scholarly journals Two different isoschizomers of the type-II restriction endonuclease Taq I (T/CGA) within the same Thermus isolate: Tsp32 I, an enzyme with similar heat stability properties to the prototype enzyme Taq I, and Tsp32 II, a hyperthermostable isoschizomer of Taq I

1995 ◽  
Vol 312 (2) ◽  
pp. 505-510 ◽  
Author(s):  
S G Welch ◽  
R A D Williams
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Cleavage Site ◽  
Heat Stability ◽  
Hot Water ◽  
Heat Inactivation ◽  
Type Ii ◽  
Magnesium Ions ◽  
Water Springs

We have recently screened 112 separate isolates of the genus Thermus, collected from neutral and alkaline hot water springs on four continents, for the presence of the Type-II restriction endonuclease Taq I (T/CGA). One particular isolate from the Azores (strain 32) was found to contain high levels of a restriction endonuclease with the same recognition and cleavage site as Taq I. Initial studies revealed that the partially purified enzyme from strain 32 was considerably more resistant to heat inactivation than the prototype enzyme Taq I, being able to withstand temperatures at least 10 degrees C higher than Taq I, before showing evidence of heat inactivation. Subsequently it became clear that the partially purified extract from strain 32 contains two separate enzymes, both of which are isoschizomers of Taq I. One of the enzymes, Tsp32 I, has similar thermal stability characteristics to Taq I, whereas the second Taq I isoschizomer, Tsp32 II, found in the same Thermus isolate as Tsp32 I, is considerably more thermostable than Taq I, retaining full enzyme activity up to a temperature of 85 degrees C. Tsp32 I and Tsp32 II were further distinguished by virtue of their different requirements for magnesium ions.

scholarly journals Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus: Tsp4C I (ACN/GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgl I (GCCNNNN/NGGC)

1995 ◽  
Vol 309 (2) ◽  
pp. 595-599 ◽  
Author(s):  
S G Welch ◽  
R A D Williams
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Thermophilic Bacteria ◽  
Hot Water ◽  
Restriction Endonucleases ◽  
Type Ii ◽  
Endonuclease Activity ◽  
Lambda Phage ◽  
Recognition Sequence ◽  
Phosphodiester Bond

Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3′-OH overhang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) [Lee, Clanton and Chirikjiam (1979) Fed. Proc. 28, 294], generating fragments with a three base 3′-OH overhang. However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases.

Heat Stability of the Bile Salt-Stimulated Lipase in Human Milk Fortified with Sodium Taurocholate1

1988 ◽  
Vol 51 (4) ◽  
pp. 310-313 ◽  
Author(s):  
H. L. PAN ◽  
C. W. DILL ◽  
E. S. ALFORD ◽  
S. L. DILL ◽  
C. A. BAILEY ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Human Milk ◽  
Bile Salt ◽  
Lipase Activity ◽  
Sodium Taurocholate ◽  
Heat Stability ◽  
Heat Inactivation ◽  
Temperature Differential ◽  
Heat Stable ◽  
Control Samples

Time-temperature relationships for heat-inactivation of the bile salt-stimulated lipase activity were compared in whole human milk and in the same product fortified to 9 mM/ml with sodium taurocholate. Heat treatments were varied from 45 to 70°C for times ranging from 15s to 40 min. Enzyme activity was more heat stable in human milk fortified with taurocholate than in control samples. The temperature required for the onset of heat inactivation at 30-min holding time was increased from 45°C for control samples to 60°C following addition of taurocholate. A temperature differential of approximately 12°C was required in the fortified milks to produce inactivation equivalent to that observed in the control milks over the heating range studied.

scholarly journals The SalGI restriction endonuclease. Purification and properties

1982 ◽  
Vol 203 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Anthony Maxwell ◽  
Stephen E. Halford
Keyword(s):  
Enzyme Activity ◽  
Restriction Endonuclease ◽  
Final Stage ◽  
Enzyme Stability ◽  
Type Ii ◽  
Optimal Conditions ◽  
Purification And Properties

The type II restriction endonuclease SalGI has been purified to near homogeneity. At least 80% of the protein remaining after the final stage of the preparation is SalGI restriction endonuclease; no contaminating nucleases remain detectable. The principal form of the protein under both native and denaturing conditions is a monomer of Mr about 29000. The optimal conditions for both enzyme stability and enzyme activity have been determined.

