scholarly journals Production, Purification, and Characterization of Extracellular Alkaline Protease From Bacillus Firmus Var. Arosia NCIB 10557

2021 ◽  
Author(s):  
Michele Francisca Dias ◽  
Zilpa Silva das Graças da Silva ◽  
Juliana Silva de Santana ◽  
Irapuan Oliveira Pinheiro ◽  
ANA CÉLIA OLIVEIRA DOS SANTOS
Keyword(s):  
Alkaline Protease ◽  
Batch Fermentation ◽  
Specific Activity ◽  
Residual Activity ◽  
Maximum Activity ◽  
Tween 20 ◽  
Partial Purification ◽  
Bacillus Firmus ◽  
Fed Batch ◽  
Feeding Profile

Abstract The most important alkaline proteases from the commercial standpoint are produced by bacteria of the genus Bacillus and used mainly in the formulation of detergents. The aim of the present study was to evaluate the production, partial purification, and characteristics of alkaline protease obtained by Bacillus firmus var. arosia NCIB 10557 in fed-batch fermentation with constant feeding profile and carbon source restriction. Firstly, it was carried out on batch fermentation and after 6.5 h of fermentation, glucose became limiting, and then the fed-batch was started with a flow rate of 0.0802 mL/min. Maximum activity (998.1 U/mL) was reached after 10.5 h of fed-batch, with a subsequent 60.91 % drop in activity after two hours. The purification steps resulted in a 1.65-fold increase in the value of the specific activity. The protease showed optimum activity at 37°C and pH 9 and residual activity above 80 % at pH 11 and 12. Residual activity was greater than 70 % at temperatures ranging from 30 to 70 °C and 90 % of this activity was maintained for 30 minutes at 70 °C until the occurrence of complete inactivation. Enzyme activity was estimated using SDS. The organic solvents Triton X-100, Tween-20, EDTA and β-mercaptoethanol and the ions Zn2+, Fe2+, Cu2+ and Ni2+ partially inhibited the activity of the protease. Ca2+, Mn2+ and Mg2+ had no stimulating action on the enzyme.

scholarly journals Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant

2017 ◽  
Vol 7 (4) ◽  
pp. 1 ◽  
Author(s):  
Sreedevi Basavaraju ◽  
Chandrasekhar Kathera ◽  
Pramoda Kumari Jasti
Keyword(s):  
Bacillus Cereus ◽  
Ammonium Sulphate ◽  
Alkaline Protease ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Tween 20 ◽  
Original Activity ◽  
Protease Enzyme

The alkaline protease produced by Bacillus cereus UV-15 mutant was purified by precipitation with ammonium sulphate and gel filtration through sephadex G-100. The enzyme has shown to have a molecular weight of 29kDa by SDS polyacrylamide gel electrophoresis. The extracted protease enzyme was purified by 16.64 fold through ammonium sulphate precipitation and chromatography separation in Sephadex G-100. The purified protease had a specific activity of 2915 (U/mg). The zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which coincided with the band obtained with SDS–PAGE. The enzyme was remained active and stable at pH 8-11, with an optimum at pH 10.0. The protease was stable in the temperature ranging from 40°C to 60°C, but gradually decreased at temperature 70°C. The optimum temperature for protease activity was determined at 60°C. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. At 1% concentration of Tween-20 and Tween-80, the enzyme retained 78% and 94% relative activity respectively. Alkaline protease retained 95% activity toward 0.5% concentration of the anionic detergent SDS. The enzyme showed compatibility at 50°C with commercial detergents such as Ariel, Surf excel, Rin, wheel, Tide and Nirma. In the presence of Ariel and Rin the enzyme retained about 72 and 75% of the original activity respectively. The supplementation of the enzyme in detergents could improve the cleansing performance towards the blood stains and suggested to be used as a detergent additive. The enzyme also removed goat hide hairs completely after 15 hr of incubation. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.

scholarly journals Purificación Parcial y Caracterización de Alfa Amilasa de granos germinados de Chenopodium quinoa (Quinua)

2018 ◽  
pp. 52-58
Keyword(s):  
Specific Activity ◽  
Chenopodium Quinoa ◽  
Maximum Activity ◽  
Alpha Amylase ◽  
Partial Purification ◽  
Chemical Hydrolysis ◽  
Germination Process ◽  
Sds Page ◽  
Global Population

