The document summarizes research on extracting and characterizing protease from the viscera of skipjack tuna fish. Protease was extracted from the intestines, stomach, pancreas, and liver of skipjack tuna using potassium phosphate solution and precipitated using either cold acetone or ammonium sulfate. The protease showed highest activity when precipitated with a 1:2 ratio of extract to acetone. The optimal temperature and pH for the acetone-precipitated protease were 50°C and 8, respectively. Tests on temperature and pH stability as well as effects of additives like NaCl, CaCl2, and EDTA were also conducted to characterize the protease.
This document summarizes research on the production of ferulic acid esterase by Streptomyces sp. Key findings include:
- Streptomyces sp. isolate S10 was identified that produced ferulic acid esterase when grown in de-starched wheat bran medium.
- Maximum ferulic acid esterase production of 2.0 mU/ml occurred in MBS medium containing 1.5% de-starched wheat bran at 30°C and pH 6.5 with agitation after 96 hours of fermentation.
- Hydrolysis experiments confirmed the enzyme produced was ferulic acid esterase as it released ferulic acid from the de-starched wheat bran substrate.
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
This document summarizes an investigation into how gut microflora affects endogenous metabolic pathways using mass spectrometry-based metabolomics. Pseudo germ-free rats were used as a model by administering antibiotics to suppress gut microflora. Urine metabolites were then analyzed and compared between control and pseudo germ-free rats using UPLC-QTOF-MS. Validation steps included repeatability testing of QC samples and a test mixture to ensure method reliability. Multivariate analysis of metabolic profiles identified differences between control and pseudo germ-free groups, suggesting gut microflora influences certain metabolic pathways.
This study developed sweet potato media (SPM) as an alternative medium for cultivating Lactobacillus bacteria using sweet potatoes as the basic nutrient component. Three SPM were formulated with varying concentrations of nitrogen sources. Ten Lactobacillus strains were cultured in MRS broth and the three SPM to analyze growth, pH, and acidity. Results showed that SPM2, containing intermediate nitrogen levels, supported Lactobacillus growth equivalent to MRS broth based on growth curves, bacterial populations, pH maintenance above 4.4, and acid production. Thus, SPM2 is a suitable low-cost alternative to MRS for cultivating Lactobacillus.
Partial purification and characterization of extracellular protease from pedi...Mushafau Adebayo Oke
This document summarizes a study that characterized and partially purified an extracellular protease produced by Pediococcus acidilactici. Key findings include:
- The protease showed optimal activity at a casein concentration of 2% and with 2.5 ml of crude enzyme.
- It had temperature and pH optima of 28°C and 4.0, respectively, indicating it is a mesophilic and acidic protease.
- Purification using gel filtration chromatography resulted in a 2.26-fold increase in purification and an estimated molecular weight between 45-66 kDa via SDS-PAGE.
1) Tomato polygalacturonase (PG) was purified using crosslinked ruredzo mucilage (CLM) as an affinity adsorbent. 2) PG was extracted from tomatoes and bound to CLM at pH 4. 3) A six-fold purification of PG was achieved in a single step using CLM. 4) The purified PG showed two main bands of 30,000 and 44,000 molecular weight by SDS-PAGE.
This document summarizes research on the purification, characterization, and antifungal activity of a chitinase enzyme from Streptomyces venezuelae P10. Key findings include:
1) The chitinase was purified using ammonium sulfate precipitation, chitin affinity chromatography, and DEAE-cellulose anion exchange chromatography, yielding a purified enzyme with a molecular weight of 66 kDa.
2) The purified chitinase demonstrated optimal activity at 35°C and pH 6-8 and showed antifungal activity against phytopathogens such as Aspergillus niger and Alternaria alternate.
3) Thin layer chromatography analysis identified the chitin
Assessing the Suitability of using Plant Latex as Immobilization Support for ...ijsrd.com
Horseradish peroxidase was immobilized onto latex from three different plants viz. Calotropis procera, Euphorbia royleana and Alstonia scholaris with 0.51 ± 0.01, 0.37 ± 0.01, 0.46 ± 0.01 mg/cm2 conjugation yield and 62.07 ± 0.85, 66.1 ± 0.85, 71.24 ± 0.80 % retention of specific activity respectively. The support, before and after addition of peroxidase was characterized using scanning electron microscopy (SEM) and Fourier transmission infra-red spectroscopy (FTIR). Optimum pH, optimum temperature and changes in kinetic parameters (Ea, Km and Vmax) for immobilized peroxidases were studied and found to differ from that of free peroxidase. Alstonia scholaris latex was most effective in stabilizing the structure of peroxidase during storage at 4°C, whereas thermal stability and reusability of peroxidase was better on Calotropis procera latex. Analytical use of Calotropis procera latex bound peroxidase for determination of phenolic content of fruit juices has also been demonstrated.
This document summarizes research on the production of ferulic acid esterase by Streptomyces sp. Key findings include:
- Streptomyces sp. isolate S10 was identified that produced ferulic acid esterase when grown in de-starched wheat bran medium.
- Maximum ferulic acid esterase production of 2.0 mU/ml occurred in MBS medium containing 1.5% de-starched wheat bran at 30°C and pH 6.5 with agitation after 96 hours of fermentation.
- Hydrolysis experiments confirmed the enzyme produced was ferulic acid esterase as it released ferulic acid from the de-starched wheat bran substrate.
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
This document summarizes an investigation into how gut microflora affects endogenous metabolic pathways using mass spectrometry-based metabolomics. Pseudo germ-free rats were used as a model by administering antibiotics to suppress gut microflora. Urine metabolites were then analyzed and compared between control and pseudo germ-free rats using UPLC-QTOF-MS. Validation steps included repeatability testing of QC samples and a test mixture to ensure method reliability. Multivariate analysis of metabolic profiles identified differences between control and pseudo germ-free groups, suggesting gut microflora influences certain metabolic pathways.
This study developed sweet potato media (SPM) as an alternative medium for cultivating Lactobacillus bacteria using sweet potatoes as the basic nutrient component. Three SPM were formulated with varying concentrations of nitrogen sources. Ten Lactobacillus strains were cultured in MRS broth and the three SPM to analyze growth, pH, and acidity. Results showed that SPM2, containing intermediate nitrogen levels, supported Lactobacillus growth equivalent to MRS broth based on growth curves, bacterial populations, pH maintenance above 4.4, and acid production. Thus, SPM2 is a suitable low-cost alternative to MRS for cultivating Lactobacillus.
Partial purification and characterization of extracellular protease from pedi...Mushafau Adebayo Oke
This document summarizes a study that characterized and partially purified an extracellular protease produced by Pediococcus acidilactici. Key findings include:
- The protease showed optimal activity at a casein concentration of 2% and with 2.5 ml of crude enzyme.
- It had temperature and pH optima of 28°C and 4.0, respectively, indicating it is a mesophilic and acidic protease.
- Purification using gel filtration chromatography resulted in a 2.26-fold increase in purification and an estimated molecular weight between 45-66 kDa via SDS-PAGE.
1) Tomato polygalacturonase (PG) was purified using crosslinked ruredzo mucilage (CLM) as an affinity adsorbent. 2) PG was extracted from tomatoes and bound to CLM at pH 4. 3) A six-fold purification of PG was achieved in a single step using CLM. 4) The purified PG showed two main bands of 30,000 and 44,000 molecular weight by SDS-PAGE.
This document summarizes research on the purification, characterization, and antifungal activity of a chitinase enzyme from Streptomyces venezuelae P10. Key findings include:
1) The chitinase was purified using ammonium sulfate precipitation, chitin affinity chromatography, and DEAE-cellulose anion exchange chromatography, yielding a purified enzyme with a molecular weight of 66 kDa.
