretions

1. Lipolytic and proteolytic activities and pH values were determined in secretions collected from innervated abomasal pouches and in abomasal contents from preruminant calves given liquid diets.2. No lipolytic activity was detected in pouch secretions collected during 1 h after feeding, though lipolytic activity was present in abomasal contents; pepsin (EC 3.4.23.1) and rennin (EC 3.4.23.4) were present in both pouch secretions and abomasal contents. The pH values of pouch secretions ranged from 1.2 to 1.8 and those of abomasal contents from 4.2 to 5.9.3. When diet was placed directly into the abomasal pouch soon after feeding, the pH values of pouch and abomasal contents decreased similarly (i.e. from 6.3 to approximately 5). Protease activity (U/ml) of pouch contents ranged from 0.1 to 0.8 and that of abomasal contents from 0.1 to 0.2. No lipolytic activity was detected in pouch contents, though abomasal contents contained 0.6 to 1.2 U/ml and when the diet contained milk-fat as the dietary fat source considerable lipolysis of triglycerides containing shorter-chain fatty acids was found.4. It is concluded that there is no significant secretion of lipolytic enzymes by the fundal mucosa and that the lipolysis of triglycerides in the abomasum of the preruminant calf is due predominantly to a lipolytic enzyme in saliva.

scholarly journals High Sensitivity Continuous Monitoring of Chloroform Gas by Using Wavelength Modulation Photoacoustic Spectroscopy in the Near-Infrared Range

2021 ◽  
Vol 11 (15) ◽  
pp. 6992
Author(s):  
Tie Zhang ◽  
Yuxin Xing ◽  
Gaoxuan Wang ◽  
Sailing He
Keyword(s):  
Photoacoustic Spectroscopy ◽  
Continuous Monitoring ◽  
Near Infrared ◽  
Resonant Cavity ◽  
High Sensitivity ◽  
Industrial Applications ◽  
Detection Sensitivity ◽  
Infrared Range ◽  
Wavelength Modulation ◽  
Linear Coefficient

An optical system for gaseous chloroform (CHCl3) detection based on wavelength modulation photoacoustic spectroscopy (WMPAS) is proposed for the first time by using a distributed feedback (DFB) laser with a center wavelength of 1683 nm where chloroform has strong and complex absorption peaks. The WMPAS sensor developed possesses the advantages of having a simple structure, high-sensitivity, and direct measurement. A resonant cavity made of stainless steel with a resonant frequency of 6390 Hz was utilized, and eight microphones were located at the middle of the resonator at uniform intervals to collect the sound signal. All of the devices were integrated into an instrument box for practical applications. The performance of the WMPAS sensor was experimentally demonstrated with the measurement of different concentrations of chloroform from 63 to 625 ppm. A linear coefficient R2 of 0.999 and a detection sensitivity of 0.28 ppm with a time period of 20 s were achieved at room temperature (around 20 °C) and atmosphere pressure. Long-time continuous monitoring for a fixed concentration of chloroform gas was carried out to demonstrate the excellent stability of the system. The performance of the system shows great practical value for the detection of chloroform gas in industrial applications.

scholarly journals Characterization of the Gene Encoding the Major Secreted Lysophospholipase A of Legionella pneumophila and Its Role in Detoxification of Lysophosphatidylcholine

2002 ◽  
Vol 70 (11) ◽  
pp. 6094-6106 ◽  
Author(s):  
Antje Flieger ◽  
Birgid Neumeister ◽  
Nicholas P. Cianciotto
Keyword(s):  
Fatty Acids ◽  
Legionella Pneumophila ◽  
Lipolytic Activity ◽  
Cholesterol Acyltransferase ◽  
Phospholipase A ◽  
Wild Type ◽  
Gene Encoding ◽  
Acyltransferase Activity ◽  
Lipolytic Enzymes ◽  
Cloned Gene

ABSTRACT We previously showed that Legionella pneumophila secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids. In this study, we identified and characterized plaA, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids. The plaA gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL. In Escherichia coli, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol. An L. pneumophila plaA mutant was generated by allelic exchange. Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of L. pneumophila. The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of plaA. Overexpression of plaA completely protected L. pneumophila from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids. The plaA mutant grew like the wild type in U937 cell macrophages and Hartmannella vermiformis amoebae, indicating that PlaA is not essential for intracellular infection of L. pneumophila. In the course of characterizing plaA, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by L. pneumophila.

Changes in lipase activity when using gelatinous agents

2021 ◽  
pp. 40-41
Author(s):  
Михаил Александрович Лаврухин ◽  
Алла Евгеньевна Баженова ◽  
Оксана Сергеевна Руденко ◽  
Николай Борисович Кодратьев
Keyword(s):  
Lipase Activity ◽  
Lipolytic Activity ◽  
Lipolytic Enzymes ◽  
Agar Solution ◽  
Confectionery Products

Липолитическая порча, приводящая к появлению мыльного привкуса, является браковочным признаком кондитерских изделий. Исследовано влияние студнеобразователей на активность липолитических ферментов, Растворы пектина и желатина не оказали влияния на липолитическую активность, а 1%-ный раствор агара увеличил ее на 10 %. Результаты работы способствуют повышению сохранности кондитерских изделий. Lipolytic spoilage, which leads to the appearance of a soapy taste, is a defective sign of confectionery products. The effect of gelatinous agents on the activity of lipolytic enzymes was studied, pectin and gelatin solutions did not affect the lipolytic activity, and 1 % agar solution increased by 10 %. The results of the work contribute to improving the safety of confectionery products.

scholarly journals Fermentative Production of Mannosylerythritol Lipids using Sweetwater as Waste Substrate by Pseudozyma antarctica (MTCC 2706)

