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Digestive enzyme stability

Digestive enzyme stability

side Digrstive of drug treatments such as treatment with metformin or those drugs used Antibacterial carpet cleaner treat the symptoms Digestive enzyme stability HIV Micronutrient deficiency and immune function autoimmune stabillty such as diabetes in which the pancreas may be compromised, obstruction e. Purification, biochemical characterization and Insilico modeling of α-amylase from Vicia faba. Lipase activity was measured after 1, 2 and 3 months of storage as shown in Table Issue Date : March


Digestive Enzymes and Disorders

Digestive enzyme stability -

Veronese FM, Sacca B, de Laureto PP, Sergi M, Caliceti P, Schiavon O, Orsolini P New PEGs for peptide and protein modification, suitable for identification of the PEGylation site.

Bioconjugate Chem 12 1 — Wang H, Lou H, Bai Y, Wang Y, Yang P, Shi P, Zhang W, Fan Y, Yao B An acidophilic β-Galactosidase from Bispora sp. MEY-1 with high lactose hydrolytic activity under simulated gastric conditions. J Agric Food Chem — Xenos K, Kyroundis S, Anagnostidis A, Papastathopoulos P Treatment of lactose intolerance with exogenous beta-D-galactosidase in pellet form.

Eur J Drug Metab 23 2 — Zheng C, Ma G, Su Z Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation. Electrophoresis 28 16 — Download references. Present address: Molecular Glycobiotechnology Group, Biochemistry, National University of Ireland, Galway, Galway, Ireland.

Industrial Biochemistry Program, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland.

Kevin M. Materials and Surface Sciences Institute, University of Limerick, Limerick, Ireland. Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, Padova, Italy. You can also search for this author in PubMed Google Scholar.

Correspondence to Kevin M. Reprints and permissions. Turner, K. et al. Stabilization of a supplemental digestive enzyme by post-translational engineering using chemically-activated polyethylene glycol.

Biotechnol Lett 33 , — Download citation. Received : 05 July Accepted : 04 November Published : 20 November Issue Date : March Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. In addition, the carnivorous fishes displayed lower amylase and alkaline protease activities than the omnivorous and herbivorous fishes.

Isoenzyme analysis and molecular weight determination of amylase and protease enzymes have been performed in bacteria, shrimp, and some species of fishes. Noticeably, bacterial enzymes display only one isoform with molecular weight ranging from 28 to 68 kDa, whereas fish and shrimp contain many isoforms 10 , 12 , 13 , 16 , 19 , 21 , 24 , 25 , 26 , 27 , The several isoforms of digestive enzymes seen in digestive tracts of fish may derive from the floral bacteria However, more experiments have to further investigate bacterial enzymes such as the expression level, biochemical properties, and molecular weights compared to enzyme extracts from digestive tracts of fish.

In the present study, the number of isoenzymes of proteolytic and amylolytic enzymes ranged from one to eight and two to eight with molecular weights between 12 and kDa and 26 and kDa, respectively. spilopterus could be detected on a zymographic gel, and the band was very faint. pH and temperature stabilities have been reported for bacterial α-amylases 10 , 11 , 33 , 34 and bacterial proteases 33 , Amylases from Bacillus showed high stability at pHs between 5.

For marine fish, the digestive enzymes from Atlantic salmon Salmo salar 36 and thornback ray Raja clavata 37 have been reported. However, little information has been reported on stability of digestive enzymes from freshwater fish.

Seven species of fishes including of P. gonionotus have been reported the biochemical properties of digestive enzyme However, molecular weight and enzyme stabilities have not been determined. Chaijareon and Thongruang determined the stability of amylase and protease from Or.

In contrast, our experiments showed that both amylase and alkaline protease from Or. niloticus and also those from P. proctozysron and P. gonionotus exhibited high stability at a wide range of pH 6.