2) The purified chitinase demonstrated optimal activity at 35°C and pH 6-8 and showed antifungal activity against phytopathogens such as Aspergillus niger and Alternaria alternate.
3) Thin layer chromatography analysis identified the chitin
Assessing the Suitability of using Plant Latex as Immobilization Support for ...ijsrd.com
Horseradish peroxidase was immobilized onto latex from three different plants viz. Calotropis procera, Euphorbia royleana and Alstonia scholaris with 0.51 ± 0.01, 0.37 ± 0.01, 0.46 ± 0.01 mg/cm2 conjugation yield and 62.07 ± 0.85, 66.1 ± 0.85, 71.24 ± 0.80 % retention of specific activity respectively. The support, before and after addition of peroxidase was characterized using scanning electron microscopy (SEM) and Fourier transmission infra-red spectroscopy (FTIR). Optimum pH, optimum temperature and changes in kinetic parameters (Ea, Km and Vmax) for immobilized peroxidases were studied and found to differ from that of free peroxidase. Alstonia scholaris latex was most effective in stabilizing the structure of peroxidase during storage at 4°C, whereas thermal stability and reusability of peroxidase was better on Calotropis procera latex. Analytical use of Calotropis procera latex bound peroxidase for determination of phenolic content of fruit juices has also been demonstrated.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
The study investigated the analgesic and anti-inflammatory properties of the ethanolic leaf extract of Syzygium guineense in rats and mice. The extract was found to have significant analgesic effects in the hot plate test at 500 mg/kg and 1000 mg/kg doses and significant anti-inflammatory and analgesic effects in the acetic acid-induced writhing test at 1000 mg/kg. The extract also significantly reduced egg albumin-induced paw edema in rats at 1000 mg/kg. Phytochemical analysis revealed the presence of flavonoids, tannins, saponins and cardiac glycosides in the extract. The results provide support for the traditional use of S. guineense in folk medicine to
Tiffany Sato investigated whether asparagus can inhibit the population growth of yeast cells. She used yeast (Saccharomyces cerevisiae) as a model organism to study the anti-proliferative effects of asparagus due to similarities between yeast and cancer cells. Yeast growth was measured using spectrophotometry and viable colony counts. Results showed decreased yeast activity and fewer colonies with asparagus compared to positive controls, indicating asparagus can minimally inhibit yeast cell growth. This supports the hypothesis that asparagus compounds may reduce cancer cell proliferation.
The document reports on a study that evaluated the antidiabetic potential of Syzygium jambolanum (Jamun). Microscopic examination showed that preadipocytes differentiated into mature adipocytes containing lipid droplets after treatment. Oil Red O staining was used to visualize lipid accumulation. MTT assays found that Jamun pulp extracts from 0.001-10% were safe for cells, while Jamun seed extracts over 10% were potentially toxic. Lipogenesis assays indicated that Jamun seed extracts from 0.01-1% and pulp extracts at 5% enhanced lipid formation in adipocytes compared to controls. Alpha-amylase inhibition assays demonstrated that Jamun seed extracts from 0.1-100% and pulp extracts at
Tiffany Sato investigated whether asparagus could inhibit the growth of yeast cells. She used yeast (Saccharomyces cerevisiae) as a model organism to study the anti-proliferative effects of asparagus due to similarities between yeast and cancer cells. Yeast was grown in media containing asparagus extract, a control lacking nutrients, and a positive control with nutrients. Growth was measured using spectrophotometry and viable colony counts. Results showed the asparagus extract resulted in lower yeast growth than the positive control, indicating it inhibited proliferation. This supported the hypothesis that compounds in asparagus may slow the growth of cells.
The Effect of Asparagus on Cancer Cell Growthtiffanysae
Tiffany Sato investigated whether asparagus can inhibit the population growth of yeast cells. Yeast cells were used as a model for cancer cells due to similarities in structure and uncontrolled cell division. Sato grew yeast in media containing asparagus extract and found it exhibited lower absorbance, indicating decreased growth, compared to a positive control with nutrients. Agar plate results also showed fewer colonies with a flatter structure for yeast grown with asparagus versus the positive control. Sato's results suggest that asparagus can inhibit yeast cell growth, supporting the hypothesis that its compounds may reduce cancer cell proliferation.
1. The document describes a protein purification procedure using ammonium sulfate precipitation and the Folin-Lowry assay to measure protein concentrations. Bovine serum albumin was used as the standard protein.
2. The Folin-Lowry assay involves two reactions - first with an alkaline copper tartrate solution and then with Folin-Ciocalteu reagent. This allows spectrophotometric measurement of protein concentrations.
3. Ammonium sulfate precipitation was used to isolate proteins based on their solubility. This increased the yield and purity of the target protein, progesterone receptors.
Glutathione S-transferase enzymes (GSTs) play central roles in phase II detoxification of both xenobiotics and endogenous compounds in almost all living organisms. The enzyme was extracted and partially purified from wheat leaves through a procedure including ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures yielded a 7.14-fold purification with 71% recovery. Optimum activity conditions-pH, temperature and ionic strength-of the enzyme were determined. Its some kinetic properties such as Vmax, KM, and kcat were calculated for GSH and CDNB substrates. The kcat/KM values of the enzyme were 603.5 for GSH and 385.3 for CDNB. The native molecular weight of the enzyme was estimated to be 52 kDa based on its mobility in gel filtration column.
Total Antioxidant Capacity of Labdane and Pimarane Diterpenoids of Juniperus ...Editor IJCATR
Although Juniperus phoenicea L is a widely distributed wild tree in the south of Saudi Arabia, but its phytochemical and physiological evaluation is still poor. The chromatographic separation of the CH2Cl2/MeOH, 1:1 extract of J, phoenicea L. fruits gave β-sitosterol, stigmasterol, four labdane and two pimarane diterpenoids, including new labdane diterpenoid. Additionally, the volatile compounds of the main petroleum ether extract, as well as the steam-volatile constituents were identified by GC/MS. The separated compounds were identified by spectral tools. The new diterpenoid was identified as 5,9,10-triepicupressic acid.
A previous false identification of sandracopimaric acid, from the fruits of Juniperus phoenicea L. grown in Egypt, was detected and declared to be revised.
Total antioxidant capacity of extracts was estimated using total antioxidant capacity kit of Biodiagnostic, based on Koracevic et al., for biological fluids, with slight modification to suit extracts. Ascorbic acid was used as a reference antioxidant compound. Reasonable results were obtained by applying the modified method on petroleum ether extract, methylene chloride extract and ethyl acetate extract of Juniperus phoenicea L.
Biotechnological applications in agriculture a new source of edibleSiamak Alizade
This document summarizes research on using the Terminalia belerica plant for various biotechnological applications. Key points:
- The plant's seeds can be used to produce a new edible oil, comprising 37% of the dry kernel weight. Analysis found the oil contains fatty acids similar to olive oil.
- Byproducts of oil extraction include an oilcake containing high levels of nitrogen (8.34%) and proteins (60% digestible) suitable for use as biofertilizer.
- Other byproducts include tannins extracted from the fruit pulp, suitable for use in leather/medicine, and antioxidants like gallic acid in the seed coat with potential to preserve vegetable oils.
