scholarly journals Purification of Wickerhamomyces anomalus Keratinase and Its Prospective Application in Poultry Feed Industries

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Ayandiran Aina ◽  
C. O Ezeamagu ◽  
S. T. Akindele ◽  
A. O. Aleshinloye
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Skim Milk ◽  
Feed Additives ◽  
Poultry Feed ◽  
Crude Fibre ◽  
Wickerhamomyces Anomalus ◽  
Ammonium Sulphate Precipitation ◽  
Promising Tool ◽  
Prospective Application

Animal wastes emanating from cow horns, hooves and feathers are keratinous in nature which can only be degraded by keratinolytic microorganisms. Consequently, the pollution resulting from the accumulation of these wastes in response to growing livestock demand is posing a significant threat to human health and the environment. This study was carried out using cow hooves as the substrate for the production of keratinase from fungal identified as Wickerhamomyces anomalus 9 (18S rDNA gene sequencing) was isolated from soil rich hooves using basal salt agar medium and potato dextrose agar. The keratinase of the isolate was assessed using skim milk agar and the enzyme was produced by solid-state fermentation.  The crude enzyme was purified using ammonium sulphate precipitation, ion exchange and gel-filtration chromatography. The specific activity of the enzyme was 0.29 U/mg with a yield of 45% and a 7.25 purification fold. The optimal pH and temperature of the enzyme were 8.0 and 60 oC respectively. The enzyme was observed to be thermo-stable at 50oC between for 30 minutes. The kinetics revealed that the Vmax was 0.384U/min with Km 86.95mg/ml. The native molecular weight of the enzyme was found to be 34KDa. There were significant differences at 95% confidence with poultry feed treated with Wickerhamomyces anomalus keratinase in moisture, ash content, crude fibre, crude fat, nitrogen content, crude protein and carbohydrate compare to the untreated feed. These results suggest an environment-friendly approach for biodegradation of cow hooves wastes for the production of keratinases, animal waste management as well as a promising tool for chicken feed additives. Keywords: Biodegradation, Chromatography, Cow hooves, Keratinase, Pollution

scholarly journals Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Kamal Uddin Zaidi ◽  
Ayesha S. Ali ◽  
Sharique A. Ali
Keyword(s):  
Agaricus Bisporus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Edible Mushroom ◽  
Total Activity ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Ammonium Sulphate Precipitation ◽  
Key Enzyme

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

Purification and Partial Characterization of Cellulase Produced by Aspergillus niger Cultured on Vitellaria paradoxa shells

2017 ◽  
Vol 6 (2) ◽  
Author(s):  
Abdulhakeem Olarewaju Sulyman ◽  
Yusuf A Iyanda ◽  
Afolabi Olaniyi Opasola ◽  
OtunOla Adedayo ◽  
Raliat Abimbola Aladodo
Keyword(s):  
Aspergillus Niger ◽  
Specific Activity ◽  
Gel Filtration ◽  
Inoculum Size ◽  
Partial Characterization ◽  
Effect Of Temperature ◽  
Vitellaria Paradoxa ◽  
Endoglucanase Activity ◽  
Ammonium Sulphate Precipitation

This research investigated the purification and partial characterization of cellulase produced by Aspergillus niger cultured on Vitellaria paradoxa shells. Cellulase (endoglucanase) from A. niger was produced under optimum fermentation conditions at 35 °C, pH 4.7, V. paradoxa, 4 g/L, inoculum size of 10 mm and the fermentation media incubated for 120 hours. The crude endoglucanase obtained were partially purified by subjecting to ammonium sulphate precipitation, dialysis and gel filtration chromatography for further purification. The effect of temperature and pH on the activity of purified endoglucanase was determined. Cellulase was purified to 734.33 folds by Sephadex G-100 column chromatography with a specific activity and yield of 4.406 U/mg and 63.03% respectively. Fractions 4 and 7 contained the highest endoglucanase activity out of 18 fractions collected and the two fractions were pooled for further analysis. The activity of purified endoglucanase was optimum at a temperature of 40 °C and pH 5. Therefore, the purified endoglucanase produced may be explored in detergent industry.

