scholarly journals Partial Purification and Characterization of a Heat Stable α-Amylase from a Thermophilic Actinobacteria, Streptomyces sp. MSC702

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Renu Singh ◽  
Vijay Kumar ◽  
Vishal Kapoor
Keyword(s):  
Enzyme Activity ◽  
Biochemical Characterization ◽  
Enzyme Stability ◽  
Potato Starch ◽  
Tween 80 ◽  
Partial Purification ◽  
Amylolytic Enzyme ◽  
Operational Conditions ◽  
Heat Stable

A partial purification and biochemical characterization of the α-amylase from Streptomyces sp. MSC702 were carried out in this study. The optimum operational conditions for enzyme substrate reaction for amylolytic enzyme activity from the strain were evaluated. The optimum pH, temperature, and incubation period for assaying the enzyme were observed to be 5.0, 55°C, and 30 min, respectively. The extracellular extract was concentrated using ammonium sulfate precipitation. It was stable in the presence of metal ions (5 mM) such as K+, Co2+, and Mo2+, whereas Pb2+, Mn2+, Mg2+, Cu2+, Zn2+, Ba2+, Ca2+, Hg2+, Sn2+, Cr3+, Al3+, Ag+, and Fe2+ were found to have inhibitory effects. The enzyme activity was also unstable in the presence of 1% Triton X-100, 1% Tween 80, 5 mM sodium lauryl sulphate, 1% glycerol, 5 mM EDTA, and 5 mM denaturant urea. At temperature 60°C and pH 5.0, the enzyme stability was maximum. α-amylase retained 100% and 34.18% stability for 1 h and 4 h, respectively, at 60°C (pH 7.0). The enzyme exhibited a half-life of 195 min at 60°C temperature. The analysis of kinetic showed that the enzyme has Km of 2.4 mg/mL and Vmax of 21853.0 μmol/min/mg for soluble potato starch. The results indicate that the enzyme reflects their potentiality towards industrial utilization.

scholarly journals Partial purification and biochemical characterization of amylase from Aeromonas caviaeNK1isolated from Industrial waste of India

2016 ◽  
Vol 7 (1) ◽  
pp. 85
Author(s):  
Kalyani Khanra ◽  
Indranil Choudhuri ◽  
Sudipta Panja ◽  
Nandan Bhattacharyya
Keyword(s):  
Enzyme Activity ◽  
Industrial Waste ◽  
Biochemical Characterization ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation ◽  
Operational Conditions ◽  
Industrial Utilization ◽  
Optimum Ph ◽  
Optimum Ph And Temperature

<p class="jbls">A partial purification and biochemical characterization of theamylase from <em>Aeromonascaviae</em>NK1 were carried out in this study. The extracellular extract was concentrated using ammonium sulfate precipitation and optimum operational conditions for the enzyme activity from the strain were evaluated. The optimum pH and temperature were observed 11.5 and37<sup>0</sup>C respectively. Ca<sup>2+</sup>, Mn<sup>2+</sup>, Mg<sup>2+</sup>, Zn<sup>2+</sup>, Pb<sup>2+</sup>, Co<sup>2+</sup> were found to have effect on amylase activity.Furthermore, the analysis of kinetic showed that the enzyme has 𝐾<sub>𝑚</sub> of 2.4mg/mL and 𝑉<sub>max</sub> of 21853.0𝜇mol/min/mg for starch. The results indicate that the enzyme reflects their potentiality towards industrial utilization.</p>

scholarly journals Partial purification and biochemical characterization of a new highly acidic NYSO laccase from Alcaligenes faecalis

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Soad A. Abdelgalil ◽  
Ahmad R. Attia ◽  
Reyed M. Reyed ◽  
Nadia A. Soliman
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl ◽  
Alcaligenes Faecalis ◽  
Maximum Activity ◽  
Sodium Oxalate ◽  
Partial Purification ◽  
Bromophenol Blue ◽  
Bromocresol Purple ◽  
Laccase Enzyme