Purificación Parcial y Caracterización de Alfa Amilasa de granos germinados de Chenopodium quinoa (Quinua) Partial Purification and Characterization of Alpha Amylase from germinated grains from Chenopopdium quinoa (Quinua) Melissa Bedón Gómez, Oscar Nolasco Cárdenas, Carlos Santa Cruz C. y Ana I. F. Gutiérrez Román Universidad Nacional Federico Villarreal, Facultad de Ciencias Naturales y Matemática, Laboratorio de Bioquímica y Biología Molecular, Jr. Río Chepén S/N, El Agustino. Telefax: 362 - 3388 DOI: https://doi.org/10.33017/RevECIPeru2013.0007/ Resumen Las alfa amilasas son las enzimas más estudiadas e importantes en el campo biotecnológico e industrial; ya que han reemplazado por completo la hidrólisis química del almidón. Estas enzimas son imprescindibles en la elaboración de productos alimenticios, combustibles, medicamentos y detergentes con la finalidad de optimizar procesos y conservar el medio ambiente. La α-amilasa puede ser purificada de diferentes organismos como plantas, animales, hongos y bacterias; actualmente un gran número de α-amilasas bacterianas en especial del género Bacillus están disponibles comercialmente y son las más utilizadas en las industrias. Sin embargo, la producción de éstas no satisfacen los requerimientos industriales en el mundo; ya que, la demanda de esta enzima se ha incrementado en los últimos dos años y el empleo de α-amilasas bacterianas ha provocado alergias afectando al 15% de la población a nivel mundial. . En este estudio, como fuente de α-amilasa se emplearon semillas de Chenopodium quinoa (quinua) var hualhuas blanca durante el proceso de germinación; esta enzima fue parcialmente purificada por precipitación con sulfato de amonio obteniendo una actividad específica final de 35.60U/mg y un grado de purificación de 5 veces. La purificación fue confirmada por SDS-PAGE, encontrando un peso molecular de 44kDa. La actividad enzimática se evaluó mediante el método de Miller mostrando máxima actividad a pH 7 y a temperatura de 37ºC. La linealización de Lineweaver-Burk nos dio un Km de 16mg/mL y Vmax de 100µM de maltosa/min. Por lo tanto, esta caracterización reúne los pre-requisitos necesarios para la aplicación en la industria. Descriptores: Chenopodium quinoa, alfa amilasa, germinación, purificación parcial. Abstract The alpha amylases are the enzymes most studied and important in biotechnology and industry; because they have completely replaced the starch’s chemical hydrolysis. These enzymes are essential in the food production, medicines and detergents in order to optimize processes and conserve the environment. The α-amylase can be isolated from different organisms such as plants, animals, fungi and bacteria, now a large number of bacterial α-amylases especially from genus Bacillus are commercially available and they are the most used in industry. However, the production of these do not meet industry requirements in the world, because the demand for this enzyme has increased in the last two years and the use of bacterial α-amilase has caused allergies affecting the 15% of the global population. In this study, as a source of α-amylase used the seeds from Chenopodium quinoa (quinoa). Var. white hualhuas during the germination process, this enzyme was partially purified by ammonium sulfate precipitation to obtain a final specific activity of 35.60U/mg, and a grade of purification of 5 times. The purification was confirmed by SDS-PAGE, where the molecular weight was 44kDa. The enzyme activity was evaluated by Miller method showing maximum activity at pH 7 and 37ºC. The Lineweaver-Burk linearization shows a Km of 16mg/mL and Vmax of 100μM the maltose / min. Therefore, these characterizations meet the prerequisites need for industry. Keywords: Chenopodium quinoa; alpha amylase; germination; partial purification

scholarly journals ENHANCED ENZYMATIC ACTIVITY OF STREPTOMYCES GRISEOPLANUS L-ASPARGINASE VIA ITS INCORPORATION IN AN OIL-BASED NANOCARRIER

2020 ◽  
pp. 203-210
Author(s):  
ABEER A. EL-HADI ◽  
HANAN MOSTAFA AHMED ◽  
RANIA A. ZAKI ◽  
AMIRA MOHAMED MOHSEN
Keyword(s):  
Thermal Stability ◽  
Oleic Acid ◽  
Enzymatic Activity ◽  
Specific Activity ◽  
Residual Activity ◽  
Tween 80 ◽  
Biochemical Properties ◽  
Lymphocytic Leukemia ◽  
Tween 20 ◽  
Ph Range

Objective: L-asparaginase (L-asp) is a vital enzyme used as a therapeutic agent in combination with other drugs in the treatment of acute lymphoma, melanosarcoma and lymphocytic leukemia. Immobilization of enzymes through loading on nanoemulsion (NE) results in some advantages such as enhancing their stability and increasing their resistance to proteases. Aim of the present study is to formulate L-asp loaded nanoemulsion to enhance its efficiency and thermal stability. Methods: Nanoemulsion loaded with L-asp crude extract (specific activity 13.23U/mg protein) was prepared employing oleic acid as oil, tween 20/tween 80 as surfactants and propylene glycol (PG) as co-surfactant. L-asp loaded NE underwent several thermodynamic stability studies and the optimized formulae were further examined for their biochemical properties and thermal stability. Results The developed formulations were spherical in shape and their sizes were in the nanometric dimensions with negatively charged zeta potential values. Upon comparing the enzyme activity of L-asp loaded NE employing tween 20 (F1) or tween80 (F4) at different concentrations, the results revealed that F4 NE showed higher enzymatic activity [323 U/ml] compared to F1 NE [197 U/ml] at the same concentration. The nanosized immobilized L-asp was more stable in the pH range from 8 to 8.5 as compared to free L-asp. The immobilized enzyme preserved about 59.11% of its residual activity at 50 °C; while free L-asp preserved about 33.84%. Conclusion: In the view of these results, NE composed of oleic acid, tween 80 and PG represents a promising dosage form for enhancing the activity and stability of Streptomyces griseoplanus L-asp.

