scholarly journals An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga

1991 ◽  
Vol 277 (2) ◽  
pp. 413-417 ◽  
Author(s):  
H D Simpson ◽  
U R Haufler ◽  
R M Daniel
Keyword(s):  
Anion Exchange ◽  
Porous Glass ◽  
Half Life ◽  
Culture Supernatant ◽  
Gel Filtration ◽  
Glass Beads ◽  
Thermostable Xylanase ◽  
Sds Page ◽  
Optimum Ph

Endo-1,4-beta-xylanase (EC 3.2.1.8) was isolated from the culture supernatant of Thermotoga sp. strain FjSS3-B.1, an extremely thermophilic anaerobic eubacterium which grows optimally at 80 degrees C. Activity was purified 165-fold by anion-exchange and hydroxyapatite chromatography. The enzyme has an Mr of 31,000 as determined by SDS/PAGE and 35,000 by analytical gel filtration. The optima for activity and stability for purified xylanase were between pH 5.0 and 5.5. At pH 5.5, which is the optimum pH for thermostability, t1/2 (95 degrees C) is 90 min. The thermostability was improved by immobilization of the xylanase on to porous glass beads; t1/2 (105 degrees C) is 10 min. Several additives, such as sorbitol and xylan, were also found to increase the thermostability. At 130 degrees C, the half-life of immobilized xylanase in the presence of 90% sorbitol was 1.3 min. At 130 degrees C in molten sorbitol half of the enzyme denatured rapidly, but the remainder appeared to have a half-life of about 60 min.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals Purification and properties of malonyl-CoA synthetase from Rhizobium japonicum

1991 ◽  
Vol 273 (3) ◽  
pp. 511-516 ◽  
Author(s):  
Y S Kim ◽  
H Z Chae
Keyword(s):  
Carbon Sources ◽  
Gel Filtration ◽  
Rhizobium Japonicum ◽  
Purification And Properties ◽  
Malonyl Coa ◽  
Sds Page ◽  
Optimum Ph ◽  
Soybean Nodule ◽  
Terminal Amino ◽  
Single Polypeptide

A novel malonyl-CoA synthetase was found in Rhizobium japonicum bacteroid of the soybean nodule. The levels of the enzyme in the free-living cells grown on a variety of carbon sources including glucose were similar, indicating that this enzyme is not inducible. The malonyl-CoA synthetase from glucose-grown Rhizobium japonicum was purified to homogeneity. The Mr of the enzyme was determined to be 58,000 by gel filtration on a Sephacryl S-300 and by SDS/PAGE respectively, indicating a single polypeptide enzyme. N-Terminal amino acid of the enzyme was methionine but the enzyme preparation contained about 40% de-methionylated protein. The enzyme catalyses the formation of malonyl-CoA, AMP and PPi directly from malonate, CoA and ATP in the presence of Mg2+. High substrate specificity on malonate and ATP was revealed, but Mn2+ could be substituted for Mg2+ without any difference in activity. Optimum pH was 7.9. Kinetic constants, Km and Vmax, for malonate, CoA and ATP were 200 microM and 21.3 mumol/min per mg, 87 microM and 41.7 mumol/min per mg, and 33.3 microM and 29.4 mumol/min per mg respectively. Succinate inhibited the enzyme noncompetitively, whereas AMP and ADP inhibited competitively. Diethylpyrocarbonate and pyridoxal-5′-phosphate severely inhibited the enzyme, but iodoacetamide, p-chloromercuriphenylsulphonate, N-acetylimidazole and phenylglyoxal did not.

scholarly journals Peroxidase from Coleus Forskohlii: Purification and Biochemical Characterization

2020 ◽  
Vol 5 (1) ◽  
pp. 9-20
Author(s):  
Yaaser Q. Almulaiky ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Ion Exchange ◽  
Peroxidase Activity ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Coleus Forskohlii ◽  
Sds Page ◽  
Optimum Ph

In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.

Production and biochemical and molecular characterization of a keratinolytic serine protease from chicken feather-degradingBacillus licheniformisRPk

2009 ◽  
Vol 55 (4) ◽  
pp. 427-436 ◽  
Author(s):  
Nahed Fakhfakh ◽  
Safia Kanoun ◽  
Laila Manni ◽  
Moncef Nasri
Keyword(s):  
Serine Protease ◽  
Gel Filtration ◽  
Chicken Feather ◽  
Feather Degradation ◽  
Subtilisin Carlsberg ◽  
Degrading Bacterium ◽  
Step Procedure ◽  
Sds Page ◽  
Optimum Ph ◽  
Optimum Ph And Temperature

