scholarly journals d-Xylose (d-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Purification and properties

1991 ◽  
Vol 277 (1) ◽  
pp. 255-261 ◽  
Author(s):  
C A Smith ◽  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Activation Energy ◽  
Soluble Protein ◽  
Protein Sequences ◽  
Glucose Isomerase ◽  
Competitive Inhibitor ◽  
Total Soluble Protein ◽  
Arrhenius Activation Energy ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Arthrobacter Strain

D-Xylose (D-glucose) isomerase was purified to homogeneity in yields of approx. 1 g/kg of wet cells from a strain of Arthrobacter that produces it as about 10% of total soluble protein. It is a tetramer of identical 43,114 Da subunits containing a preponderance of acidic residues and no cysteine. Partial protein sequences were determined as a step to gene cloning. It requires Mg2+, Co2+ or Mn2+ for activity, Mg2+ being best; Ca2+ is an inhibitor, competitive with Mg2+. It is a good D-glucose isomerase with kcat. 1200 min-1 at pH 8 at 60 degrees C, which is higher than that of any other enzyme of this class. L-Arabinose, D-ribose and D-lyxose are poor substrates, with kcat. 78, 31 and 3.7 min-1 respectively at pH 8 at 30 degrees C, compared with 533 min-1 for D-xylose. Xylitol is a true competitive inhibitor for D-xylose (Ki 0.3 mM), but D-sorbitol shows mixed inhibition (Ki 6.5 mM). For D-fructose the pH optimum at 60 degrees C is 8, and at pH 7 the Arrhenius activation energy is 75 kJ/mol over the range 30-70 degrees C.

β-Galactosidase from Bacillus stearothermophilus

1976 ◽  
Vol 22 (6) ◽  
pp. 817-825 ◽  
Author(s):  
Richard E. Goodman ◽  
Dennis M. Pederson
Keyword(s):  
Activation Energy ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Thermal Inactivation ◽  
Ph Dependence ◽  
Temperature Sensitive ◽  
Optimum Temperature ◽  
Arrhenius Activation Energy ◽  
Ph Optimum

Several strains of thermophilic aerobic spore-forming bacilli synthesize β-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The β-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 °C and the Arrhenius activation energy is about 24 kcal/mol below 47 °C and 16 kcal/mol above that temperature. At 55 °C the Km is 0.11 M for lactose and 9.8 × 10−3 M for o-nitrophenyl-β-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki = 2.5 × 10−3 M). It is relatively stable at 50 °C, losing only half of its activity after 20 days at this temperature. At 60 °C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 °C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.

scholarly journals The purification and properties of Escherichia coli methylglyoxal synthase

1972 ◽  
Vol 128 (2) ◽  
pp. 321-329 ◽  
Author(s):  
D. J. Hopper ◽  
R. A. Cooper
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Ph Optimum ◽  
E Coli ◽  
Atp Formation ◽  
Purification And Properties ◽  
K 12 ◽  
Methylglyoxal Synthase

1. Methylglyoxal synthase was purified over 1500-fold from glycerol-grown Escherichia coli K 12 strain CA 244. The purified enzyme was inactivated by heat or proteolysis, had a molecular weight of approx. 67000, a pH optimum of 7.5 and was specific for dihydroxyacetone phosphate with Km 0.47mm. 2. The possibility that a Schiff-base intermediate was involved in the reaction mechanism was investigated but not confirmed. 3. The purified enzyme lost activity, especially at low temperature, but could be stabilized by Pi. Two binding sites for Pi may be present on the enzyme. Of other compounds tested only the substrate, dihydroxyacetone phosphate, and bovine serum albumin showed any significant stabilizing effect. 4. Phosphoenolpyruvate, 3-phosphoglycerate, PPi and Pi were potent inhibitors of the enzyme. Kinetic experiments showed that PPi was apparently a simple competitive inhibitor, but inhibition by the other compounds was more complex. In the presence of Pi the enzyme behaved co-operatively, with at least three binding sites for dihydroxyacetone phosphate. 5. It is proposed that methylglyoxal synthase and glyceraldehyde 3-phosphate dehydrogenase play important roles in the catabolism of the triose phosphates in E. coli. Channelling of dihydroxyacetone phosphate via methylglyoxal would not be linked to ATP formation and could be involved in the uncoupling of catabolism and anabolism.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

Biosynthesis of the Diguanosine Nucleotides. I. Purification and Properties of an Enzyme from Yolk Platelets of Brine Shrimp Embryos

