scholarly journals Production and Partial Characterization of α-Amylase Enzyme from Marine Actinomycetes

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Mohamed H. Al-Agamy ◽  
Mohammad R. Alhuzani ◽  
Mahmoud S. Kelany ◽  
Moaz M. Hamed
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Culture Conditions ◽  
Activity Measurement ◽  
Terrestrial Environment ◽  
Box Behnken Design ◽  
Thermal Stability Of

Amylase producing actinobacteria were isolated and characterized from terrestrial environment. There are a limited number of reports investigating the marine environment; hence, in the present study, four marine enzymes were tested for their amylase production ability. On starch agar plates, the Streptomyces rochei strain showed a higher hydrolytic zone (24 mm) than the other isolates. Growth under optimized culture conditions using Plackett-Burman’s experimental design led to a 1.7, 9.8, 7.7, and 3.12-fold increase for the isolates S. griseorubens, S. rochei, S. parvus, and Streptomyces sp., respectively, in the specific activity measurement. When applying the Box-Behnken design on S. rochei using the most significant parameters (starch, K2HPO4, pH, and temperature), there was a 12.22-fold increase in the specific activity measurement 7.37 U/mg. The α-amylase was partially purified, and its molecular weight was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. α-Amylase was particularly active at pH 6 and 65°C. The purified enzyme was most active at 65°C and pH 6, thermal stability of 70°C for 40 min, and salt concentration of 1 M with Km and Vmax of 6.58 mg/ml and 21.93 μmol/ml/min, respectively. The α-amylase was improved by adding Cu+2, Zn+2, and Fe+2 (152.21%, 207.24%, and 111.89%). Increased production of α-amylase enzyme by S. rochei KR108310 leads to production of significant industrial products.

scholarly journals Production optimization using Plackett-Burman and Box-Behnken designs with partial characterization of amylase from marine actinomycetes

2021 ◽  
Author(s):  
Sarah I Bukhari ◽  
Mohamed H Al-Agamy ◽  
Mahmoud S Kelany ◽  
Mohammad R Al Hazani ◽  
Moaz M Hamed
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Fermentation Medium ◽  
Culture Conditions ◽  
Production Optimization ◽  
Activity Measurement

Abstract Amylase is an industrial enzyme that is used in the food and biofuel industries. We screened four actinomycetes strains for amylase biosynthesis. The Streptomyces rochei strain had a larger hydrolytic zone (24 mm) on starch agar plates, than the other isolates. Plackett-Burman’s experimental design was implemented to optimize the conditions for amylase production by the selected strains. Growth under optimized culture conditions led to 1.7, 9.8, 7.7, and 3.12 -fold increases for the isolates S. griseorubens, S. rochei, S. parvus, and Streptomyces sp., respectively, in the specific activity measurement in comparison with growth under primary conditions. When applying the Box-Behnken design on S. rochei using the most significant parameters starch, K2HPO4, pH, and temperature, there was a 12.22-fold increase in the specific activity measurement: 7.37 U/mg. The optimal fermentation medium formula was kept at 30.6°C for seven days. The amylase from S. rochei was partially purified, and its molecular weight was determined using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight was found to be 45, 43, and 53 kDa. Amylase was particularly active at pH 6 and 65°C. The purified enzyme was most active at 65°C and a pH of 6, thermal stability of 70°C for 40 min and salt concentration of 1 M with a Km and Vmax of 6.58 mg/ml and 21.93 mg/ml/min, respectively. The amylase improved by adding Cu + 2, Zn + 2, and Fe + 2 (152.21%, 207.24%, and 111.89%). Increased production of amylase enzyme by Streptomyces rochei KR108310 attracts the production of industrially significant products.

scholarly journals Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium byLeuconostoc mesenteroides NRRL B-1299

1998 ◽  
Vol 64 (4) ◽  
pp. 1298-1302 ◽  
Author(s):  
Marguerite Dols ◽  
M. Remaud-Simeon ◽  
R. M. Willemot ◽  
M. Vignon ◽  
P. Monsan
Keyword(s):  
Gel Electrophoresis ◽  
Leuconostoc Mesenteroides ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Traditional Culture ◽  
Culture Conditions ◽  
Acceptor Reaction ◽  
Sucrose Medium ◽  
Molecular Masses

ABSTRACT When grown in glucose or fructose medium in the absence of sucrose,Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% α(1→2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of α(1→6) linkages.

scholarly journals Purification and characterization of 3-dehydroquinase from Escherichia coli

1986 ◽  
Vol 239 (3) ◽  
pp. 699-704 ◽  
Author(s):  
S Chaudhuri ◽  
J M Lambert ◽  
L A McColl ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.