This study examined the glucose and lactic acid contents of tube paste flour with additions of the amylolytic bacteria Lactobacillus bulgaricus FNCC 004P and Streptococcus thermophillus FNCC 1.9.03. The bacteria were added to wheat, tapioca, and taka tube pastes at concentrations from 0.2x108 to 10x108 CFU/mL. Glucose content was highest in wheat paste with 2x108 CFU/mL of S. thermophillus and in tapioca and taka pastes with 2x108 CFU/mL of L. bulgaricus. Lactic acid contents were similar between the bacteria in wheat and tapi
Rare earth elements in Animal nutritionMahesh Tambe
The document summarizes information on rare earth elements (REE), including their discovery, occurrence, properties, applications, and implications in animal nutrition. Some key points:
- REE are a group of 17 elements found in the periodic table. They are found in small amounts in soils and plants and have various industrial and biological applications.
- Studies have shown that supplementing REE like lanthanum and cerium in animal diets can improve growth performance, nutrient digestibility, blood mineral levels, antioxidant status, and rumen fermentation.
- Toxicity studies found no negative effects at supplementation levels up to 300mg/kg, though higher levels may increase some blood parameters like albumin and decrease mal
This document provides instructions for quantifying biotin labeling of insulin-like growth factor 1 (IGF-1) and measuring its binding to different protein gels. The process involves biotinylating IGF-1, diluting and incubating it with protein gels coated in wells, detecting bound biotinylated IGF-1 using avidin-HRP and a colorimetric substrate, and measuring absorption to quantify relative binding to each protein gel. The assay is carried out over 3 days, with biotinylation on day 1, an overnight incubation on day 2, and binding, detection and quantification on day 3. Positive and negative controls are also used to validate the assay measurements.
Tsujita (2007) Lipolysis Induced By Segment Wallx2006nvu
This document summarizes a study that investigated the lipolytic (fat-breaking down) effects of extracts from different parts of Satsuma mandarin oranges (Citrus unshu Mark). The study found that extracts from the peel and segment wall (the white part between segments) induced lipolysis in rat fat cells, while extracts from the juice sac did not. High levels of the lipolytic amine synephrine were detected in the peel and segment wall extracts but not the juice sac extract. Segment wall extracts from some other citrus fruits also induced lipolysis, but extracts from grapefruit and lemon did not. The lipolysis induced by Satsuma mandarin segment wall extract was inhibited by a beta-antagon
Tenderization of camel meat by using fresh ginger (zingiber officinale) extractAlexander Decker
- The document examines using ginger extract to tenderize aged camel meat. Camel meat chunks were marinated with different concentrations (0, 15, 30, 45% v/w) of ginger extract for 48 hours.
- The 30% ginger extract treatment showed the best results in improving water holding capacity, tenderness, and sensory attributes compared to the control.
- Samples treated with 30% ginger extract were then gamma irradiated at doses from 0-4.5 kGy to extend shelf-life while maintaining quality and safety during refrigerated storage.
The document summarizes a study that investigated the antioxidant and anti-inflammatory activities of Pterospermum acerifolium. The study found that the ethyl acetate fraction of P. acerifolium showed the highest free radical scavenging activity in various in vitro antioxidant assays. This fraction also demonstrated significant anti-inflammatory effects in both in vivo and in vitro models of inflammation. The results support the traditional use of P. acerifolium for reducing oxidative stress and inflammation.
TSK-GEL STAT Cation Exchange Columns (TP132)guest149f116
Tosoh scientists developed novel non-porous cationic exchange resins, marketed as TSK-GEL SP-STAT and TSK-GEL CM-STAT columns. The cation exchange columns are packed with 7 and 10μm spherical, mono-disperse, non-porous particles of which the surface has been modified with open access multi-layered cation exchange groups. The cation exchangers show higher adsorption capacities and require lower pressures compared with conventional non-porous columns of the same column dimension. Rapid separations of proteins were achieved within 1 minute on short columns (3.5cm) packed with 10μm resin and high resolution analyses were achieved on a 10cm length column packed with 7μm resin. The basic properties of the novel cation exchange columns and how they apply to the separation of proteins, antibodies and peptides are reported in comparison with commercially available non-porous CIEX columns.
Heartburn, also known as acid indigestion, is a burning feeling in the chest or throat caused by stomach acid backing up into the esophagus. Gastroesophageal reflux disease (GERD) is the most common cause of heartburn. Common triggers for heartburn include fatty foods, caffeine, alcohol, carbonated drinks, overeating, and eating close to bedtime. Lifestyle changes like eating more slowly, increasing fiber intake, losing weight, and exercising can help prevent heartburn. Bing Han ginseng powder can be used to treat both mild and severe heartburn by dissolving it under the tongue or in warm water.
Heartburn is caused by excess stomach acid and can be relieved through home remedies such as baking soda, aloe verra juice, chewing gum, limiting acidic foods, sleeping elevated, apple cider vinegar, bananas, apples, and mustard. The full article provides more details on effective home remedies for heartburn.
Heartburn is a common condition that causes a burning pain in the chest area. It occurs when stomach acid backs up into the esophagus. Risk factors include eating fatty or acidic foods, drinking alcohol, lying down after eating, and taking certain medications. Symptoms include chest pain and a sour taste in the mouth. Most cases can be treated with over-the-counter antacids and lifestyle changes. But severe or frequent heartburn may indicate gastroesophageal reflux disease (GERD), which requires prescription medication to reduce acid production and heal damage to the esophagus.
Create online video resources for students. These on-demand lessons allow students to review content as needed, and effectively extend how long you can teach a particular lesson.
Heartburn and gastroesophageal reflux disease (GERD) are caused by stomach acid backing up into the esophagus. Symptoms of GERD include frequent heartburn and acid indigestion. Treatment involves lifestyle changes like losing weight and avoiding trigger foods, as well as medications like antacids, H2 blockers, and proton pump inhibitors. Surgery may be considered for severe cases that do not respond to other treatments.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
The study investigated the analgesic and anti-inflammatory properties of the ethanolic leaf extract of Syzygium guineense in rats and mice. The extract was found to have significant analgesic effects in the hot plate test at 500 mg/kg and 1000 mg/kg doses and significant anti-inflammatory and analgesic effects in the acetic acid-induced writhing test at 1000 mg/kg. The extract also significantly reduced egg albumin-induced paw edema in rats at 1000 mg/kg. Phytochemical analysis revealed the presence of flavonoids, tannins, saponins and cardiac glycosides in the extract. The results provide support for the traditional use of S. guineense in folk medicine to
Tiffany Sato investigated whether asparagus can inhibit the population growth of yeast cells. She used yeast (Saccharomyces cerevisiae) as a model organism to study the anti-proliferative effects of asparagus due to similarities between yeast and cancer cells. Yeast growth was measured using spectrophotometry and viable colony counts. Results showed decreased yeast activity and fewer colonies with asparagus compared to positive controls, indicating asparagus can minimally inhibit yeast cell growth. This supports the hypothesis that asparagus compounds may reduce cancer cell proliferation.
The document reports on a study that evaluated the antidiabetic potential of Syzygium jambolanum (Jamun). Microscopic examination showed that preadipocytes differentiated into mature adipocytes containing lipid droplets after treatment. Oil Red O staining was used to visualize lipid accumulation. MTT assays found that Jamun pulp extracts from 0.001-10% were safe for cells, while Jamun seed extracts over 10% were potentially toxic. Lipogenesis assays indicated that Jamun seed extracts from 0.01-1% and pulp extracts at 5% enhanced lipid formation in adipocytes compared to controls. Alpha-amylase inhibition assays demonstrated that Jamun seed extracts from 0.1-100% and pulp extracts at
Tiffany Sato investigated whether asparagus could inhibit the growth of yeast cells. She used yeast (Saccharomyces cerevisiae) as a model organism to study the anti-proliferative effects of asparagus due to similarities between yeast and cancer cells. Yeast was grown in media containing asparagus extract, a control lacking nutrients, and a positive control with nutrients. Growth was measured using spectrophotometry and viable colony counts. Results showed the asparagus extract resulted in lower yeast growth than the positive control, indicating it inhibited proliferation. This supported the hypothesis that compounds in asparagus may slow the growth of cells.