Moreover, the enzymes were stable on incubation in buffer pH 8. High stability of amylase and alkaline protease, digestive enzymes from freshwater fishes, was found in this study and is good evidence for further study and application.

Casein sodium salt, from bovine milk, cat. C and starch from rice, cat. S were purchased from Sigma-Aldrich, USA. Acrylamide cat. Sodium dodecylsulfate cat. DB , soluble starch cat.

SB and β-mercaptoethanol cat. MB were from Bio Basic Canada Inc. Chemical reagents for preparation of buffers were purchased from Carlo Erba France , Scharlau Barcelona, Spain , and Ajax Finechem MA, USA. Samples were collected as part of a fish faunal survey in Lam Nam Choen swamp, Nongruea district, Khon Kaen province, Thailand, by using nets.

Fish were killed in ice without using any chemical agent. The condition did not severely distress or cause lasting harm to sentient fish.

The body weight and body length were recorded. The digestive tracts were subsequently rinsed with distilled water and then homogenized on ice by uses of pestle and mortar with a buffer 0.

Determination of protein content was performed by using Bradford reagent Bovine serum albumin was used for the calibration curve of protein concentration. All methods were carried out in accordance with relevant guidelines and regulations.

All experimental protocols and the care and use of experimental animals complied with animal welfare laws of Thailand, and guidelines and policies approved by ThaiIACUC permit reference number U, To determine the initial velocity of enzymatic reaction, the enzyme activity was observed after incubation of enzyme with substrate for 5 to 30 min.

One reaction for one time point of amylase assay contained 20 μl of supernatant containing digestive enzymes or crude enzyme extract, μl of 0. The reactions were incubated at room temperature 23 °C for 5, 10, 15, 20, 25, or 30 min.

Supernatants containing the catalytic products of amylase and protease were taken to measure absorbance at and nm, respectively. The absorbance values were plotted against the incubation time. The activities of amylase and protease were observed at pH 5—11 and pH 1—11, respectively.

Buffers used 0. One milliliter of reaction for amylase assay contained 20 μl of crude enzyme extract, μl of 0. Absorbance of the supernatants was measured at nm. The reactions were incubated on ice for 30 min then centrifuged to separate denatured casein and enzyme. Absorbance of the resulting supernatants was measured at nm.

Amylase and protease reactions were monitored at 29, 35, 40, 45, 50, 55, 60, and 70 °C. The reactions were incubated for 7 min at various temperatures listed above.

The supernatants were measured at and nm to determine the catalytic products of amylase and protease, respectively. Five to eight specimens of each fish species were determined for both amylase and protease activities.

One milliliter of amylase reaction contained 20 μl of crude enzyme extract, μl of 0. The reactions were incubated for 10 min at the optimum temperature for each enzyme.

The resulting supernatants were taken to measure the absorbance at and nm, respectively. The contents of product released from substrate were calculated from tyrosine and maltose standard curves, respectively.

The enzyme activity and specific activity were subsequently analyzed and compared between fish species. One unit of protease activity was defined as the amount of enzyme required to generate one nmol of tyrosine equivalents in 1 min; one unit of amylase activity was defined as the amount of enzyme required to produce one mmol of maltose in 1 min.

Specific activity is the enzyme activity nmol tyrosine equivalents min -1 for proteases or mmol maltose min -1 for amylase per mg of total protein and therefore it was reported as units mg -1 protein. The activities of both digestive enzymes were also determined at swamp temperature; the reaction components and procedure were similar to reactions in optimum conditions, but the assay temperature was changed to 29 °C for all samples.

Control reactions were also conducted for all samples. The obtained absorbance values were subtracted from those for catalytic reactions. Data were analyzed by one-way analysis of variance IBM SPSS statistics software v.

Total crude enzymes 25 and μg respectively were used to analyze amylase and protease isoenzymes, by using sodium dodecyl sulfate SDS -polyacrylamide gel electrophoresis and zymography.

