scholarly journals Novel Coprinopsis cinerea Polyesterase That Hydrolyzes Cutin and Suberin

2009 ◽  
Vol 75 (7) ◽  
pp. 2148-2157 ◽  
Author(s):  
Hanna Kontkanen ◽  
Ann Westerholm-Parvinen ◽  
Markku Saloheimo ◽  
Michael Bailey ◽  
Marjaana Rättö ◽  
...  
Keyword(s):  
Residual Activity ◽  
Fatty Acid Esters ◽  
Coprinopsis Cinerea ◽  
Outer Bark ◽  
Metal Affinity ◽  
Wide Ph Range ◽  
Ph Range ◽  
Laboratory Fermentor ◽  
Acid Esters

ABSTRACT Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter−l purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2 to C10) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.

Polarographic and spectrophotometric behaviour of some N-p-phenyl substituted benzamidines

1991 ◽  
Vol 56 (12) ◽  
pp. 2791-2799 ◽  
Author(s):  
Juan A. Squella ◽  
Luis J. Nuñez-Vergara ◽  
Hernan Rodríguez ◽  
Amelia Márquez ◽  
Jose M. Rodríguez-Mellado ◽  
...  
Keyword(s):  
Spectrophotometric Study ◽  
Absorption Bands ◽  
Electrochemical Parameters ◽  
Wide Ph Range ◽  
Group A ◽  
Substituent Constants ◽  
Ph Range ◽  
The Waves

Five N-p-phenyl substituted benzamidines were studied by DC and DP polarography in a wide pH range. Coulometric results show that the overall processes are four-electron reductions. Logarithmic analysis of the waves indicate that the process are irreversible. The influence of the pH on the polarographic parameters was also studied. A UV spectrophotometric study was performed in the pH range 2-13. In basic media some variations in the absorption bands were observed due to the dissociation of the amidine group. A determination of the pK values was made by deconvolution of the spectra. Correlations of both the electrochemical parameters and spectrophotometric pK values with the Hammett substituent constants were obtained.

scholarly journals Digestive Proteases From Fish Processing Wastes: Their Partial Characterization and Comparison

2020 ◽  
Author(s):  
Ivana Soledad Friedman ◽  
Leonel Agustín Behrens ◽  
Nair A Pereira ◽  
Edgardo Contreras ◽  
Analia Verónica Fernández-Gimenez
Keyword(s):  
Residual Activity ◽  
Fish Processing ◽  
Intestinal Enzymes ◽  
Optimum Ph ◽  
Digestive Proteinases ◽  
Wide Ph Range ◽  
Processing Wastes ◽  
Reducing Costs ◽  
Ph Range ◽  
Percophis Brasiliensis

Abstract Fish processing generates a lot of wastes which are discarded resulting in environmental problems. However, this material represents a significant source of high-value bioproducts with potential biotechnological applications. The objective of this study was to characterize and to compare specific activities of acid and alkaline proteases recovered from the viscera of Merluccius hubbsi (Mh), Percophis brasiliensis (Pb), Urophyis brasiliensis (Ub), and Cynoscion guatucupa (Cg) under different pH and temperature conditions. Stomach proteinases from four species had a higher activity at pH 2, with stability in the range of pH 2-4. Optimum pH from intestinal enzymes of Cg was 11.5, while for the crude extract of Mh, Pb, and Ub catalytic activity was registered over a wide pH range range from 7 to 11.5. Stomach proteinases from four studied species had a higher activity at 30 °C and 50 °C, with stability at 10 °C and 30 °C. Optimum temperature from intestinal enzymes of the four tested species was 50 °C with high stability at 10 °C and 30 °C. Alkaline proteinase from all species and acid proteinases from Cg was inactivated at 70ºC, while stomach enzymes of Mh, Pb, and Ub had a residual activity lower than 5% at 80 °C after 5, 10 y 20 minutes of pre-incubation, respectively. Digestive proteinases recovered in this study could be used as biocatalysts in industrial processes, reducing costs, adding value to the fishery waste, and contributing to the reduction of environmental pollution.