The Effect of Asparagus on Cancer Cell Growthtiffanysae
Tiffany Sato investigated whether asparagus can inhibit the population growth of yeast cells. Yeast cells were used as a model for cancer cells due to similarities in structure and uncontrolled cell division. Sato grew yeast in media containing asparagus extract and found it exhibited lower absorbance, indicating decreased growth, compared to a positive control with nutrients. Agar plate results also showed fewer colonies with a flatter structure for yeast grown with asparagus versus the positive control. Sato's results suggest that asparagus can inhibit yeast cell growth, supporting the hypothesis that its compounds may reduce cancer cell proliferation.
1. The document describes a protein purification procedure using ammonium sulfate precipitation and the Folin-Lowry assay to measure protein concentrations. Bovine serum albumin was used as the standard protein.
2. The Folin-Lowry assay involves two reactions - first with an alkaline copper tartrate solution and then with Folin-Ciocalteu reagent. This allows spectrophotometric measurement of protein concentrations.
3. Ammonium sulfate precipitation was used to isolate proteins based on their solubility. This increased the yield and purity of the target protein, progesterone receptors.
Glutathione S-transferase enzymes (GSTs) play central roles in phase II detoxification of both xenobiotics and endogenous compounds in almost all living organisms. The enzyme was extracted and partially purified from wheat leaves through a procedure including ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures yielded a 7.14-fold purification with 71% recovery. Optimum activity conditions-pH, temperature and ionic strength-of the enzyme were determined. Its some kinetic properties such as Vmax, KM, and kcat were calculated for GSH and CDNB substrates. The kcat/KM values of the enzyme were 603.5 for GSH and 385.3 for CDNB. The native molecular weight of the enzyme was estimated to be 52 kDa based on its mobility in gel filtration column.
Total Antioxidant Capacity of Labdane and Pimarane Diterpenoids of Juniperus ...Editor IJCATR
Although Juniperus phoenicea L is a widely distributed wild tree in the south of Saudi Arabia, but its phytochemical and physiological evaluation is still poor. The chromatographic separation of the CH2Cl2/MeOH, 1:1 extract of J, phoenicea L. fruits gave β-sitosterol, stigmasterol, four labdane and two pimarane diterpenoids, including new labdane diterpenoid. Additionally, the volatile compounds of the main petroleum ether extract, as well as the steam-volatile constituents were identified by GC/MS. The separated compounds were identified by spectral tools. The new diterpenoid was identified as 5,9,10-triepicupressic acid.
A previous false identification of sandracopimaric acid, from the fruits of Juniperus phoenicea L. grown in Egypt, was detected and declared to be revised.
Total antioxidant capacity of extracts was estimated using total antioxidant capacity kit of Biodiagnostic, based on Koracevic et al., for biological fluids, with slight modification to suit extracts. Ascorbic acid was used as a reference antioxidant compound. Reasonable results were obtained by applying the modified method on petroleum ether extract, methylene chloride extract and ethyl acetate extract of Juniperus phoenicea L.
Biotechnological applications in agriculture a new source of edibleSiamak Alizade
This document summarizes research on using the Terminalia belerica plant for various biotechnological applications. Key points:
- The plant's seeds can be used to produce a new edible oil, comprising 37% of the dry kernel weight. Analysis found the oil contains fatty acids similar to olive oil.
- Byproducts of oil extraction include an oilcake containing high levels of nitrogen (8.34%) and proteins (60% digestible) suitable for use as biofertilizer.
- Other byproducts include tannins extracted from the fruit pulp, suitable for use in leather/medicine, and antioxidants like gallic acid in the seed coat with potential to preserve vegetable oils.
This study examined the glucose and lactic acid contents of tube paste flour with additions of the amylolytic bacteria Lactobacillus bulgaricus FNCC 004P and Streptococcus thermophillus FNCC 1.9.03. The bacteria were added to wheat, tapioca, and taka tube pastes at concentrations from 0.2x108 to 10x108 CFU/mL. Glucose content was highest in wheat paste with 2x108 CFU/mL of S. thermophillus and in tapioca and taka pastes with 2x108 CFU/mL of L. bulgaricus. Lactic acid contents were similar between the bacteria in wheat and tapi
Rare earth elements in Animal nutritionMahesh Tambe
The document summarizes information on rare earth elements (REE), including their discovery, occurrence, properties, applications, and implications in animal nutrition. Some key points:
- REE are a group of 17 elements found in the periodic table. They are found in small amounts in soils and plants and have various industrial and biological applications.
- Studies have shown that supplementing REE like lanthanum and cerium in animal diets can improve growth performance, nutrient digestibility, blood mineral levels, antioxidant status, and rumen fermentation.
- Toxicity studies found no negative effects at supplementation levels up to 300mg/kg, though higher levels may increase some blood parameters like albumin and decrease mal
This document provides instructions for quantifying biotin labeling of insulin-like growth factor 1 (IGF-1) and measuring its binding to different protein gels. The process involves biotinylating IGF-1, diluting and incubating it with protein gels coated in wells, detecting bound biotinylated IGF-1 using avidin-HRP and a colorimetric substrate, and measuring absorption to quantify relative binding to each protein gel. The assay is carried out over 3 days, with biotinylation on day 1, an overnight incubation on day 2, and binding, detection and quantification on day 3. Positive and negative controls are also used to validate the assay measurements.
Tsujita (2007) Lipolysis Induced By Segment Wallx2006nvu
This document summarizes a study that investigated the lipolytic (fat-breaking down) effects of extracts from different parts of Satsuma mandarin oranges (Citrus unshu Mark). The study found that extracts from the peel and segment wall (the white part between segments) induced lipolysis in rat fat cells, while extracts from the juice sac did not. High levels of the lipolytic amine synephrine were detected in the peel and segment wall extracts but not the juice sac extract. Segment wall extracts from some other citrus fruits also induced lipolysis, but extracts from grapefruit and lemon did not. The lipolysis induced by Satsuma mandarin segment wall extract was inhibited by a beta-antagon
Tenderization of camel meat by using fresh ginger (zingiber officinale) extractAlexander Decker
- The document examines using ginger extract to tenderize aged camel meat. Camel meat chunks were marinated with different concentrations (0, 15, 30, 45% v/w) of ginger extract for 48 hours.
- The 30% ginger extract treatment showed the best results in improving water holding capacity, tenderness, and sensory attributes compared to the control.
- Samples treated with 30% ginger extract were then gamma irradiated at doses from 0-4.5 kGy to extend shelf-life while maintaining quality and safety during refrigerated storage.
The document summarizes a study that investigated the antioxidant and anti-inflammatory activities of Pterospermum acerifolium. The study found that the ethyl acetate fraction of P. acerifolium showed the highest free radical scavenging activity in various in vitro antioxidant assays. This fraction also demonstrated significant anti-inflammatory effects in both in vivo and in vitro models of inflammation. The results support the traditional use of P. acerifolium for reducing oxidative stress and inflammation.