2021 ◽  
Vol 58 (4) ◽  
pp. 246-258
Author(s):  
Jayata Mawani ◽  
Jagruti Jadhav ◽  
Amit Pratap
Keyword(s):  
Soybean Oil ◽  
Industrial Applications ◽  
Commercial Production ◽  
Liquid Chromatography Mass Spectrometry ◽  
Mannosylerythritol Lipids ◽  
Pseudozyma Antarctica ◽  
Fermentative Production ◽  
Oil Concentration ◽  
High Production ◽  
High Production Cost

Abstract Mannosylerythritol lipids are glycolipid biosurfactants with promising industrial applications. However, their commercial production is hindered due to its high production cost. The current study investigates the use of sweetwater, a by-product of the fat-splitting industry in combination with soybean oil for the production of mannosylerythritol lipids using Pseudozyma antarctica (MTCC 2706). The optimum sweetwater and soybean oil concentration of 22% and 7% (w/v) yielded 7.52 g L–1and 21.5 g L–1 mannosylerythritol lipids at shake flask and fermenter level respectively. The structure and functional groups of mannosylerythritol lipids were confirmed by fourier transform infrared (FTIR) spectroscopy, liquid chromatography-mass spectrometry (LC/MS) and 1H- and 13C-nuclear magnetic resonance (NMR) analysis. Surfactant properties, such as surface tension, critical micelle concentration, foaming and emulsification of mannosylerythritol lipids were also explored.

Targeted analysis of phenolic compounds by LC-MS v1

2021 ◽  
Author(s):  
Bashar Amer ◽  
Ramu Kakumanu ◽  
yangtian not provided ◽  
Aymerick Eudes ◽  
Edward EK Baidoo
Keyword(s):  
Phenolic Compounds ◽  
Plant Biomass ◽  
Reversed Phase ◽  
Industrial Applications ◽  
Liquid Chromatography Mass Spectrometry ◽  
Reversed Phase Liquid Chromatography ◽  
Sample Matrix ◽  
Targeted Analysis ◽  
Challenges And Opportunities ◽  
Phase Liquid

Cell-wall-bound (CWB) aromatics such as ferulate and p-coumarate play important physiological roles in plant development and response to stresses. Their presence also poses some challenges and opportunities during processing of plant biomass in various agro-industrial applications. To this end, we have developed a robust high-throughput reversed-phase liquid chromatography mass spectrometry method for quantifying CWB phenolic compounds. The method showed excellent linearity (R2 = ≥0.999) and intraday retention time repeatability (≤ 0.31 %RSD) for ferulate and p-coumarate. The limits of detection and quantitation for these analytes were ≤ 39 nM and 130 nM, respectively. Furthermore, there was very little effect of the CWB sample matrix on the retention times of the analytes and analyte percent recoveries from the CWB sample matrix was ≥83.91%.

Cardiac triacylglycerol content and lipase activity during recovery from exercise

1989 ◽  
Vol 66 (3) ◽  
pp. 1099-1103 ◽  
Author(s):  
B. Podbielski ◽  
W. K. Palmer
Keyword(s):  
Lipase Activity ◽  
Temporal Relationship ◽  
Lipolytic Activity ◽  
Control Level ◽  
Female Rats ◽  
Lipolytic Enzyme ◽  
Glycogen Level ◽  
Control Activity ◽  
Triacylglycerol Content ◽  
Plasma Ffa

Female rats swam for 2-h to determine the temporal relationship between triglyceride (TG) repletion and TG lipase activity in the heart during recovery from exercise. Immediately after the exercise, plasma free fatty acids (FFA) had increased from a resting value of 0.44 +/- 0.04 to 0.84 +/- 0.04 mM. Heart TG concentration was reduced 75%, whereas the glycogen level was decreased 34% below control. TG lipase activity was elevated 33% above control activity. One hour after the end of the exercise, lipolytic activity was still 26% above control and did not return to the resting level until the 4th h of recovery. The cardiac TG concentration was back to control levels by the 2nd h after the swim. Plasma FFA concentrations remained elevated during the first 4 h of recovery and were back to the control level by h 8. Cardiac glycogen was “supercompensated” during recovery h 1 and 2 and returned to the preexercise level by h 4. These data indicate that TG is being synthesized in the heart while lipolytic enzyme activity is elevated above control levels. This points out that the rate of TG synthesis is in excess of the hydrolysis. Since plasma FFA concentrations are elevated during periods of augmented TG synthesis, substrate availability, namely plasma FFA, may play a key role in regulating the size of the intracellular TG pool.

scholarly journals Development of an Automated High-Throughput Analytical Characterization System for Support of a Central Sample Collection Resource

2003 ◽  
Vol 8 (3) ◽  
pp. 55-61
Author(s):  
Bernard K. Choi ◽  
Nathan A. Yates ◽  
James P. Springer ◽  
Robert E. Schwartz ◽  
Andrew Roberts
Keyword(s):  
Small Sample ◽  
Continuous Operation ◽  
Detection Sensitivity ◽  
Liquid Chromatography Mass Spectrometry ◽  
Sample Collection ◽  
Information Characteristic ◽  
Analytical Characterization ◽  
On Line ◽  
Analytical Instrumentation ◽  
Relative Purity

Ahigh-throughput analytical characterization system was developed for quality control support of a central sample collection resource. This system utilizes liquid chromatography mass spectrometry with in-house developed data automation applications. Continuous operation of analytical instrumentation is accomplished by fully automating sample submission and report processing functions. Comprehensive analytical information characteristic of quality, chemical, and physical properties (e.g. relative purity, detection sensitivity, LogD) are automatically transferred to an on-line database. The application of this database for detailed quality assessment of a small sample library (ca. 24,000 compounds) is demonstrated.

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