Crude enzyme samples were mixed with SDS sample buffer without any reducing agent Electrophoresis was performed at V and 4 °C using Mini-PROTEAN Tetra Cell apparatus Bio-Rad. After electrophoresis, two gels with a similar pattern of sample loading were separately examined.

The first gel was stained with Coomassie Brilliant Blue [0. The protein bands were dark blue. For in-gel analysis of amylase activity, the second gel was soaked for 20 min in 0.

The enzyme was allowed to react in-gel for 1 h. The starch solution was discarded and the gel was rinsed with water. Iodine solution was used to stain the gel. The presence of starch produced a brownish—purple color on the gel.

A clear band thus showed the position of amylase i. Protein markers with different molecular weights were used in this study.

M1 was Pink Plus-prestained protein ladder from GeneDirex and contained 11 proteins in the range 10— kDa. The manufacturer does not provide any detail about the specific proteins in this marker mixture.

M2 was unstained protein molecular weight marker from Thermo Scientific, containing chicken egg white lysozyme coli β-galactosidase To detect protease activity in-gel, the procedure was similar to that described above for amylase. SDS was removed by using 0.

Gels were then incubated with gentle shaking at 45 °C for 1 h. Casein solution was removed and the gels were rinsed with water before staining with Coomassie Brilliant Blue R Protein staining was performed at room temperature for 1 to 2 h.

Destaining was carried out for 30 min or until a clear band was visualized against the dark blue background. The molecular weight of proteases was analyzed as described above for amylases. Enzyme activity was determined after incubation of crude enzyme in 0. At each time point, enzyme mixture was aliquoted and substrate solution was added 0.

The reaction was incubated at room temperature 23 °C. The enzymatic product was detected by measuring the absorbance as described above. The effect of pH on enzyme activity was investigated. Crude enzyme was incubated for 1 h at room temperature 23 °C in buffer at pH 5— Enzyme solution was aliquoted and mixed with the optimum buffer and substrate for activity testing.

To determine the effect of temperature, crude enzyme mixed with 0. Substrate and the optimum buffer for reaction were mixed with the enzyme solution before assay at the optimum reaction temperature.

Tokes, Z. Digestive enzymes secreted by the carnivorous plant Nepenthes macferlanei L.. Planta Ber. Article CAS Google Scholar. Amagase, S. Enzymatic digestion of insects by Nepenthes secretion and Drosera peltata extract: proteolytic and chitinolytic activities.

a Article CAS PubMed Google Scholar. Gopinath, S. et al. Biotechnological processes in microbial amylase production. Article PubMed PubMed Central Google Scholar. Kirk, O. Industrial enzyme applications.

Singh, K. Purification, biochemical characterization and Insilico modeling of α-amylase from Vicia faba. Ghalati, R. Guava Psidium guajava L. leaf protease activity enriched by controlled stress and putrescine application.

Sun, Q. Comparative analysis on the distribution of protease activities among fruits and vegetable resources. Food Chem. Zeng, F. Comparison of α-amylase and protease activities of a zoophytophagous and two phytozoophagous Heteroptera.

Article ADS CAS Google Scholar. Darvishzadeh, A. Identification and biochemical characterisation of α-amylase in the alimentary tract of Mediterranean fruit fly, Ceratitis capitata Wiedemann Diptera: Tephritidae.

Plant Protect. Du, R. Purification and characterization of novel thermostable and Ca-independent α-amylase produced by Bacillus amyloliquefaciens BH Wu, X. Purification and biochemical characterization of a thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4— Falcon-Hidalgo, B.

Digestive enzymes of two freshwater fishes Limia vittata and Gambusia punctata with different dietary preferences at three developmental stages. B , — Ji, H. Studies on activity, distribution, and zymogram of protease, α-amylase, and lipase in the paddlefish Polyodon spathula.

