Purified proteases of two Antarctic bacteria: from screening to characterization

2021 ◽  
pp. 1-12
Author(s):  
Christian Peralta-Figueroa ◽  
José Martínez-Oyanedel ◽  
Marta Bunster ◽  
Gerardo González-Rocha
Keyword(s):  
Positive Impact ◽  
Industrial Processes ◽  
Geobacillus Stearothermophilus ◽  
Purification And Characterization ◽  
Cold Adapted ◽  
Optimum Ph ◽  
Different Temperatures ◽  
Growing Conditions ◽  
Alkaline Range

Abstract Proteases are widely used in industrial processes, and the discovery of new, more kinetically efficient proteases can have a positive impact on industry. Enzymes from Antarctic microorganisms exhibit cold-adaptive properties, making them useful in biotechnology. The cold and harsh environment of Antarctica makes it a valuable source for new biotechnologically related enzymes. In this study, we characterized two cold-adapted proteases purified from Pseudoalteromonas issachenkonii P14M1-4 and Flavobacterium frigidimaris ANT34-7, isolated from King George Island, Antarctica, and compared these with proteases from the non-cold-adapted bacteria Bacillus licheniformis and Geobacillus stearothermophilus. The best temperature growing conditions were used for protease purification and characterization. The protease from P. issachenkonii P14M1-4 was identified as a 40–43 kDa metal-dependent subtilisin-like serine protease and the protease from F. frigidimaris ANT34-7 was identified as a 28 kDa metalloprotease. The enzymes showed an optimum temperature of between 35°C and 40°C and an optimum pH in the neutral to alkaline range. Their activation energies, catalytic constants and growth capacities at different temperatures categorize them as cold-adapted enzymes. We conclude that the characteristics exhibited by these proteases make them useful for biotechnological purposes requiring high activity at low temperatures. Moreover, to the best of our knowledge, this is the first characterization of a cold-adapted protease from F. frigidimaris.

scholarly journals Purification and Characterization of Asparaginase fromPhaseolus vulgarisSeeds

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Saleh A. Mohamed ◽  
Mohamed F. Elshal ◽  
Taha A. Kumosani ◽  
Alia M. Aldahlawi
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Potential Candidate ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Wide Range ◽  
Optimum Ph ◽  
Glutaminase Activity ◽  
Low Activity

L-asparaginase from bacteria has been used in treatment of acute lymphoblastic leukemia. The aim of this study was to purify and characterize L-asparaginase fromPhaseolus vulgarisseeds instead of microbial sources. L-asparaginase was purified to apparent homogeneity. The enzyme has molecular mass of 79 kDa. The purified asparaginase had very low activity toward a number of asparagine and glutamine analogues. L-asparaginase was free from glutaminase activity. Kinetic parameters, Km andVmax of purified enzyme, were found to be 6.72 mM and 0.16 μM, respectively. The enzyme had optimum pH at 8.0. The enzyme showed high stability at alkaline pH (pH 7.5–9.0) when incubated for up to 24 h. L-asparaginase had the same temperature optimum and thermal stability at 37°C. K+was able to greatly enhance the activity of asparaginase by 150% compared with other metals tested. In conclusion, L-asparaginase showed no glutaminase activity and good stability over a wide range of physiological conditions, and thus it could be used as a potential candidate for treatment of acute lymphoblastic leukemia.

scholarly journals Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
Author(s):  
I Ishii-Karakasa ◽  
H Iwase ◽  
K Hotta ◽  
Y Tanaka ◽  
S Omura
Keyword(s):  
Enzyme Activity ◽  
Culture Medium ◽  
Gel Filtration ◽  
Partial Purification ◽  
Gastric Mucus ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Optimum Ph ◽  
New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

scholarly journals Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain

2015 ◽  
Vol 46 (3) ◽  
pp. 683-690 ◽  
Author(s):  
Jiang Li ◽  
Qiushi Hu ◽  
Yuquan Li ◽  
Yuan Xu
Keyword(s):  
Purification And Characterization ◽  
Cold Adapted

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

Purification and characterization of ferro- and cobalto-chelatases

1988 ◽  
Vol 66 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Eduardo T. Cánepa ◽  
Elena B.C. Llambías
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Purification And Characterization ◽  
Degree Of Unsaturation ◽  
Apparent Homogeneity ◽  
Optimum Ph ◽  
Single Protein ◽  
Metal Insertion

Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40 000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240 000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h. but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 μM for the metal and 30.3 μM for mesoporphyrin. and for cobaltochelatase activity. 27 and 45.5 μM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.

EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF COLD-ADAPTED INORGANIC PYROPHOSPHATASE FROM PSYCHROPHILICShewanellasp. AS-11

2014 ◽  
Vol 44 (5) ◽  
pp. 480-492 ◽  
Author(s):  
E. L. Ginting ◽  
S. Iwasaki ◽  
C. Maeganeku ◽  
H. Motoshima ◽  
K. Watanabe
Keyword(s):  
Inorganic Pyrophosphatase ◽  
Purification And Characterization ◽  
Cold Adapted

scholarly journals Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

scholarly journals Optimization of Growth Conditions for Purification and Production of L-Asparaginase bySpirulina maxima

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Hanaa H. Abd El Baky ◽  
Gamal S. El Baroty
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Growth Conditions ◽  
Partial Purification ◽  
Specific Enzyme ◽  
Purification And Characterization ◽  
Spirulina Maxima ◽  
Optimum Ph ◽  
Biomass Yields

L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA fromSpirulina maxima(SM) were tested. SM cultures grown in Zarrouk medium containing different N2(in NaNO3form) concentrations (1.25, 2.50, and 5.0 g/L) for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2concentration. The cultures of SM at large scales (300 L medium, 5 g/L N2) showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production inS. maxima.

scholarly journals Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes' strain)

1978 ◽  
Vol 169 (3) ◽  
pp. 633-641 ◽  
Author(s):  
S A Baldwin ◽  
R N Perham ◽  
D Stribling
Keyword(s):  
Escherichia Coli ◽  
Common Ancestor ◽  
Class Ii ◽  
Purification And Characterization ◽  
Thiol Compounds ◽  
E Coli ◽  
Optimum Ph ◽  
Class Ii Aldolase ◽  
Fructose 1,6 Bisphosphate

A new form of the class-II D-fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) of Escherichia coli (Crookes' strain) was isolated from an extract of glycerol-grown bacteria. It has a higher molecular weight (approx. 80000)than previous preparations of the enzyme and closely resembles the typical class-II aldolase from yeast in size and amino acid composition. On the other hand, its kinetic behaviour is not typical of a class-II aldolase. The enzyme has no requirement for thiol compounds either for stability or activity, added K+ ions have no effect, and the optimum pH for the cleavage activity is unusually high. The class-II enzymes from the prokaryote E. coli and the eukaryote yeast show no immunological identity. However, the similarity of their structures suggests that they have evolved from a common ancestor.

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