scholarly journals Statistical Optimization, Partial Purification, and Characterization of Phytase Produced from Talaromyces purpureogenus NSA20 Using Potato Peel Waste and its Application in Dyes De-colorization

2021 ◽  
Vol 12 (4) ◽  
pp. 4417-4431
Keyword(s):  
Crystal Violet ◽  
Specific Activity ◽  
Marine Fungus ◽  
Maximum Activity ◽  
Statistical Optimization ◽  
Partial Purification ◽  
Methyl Red ◽  
Potato Peel ◽  
Phytase Production

In this study, Talaromyces purpureogenus NSA20 as phytase-producing marine fungus was isolated and identified morphologically and genetically and deposited in Gene Bank with accession number MW031769.1. One factor at a time (OFAT) optimization was performed, where the result revealed that potato peel waste (1.5%) as a substrate was the highest for phytase production at 6 days, where the maximum activity of phytase was 138.4 U/ml. Moreover, Box–Behnken design as response surface methodology was carried out for statistical optimization of phytase production by T. purpureogenus NSA20. Statistical optimization illustrated that the optimized medium for phytase production increased 1.57 fold compared to the OFAT optimized medium. Partial purification of phytase was carried out, where the enzyme after precipitation with ammonium sulfate (80%) was 2.6-fold purified phytase, and the yield was 39.8 %., the specific activity was 31.19 U/mg proteins. Additionally, partially purified phytase was characterized; the maximum activity of phytase at Fe++ 0.1% and pH 5.5 at 37 oC was 350 U/ml. Eventually, phytase was applied for crystal violet and methyl red decolorization, where decolorization percentages of crystal violet and methyl red were 85.5% and 75% at 120 min, respectively.

scholarly journals Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system

1975 ◽  
Vol 146 (3) ◽  
pp. 713-722 ◽  
Author(s):  
K P Wheeler ◽  
J A Walker ◽  
D M Barker
Keyword(s):  
Adenosine Triphosphatase ◽  
Specific Activity ◽  
Total Phospholipid ◽  
Kidney Tissue ◽  
Residual Activity ◽  
Fold Increase ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Phospholipid Content ◽  
Total Phospholipid Content

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.

Secretion of Plasminogen Activators by Cultured Bovine Endothelial Cells: Partial Purification, Characterization and Evidence for Multiple Forms

1981 ◽  
Vol 45 (03) ◽  
pp. 219-224 ◽  
Author(s):  
W E Laug
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Plasminogen Activators ◽  
Maximum Activity ◽  
Partial Purification ◽  
Diisopropyl Fluorophosphate

SummaryEndothelial cells were obtained from the aortae of newborn calves and cloned. High plasminogen activator (PA) activity was detected in the supernatant medium and the cell lysates of confluent cultures. The PA activity in the growth medium increased steadily during 12 hrs of incubation, indicating active enzyme secretion by these cells. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the concentrated medium demonstrated the presence of four plasminogen activators with apparent molecular weights of 77,000 (±3000), 43,000 (±2000), 26,000 (±1500) and 14,500 (±1500) respectively. The 43,000, 26,000 and 14,500 molecular weight forms could be converted to radioactive derivates by active site labeling with 3H diisopropyl fluorophosphate (3H DFP) while the 77,000 Dalton form took up only traces of this radioactively labeled compound. The 43,000 molecular weight form was partially purified by means of salt precipitation and gel filtration. This enzyme preparation activated plasminogen by proteolytic cleavage with maximum activity at pH 7.5-8.5 and demonstrated a specific activity of 80,000 CTA (Committee on Thrombolytic Agents) units/mg protein when tested on 125I-fibrin in the presence of plasminogen. This PA was rapidly and irreversibly inhibited by diisopropyl fluorophosphate (DFP), suggesting that it was a serine protease. The partially purified enzyme was extremely labile at temperatures from 0-60° C, but could be stabilized by lowering the pH to 3 or by the addition of albumin.

FED-BATCH FERMENTATION OFBACILLUS CLAUSIIFOR EFFICIENT PRODUCTION OF ALKALINE PROTEASE USING DIFFERENT FEEDING STRATEGIES

2011 ◽  
Vol 198 (9) ◽  
pp. 1063-1074 ◽  
Author(s):  
F. Tabandeh ◽  
H. R. Hosseinian Moghaddam ◽  
B. Yakhchali ◽  
P. Shariati ◽  
M. T. Hamed Mousavian ◽  
...  
Keyword(s):  
Alkaline Protease ◽  
Batch Fermentation ◽  
Feeding Strategies ◽  
Efficient Production ◽  
Fed Batch ◽  
Fed Batch Fermentation
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