scholarly journals Isolation and characterization of three distinct forms of lipases from Candida rugosa produced in solid state fermentation

2001 ◽  
Vol 44 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Sailas Benjamin ◽  
Ashok Pandey
Keyword(s):  
Solid State ◽  
Solid State Fermentation ◽  
Specific Activity ◽  
Candida Rugosa ◽  
Gel Filtration ◽  
Coconut Oil ◽  
Apparent Molecular Weight ◽  
Ammonium Sulphate Precipitation ◽  
Isolation And Characterization ◽  
Ultra Filtration

Three distinct forms (Lip A, Lip B and Lip C) of extra-cellular lipases (EC- 3.1.1.3), produced by Candida rugosa in solid state fermentation (SSF) were purified and characterised. SSF was carried out in glass columns using coconut oil cake and wheat bran. The enzyme was purified from the aqueous extract of fermented matter by ammonium sulphate precipitation, dialysis, ultra-filtration and gel filtration using Sephadex-200 to a 43-fold purification and 64.35-mg/ml specific activity. SDS-PAGE of purified enzyme revealed three distinct bands, indicating the existence of three iso-forms, Lip A, Lip B and Lip C with apparent molecular weight about 64,000, 62,000 and 60,000 Da, respectively. All the three iso-forms were optimally active at 35-40°C and pH 7-8. They showed marked differences in their Km values with different saturated and unsaturated triacyl glycerols. Ag++ and Hg++ strongly inhibited enzyme activity of all the iso-forms, Mn++ has no effect and Ca++ and Mg++ enhanced the activity. EDTA also strongly inhibited the enzyme activities of iso-forms. However, activities of all the three lipases were completely inhibited by serine protease inhibitors such as 3,4-dichloroisocoumarin, pefabloc and partially by phenylmethanesulphonyl fluoride. To the best of our knowledge, this is the first report describing the purification and characterisation of C. rugosa lipase iso-forms from solid cultures. These lipase iso-forms with diverse characteristics produced in solid cultures may find potential application in biomedical field.

scholarly journals New Lipase for Biodiesel Production: Partial Purification and Characterization of LipSB 25-4

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Aysel Ugur ◽  
Nurdan Sarac ◽  
Rukiye Boran ◽  
Berk Ayaz ◽  
Ozgur Ceylan ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Nitrogen Sources ◽  
Biodiesel Production ◽  
Partial Purification ◽  
Period Effect ◽  
Carbon And Nitrogen ◽  
Ammonium Sulphate Precipitation ◽  
Thermophilic Conditions

The lipolytic activities of 300 Streptomyces isolates were determined in Tributyrin and Rhodamine-B Agar. Lipase activities were also measured with p-nitrophenyl palmitate (p-NPP) as a substrate. The strain of Streptomyces bambergiensis OC 25-4 used in this study was selected among 300 strains of Streptomyces from MUCC as the best lipase producer. The incubation conditions were optimized and the inoculum amount, incubation period, effect of carbon and nitrogen sources, and rates of MgSO4 and CaCO3 were investigated. LipSB 25-4 (the lipase produced by S. bambergiensis OC 25-4 strain) was partially purified with ammonium sulphate precipitation, dialysis, and gel filtration chromatography 2.73-fold and with 92.12 U/mg specific activity. The optimal pH and temperature for LipSB 25-4 were determined as 8.0 and 50°C, respectively. The lipase has high stability in all pH and temperature values used in this study. While LipSB 25-4 was slightly activated in the presence of β-mercaptoethanol, it was slightly reduced by PMSF. The enzyme conserved approximately 75% of its activity at the end of 60 h, in the presence of methanol and ethanol. Since LipSB 25-4 displays high activity in the thermophilic conditions and stability in the presence of organic solvents, this lipase can catalyse the biodiesel production from olive oil by the transesterification reactions.