TSK-GEL STAT Cation Exchange Columns (TP132)guest149f116
Tosoh scientists developed novel non-porous cationic exchange resins, marketed as TSK-GEL SP-STAT and TSK-GEL CM-STAT columns. The cation exchange columns are packed with 7 and 10μm spherical, mono-disperse, non-porous particles of which the surface has been modified with open access multi-layered cation exchange groups. The cation exchangers show higher adsorption capacities and require lower pressures compared with conventional non-porous columns of the same column dimension. Rapid separations of proteins were achieved within 1 minute on short columns (3.5cm) packed with 10μm resin and high resolution analyses were achieved on a 10cm length column packed with 7μm resin. The basic properties of the novel cation exchange columns and how they apply to the separation of proteins, antibodies and peptides are reported in comparison with commercially available non-porous CIEX columns.
Heartburn, also known as acid indigestion, is a burning feeling in the chest or throat caused by stomach acid backing up into the esophagus. Gastroesophageal reflux disease (GERD) is the most common cause of heartburn. Common triggers for heartburn include fatty foods, caffeine, alcohol, carbonated drinks, overeating, and eating close to bedtime. Lifestyle changes like eating more slowly, increasing fiber intake, losing weight, and exercising can help prevent heartburn. Bing Han ginseng powder can be used to treat both mild and severe heartburn by dissolving it under the tongue or in warm water.
Heartburn is caused by excess stomach acid and can be relieved through home remedies such as baking soda, aloe verra juice, chewing gum, limiting acidic foods, sleeping elevated, apple cider vinegar, bananas, apples, and mustard. The full article provides more details on effective home remedies for heartburn.
Heartburn is a common condition that causes a burning pain in the chest area. It occurs when stomach acid backs up into the esophagus. Risk factors include eating fatty or acidic foods, drinking alcohol, lying down after eating, and taking certain medications. Symptoms include chest pain and a sour taste in the mouth. Most cases can be treated with over-the-counter antacids and lifestyle changes. But severe or frequent heartburn may indicate gastroesophageal reflux disease (GERD), which requires prescription medication to reduce acid production and heal damage to the esophagus.
Create online video resources for students. These on-demand lessons allow students to review content as needed, and effectively extend how long you can teach a particular lesson.
Heartburn and gastroesophageal reflux disease (GERD) are caused by stomach acid backing up into the esophagus. Symptoms of GERD include frequent heartburn and acid indigestion. Treatment involves lifestyle changes like losing weight and avoiding trigger foods, as well as medications like antacids, H2 blockers, and proton pump inhibitors. Surgery may be considered for severe cases that do not respond to other treatments.
Collection,Dispatch & Processing of toxicological samples.فهيم سلطان
Collection,Dispatch & Processing of toxicological samples.
Dr Faheem Sultan
Division of Veterinary Pharmacology & Toxicology
Indian Veterinary Research Institute
Forensic toxicology is the study of poisons and their effects on the human body. Samples must be collected and stored properly to allow for accurate analysis. The most useful samples are blood and urine, as they allow for quantification of drugs and toxins. However, tissue samples like liver and bile can also be useful in cases where blood is not available. Proper containers and preservatives are needed to transport samples to a laboratory without degradation or contamination.
Heartburn is a symptom of gastroesophageal reflux disease (GERD) that is caused by stomach acid regurgitating into the esophagus, resulting in a burning sensation in the chest. GERD occurs when the lower esophageal sphincter relaxes inappropriately, allowing acid to flow back into the esophagus. Lifestyle changes such as avoiding trigger foods, eating smaller meals, and elevating the head of the bed can help control symptoms. If symptoms persist, medications like antacids, H2 blockers, and proton pump inhibitors may be used to reduce acid production. Untreated GERD can lead to complications like esophageal ulcers, strictures, and Barrett's esophagus
This document discusses various types of agricultural poisons including herbicides, insecticides, rodenticides, and fungicides. It describes the properties and uses of halogenated insecticides such as DDT and lindane. The toxicity and treatment of organophosphate and carbamate insecticides are covered. These cholinesterase inhibitor insecticides work by inhibiting the enzyme acetylcholinesterase in the nervous system. Symptoms of poisoning include excessive salivation, urination, diarrhea, nausea, and tremors that can progress to seizures and respiratory failure without treatment. The document also provides an overview of miscellaneous pesticides.
This document provides a protocol for the proper collection, preservation, packaging, and documentation of visceral samples during autopsies in suspected poisoning cases. It details which samples should be collected based on the suspected poison, such as stomach contents, liver, kidneys, blood, urine, etc. It describes techniques for safely extracting these samples. The samples should be preserved in appropriate chemicals to prevent decomposition during transport and labeled with case information. Relevant documentation like the autopsy report and request from investigators should accompany the samples to the forensic laboratory. Overall, the protocol aims to ensure visceral evidence is properly handled for toxicological analysis in determining cause of death.
Forensic toxicologists identify and analyze poisons, drugs, and chemicals found in the body to help determine causes of death. They use various tests like chromatography, mass spectrometry, and spectrophotometry to separate and identify unknown chemical compounds in blood and tissue samples. Crime scene technicians can also use field tests to detect chemical residues like accelerants, explosives, drugs, and metals that can provide evidence found at crime scenes or on clothing.
Postmortem toxicology examines whether alcohol, drugs, or poisons caused or contributed to a person's death. Investigating officers provide toxicologists with case details to plan analyses and interpret results. Every submitted case requires an accompanying form with information to assist toxicologists in selecting appropriate analysis methods and later interpreting results. Forensic toxicology involves detecting and identifying poisons in legal investigations and relating concentrations to toxicity. Poisons are classified by extraction method for analytical purposes.
Forensic toxicology focuses on the medical-legal aspects of chemical exposure and toxic injury. It involves analyzing samples like urine, blood, hair, oral fluid, and other bodily tissues or fluids to detect and quantify the presence of toxins and drugs. The concentration and type of substance present can provide information about factors like dosage and timing of exposure. A variety of analytical techniques are used, including chromatography methods and mass spectrometry, to screen for and confirm the identity of substances in biological samples as part of a forensic investigation.
- Alkaloids are basic nitrogen-containing compounds found in plants, animals, and microorganisms that often have physiological effects.
- They are classified based on their biological origin, biosynthetic pathway, and chemical structure, with the main classes being proto, typical, and pseudo alkaloids.
- Common alkaloids include morphine, codeine, caffeine, and cocaine. Extraction methods take advantage of alkaloids' basic properties, using organic solvents to extract them from plant materials into aqueous solutions as salts.
This document discusses the extraction of alkaloids from plant materials. It describes the Stas-Otto method, which involves distributing alkaloidal bases between an acid or aqueous solution and an immiscible organic solvent. The method involves rendering the plant material alkaline, extracting with an organic solvent, shaking with dilute sulfuric acid to form alkaloidal salts, making the solution alkaline to precipitate the free alkaloidal bases. The Stas-Otto method allows for the separation and isolation of alkaloids from other plant compounds.
The document discusses antidotes for poisoning treatment. It defines an antidote as a therapeutic substance used to counteract the toxic effects of a xenobiotic. Antidotes are classified based on their mode and site of action, and include physical, chemical, and pharmacological antidotes. Physical antidotes like activated charcoal work by adsorption. Chemical antidotes form non-toxic complexes with poisons or accelerate detoxification. Pharmacological antidotes counteract poisons by producing opposite effects, competing for receptors, blocking toxic receptors, or aiding normal function restoration. The proper use of antidotes with supportive care can significantly reduce morbidity and mortality from poisonings.
This document provides information on commonly used emergency drugs, including their initial doses and indications. It also lists common antidotes for various agents. Additionally, it discusses drug name endings and what they may suggest about a drug's class or action. Finally, it provides a table of frequently asked medications including their classifications, desired effects, best times for administration, and other considerations.
The document discusses clinical toxicology and the approach to treating poisoned patients. It covers the learning objectives of understanding factors that influence toxicity, evaluating patients, reducing poison absorption and burden, providing supportive care, and using specific antidotes. The key goals are stabilizing vital signs, preventing further absorption if possible, counteracting toxic effects, and treating the patient rather than the poison.