Fish Physiol. Gomez, A. Digestive aspartic proteases from sábalo Prochilodus lineatus : Characterization and application for collagen extraction. Nasri, R. Calcium dependent, alkaline detergent-stable trypsin from the viscera of Goby Zosterisessor ophiocephalus : Purification and characterization.

Process Biochem. Champasri, C. Biochemical characterization, activity comparison and isoenzyme analysis of amylase and alkaline proteases in seven cyprinid fishes. Fish Aquat. Crambe meal in diets supplemented with enzyme complex SSF solid state fermentation for Nile tilapia.

African Journal of Agricultural Research However, studies on the stability of enzymes present in this complex should be performed, because this is subjected to various physical and chemical factors of the diet during the processing, storage, and digestive processes, which can reduce or inactivate its catalytic activity.

The effects of processing and storage time on the stability of enzymes of the enzyme complex SSF in pelleted diets for animals were evaluated. Two isonutritive diets were formulated containing g kg -1 of crude protein CP and 2, kcal kg -1 of digestible energy DE in the diet Table 1.

This diet was formulated according to the requirements recommended for omnivore fish, but the type of processing and the enzyme activity can be applied to any animal species.

The control diet was formulated without SSF and 50 g kg -1 of enzyme complex was added to the other experimental diet. The ingredients were weighed and placed in a plastic bag for mixing. These bags were shaken for 5 min, providing a homogeneous mixture of ingredients. This mixture was placed in a bowl and water at 55 °C was added until the dough reached the point alloy.

Soon after, the pellet machine received this dough and the wet pellets were produced. These pellets remained in forced ventilation oven at 55 °C for 14 h, which promoted its drying.

The trial was started at the moment of the processing of experimental diets and the samples were collected during the following steps: mixing, then pelleting, and then drying in an oven at 55 °C for 14 h.

To evaluate the storage time, the diet ready after drying was regarded as day 1. On this day, two samples were taken, one kept at room temperature at 25 °C and one kept in a freezer at °C. At 15, 30, 45, and 60 days, sub-samples were taken to the two kinds of storage.

All samples, the processing steps, and storage time were submitted to the laboratory and the activity of the following enzymes were measured: α-galactosidase, endoglucanase carboxymethyl cellulase , xylanase, sucrase invertase , α-amylase, lipase, and Trypsin.

To assess α-galactosidase, endoglucanase, xylanase, and sucrase, mg of each sample of feed were macerated in 10 mL of buffer solution of mM sodium acetate. This mixture was centrifuged at 13, rpm for 2 min and the supernatant extract was removed and stored in a freezer at °C for enzyme analysis.

The activity of α-galactosidase was determined by measuring the amount of reducing sugar produced through the use of dinitrosalicylic acid DNS reagent according to Miller Miller, G. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry The reaction mixture was composed of µL sodium acetate buffer mM, pH 5 , µL of a solution of 10 mM sucrose, and 50 µL of enzyme extract.

The trial was conducted in a water bath for 15 min at 40 o C. To stop the reaction, 1 mL of DNS reagent was added, followed by immersion of the test tube in boiling water bath for 5 min.

The spectrophotometric measurements were taken at nm and absorbance values converted into µmoles of reducing sugar, using a standard curve constructed from glucose amounts ranging from 0.

For endoglucanase assay, 30 µL of the enzyme solution was mixed with µL of carboxymethyl cellulose 0.

This solution was placed in a water bath at 50 °C for 30 min and the reaction stalled according to Miller Miller, G.

Afterwards, the spectrophotometric measurements were made at nm. The xylanase activity was determined using 70 µL sodium acetate buffer mM, pH 5 , 30 µL of enzyme extract, and µL of birch wood xylan solution 1. The reaction was conducted for 20 min at 40 °C and paralyzed with µL of DNS.

This solution was incubated in boiling water bath for 5 min for color development. The activity was determined at nm using a standard glucose curve. For sucrase activity, 15 µL of enzyme extract was added to µL of sucrase solution 2 g sucrose for analysis in 50 mL of sodium acetate buffer, mM, pH 5 and µL sodium acetate buffer solution mM, pH 5.