scholarly journals Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

1996 ◽  
Vol 314 (2) ◽  
pp. 587-593 ◽  
Author(s):  
Antonio del CASTILLO-OLIVARES ◽  
Miguel A. MEDINA ◽  
Ignacio NÚÑEZ de CASTRO ◽  
Javier MÁRQUEZ
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Nadh Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumour Cells ◽  
Ammonium Sulphate Precipitation ◽  
Apparent Homogeneity

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis–Menten kinetics for the substrates, with apparent Km values of 4.3×10-5 M and 6.7×10-5 M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thiol-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

scholarly journals Biobleaching of Industrial Important Dyes with Peroxidase Partially Purified from Garlic

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Akudo Chigozirim Osuji ◽  
Sabinus Oscar O. Eze ◽  
Emmanuel Emeka Osayi ◽  
Ferdinand Chiemeka Chilaka
Keyword(s):  
Ammonium Sulphate ◽  
Specific Activity ◽  
Low Cost ◽  
Gel Filtration ◽  
Enzyme Concentration ◽  
Gel Filtration Chromatography ◽  
Vat Dyes ◽  
Ammonium Sulphate Precipitation ◽  
Ph Range ◽  
Better Than

An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km andVmaxfor H2O2and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2concentration, and enzyme concentration of 4.5–5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources) show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.

scholarly journals PURIFICATION OF β-GLUCOSIDASE PRODUCED FROM Trichoderma viride USING COW DUNG AS CARBON SOURCE

2020 ◽  
Vol 4 (2) ◽  
Author(s):  
S. T. Tyohemba ◽  
S. Aliyu ◽  
N. N. Ndukwe ◽  
G. G. Memi ◽  
U. O. Edem
Keyword(s):  
Specific Activity ◽  
Trichoderma Viride ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Broad Peak ◽  
Cow Dung ◽  
Elution Profile ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Peak Fraction

β-glucosidases have characteristics of biotechnological interest and have thus become important industrial enzymes.In this study, β-glucosidase produced by Trichoderma viride from cow dung was subjected to a three step purification process involving ammonium sulphate precipitation, gel filtration by Sephadex G-100 and ion exchange chromatography by DEAE-Sephadex A-25. The elution profile on Sephadex G-100 resulted in a single broad peak (fractions 9-21) which had a yield of 3.7% and a purification fold of 4.29 with a specific activity of 25.70 µmol/min/mg proteins while the elution profile on DEAE-Sephadex A-25 resulted in a single broad peak (fraction 8-14) which had a yield of 2.76% and a purification fold of 22.14 with a specific activity of 132.41µmol/min/mg of protein. The purified enzyme was obtained as a single band and had a molecular mass of 51.8 kDa on SDS-PAGE. This results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

scholarly journals Isolation and characterization of three distinct forms of lipases from Candida rugosa produced in solid state fermentation

2000 ◽  
Vol 43 (5) ◽  
pp. 453-460 ◽  
Author(s):  
Sailas Benjamin ◽  
Ashok Pandey
Keyword(s):  
Solid State ◽  
Solid State Fermentation ◽  
Specific Activity ◽  
Candida Rugosa ◽  
Gel Filtration ◽  
Coconut Oil ◽  
Apparent Molecular Weight ◽  
Ammonium Sulphate Precipitation ◽  
Isolation And Characterization ◽  
Ultra Filtration

Three distinct forms (Lip A, Lip B and Lip C) of extra-cellular lipases (EC- 3.1.1.3), produced by Candida rugosa in solid state fermentation (SSF) were purified and characterised. SSF was carried out in glass columns using coconut oil cake and wheat bran. The enzyme was purified from the aqueous extract of fermented matter by ammonium sulphate precipitation, dialysis, ultra-filtration and gel filtration using Sephadex-200 to a 43-fold purification and 64.35-mg/ml specific activity. SDS-PAGE of purified enzyme revealed three distinct bands, indicating the existence of three iso-forms, Lip A, Lip B and Lip C with apparent molecular weight about 64,000, 62,000 and 60,000 Da, respectively. All the three iso-forms were optimally active at 35-40ºC and pH 7-8. They showed marked differences in their Km values with different saturated and unsaturated triacyl glycerols. Ag++ and Hg++ strongly inhibited enzyme activity of all the iso-forms, Mn++ has no effect and Ca++ and Mg++ enhanced the activity. EDTA also strongly inhibited the enzyme activities of iso-forms. However, activities of all the three lipases were completely inhibited by serine protease inhibitors such as 3,4-dichloroisocoumarin, pefabloc and partially by phenylmethanesulphonyl fluoride. To the best of our knowledge, this is the first report describing the purification and characterisation of C. rugosa lipase iso-forms from solid cultures. These lipase iso-forms with diverse characteristics produced in solid cultures may find potential application in biomedical field.