2-Monochloropropanediol (2-MCPD), 3-Monochloropropanediol (3-MCPD), and Glycidol in Infant and Adult/Pediatric Nutritional Formula: Single-Laboratory Validation, First Action 2018.12

2019 ◽  
Vol 102 (4) ◽  
pp. 1205-1220 ◽  
Author(s):  
Jan Kuhlmann
Keyword(s):  
Fatty Acid ◽  
Infant Formula ◽  
Edible Oils ◽  
Accurate Determination ◽  
Fatty Acid Esters ◽  
Method Performance ◽  
Relative Standard ◽  
Single Laboratory ◽  
Acid Esters

Abstract Background: Fatty acid esters of glycidol, 2-Monochloropropanediol (MCPD), and 3-MCPD are heat-induced foodborne processing contaminants with possible adverse health effects. These compounds occur frequently in refined edible oils. Consequently, glycidyl esters and 2- and 3-MCPD esters might also be present in foods that contain refined edible oils. Objective: This manuscript describes the single-laboratory validation of an analytical method for the quantitative determination of glycidol, 2-MCPD, and 3-MCPD present as fatty acid esters or as free 2- or 3-MCPD in infant and adult/pediatric nutritional formula. Methods: Technically, the presented method is based on the combination of a Heat-Ultrasound Pressure-supported Solvent Extraction and a GC–MS determination of glycidol, 2-MCPD, and 3-MCPD. From a chemical perspective, the method includes an alkaline catalyzed transesterification, conversion of the unstable glycidol into monobromopropanediol, and the parallel derivatization of all analytes with phenylboronic acid. Results: Validation results showed that method linearity for all analytes in powdered and liquid infant formula ranged from 0.9981 to 0.9999 (n = 18). Repeatability relative standard deviation values for concentration levels between 1.3 μg/kg and 331 μg/kg were in the range of 1 to 12%. Relative recoveries were found to be between 93 and 107%. The analytes were quantifiable down to 5–10 μg/kg in powdered samples and 1–2 μg/kg in liquid samples. Conclusions: The reported results met actual AOAC Standard Method Performance Requirements. Highlights: In terms of consumer protection, the presented method is a novel approach for the sensitive and accurate determination of glycidol, 2-MCPD, and 3-MCPD in infant formula and related foodstuffs.

A Simple Method for the Determination of Lipoprotein Lipase

1968 ◽  
Vol 14 (10) ◽  
pp. 1023-1025
Author(s):  
Leonard J Stutman ◽  
Marilyn Dolliver
Keyword(s):  
Fatty Acids ◽  
Lipoprotein Lipase ◽  
Fatty Acid Esters ◽  
Simple Method ◽  
Intravenous Heparin ◽  
Dense Band ◽  
Layer Chromatography ◽  
Acid Esters

Abstract A rapid method for determining the in-vivo effect of small amounts of intravenous heparin utilizing thin-layer chromatography is presented. A dense band of stainable fatty acids appears and represents nonesterified acids after the fatty acid esters have been hydrolyzed by lipoprotein lipase and determines, therefore, whether the enzyme is activated by heparin.

scholarly journals Quantitative Determination of Estradiol Fatty Acid Esters in Lipoprotein Fractions in Human Blood

2003 ◽  
Vol 88 (6) ◽  
pp. 2552-2555 ◽  
Author(s):  
Veera Vihma ◽  
Aila Tiitinen ◽  
Olavi Ylikorkala ◽  
Matti J. Tikkanen
Keyword(s):  
Fatty Acid ◽  
Quantitative Determination ◽  
Human Blood ◽  
Fatty Acid Esters ◽  
Acid Esters

scholarly journals Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

2015 ◽  
Vol 81 (19) ◽  
pp. 6938-6944 ◽  
Author(s):  
Tao Tu ◽  
Huiying Luo ◽  
Kun Meng ◽  
Yanli Cheng ◽  
Rui Ma ◽  
...  
Keyword(s):  
Rational Design ◽  
Residual Activity ◽  
Maximal Activity ◽  
Wild Type ◽  
Content Type ◽  
Highly Active ◽  
Wide Ph Range ◽  
Enzyme Thermal Stability ◽  
Ph Range ◽  
Charge Interactions

ABSTRACTImproving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) fromAchaetomiumsp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues—D244 and D299—were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (Tmax), temperature at which 50% of the maximal activity of an enzyme is retained (T50), and melting temperature (Tm), of about 10, 17, and 10.2°C upward, respectively, with the half-life (t1/2) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ± 130 U/mg versus 28,000 ± 293 U/mg); on the other hand, it showed more residual activity (8,400 ± 83 U/mg versus 1,400 ± 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability.

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