Application of glycerol to freezing bovine pancreas. Part 1. Effect of cryoprotector on activity of certain enzymes in frozen minced pancreas

Hajduk, Ewa; Fik, M.

doi:10.1002/food.19900340117

Cited by 5 publications

(4 citation statements)

References 9 publications

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“…In fact, the addition of glycerol reveals positive effects in the −80 °C storage, independently, from the used buffer registering a Tm increasing in range from 3 to 4 °C. This behavior has already been observed for proteins such as β-lactoglobulin, lysozyme, and amylase, among others [ 11 , 51 , 52 ].…”

Section: Resultssupporting

confidence: 60%

“…Specifically, Lerbret and coworkers observed the “destructuring” effect of trehalose upon the water network by which the water molecules are ordered next to the sugar molecule (as a kosmotrope), preventing ice formation [ 9 ]. Glycerol is also known for its cryoprotectant role [ 10 , 11 ]. This polyol enhances protein stability by increasing its hydration.…”

Section: Introductionmentioning

confidence: 99%

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Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Gonçalves

Pedro

Oliveira

et al. 2022

IJMS

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Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson’s disease. Currently, Parkinson’s disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography—IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 °C and −80 °C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.

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Section: Resultssupporting

confidence: 60%

Section: Introductionmentioning

confidence: 99%

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Gonçalves

Pedro

Oliveira

et al. 2022

IJMS

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“…In order to use pooled gastroduodenal juices in in vitro digestion studies, the stability of the individual enzyme activities needs to be monitored and preferably stabilised to avoid substantial decrease in activities. Glycerol has by many been used as a cryoprotector to avoid loss of enzyme activities in stored gastric and duodenal juice [ 20 , 44 , 45 ]. The addition of 13% glycerol to freshly isolated gastric and duodenal juices only marginally protected the enzymes from degradation compared to the control in this study.…”

Section: Discussionmentioning

confidence: 99%

Human Gastrointestinal Juices Intended for Use in In Vitro Digestion Models

et al. 2011

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The aim of this study was to characterise the individual human gastric and duodenal juices to be used in in vitro model digestion and to examine the storage stability of the enzymes. Gastroduodenal juices were aspirated, and individual variations in enzymatic activities as well as total volumes, pH, bile acids, protein and bilirubin concentrations were recorded. Individual pepsin activity in the gastric juice varied by a factor of 10, while individual total proteolytic activity in the duodenal juice varied by a factor of 5. The duodenal amylase activity varied from 0 to 52.6 U/ml, and the bile acid concentration varied from 0.9 to 4.5 mM. Pooled gastric and duodenal juices from 18 volunteers were characterised according to pepsin activity (26.7 U/ml), total proteolytic activity (14.8 U/ml), lipase activity (951.0 U/ml), amylase activity (26.8 U/ml) and bile acids (4.5 mM). Stability of the main enzymes in two frozen batches of either gastric or duodenal juice was studied for 6 months. Pepsin activity decreased rapidly and adjusting the pH of gastric juice to 4 did not protect the pepsin from degradation. Lipase activity remained stable for 4 months, however decreased rapidly thereafter even after the addition of protease inhibitors. Glycerol only marginally stabilised the survival of the enzymatic activities. These results of compositional variations in the individual gastrointestinal juices and the effect of storage conditions on enzyme activities are useful for the design of in vitro models enabling human digestive juices to simulate physiological digestion.

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“…In order to figure out the best method to store rEla for a longer period of time, we incubated the dissolved enzyme at 4, 25 and − 20 °C and also lyophilized enzyme at 4 °C for 100 days. Glycerol at final concentration www.nature.com/scientificreports/ of 20% was added to half of the -20 °C samples as it has anti-destruction effect on native protein structures while freezing, by forming hydrogen bonds with water molecules but it caused negative outcome, the enzyme concentration decreased and it showed 15% less activity in comparison with samples kept in − 20 °C without glycerol 52 . Altogether the results indicate that rEla has remarkable stability and enzyme half-life at room temperature is about 100 days.…”

Section: Discussionmentioning

confidence: 99%

Cohnella 1759 cysteine protease shows significant long term half-life and impressive increased activity in presence of some chemical reagents

Saghian

Mokhtari

Aminzadeh

2021

Sci Rep

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Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme Km and Vmax values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme’s co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

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Application of glycerol to freezing bovine pancreas. Part 1. Effect of cryoprotector on activity of certain enzymes in frozen minced pancreas

Cited by 5 publications

References 9 publications

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase

Human Gastrointestinal Juices Intended for Use in In Vitro Digestion Models

Cohnella 1759 cysteine protease shows significant long term half-life and impressive increased activity in presence of some chemical reagents

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