33.Expression, Production and Purification of Proteinases from Aspergillus spp.Annadurai B
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Kinetic study of free and immobilized protease from Aspergillus sp.IOSR Journals
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Expression and Purification of His-tag β-galactosidase Enzyme from E.coliSurayya Sana
Protein expression and purification were used to study β-galactosidase. β-gal activity increased over time after inducing expression, reaching a maximum at 2 hours. Purification using affinity chromatography captured β-gal in elute fractions as expected. Activity assays on fractions found the highest specific activity and fold purification in elute 2, indicating it contained the purest β-gal. SDS-PAGE and immunoblotting confirmed β-gal was most abundant in lysate and elute fractions, supporting a successful expression and purification.
The researchers aimed to purify cellular retinol binding protein (CRBP) from bovine liver. Through a process involving homogenization, centrifugation, cation exchange chromatography, gel filtration, and concentration, they obtained a final product. However, characterization through SDS-PAGE and absorption spectroscopy identified the protein as catalase rather than CRBP. Despite initial absorption at 350nm for CRBP, the maximal absorption and thermal/pH profiles matched those of catalase. The purification resulted in the isolation of catalase rather than the intended CRBP.
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The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay
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46.Purification of indole acetic acid oxidase produced by Alternaria cepulae ...Annadurai B
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This document describes the purification and characterization of a chitinase enzyme from Streptomyces venezuelae P10. The enzyme was purified using ammonium sulfate precipitation, chitin affinity chromatography, and DEAE-cellulose ion exchange chromatography. SDS-PAGE analysis showed the purified chitinase has a molecular weight of 66 kDa. The chitinase demonstrated optimal activity at pH 7.5 and 35°C. It was able to hydrolyze chitin into N-acetylglucosamine and N,N'-diacetylchitobiose. The purified enzyme also showed antifungal activity against phytopathogens such as Aspergillus niger and Alternaria altern
The objective was to isolate acid phosphatase from wheat germ and determine its concentration. Wheat germ was purified through centrifugation and fractions were analyzed using gel electrophoresis and a protein assay. The results were unexpected and inconsistent with expected patterns of decreasing concentration and bands as purification increased. Multiple errors could have occurred during ammonium sulfate addition or sample dilution. The experiment needs to be repeated to properly isolate the phosphatase for further study.
This document summarizes a study characterizing an α-amylase enzyme from the halotolerant bacteria Bacillus aquimaris. The enzyme was found to be metal ion dependent, with calcium ions enhancing its activity. It exhibited optimal activity at pH 8.0 and a temperature of 40°C, and was highly thermostable. The enzyme was also detergent activated and tolerant of high salt concentrations, with potential industrial applications. Purification of the enzyme was in progress to further characterize its properties.
Lipase production and purification Likhith KLIKHITHK1
Lipase (tri acyl glycerol acyl hydrolase, EC 3.1.1.3) catalyzes the hydrolysis of the carboxyl ester bonds in tri acyl glycerols to produce di acyl glycerols, mono acyl glycerols, fatty acids and glycerol under aqueous conditions and the synthesis of esters in organic solvents.
Under the controlled conditions, lipases are able to catalyze a large number of reactions. Lipases of microbial origin are of considerable commercial importance, because of the high versatility and high stability, moreover, the advantage of being readily produced in high yields.
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1. The document reports on the isolation, identification, and production of alkaline protease enzymes from Bacillus species for industrial applications.
2. Six alkaline protease producing Bacillus species were isolated from soil samples and identified using biochemical tests. The enzyme was partially purified and optimized.
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This document summarizes research on the production of ferulic acid esterase by Streptomyces sp. Key findings include:
- Streptomyces sp. isolate S10 was identified that produced ferulic acid esterase when grown in de-starched wheat bran medium.
- Maximum ferulic acid esterase production of 2.0 mU/ml occurred in MBS medium containing 1.5% de-starched wheat bran at 30°C and pH 6.5 with agitation after 96 hours of fermentation.
- Hydrolysis experiments confirmed the enzyme produced was ferulic acid esterase as it released ferulic acid from the de-starched wheat bran substrate.
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The document discusses the utilization of collagen extracted from catla fish waste in wine fining and clarification. Fish processing produces significant waste (20-80%) that causes pollution. The study extracted collagen from fish scales and bones using acid soluble methods. The collagen was transformed into gelatin then analyzed. Gelatin extraction was efficient as the product had high protein and low moisture. Gelatin was then used in wine fining, showing enhanced clarification properties as its concentration increased.
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1) B. mojavensis 5-SM produced the maximum amount of lipase extracellularly at 72 hours of growth at pH 11 and 60°C.
2) The lipase was stable from pH 5-12 and temperatures from 30-80°C, retaining optimal activity for 2 hours at 60°C.
3) The lipase showed stability in organic solvents, surfactants, and reducing/oxidizing agents on the first day of testing, though activity decreased on the second day in organic
Effect of centrifugal pretreatment, p h and water activity on the production ...Mushafau Adebayo Oke
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Similar to Extraction And Characterization Of Protease From The Viscera Of Skipjack Tuna Fish (20)
Extraction And Characterization Of Protease From The Viscera Of Skipjack Tuna Fish
1. EXTRACTION AND CHARACTERIZATION OF PROTEASE FROM
THE VISCERA OF SKIPJACK TUNA FISH
(Katsuwonus pelamis Linnaeus)
ABSTRACT
Extraction and characteristics of protease from the viscera of skipjack tuna fish were studied.
Viscera of skipjack tuna fish (consist of intestines, stomach, pancreas, and liver) was extracted using
potassium phosphate solution 20mM (pH 7.5) and precipitated by using cold acetone (ratio protease
extract to cold acetone were 1:1 and 1:2) and ammonium sulfate (30%, 40%, 50%, and 60%, w/v).
The enzyme showed the highest activity when precipitated by using acetone 1:2. The optimal
temperature and pH of the cold acetone (1:2) precipitated were 50 oC and 8 respectively. The enzyme
was stable in 40oC for 3 hours incubation but less stable in 70oC. The enzyme retained more than 50%
of its activity after heating 50 oC for 30 minutes. The enzyme activity was decreased to 11.29% when
incubated in 70oC for 30 minutes. The enzyme was more stable in pH 7 and less stable in pH 10. The
enzyme activity was 5.40, 0.29, and 0.15 units/mg protein in casein (0.65%, w/v), BSA (0.65%, w/v)
and chicken feather powder (0.65%, w/v) substrate respectively. The presence of NaCl 4.6 mM
concentration increased the activity of enzyme (control activity was 100%) to 104.10% whereas the
presence of CaCl2 4.6 mM didn’t increase the activity of enzyme significantly. The presence of EDTA
4.6 mM concentration decreased the activity to 84.43%.
Keywords: Skipjack tuna, protease, extraction, characterization
INTRODUCTION
Skipjack/cakalang fish (Katsuwonus pelamis Linnaeus) is one of the most species tuna caught
in Indonesia. Fish viscera are rich of many kinds of enzyme (Venugopal, 2006). Enzyme is commonly
used in food or chemical industry and also as food supplement to help food digestion. Protease is the
highest production among the other kinds of enzyme widely used. The objectives of this research were
to study the efficiency of protease precipitation by using acetone (ratio protease extract to acetone) or
ammonium sulfate and to characterize protease from viscera of skipjack tuna such as optimum pH,
optimum temperature, pH stability, temperature stability, substrate specificity, and effect of EDTA,
NaCl, and CaCl2 on protease activity.