A Novel Heterologous Expression System for Characterization of Individual NADH-Cytochrome b5 Reductase Variants in Recessive Congenital Methemoglobinemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1676-1676
Author(s):  
Melanie J. Percy ◽  
L. J. Crowley ◽  
C. A. Davis ◽  
J. Boudreaux ◽  
D. M. Layton ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Heterologous Expression ◽  
Expression System ◽  
Cytochrome B5 ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Heterologous Expression System ◽  
Nadh Cytochrome

Abstract Deficiency of NADH-cytochrome b5 reductase (cb5r) causes two clinically distinct phenotypes of recessive congenital methemoglobinemia (RCM). Type I patients often manifest cyanosis from birth, and in type II patients the cyanosis is accompanied by severe neurological impairment. The mechanisms responsible for the phenotypic differences between the two subgroups remain to be defined. The majority of patients harbor two different mutant alleles. To date 39 mutant variants of cb5r have been identified, 2 of which are common to both types of RCM. In order to characterize the individual cb5r variant proteins we have developed a novel heterologous expression system based on the structures of the rat and human proteins derived by X-ray crystallography. The system permits the investigation of the catalytic efficiencies, protein thermostability, FAD cofactor properties and substrate (NADH/NAD+) affinities of the variants. We have investigated four patients with type I RCM, one of whom was homozygous for the D239G mutation. The other three were compound heterozygous: R159-/D239G; G75S/V252M; and P275L/G291D, and one mutation, P275L, was novel. All patients showed reduced enzyme activity, in the range 0.5 to 5.8 IU/g Hb compared to normal values of 7.2 to 26.9 IU/g Hb. Individual variant proteins were prepared and the analytical data are summarised in the Table below. Variant Catalytic Efficiency (% of normal) Thermal Stability (T50°C) NADH affinity (Km) NAD+ affinity (Ks) ND - not determined G75S 11 48 Normal 9-fold ↑ R159- 0 ND ND ND D239G 2 56 40-fold ↓ ND V252M 9 53 9-fold ↓ 18-fold ↑ E255- 0.4 51 100-fold ↓ ND P275L 0.2 53 437-fold ↓ ND G291D 43 49 1.3-fold ↓ 1.1-fold ↑ Wild type 100 57 normal normal As expected all of the variants generated had decreased enzyme activity compared to wild type heterologous protein, supporting the validity of this approach. Thermal stability was decreased in the G75S, V252M and G291D variants. G75 is present in a highly conserved region in the FAD-binding lobe. Although it does not interact directly with the FAD prosthetic group it is important for association with cytochrome b5. Substitution of glycine at residue 75 by serine resulted in decreased enzyme activity and stability, with a marginal decrease in NADH affinity. The R159- variant protein was unstable and could not be isolated. Both the D239G and P275L mutations significantly reduced the affinity of cb5r for NADH, by 40-fold and 437-fold respectively. The rat cb5r model suggests that residue D239 is key for selecting between the NADPH and NADH pyridine nucleotides. This was confirmed by the 40-fold decrease in affinity for NADH and a 125-fold increase in affinity for NADPH. Residue P275 is located in a highly conserved region, which is important for the correct positioning and binding of NADH. Consequently, substitution of proline at 275 would affect the affinity of cb5r towards NADH, which was confirmed by the affinity constant measurements. These studies provide important information about the structure-function relationships of the variant cb5r proteins which may impart useful insights into the pathophysiological differences between type I and type II RCM.

scholarly journals A Novel D-Allulose 3-Epimerase Gene from the Metagenome of a Thermal Aquatic Habitat and D-Allulose Production by Bacillus subtilis Whole-Cell Catalysis

2019 ◽  
Vol 86 (5) ◽  
Author(s):  
Satya Narayan Patel ◽  
Girija Kaushal ◽  
Sudhir P. Singh
Keyword(s):  
Thermal Stability ◽  
Bacillus Subtilis ◽  
Water Reservoir ◽  
Heat Stability ◽  
Thermal Spring ◽  
Hot Water ◽  
Whole Cell ◽  
Content Type ◽  
Extraction And Purification ◽  
Whole Cell Catalysis