A novel feather-degrading bacterium was isolated from a polluted river and identified as Bacillus licheniformis RPk. The isolate exhibited high proteinase production when grown in chicken-feather media. Complete feather degradation was achieved during cultivation. Maximum protease activity (4150 U/mL with casein as a substrate and 37.35 U/mL with keratin as a substrate) was obtained when the strain was grown in a medium containing 7.5 g/L chicken feathers, 2 g/L yeast extract, 0.5 g/L NaCl, 0.1 g/L MgSO4·7H2O, 0.7 g/L KH2PO4, and 1.4 g/L K2HPO4for 48 h with agitation of 200 rev/min at 37 °C. The major protease produced by B. licheniformis RPk was purified to homogeneity by a 3-step procedure. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS–PAGE and gel filtration. The optimum pH and temperature for the caseinolytic activity were around 11.0 and 65 °C, respectively. The optimum pH and temperature for the keratinolytic activity were 9.0 and 60 °C, respectively. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, which suggests that the purified enzyme is a serine protease. The thermostability of the enzyme was considerably enhanced in the presence of Ca2+at temperatures >50 °C. The kerRP gene, which encodes the keratinolytic protease, was isolated, and its DNA sequence was determined. The deduced amino acid sequence revealed that the keratinase KerRP differs from KerA of B. licheniformis PWD-1, subtilisin Carlsberg, and keratinase of B. licheniformis by 2, 4, and 62 amino acids, respectively.

Purification and partial biochemical–genetic characterization of trehalose 6-phosphate synthase from muscles of adult femaleAscaris suum

2012 ◽  
Vol 87 (2) ◽  
pp. 212-221 ◽  
Author(s):  
M. Dmitryjuk ◽  
M. Dopieralska ◽  
E. Łopieńska-Biernat ◽  
R.J. Frączek
Keyword(s):  
Genetic Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sds Page ◽  
Optimum Ph ◽  
Genes Encoding ◽  
Ammonium Sulphate Fractionation ◽  
Biochemical Genetic

AbstractTrehalose 6-phosphate (T6P) synthase (TPS;EC2.4.1.15) was isolated from muscles ofAscaris suumby ammonium sulphate fractionation, ion-exchange DEAE SEPHACELTManion exchanger column chromatography and Sepharose 6B gel filtration. On sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), 265-fold purified TPS exhibited a molecular weight of 66 kDa. The optimum pH and temperature of the purified enzyme were 3.8–4.2 and 35°C, respectively. The isoelectric point (pI) of TPS was pH 5.4. The studied TPS was not absolutely substrate specific. Besides glucose 6-phosphate, the enzyme was able to use fructose 6-phosphate as an acceptor of glucose. TPS was activated by 10 mMMgCl2, 10 mMCaCl2and 10 mMNaCl. In addition, it was inhibited by ethylenediaminetetra-acetic acid (EDTA), KCl, FeCl3and ZnCl2. Two genes encoding TPS were isolated and sequenced from muscles of the parasite. Complete coding sequences fortps1(JF412033.2) andtps2(JF412034.2) were 3917 bp and 3976 bp, respectively. Translation products (AEX60788.1 and AEX60787.1) showed expression to the glucosyltransferase-GTB-type superfamily.

scholarly journals Thermostable cellobiohydrolase from the thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1. Purification and properties

1991 ◽  
Vol 277 (3) ◽  
pp. 887-890 ◽  
Author(s):  
L D Ruttersmith ◽  
R M Daniel
Keyword(s):  
Filter Paper ◽  
Half Life ◽  
Culture Supernatant ◽  
Maximal Activity ◽  
Amorphous Cellulose ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Sds Page ◽  
Thermostable Cellulase ◽  
Hydrolysis Of

Exo-1,4-beta-cellobiohydrolase (EC 3.2.1.91) was isolated from the culture supernatant of Thermotoga sp. strain FjSS3-B.1, an extremely thermophilic eubacterium that grows optimally at 80 degrees C. The enzyme was purified to homogeneity as determined by SDS/PAGE and has an Mr of 36,000. The enzyme is the most thermostable cellulase reported to date, with a half-life at 108 degrees C of 70 min in buffer. In a 40 min assay, maximal activity was recorded at 105 degrees C. Cellobiohydrolase from strain FjSS3-B.1 is active against amorphous cellulose and CM-cellulose but only effects limited hydrolysis of filter paper or Sigmacell 20. The only product identified by h.p.l.c. is the disaccharide cellobiose. The enzyme has a pH optimum around neutral and is stabilized by the presence of 0.8 M-NaCl.

scholarly journals A highly inducible β-galactosidase from Enterobacter sp.