MATERIALS AND METHODS
Materials
Skipjack tuna viscera were obtained from Muara Baru Harbor, North Jakarta, Indonesia,
frozen at ±-20oC. Chemicals used were from Merck such as ethanol, cold acetone (pa), ammonium
sulfate, potassium phosphate (pa), casein, sodium hydroxide (pa), hydrochloric acid (pa),
Trichloroacetic Acid (pa), Folin & Ciocalteu’s phenol reagent, Sodium carbonate anhydrous (pa), L-
Tyrosin, Bovine Serum Albumin, ortho-phosphoric acid 85% (pa), Coomassive brilliant blue G-250,
chicken feather, calcium chloride, sodium chloride, EDTA, citric acid, and boric acid; screen fabric 60
mesh, and demin. water.
METHODS
1. Crude Protease Extraction (Yaneza et al. (2004), modified): Viscera were washed by using water
and cold acetone 70%. Viscera were blended in cold potassium phosphate 20 mM solutions (pH
7.5) for 1 min, filtered by using screen fabric 60 mesh, refrigerated (±4oC) and centrifuged at
6000rpm for 20 min. Supernatant was precipitated over night at ±4oC by using cold acetone (ratio
protease extract to cold acetone, 1:1 and 1:2) or by using ammonium sulfate (30, 40, 50, and 60%)
and then centrifuged at 6000rpm for 30 min. Crude protease with the highest activity would be
used for calculating purification and characterization.
2. Optimum Temperature and pH (El-Beltagy et al. (2004), Bougatef et al. (2007), Lopez and
Norman, 2007) modified): measuring the activity of enzyme extracts using casein (0.65%) as
substrate at pH 7.5 and various temperatures (10, 30, 40, 50, and 70oC) for 10 min (for temperature
optimizing); and at 50oC and various pH (3, 5, 7, 8 and 10) in buffer universal for 10 min (for pH
optimizing). Percentage of enzyme activity was estimated at the highest activity detected.
1
2. 3. Temperature and pH Stability (El-Beltagy et al. (2004), modified): estimated by incubating
enzyme extracts at various temperatures (40, 50, and 70oC) for 0, 30, 60, 90, and 180 min at pH
7.5 (for temperature stabilization analyses); and incubating enzyme extracts and various pH (7, 8,
and 10) using buffer universal for 0, 30, 60, 90, and 180 min at 37 oC (for pH stabilization
analyses). Residual activities were estimated at 37oC and pH 7.5 for 10 min of incubation. The
100% of the enzyme activity was the activity of enzyme without incubation.
4. Substrate specificity (Guangrong et al. (2006) and Syed et al. (2008), modified): The hydrolysis
activity toward a variety of protein including casein (0.65%, w/v), BSA (0.65%, w/v), and chicken
feather powder (0.65%, w/v) were estimated. Protease activity was estimated by incubating
enzyme extract in each substrate at 50oC (pH 8) for 10 min. The 100% of the enzyme activity was
the activity of enzyme on casein substrate.
5. Effect of EDTA, NaCl, dan CaCl2 (Bougatef et al. (2007), Balti et al. (2008), and Syed et al.
(2008), modified): estimated and evaluated at 50oC, pH 8. 50mM of solution (0.6mL) was added
to the mixture of enzyme extract and substrate for 10 min of incubation. The activity of enzyme
was calculated and the 100% of the enzyme activity was the activity of enzyme without addition.
6. Protein Evaluation (Bradford (1976): using Bovine Serum Albumin standard (1.25mg/ml).
7. Assay of Protease Activity ( colorimetric methods) by using casein as substrate: Casein substrate
5ml (0.65%, w/v) was mixed with 1ml enzyme extract and incubated at 37 oC, pH 7.5 for 10 min.
The same procedure was done for blank but enzyme extract addition was added after 10 min of
incubation. The reaction was stopped by adding 5ml TCA 110mM solution and mixture was
incubated at 37oC, pH 7.5 for 30 min. The mixture was centrifuged at 6000rpm for 10 min.
Supernatant was mixed with 5ml Na2CO3 500mM solution and 1ml F-C solution (F-C solution
have been thinned by using demin. water). The mixture was incubated for 30 min at 37 oC and
centrifuged at 6000rpm for 10 min. The absorbance of supernatant at 660nm. One unit of activity
was defined as the amount hydrolyzing casein to produce color equivalent to 1.0 μmol of tyrosine
per minute at assayed temperature and pH.
8. Statistical Analysis: A completely randomized design was used throughout this study and
experiments were done in duplicate. Data were subjected to ANOVA and t-test for 2 sample
comparison and further analyses of Tukey and Dunnet multiple range test. Statistical analysis was
performed using the SPSS version 16.
RESULTS AND DISCUSSION
Crude Protease Extraction
1.200
1.12e
1.021d
Specific activity (unit/mg protein)
1.000
0.800
0.61c
0.600 0.52b
0.37a 0.37a
0.400
0.200
0.000
1:1 1:2 30 40 50 60
Ratio extract:cold acetone %Ammonium sulfate
Different notation indicate value have significant difference at α=0.05
Figure 1. Crude protease activity after precipitation using acetone and ammonium sulfate
The highest enzyme activity was showed at precipitation by using cold acetone (ratio protease
extract to cold acetone, 1:2) and this cold acetone precipitation method was used to purify enzyme
extract henceforth. Extracted enzyme could be in the inactive form (zymogen) but activation of
enzyme could happen cause of other enzyme consisted in intestine mucosa. This enzyme could
2
3. activate zymogen likes trypsinogen and trypsinogen that has been activated could activate the other
zymogen likes chymotrypsinogen. So, the presence of inactive form of enzyme in enzyme extract
could be minimized.
Protein concentration was decreased but the enzyme specific activity was increased after the
precipitation (Table 1.). Generally, these data are in agreement with those reported by Balti et al. (2008)
that studied about protease from Cuttlefish hepatopancreas, Bougatef et al. (2007) that studied about
protease from sardine viscera, and Yaneza et al. (2004) that studied about protease from Monterey
sardine viscera. Protein concentration might decrease caused by soluble protein that castaway with
supernatant after centrifugation. But, specific activity was increase caused by the increasing of
purification level after precipitation using cold acetone.
Table 1. Protein concentration and crude protease activity before and after precipitation
Protein concentration Activity of crude protease
Specific activity Purification
Precipitation (mg/ml crude (unit/ml crude protease
(unit/mg protein)* fold*
protease extract)* extract)*
Before 8.48 8.54 1.007 1.00
After 8.25 12.1 1.47 1.46
* indicate value have significant difference at α= 0.05
Crude Protease Characterization
Optimum Temperature and pH
120.00
120.00 d
100.00
100.00 c
d 92.38 c
100.00 100.00 Relative activity (%) 94.00
Relative activity (%)
80.00
80.00 83.91c
60.00
60.00
b
49.14
b 51.69 40.00
40.00
20.00
b
20.00 0.97
a 13.64
8.90 a 0.00
0.00 0 2 4 6 8 10 12
0 10 20 30 40 50 60 70 80 pH
Temperature (oC) Different notation indicate value have significant difference at α=0.05
Different notation indicate value have significant difference at α=0.05
Figure 2. Optimum temperature (left) and pH (right) of skipjack tuna viscera crude protease
The partially purified protease had the highest activity at 50oC, pH 7.5 and it decreased with
the increasing of temperature (Figure 2, left). Enzyme activity was increased caused by the increasing
of reaction constanta when the temperature was increased but the activity was decrease after optimum
temperature caused by denaturation of protein at high temperature. Optimum temperature of each kind
of protease was varying according to its temperature endurance (Reed a, 1975). This protease has
higher optimum temperature than protease from Bolti fish viscera (35oC) (El-Beltagy et al., 2004),
papaya and pineapple (30-40oC) (Aurand et al., 1987) but it has lower optimum temperature than
protease from sardine viscera (Bougatef et al., 2007), skipjack tuna spleen (Klomklao a et al., 2007),
and Colossoma macropomum viscera (Esposito et al., 2008) which have optimum temperature at 60 oC,
and protease from cuttlefish hepatopancreas at 70oC (Balti et al., 2008).