ABSTRACT A novel d-allulose 3-epimerase gene (daeM) has been identified from the metagenomic resource of a hot-water reservoir. The enzyme epimerizes d-fructose into d-allulose, a functional sugar of rare abundance in nature. The metagenomic DNA fragment was cloned and expressed in Escherichia coli. The purified recombinant protein (DaeM) was found to be metal dependent (Co2+ or Mn2+). It displayed the maximal levels of catalytic activity in a pH range of 6 to 11 and a temperature range of 75°C to 80°C. The enzyme exhibited remarkably high thermal stability at 60°C and 70°C, with half-life values of 9,900 and 3,240 min, respectively. To the best of our knowledge, this is the highest thermal stability demonstrated by a d-allulose 3-epimerase that has been characterized to date. The enzymatic treatment of 700 mg·ml−1 d-fructose yielded about 217 mg·ml−1 d-allulose, under optimal condition. The catalytic product was purified, and its nuclear magnetic resonance (NMR) spectra were found to be indistinguishable from those of standard d-allulose. For biomolecule production, the whole-cell catalysis procedure avoids the tedious process of extraction and purification of enzyme and also offers better biocatalyst stability. Further, it is desirable to employ safe-grade microorganisms for the biosynthesis of a product. The daeM gene was expressed intracellularly in Bacillus subtilis. A whole-cell catalysis reaction performed with a reaction volume of 1 liter at 60°C yielded approximately 196 g·liter−1 d-allulose from 700 g·liter−1 d-fructose. Further, the whole recombinant cells were able to biosynthesize d-allulose in apple juice, mixed fruit juice, and honey. IMPORTANCE d-Allulose is a noncaloric sugar substitute with antidiabetes and antiobesity potential. With several characteristics of physiological significance, d-allulose has wide-ranging applications in the food and pharmacology industries. The development of a thermostable biocatalyst is an objective of mainstream research aimed at achieving industrial acceptability of the enzyme. Aquatic habitats of extreme temperatures are considered a potential metagenomic resource of heat-tolerant biocatalysts of industrial importance. The present study explored the thermal-spring metagenome of the Tattapani geothermal region, Chhattisgarh, India, discovering a novel d-allulose 3-epimerase gene, daeM, encoding an enzyme of high-level heat stability. The daeM gene was expressed in the microbial cells of a nonpathogenic and safe-grade species, B. subtilis, which was found to be capable of performing d-fructose to d-allulose interconversion via a whole-cell catalysis reaction. The results indicate that DaeM is a potential biocatalyst for commercial production of the rare sugar d-allulose. The study established that extreme environmental niches represent a genomic resource of functional sugar-related biocatalysts.

Effect of the hydrophilicity of the polymer matrix on immobilized α-chymotrypsin

1984 ◽  
Vol 49 (6) ◽  
pp. 1552-1556
Author(s):  
Minoru Kumakura ◽  
Isso Kaetsu
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Pore Size ◽  
Polymer Matrix ◽  
Low Temperatures ◽  
Radiation Polymerization ◽  
Thermal Stability Of

α-Chymotrypsin was immobilized by radiation polymerization at low temperatures and the effect of the hydrophilicity of the polymer matrix on the enzyme activity and thermal stability was studied. The activity and thermal stability of immobilized chymotrypsin increased with the increasing hydrophilicity of the polymer matrix or monomer. The thermal stability was affected by the form and pore size of the polymer matrix; chymotrypsin immobilized on a soft-gel polymer matrix exhibited an enhanced thermal stability.

Assessment of Physicochemical Characteristics and Thermophilic Bacterial Enumeration from Hot Water Springs of Himachal Pradesh

2018 ◽  
Vol 41 (5) ◽  
pp. 285-287
Author(s):  
Poonam Shirkot ◽  
Ambika Verma ◽  
Monika Gupta ◽  
Anshul Sharma Manjul ◽  
Ruchika Sharma
Keyword(s):  
Physicochemical Characteristics ◽  
Himachal Pradesh ◽  
Hot Water ◽  
Bacterial Enumeration ◽  
Water Springs

scholarly journals DNA Genome Size Affects the Stability of the Adenovirus Virion

2008 ◽  
Vol 83 (4) ◽  
pp. 2025-2028 ◽  
Author(s):  
Adam C. Smith ◽  
Kathy L. Poulin ◽  
Robin J. Parks
Keyword(s):  
Genome Size ◽  
Critical Role ◽  
Heat Stability ◽  
Helper Virus ◽  
Heat Inactivation ◽  
Similar Relationship ◽  
Viral Dna ◽  
A Genome ◽  
The Stability ◽  
Replication Defective Adenovirus

ABSTRACT Replication-defective adenovirus (Ad) vectors can vary considerably in genome length, but whether this affects virion stability has not been investigated. Helper-dependent Ad vectors with a genome size of ∼30 kb were 100-fold more sensitive to heat inactivation than their parental helper virus (>36 kb), and increasing the genome size of the vector significantly improved heat stability. A similar relationship between genome size and stability existed for Ad with early region 1 deleted. Loss of infectivity was due to release of vertex proteins, followed by disintegration of the capsid. Thus, not only does the viral DNA encode all of the heritable information essential for virus replication, it also plays a critical role in maintaining capsid strength and integrity.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

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