Purification and preliminary characterization of 2-monoacylglycerol acyltransferase from rat intestinal villus cells

1985 ◽  
Vol 63 (5) ◽  
pp. 341-347 ◽  
Author(s):  
F. Manganaro ◽  
A. Kuksis
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cell Protein ◽  
Racemic Mixture ◽  
Small Intestinal Mucosa ◽  
Small Intestinal ◽  
Monoacylglycerol Acyltransferase

We have purified the monoacylglycerol acyltransferase from rat small intestinal mucosa to homogeneity by a combination of hydrophobic absorption, guanidine dissociation, and gel filtration. The purified enzyme gives a single band of 37 000 daltons on sodium dodecyl sulphate – polyacrylamide gel electrophoresis. The enzyme has a specific activity of about 5900 nmol/mg per hour and represents 0.12% of total cell protein, corresponding to about a 600-fold purification. The enzyme does not acylate diacylglycerols to triacylglycerols, which is consistent with the separate physical existence of the mono- and di-acylglycerol acyltransferases. The enzyme acylates the 2-monoacylglycerols to yield an essentially racemic mixture of diacylglycerols. It does not acylate glycerol 3-phosphate.

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

scholarly journals Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

1984 ◽  
Vol 219 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A A Davies ◽  
N M Wigglesworth ◽  
D Allan ◽  
R J Owens ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Insoluble Fraction ◽  
Insoluble Residue ◽  
Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

scholarly journals Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

2010 ◽  
Vol 192 (9) ◽  
pp. 2407-2413 ◽  
Author(s):  
Jacalyn M. Green ◽  
Ryan Hollandsworth ◽  
Lenore Pitstick ◽  
Eric L. Carter
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Size Exclusion ◽  
Breakdown Product ◽  
Purification And Characterization ◽  
Sds Page

ABSTRACT The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a Km value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min−1 mg−1. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

scholarly journals Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

2001 ◽  
Vol 67 (8) ◽  
pp. 3746-3749 ◽  
Author(s):  
Yu-Huan Liu ◽  
Ying-Cheng Chung ◽  
Ya Xiong
Keyword(s):  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Degrading Enzyme

ABSTRACT A dimethoate-degrading enzyme from Aspergillus nigerZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu2+. The Michaelis constant (K m ) andV max for dimethoate were 1.25 mM and 292 μmol min−1 mg of protein−1, respectively.

scholarly journals Characterization of the d-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) from Bifidobacterium lactis

2001 ◽  
Vol 183 (9) ◽  
pp. 2929-2936 ◽  
Author(s):  
Leo Meile ◽  
Lukas M. Rohr ◽  
Thomas A. Geissmann ◽  
Monique Herensperger ◽  
Michael Teuber
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Guanosine Monophosphate ◽  
Bifidobacterium Lactis ◽  
Terminal Amino ◽  
Cloned Gene

ABSTRACT A d-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (Xfp) from the probioticBifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with d-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme.Km values for d-xylulose 5-phosphate and d-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (namedxfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta andguaA, were localized adjacent to xfp on theB. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes inMycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.

scholarly journals Purification and characterization of β-N-acetylglucos-aminidase from Grifola frondosa

BioResources ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. 7234-7248
Author(s):  
Yi-Cheng Wang ◽  
Te-Sheng Lien ◽  
Nan-Yin Chen ◽  
Tai-Hao Hsu
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrolysis Product ◽  
Chitinase Activity ◽  
Grifola Frondosa ◽  
Purification And Characterization ◽  
Carbohydrate Utilization ◽  
Highly Active

Using commercial API-ZYM screening kits, highly active α-glucosidase, β-glucosidase, and β-N-acetylglucosaminidase were found in Grifola frondosa, having potential for carbohydrate utilization. Of these, β-N-acetylglucosaminidase, which converts chitin to N-acetylglucosamine, was purified and characterized. The recovery was 24.5%, and the purified enzyme had a specific activity 0.67 U/mg protein. Chitinase activity was confirmed by zymogram analysis. The enzyme was also shown to be β-N-acetylglucosaminidase, as N-acetylglucosamine was the main hydrolysis product from colloidal chitin. Thus, the molecule was named NAG38, to indicate β-N-acetylglucosaminidase activity and a molecular weight of 38 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity was optimal at pH 7.0 and 50 °C, with Km and Vmax values of 0.112 mM and 0.570 μmol/min/mg protein against p-nitrophenyl N-acetyl-β-D-glucosaminide. The bioactivity was inhibited by Hg2+, Ag+, Mg2+, Zn2+, Ca2+, and Mn2+, with residual enzyme bioactivity only 11.1% after incubation in Hg2+, but was not substantially inhibited by Ba2+, K+, and Na+.

Sign in / Sign up

Export Citation Format

Share Document