Abstract Background Due to the multitude industrial applications of ligninolytic enzymes, their demands are increasing. Partial purification and intensive characterization of contemporary highly acidic laccase enzyme produced by an Egyptian local isolate designated Alcaligenes faecalis NYSO were studied in the present investigation. Results Alcaligenes faecalis NYSO laccase has been partially purified and intensively biochemically characterized. It was noticed that 40–60% ammonium sulfate saturation showed maximum activity. A protein band with an apparent molecular mass of ~ 50 kDa related to NYSO laccase was identified through SDS-PAGE and zymography. The partially purified enzyme exhibited maximum activity at 55 °C and pH suboptimal (2.5–5.0). Remarkable activation for enzyme activity was recognized after 10-min exposure to temperatures (T) 50, 60, and 70 °C; time elongation caused inactivation, where ~ 50% of activity was lost after a 7-h exposure to 60 °C. Some metal ions Cu2+, Zn2+, Co2+, Ni2+, Mn2+, Cd2+, Cr2+, and Mg2+ caused strong stimulation for enzyme activity, but Fe2+ and Hg2+ reduced the activity. One millimolar of chelating agents [ethylenediamine tetraacetic acid (EDTA), sodium citrate, and sodium oxalate] caused strong activation for enzyme activity. Sodium dodecyl sulfate (SDS), cysteine-HCl, dithiothreitol (DTT), β-mercaptoethanol, thioglycolic acid, and sodium azide caused strong inhibition for NYSO laccase activity even at low concentration. One millimolar of urea, imidazole, kojic acid, phenylmethylsulfonyl fluoride (PMSF), H2O2, and Triton X-100 caused activation. The partially purified NYSO laccase had decolorization activity towards different dyes such as congo red, crystal violet, methylene blue, fast green, basic fuchsin, bromophenol blue, malachite green, bromocresol purple eriochrome black T, and Coomassie Brilliant Blue R-250 with various degree of degradation. Also, it had a vast range of substrate specificity including lignin, but with high affinity towards p-anisidine. Conclusion The promising properties of the newly studied laccase enzyme from Alcaligenes faecalis NYSO strain would support several industries such as textile, food, and paper and open the possibility for commercial use in water treatment. It will also open the door to new applications due to its ligninolytic properties in the near future.

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 Isolation and characterization of bleomycin-resistant clones of CHO cells

1979 ◽  
Vol 34 (3) ◽  
pp. 269-279 ◽  
Author(s):  
Sylvia Brabbs ◽  
J. R. Warr
Keyword(s):  
Enzyme Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Tween 80 ◽  
The Other ◽  
Resistant Clone ◽  
Ovary Cells ◽  
Isolation And Characterization ◽  
Significant Difference

SUMMARYFive clones of Chinese hamster ovary cells with increased resistance to bleomycin have been isolated following ethylmethanesulphonate mutagenesis. Resistance was stable in three of the clones, but unstable in the other two. One of the stably resistant clones was cross resistant to unrelated drugs, and in contrast to the parental cells, its response to bleomycin was potentiated by tween 80. These two observations suggested a membrane alteration in the resistant clone. There was no significant difference in bleomycin-inactivating enzyme activity between the parental and resistant clones.

scholarly journals Biochemical Characterization of Laboratory Mutants of Extended-Spectrum β-Lactamase TEM-60

2004 ◽  
Vol 48 (9) ◽  
pp. 3579-3582 ◽  
Author(s):  
Bibiana Caporale ◽  
Nicola Franceschini ◽  
Mariagrazia Perilli ◽  
Bernardetta Segatore ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Extended Spectrum

ABSTRACT Three mutants of the extended-spectrum β-lactamase TEM-60, the P51L, K104E, and S164R mutants, were constructed by site-directed mutagenesis. The kinetic parameters of the mutated enzymes and interactions of inhibitors were significantly different from those of TEM-60, revealing that the L51P mutation plays an important role in enzyme activity and stability in the TEM-60 background.

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 Biochemical Characterization of Cellulase From Bacillus subtilis Strain and its Effect on Digestibility and Structural Modifications of Lignocellulose Rich Biomass

2021 ◽  
Vol 9 ◽  
Author(s):  
Waseem Ayoub Malik ◽  
Saleem Javed
Keyword(s):  
Bacillus Subtilis ◽  
Biochemical Characterization ◽  
Biomass Conversion ◽  
Industrial Applications ◽  
Partial Purification ◽  
Subtilis Strain ◽  
Complex Nature ◽  
Structural Modifications ◽  
Optimum Activity

Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. The present study reports the partial purification and characterization of cellulase from Bacillus subtilis CD001 and its application in biomass saccharification. Out of four different substrates, carboxymethyl cellulose, when amended as fermentation substrate, induced the highest cellulase production from B. subtilis CD001. The optimum activity of CMCase, FPase, and amylase was 2.4 U/ml, 1.5 U/ml, and 1.45 U/ml, respectively. The enzyme was partially purified by (NH4)2SO4 precipitation and sequenced through LC-MS/MS. The cellulase was found to be approximately 55 kDa by SDS-PAGE and capable of hydrolyzing cellulose, as confirmed by zymogram analysis. The enzyme was assigned an accession number AOR98335.1 and displayed 46% sequence homology with 14 peptide-spectrum matches having 12 unique peptide sequences. Characterization of the enzyme revealed it to be an acidothermophilic cellulase, having an optimum activity at pH 5 and a temperature of 60°C. Kinetic analysis of partially purified enzyme showed the Km and Vmax values of 0.996 mM and 1.647 U/ml, respectively. The enzyme activity was accelerated by ZnSO4, MnSO4, and MgSO4, whereas inhibited significantly by EDTA and moderately by β-mercaptoethanol and urea. Further, characterization of the enzyme saccharified sugarcane bagasse, wheat straw, and filter paper by SEM, ATR-FTIR, and XRD revealed efficient hydrolysis and structural modifications of cellulosic materials, indicating the potential industrial application of the B. subtilis CD001 cellulase. The findings demonstrated the potential suitability of cellulase from B. subtilis CD001 for use in current mainstream biomass conversion into fuels and other industrial processes.

Partial purification and characterization of a non-cyanobacterial cyanophycin synthetase from Acinetobacter calcoaceticus strain ADP1 with regard to substrate specificity, substrate affinity and binding to cyanophycin

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2599-2608 ◽  
Author(s):  
Martin Krehenbrink ◽  
Alexander Steinbüchel
Keyword(s):  
Aspartic Acid ◽  
Biochemical Characterization ◽  
Acinetobacter Calcoaceticus ◽  
Partial Purification ◽  
Atp Binding ◽  
Substrate Affinity ◽  
Two Dimensional Gel Electrophoresis ◽  
Atp Binding Site ◽  
Cyanophycin Synthetase

This study reports, for the first time, purification and biochemical characterization of a cyanophycin synthetase from a non-cyanobacterial strain. Cyanophycin synthetase of Acinetobacter calcoaceticus strain ADP1 was purified 69-fold from recombinant Escherichia coli by two chromatographic steps and one novel affinity step utilizing the Mg2+-dependent binding of the enzyme to cyanophycin. Unlike cyanobacterial cyanophycin synthetases characterized so far, the purified enzyme from A. calcoaceticus strain ADP1 did not accept lysine as an alternative substrate to arginine. The apparent K m-values for arginine (47 μM) and aspartic acid (240 μM) were similar to those of known cyanophycin synthetases from cyanobacteria, but this enzyme had a slightly higher affinity for aspartic acid. In addition, the two different ATP-binding sites of the enzyme were characterized independently of each other with respect to K m values for ATP. The ATP-binding site responsible for the addition of arginine was found to have a much higher affinity for ATP (38 μM) than that responsible for the addition of aspartate (210 mM). Furthermore, the binding of the enzyme to the two possible forms of cyanophycin granule polypeptide (CGP), CGP-Asp and CGP-Arg, was studied. While both forms bound around 30–40 % of the enzyme activity present under the assay conditions, binding was Mg2+-dependent in the case of CGP-Asp. Two-dimensional gel electrophoresis revealed that both forms of cyanophycin were equally abundant in cyanophycin-accumulating cells of A. calcoaceticus ADP1.

scholarly journals Production and Biochemical Characterization of a High Maltotetraose (G4) Producing Amylase fromPseudomonas stutzeriAS22

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Hana Maalej ◽  
Hanen Ben Ayed ◽  
Olfa Ghorbel-Bellaaj ◽  
Moncef Nasri ◽  
Noomen Hmidet
Keyword(s):  
Culture Medium ◽  
Biochemical Characterization ◽  
Amylase Activity ◽  
Potato Starch ◽  
Pseudomonas Stutzeri ◽  
Crude Enzyme Preparation ◽  
Amylase Production ◽  
Initial Ph ◽  
Very High

Amylase production and biochemical characterization of the crude enzyme preparation fromPseudomonas stutzeriAS22 were evaluated. The highestα-amylase production was achieved after 24 hours of incubation in a culture medium containing 10 g/L potato starch and 5 g/L yeast extract, with initial pH 8.0 at 30°C under continuous agitation at 200 rpm. The optimum temperature and pH for the crudeα-amylase activity were 60°C and 8.0, respectively. The effect of different salts was evaluated and it was found that bothα-amylase production and activity were Ca2+-dependent. The amylolytic preparation was found to catalyze exceptionally the formation of very high levels of maltotetraose from starch (98%, w/w) in the complete absence of glucose since the initial stages of starch hydrolysis (15 min) and hence would have a potential application in the manufacturing of maltotetraose syrups.

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