1974 ◽  
Vol 52 (3) ◽  
pp. 231-240 ◽  
Author(s):  
A. H. Warner ◽  
P. C. Beers ◽  
F. L. Huang
Keyword(s):  
Molecular Weight ◽  
Brine Shrimp ◽  
Artemia Salina ◽  
Temperature Optimum ◽  
Small Molecular Weight ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Purification And Properties

An enzyme that catalyzes the synthesis of P1P4-diguanosine 5′-tetraphosphate (Gp4G) has been isolated and purified from yolk platelets of encysted embryos of the brine shrimp, Artemia salina. The enzyme GTP:GTP guanylyltransferase (Gp4G synthetase) utilizes GTP as substrate, has a pH optimum of 5.9–6.0, a temperature optimum of 40–42 °C, and requires Mg2+ and dithiothreitol for optimal activity. The synthesis of Gp4G is inhibited markedly by pyrophosphate, whereas orthophosphate has no effect on the reaction. In the presence of GDP the enzyme also catalyzes the synthesis of P1,P3-diguanosine 5′-triphosphate (Gp3G), but the rate of synthesis is low compared with Gp4G synthesis and dependent upon other small molecular weight components of yolk platelets.

Tissue culture and genetic analysis of somaclonal variations of Solanum melongena L. cv. Nirrala

2014 ◽  
Vol 9 (12) ◽  
pp. 1182-1195
Author(s):  
Samar Naseer ◽  
Tariq Mahmood
Keyword(s):  
Tissue Culture ◽  
Acetic Acid ◽  
Soluble Protein ◽  
Solanum Melongena ◽  
Total Soluble Protein ◽  
Naphthalene Acetic Acid ◽  
Fresh Tissue ◽  
Somaclonal Variations ◽  
Random Amplification ◽  
Plant Grown

AbstractThe present study was designed to analyze genetically somaclonal variants using biochemical and molecular markers. Efficient tissue culture protocol for Solanum melongena L. cv. Nirrala was developed. Maximum callus induction (100%) was observed for Murashige and Skoog (MS) media supplemented with 2.0 mg L−1 naphthalene acetic acid +0.5 mg L−1 6-benzylaminopurine; and nodal explants gave best callusing response (88.8%) as compared to internodes (88.3%) and leaves (87.7%). The best shooting was induced on nodal and internodal callus in the presence of 2.0 mg L−1 6-benzylaminopurine. Total soluble protein content of callus and regenerated variant plants was estimated for biochemical analysis, and largest amount of soluble protein was found in callus (6.54 mg g−1 fresh tissue) followed by variant plant grown on 2.0 mg L−1 6-benzylaminopurine (5.96 mg g−1 fresh tissue). Random amplification of polymorphic DNA technique was done with five decamer primers (OPC1-OPC5) and maximum polymorphism was detected by OPC 2 (26.99%) among all samples, whereas nodal callus on media containing 1.0 mg L−1 naphthalene acetic acid +1.0 mg L−1 6-benzylaminopurine showed highest polymorphism producing 22 bands, out of which 8 bands were polymorphic. The study shows that this marker system can provide better evaluation of genetic variation induced by tissue culture.

scholarly journals Biochemical changes in cultured foetal rat liver explants

1975 ◽  
Vol 150 (2) ◽  
pp. 269-273 ◽  
Author(s):  
P C MacDonnel ◽  
E Ryder ◽  
J A Delvalle ◽  
O Greengard
Keyword(s):  
Soluble Protein ◽  
Cell Damage ◽  
Tyrosine Aminotransferase ◽  
Culture Conditions ◽  
Total Soluble Protein ◽  
Mitochondrial Enzymes ◽  
Stage Of Development ◽  
Foetal Rat ◽  
Total Soluble Protein Content ◽  
Do So

Liver explants from 20-day-old foetuses cultured for 48h in the absence of serum released 70% of their total soluble protein content into the medium. In the presence of serum this loss still amounted to 60%. The concentration of total particulate protein remained unchanged but there was some translocation of mitochondrial enzymes to the cytosol, and enzymes expected to increase during this stage of development failed to do so. The addition of cortisol plus glucagon (to serum-containing media) did not decrease the loss of total soluble protein from the explants but induced considerable tyrosine aminotransferase activity which was not released into the medium. The observations suggest that under the usual culture conditions a minority of the cells retain their functional integrity. The extent of deterioration, not reflected in histologically visible necrosis or cell damage, can be conveniently monitored by the malate dehydrogenase activity released to the medium.

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