2020 ◽  
Vol 85 (5) ◽  
pp. 609-622
Author(s):  
Bestoon Shaikhan ◽  
Kemal Güven ◽  
Fatma Bekler ◽  
Ömer Acer ◽  
Reyhan Güven
Keyword(s):  
Gel Filtration ◽  
Gel Permeation Chromatography ◽  
Inhibitory Effect ◽  
Native Page ◽  
Environment And Health ◽  
Low Concentrations ◽  
Sds Page ◽  
Gel Permeation ◽  
Optimum Ph ◽  
Strong Inhibitory Effect

Enterobacter sp. 3TP2A isolated from a petroleum station was found to produce a novel, highly inducible mesophilic intracellular ?-galactosidase in the presence of lactose up to 76.5 U mg-1. The enzyme was purified to 17.3- -fold after gel permeation chromatography with a yield of approximately 11 %. The optimum pH and temperature values of the purified enzyme were found to be 8.0?9.0 and 35 ?C, respectively. The molecular weight of the enzyme was approx. 60 kDa with a single band by both SDS-PAGE and native-PAGE, and estimated by gel filtration chromatography. The enzyme was inhibited by Zn2+ and EDTA, while Cu2+ had strong inhibitory effect even at low concentrations. Activation by Mg2+ and inhibition by EDTA show that the enzyme is metal- -dependent or a metalloenzyme. The enzyme was slightly activated by 2-mercaptoethanol, while slightly inhibited by iodoacetamide. On the other hand, PCMB inhibited the enzymatic activity to a great extent, whereas it was completely inhibited by N-ethylmaleimide. The Vmax and Km values were calculated as 0.701 ?mol min-1 and 0.104 mM, respectively. The results indicated that the ?-galactosidase Enterobacter sp. 3TP2A might well be a good candidate for use in biotechnology, particularly in the area of environment and health.

scholarly journals Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli

1991 ◽  
Vol 276 (2) ◽  
pp. 541-546 ◽  
Author(s):  
K Aisaka ◽  
A Igarashi ◽  
K Yamaguchi ◽  
T Uwajima
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Genetically Engineered ◽  
E Coli ◽  
Sds Page ◽  
Optimum Ph ◽  
Molecular Masses ◽  
Related Compounds ◽  
Optimum Ph And Temperature

N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.

scholarly journals Purification, Characterization of L-Methioninase from Candida tropicalis, and Its Application as an Anticancer

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Mohsen Helmy Selim ◽  
El-Zahraa Karm Eldin ◽  
Moataza Mahmoud Saad ◽  
El-Sayed Eliwa Mostafa ◽  
Yosrea Hassan Shetia ◽  
...  
Keyword(s):  
Candida Tropicalis ◽  
Anticancer Agent ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumor Cell Lines ◽  
Sds Page ◽  
Optimum Ph ◽  
Ph Range

The aim of the present study is to purify L-methioninase from Candida tropicalis 34.19-fold with 27.98% recovery after ion exchange chromatography followed by gel filtration. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular weight of 46 KDa. Its optimum temperature was 45 to 55 and thermal stability was 55°C for 15 min. The enzyme had optimum pH at 6.5 and stability at a pH range of 5.5 to 7.0 for 24 hr. The maximum activity was observed with substrate concentration of 30 µM and Km was 0.5 mM. The enzyme was strongly inhibited by Cd+2 and Cu+2 while it was enhanced by Na+, Ni+2, and Mg+2 at 10 mM while Ca+2 had slight activation at 20 mM. In addition, the potential application of the L-methioninase as an anticancer agent against various types of tumor cell lines is discussed.

scholarly journals Isolasi dan Karakterisasi Inulinase dari Aspergillus niger Gmn11.1 Galur Lokal

2012 ◽  
Vol 11 (1) ◽  
pp. 19 ◽  
Author(s):  
Saryono Saryono
Keyword(s):  
Molecular Weight ◽  
Aspergillus Niger ◽  
Incubation Time ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Salt Precipitation ◽  
Sds Page ◽  
Optimum Ph ◽  
Relative Molecular Weight ◽  
Optimum Ph And Temperature

Inulin is a naturally potential polysaccharide used to produced fructose and fructooligosaccharide. Inulinaseknown also as ß-fructosidase can hydrolise inulin to fructose or fructooligosaccharide. Inulinase production fromAspergillus niger Gmn11.1 isolated from dahlia tubers is conducted using medium containing 1% inulin and 0,2%yeast extract. The crude enzyme (filtrate culture) is purified by means of ammonium sulphate salt precipitation,followed by Sephadex G25 gel filtration column chromatography and DEAE cellulose anion exchanger columnchromatography. The result indicated that the enzyme had optimum pH and temperature of 4,6 and 450C, respectivelywith incubation time of 15 hours. The Km and Vmaxs values obtained from this experiment are 20 mg/ml and 0,769mg/ml/hours, respectively. Whereas the relative molecular weight of inulinase was monitored by SDS PAGE is 63KDa.

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