Optimum pH of partially purified skipjack tuna protease was 8 at 50 oC (shown in Figure 3).
The protease activity was low in low pH value and shown higher activity in neutral and high pH value
(7-10) so this kind of protease could be employed at neutral to alkalis condition. This might be caused
by ionic group in active site that more active in neutral to alkalis condition. Similar result was reported
for Colossoma macropomum protease (Esposito et al., 2008). Different results were reported for
Monterey sardine viscera which has optimum pH 2.5 (Yaneza et al., 2004), bolti fish viscera which has
optimum pH 2.5 (El-Beltagy et al., 2004), and hepatopancreas of Jumbo squid which has optimum pH
4.5 (Lopez and Norman, 2007). Protease that was produced by papaya and pineapple were more active
in acid to neutral pH condition. Generally, proteases application at 50 oC and pH 8 were used in
production of protein hydrolysates from soybean (Suhartono, 1989), production of gelatin hydrolysates
for low-calories beverages production (Suhartono, 1989), production of Fish Protein Hydrolysates
(FPH) (Gomez et al., 2007), production of amino acid from fish waste (Gomez et al., 2007), and
extraction of astaxanthin pigment from shrimp waste (Armenta and Isabel, 2008). Besides that,
3
4. protease also could be employed to repair the functional properties of protein (Gomez et al., 2007) and
to overcome waste from food industry likes chicken feather or animal skin (Suhartono, 1989).
Temperature and pH Stability
Skipjack tuna protease was more stable at 40 oC and less stable at 70oC which is shown in
Figure 4. Similar result was shown for bolti fish viscera (El-Beltagy et al., 2004). So, this enzyme was
better employed at room temperature or temperature below 40 oC if it needs a longer process time in
application. Protease from Colossoma macropomum viscera was reported had better temperature
stability at optimum temperature (Esposito et al., 2008). Protease from cuttlefish hepatopancreas was
reported stable at 50oC in 1 hour incubation (Balti et al., 2008). Generally, proteases that were
produced from fish viscera were stable at temperature 30 until 50oC (Klomklaoa et al. (2007), Yaneza
et al. (2004), El-Beltagy et al. (2004), Bougatef et al. (2007), dan Yanezb et al. (2008)).
120.00 120.00
100.00E
100.00 100.00d 100.00e
100.00
Residual activity (%)
100.005 100.005 100.00
E
Residual activity (%)
80.00
80.00
64.63c
60.00
D b b
50.43 53.99 53.06 60.00
48.25a 53.56d
40.00 50.27D
25.54C 40.00
17.77B 31.98c
20.00
4 A 28.071C 23.15b
11.29 3 5.65 20.00 20.98B 12.67a
6.11 16.844
0.00 2.692 1.921 3
10.29 7.402 8.041A
0 30 60 90 180
Time (minute s) 0.00 5.041
Different notation indicate value have significant difference at α=0.05 0 30 60 90 180
Keterangan: notation a, b, c, and d for temperature 40o C (♦) Time (minutes)
Different notation indicate value have significant difference at α=0.05
Keterangan: notation A, B, C, D, and E for temperature 50o C (■)
Keterangan: notation a, b, c, d, and e for pH 7 (♦)
Keterangan: notation 1, 2, 3, 4, and 5 for temperature 70o C (▲) Keterangan: notation A, B, C, D, and E for pH 8 (■)
Keterangan: notation 1, 2, 3, 4, and 5 for pH 10 (▲)
Figure 3. Temperature (left) and pH (right) stability of skipjack tuna viscera crude protease
Protease skipjack tuna viscera had less stability for longer incubation time in neutral to
alkaline pH condition (Figure 5). Incubation for 30 min in pH 7 and 8 still remained more than 50%
residual activity. Protease was not stable in pH 10. Similar results were reported for Monterey sardine
viscera (Yanez et al., 2004) and bolti fish viscera (El-Beltagy et al., 2004).
Substrate Specificity
120.00
c
100.00
100.00
Relative activity (%)
80.00
60.00
40.00
20.00 b
5.34 2.85a
0.00
Kasein BSA Bulu ayam
Substrate
Different notation indicate value have significant difference at α=0.05
Figure 4. Substrate specificity of skipjack tuna viscera crude protease
The enzyme showed the highest activity on casein substrate 0.65% (w/v) at 50oC and pH 8.
The enzyme activity was 5.40, 0.29, and 0.15 units/mg protein in casein (0.65%, w/v), BSA (0.65%,
w/v) and chicken feather (bulu ayam) powder (0.65%, w/v) substrate respectively. Protease from
Thunnus obesus and Thunnus albacore stomach had high activity on casein substrate (Daulay et al.,
1996) and Hartono, 1994). Alkaline protease from Bacillus sp bacteria was reported can be used in
production of protein hydrolysates and gelatin hydrolysates (Suhartono, 1989). This protease from
skipjack tuna viscera might be used in those production processes too.
4
5. Effect of EDTA, NaCl, and CaCl2
120.00
104.10* 101.48
100.00
100.00
84.43*
Relative activity (%)
80.00
60.00
40.00
20.00
0.00
Control EDTA NaCl CaCl2
* indicate value have significant difference with control at α=0.05
Figure 5. Effect of EDTA, NaCl, dan CaCl2 on protease activity
The activity of skipjack tuna viscera protease was decrease by the addition of EDTA 4.6 mM.
Soybeans trypsin inhibitor and EDTA did not affect Monterey sardine viscera enzyme activity (Yanez
et al., 2004). The enzyme activity was decrease because EDTA could chelate the ion required for
activity of enzyme. The presence of NaCl 4.6mM could increase enzyme activity. The activity of
protease from skipjack tuna spleen decreased with NaCl addition (Klomklaoa et al, 2006). But, similar
result was reported for protease from bolti fish viscera (El-Beltagy et al., 2004). Addition of 4.6mM
CaCl2 was not significantly effect the enzyme activity. The activity of protease from bolti fish viscera
and true sardine viscera increase when CaCl2 was added to enzyme extract (El-Beltagy et al. 2004) and
Klomklaob et al. 2008). Protease from skipjack tuna viscera might be categorized to serine or metalo
protease but generally serine protease was the commonly protease that found in fish viscera.
CONCLUSION
The enzyme showed the highest activity when precipitated by using cold acetone 1:2, at pH 8
and temperature 50oC. The enzyme was stable in 40oC for 3 hours of incubation but less stable in 70 oC.
The enzyme was more stable in pH 7 and less stable in pH 10. The enzyme showed the highest activity
on casein substrate (0.65%, w/v) compare to BSA and chicken feather powder substrate (0.65%, w/v).
The presence of NaCl 4.6 mM increased the enzyme activity (control was 100%) to 104.10% whereas
the presence of CaCl2 4.6 mM didn’t increase the enzyme activity. The presence of EDTA 4.6 mM
decreased the enzyme activity. This study needs to study about identification of protease, effect of
activator and inhibitor, storage stability, application to food production, effect of extraction methods
and maximizing activation of inactivated form of enzyme in viscera.
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