scholarly journals Effects of Tannin Extract fromGongronema latifoliumLeaves on LipoxygenaseCucumeropsis maniiSeeds

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Sabinus O. O. Eze ◽  
Bennett C. Nwanguma
Keyword(s):  
Ascorbic Acid ◽  
Propyl Gallate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Oxidative Enzymes ◽  
Kinetic Properties ◽  
Tannin Extract ◽  
Ammonium Sulphate Precipitation ◽  
Show Maximum Activity

Lipoxygenase (EC 1.13.11.12) was partially purified from germinated seeds ofCucumeropsis maniito a purification fold of 47.14, enzyme activity recovery of 72.18%, and specific activity of 326.25 units/mg protein, using a three-step process of centrifugation, ammonium sulphate precipitation and gel filtration. Kinetic properties show maximum activity at pH 6.0 and at optimum temperature of 40°C. Inhibitory effects of the extract fromGongronema latifoliumand two other known antioxidants: ascorbic acid and propyl gallate on lipoxygenase fromCucumeropsis maniiwere studied. Result shows presence of inhibition with IC50 of4.2×10−3±0.09×10−3 g/L,4.3×10−2±0.11×10−2 g/L and7.9×10−2±0.11×10−2 g/L for the extract from ascorbic acid and propyl gallate, respectively. The extract when compared to the other antioxidants exhibits a competitive mechanism of inhibition. This tannin extract could be included during food processing as preservative against food deterioration that might be caused by oxidative enzymes such as lipoxygenase.

scholarly journals Preparation of antibodies type IgY against Salmonella typhi lipopolysaccharide in chicken eggs

2012 ◽  
Vol 6 (2) ◽  
pp. 15-22
Author(s):  
Mouruj A. Alaubydi ◽  
Susan Ahmad ◽  
Muayad Sabri
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Gel Filtration ◽  
Salmonella Typhi ◽  
Dilution Method ◽  
Gel Filtration Chromatography ◽  
Chromatography Method ◽  
Ammonium Sulphate Precipitation ◽  
Chicken Eggs ◽  
Water Dilution

ipopolysaccharide was extracted from local isolate Sallmonella typhi (previously isolated and characterized) by hot EDTA method, and the extract was partially purified by gel filtration chromatography on sepharose Cl-6B gel. The results showed that the percentage of the carbohydrates amount in the partially purified LPS extract was 43.7%, while the percentage of binding proteins in the same extract was 0.7% with no nucleic acids was found. The molecular weight for the LPS was measured by the gel filtration chromatography method using Sepharose Cl-6B gel and was equivalent to 263000 Dalton. The LD¬50 of LPS was determined by injection of chicken embryos type Ice Brown in the charioallantoic membrane, and was 14.66 µg/Kg. In order to obtain anti S. typhi IgY antibodies, chickens were immunized with the partially purified S. typhi subcutaneously. The IgY antibodies were extracted from eggs yolk by water dilution method and the extract was partially purified by ammonium sulphate precipitation at ratio 60% saturation, and gel filtration chromatography on sepharose Cl-6B gel. The results showed that the protein amount was equivalent to 23.5 mg/ml; specific activity was 0.268, and an overall yield of 70%. The molecular weight for the IgY antibodies was measured by the gel filtration chromatography method using Sepharose Cl-6B gel and was found to be 178000 dalton. The concentration of anti S.typhi LPS IgY antibodies in chicken eggs were investigated by ELISA and was found to be 6.3 mg/ml, and there is a significant differences (P<0.01).

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