This solution was immediately placed in a water bath at 30 °C for 30 min and the reaction terminated with µL DNS. Subsequently, the sample was taken to a bath in boiling water for 5 min and cooled in ambient temperature.

Then, the activities were read in a spectrophotometer at nm. For α-amylase, trypsin, and lipase, 0. This material was placed into polyethylene tubes and centrifuged at 12, rpm for 10 min.

Thus, the supernatant was removed for determination of enzyme activity. The α-amylase activity was based in the starch hydrolysis with release of dextrin and maltose molecules. By adding iodine, unhydrolysed starch acquires blue color. The amylase activity is inversely proportional to the intensity of blue color and is calculated by comparison with a control substrate.

The α-amylase activity was determined in spectrophotometer at nm wavelength, using the amylase of Bioclin colorimetric kit according to Caraway Caraway, W.

A stable starch substrate for the determination of amylase in serum and other body fluids. American Journal of Clinical Pathology The trypsin activity was obtained using N-Benzoyl-D-p-nitroanilide L-arginine D, L-BApNA as substrate according to the method described by Erlanger et al. and Chen, N.

The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics Ten µL of enzyme extract was added, and immediately, the initial velocity was obtained by forming the p-nitroalinine. This reaction was determined in absorbance at nm as a function of time.

For calculations, the molar extinction coefficient of 8, M -1 cm -1 was used for the product. For lipase activity, we used the Bioclin kit with the modified methodology of Cherry Cherry, I.

and Crandall, L. The specificity of pancreatic lipase: it's appearance in the blood after pancreatic injury. American Journal of Physiology This methodology evaluated the lipase action present in the extract of the diet on a glycerol ester, releasing a chromogen, which was quantitatively determined in a spectrophotometer at nm.

The intensity of color formed was proportional to lipase activity. All enzyme analyzes were performed with three replicates in duplicate. For statistical analysis we used the SAS software Statistical Analysis System, version 9.

The means were compared by SNK Student Newman Kells test at 0. Through the F test at 0. Enzyme activities were not observed in all the samples of the control treatment. Thus, only the means of enzyme activities of the treatment with SSF subjected to the processing and storage time at different temperatures were demonstrated.

Over the processing Table 2 , the mixture was subjected to the pelletizing temperature of 55 °C and 14 h in forced-ventilation oven at the same temperature. In general, the enzyme complex SSF was stable in relation to chemical and physical adversities of the trial.

The activity results of the enzymes involved in this study were consistent with Spring et al. and Vukicvranjes, M. Effect of pelleting temperature on the activity of different enzymes. Poultry Science The authors concluded that the enzyme activity was maintained at a pellet temperature of 80 °C.

Close values were obtained by Silveira et al. and Nunes, J. Efeito da peletização em dietas contendo complexo enzimático para frangos de corte. Ciência Animal Brasileira Moreover, Colier and Hardy Colier, B. and Hardy, B.

Many enzymes used as digestive Water retention reduction tips exhibit, Digestive enzyme stability best, moderate Dibestive when incubated ehzyme gastrointestinal conditions. A supplemental β-galactosidase administered Post-workout recovery to treat ennzyme intolerance was conjugated to Mouth kDa, branched polyethylene sgability PEG. Conjugation also provided significant protection against Digestive enzyme stability proteolytic component of pancreatin. Overall, the PEGylated enzyme retained over twice the levels of residual activity recorded for non-PEGylated enzyme after exposure to complete simulated gastrointestinal conditions. This appears to be the first report of the use of a conjugated PEG to stabilize a digestive enzyme and the first report of the ability of conjugated PEG to stabilize a protein at low pH. This is a preview of subscription content, log in via an institution to check access. Rent this article via DeepDyve. Digestive enzyme stability

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