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.

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.

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 Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′

1990 ◽  
Vol 18 (8) ◽  
pp. 2061-2064 ◽  
Author(s):  
Janise Meyertons Nelson ◽  
Sheila M. Miceli ◽  
Mary P. Lechevalier ◽  
Richard J. Roberts
Keyword(s):  
Restriction Endonuclease ◽  
Type Ii ◽  
New Type

scholarly journals Elevated lymphotoxin-α (TNFβ) is associated with intervertebral disc degeneration

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhu Guo ◽  
Chensheng Qiu ◽  
Christina Mecca ◽  
Yang Zhang ◽  
Jiang Bian ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Cell Viability ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Caspase 3 ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Optimal Conditions ◽  
Cell Degeneration ◽  
Lymphotoxin Α

Abstract Background Intervertebral disc degeneration (IVDD) is a primary cause of degenerative disc diseases; however, the mechanisms underlying the degeneration remain unclear. The immunoinflammatory response plays an important role in IVDD progression. The inflammatory cytokine lymphotoxin-α (LTα), formerly known as TNFβ, is associated with various pathological conditions, while its role in the pathogenesis of IVDD remains elusive. Methods Real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and enzyme-linked immunosorbent assays were used to assess the levels of LTα in human nucleus pulposus (NP) tissues between degeneration and control groups. The plasma concentrations of LTα and C-reactive protein (CRP) were compared between healthy and IVDD patients. Rat primary NP cells were cultured and identified via immunofluorescence. Methyl-thiazolyl-tetrazolium assays and flow cytometry were used to evaluate the effects of LTα on rat NP cell viability. After NP cells were treated with LTα, degeneration-related molecules (Caspase-3, Caspase-1, matrix metalloproteinase (MMP) -3, aggrecan and type II collagen) were measured via RT-qPCR and WB. Results The levels of both the mRNA and protein of LTα in human degenerated NP tissue significantly increased. Plasma LTα and CRP did not differ between healthy controls and IVDD patients. Rat primary NP cells were cultured, and the purity of primary NP cells was > 90%. Cell experiments showed inversely proportional relationships among the LTα dose, treatment time, and cell viability. The optimal conditions (dose and time) for LTα treatment to induce rat NP cell degeneration were 5 μg/ml and 48 ~ 72 h. The apoptosis rate and the levels of Caspase-3, Caspase-1, and MMP-3 significantly increased after LTα treatment, while the levels of type II collagen and aggrecan were decreased, and the protein expression levels were consistent with their mRNA expression levels. Conclusions This study demonstrated that elevated LTα is closely associated with IVDD and that LTα may induce NP cell apoptosis and reduce important extracellular matrix (ECM) proteins, which cause adverse effects on IVDD progress. Moreover, the optimal conditions for LTα treatment to induce NP cell degeneration were determined.

Enantioselective Resolution of (R, S)-DMPM by a Adsorption-Covalent Crosslinked Esterase PAE07 from Pseudochrobactrum Asaccharolyticum WZZ003

2021 ◽  
Author(s):  
Mingpeng Zhou ◽  
Yuandan Xia ◽  
Hongjun Zhang ◽  
Xinjun Yu ◽  
Yinjun zhang
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Sem Analysis ◽  
Optimal Conditions ◽  
Synthetic Resin ◽  
Adsorption Time ◽  
Enantioselective Resolution ◽  
Primary Amino ◽  
Repeated Use ◽  
Amino Resin

Abstract (R)-N-(2,6-dimethylphenyl) alanine ((R)-MAP-acid) is an important chiral intermediate of the Fungicide (R)-Metalaxyl. In this study, ten kinds of immobilized resins(XAD1180N, H103, HAD7HP, D3520, NKA, D101 , DM11,850 JinKai, Primary amino resin and 850 synthetic resin) were used to adsorption-covalent crosslinked esterase PAE07 for splitting (R, S)-DMPM. The resin D3520 with porous structure and hydrophobic polystyrene was selected for immobilization as the carrier, after optimization of the immobilization conditions, the enzyme load is 20:1 (mg/g), the adsorption time is 4h, and the adsorption buffer pH is 7.0 . The Km and Vmax of the free esterases were 35.66 mM and 4.46 mM/mg·min, respectively, The Km and Vmax of the immobilized PAE07 were 19.05 mM and 2.84 mM/mg·min. The SEM analysis showed that the immobilized esterase PAE07 had higher thermal stability, pH stability and substrate specifity than those from the free esterase. Under the optimal conditions,the reaction was carried out at 35°C and 200 rpm for resolution of 350 mM substrate for 14 hours, the conversion rate reached 48%, and the e.e.p was 99.5%.The repeatability of immobilized esterase PAE07 was evaluated by continuous catalytic resolution of (R, S)-DMPM. The results showed that after 15 times of repeated use, 86.2% of the relative enzyme activity was retained. These results proved that immobilized esterase PAE07 as a new catalyst had great potential for the application and industrial enzymatic resolution of (R, S)-DMPM to prepare (R)-metalaxyl.

Extracellular alkaline phosphatase from marine bacteria: purification and properties of extracellular phosphatase from a marine Pseudomonas sp.

1980 ◽  
Vol 26 (7) ◽  
pp. 833-838 ◽  
Author(s):  
Hiromi Kobori ◽  
Nobuo Taga
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Marine Bacteria ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Pseudomonas Sp ◽  
Purification And Properties ◽  
Extracellular Alkaline Phosphatase ◽  
Increased Pressure

Extracellular alkaline phosphatase produced by a marine Pseudomonas was purified to electrophoretic homogeneity. The molecular weight of the enzyme was estimated to be 100 000. The enzyme had maximal activity at pH 11.5. The enzyme was completely inhibited by 1 mM EDTA. However, divalent cations reversed the enzyme inhibition and their order of effectiveness on the reaction was Zn2+ > Ca2+ > Mn2+ > Mg2+ > Sr2+ > Co2+. The enzyme activity was affected by the species of anion whose order of effectiveness was demonstrated to follow the lyotrophic series, Cl− > Br− > NO3−> ClO4− > SCN−. The activity of phosphatase was accelerated linearly by increased pressure until up to 1000 atm (1 atm = 101.325 kPa), and the enzyme activity at 1000 atm was 3.2 times higher than that at 1 atm.

Isolation and characterization of a newly identified type II restriction endonuclease from a local streptomyces sp. in Taiwan

1998 ◽  
Vol 73 (2-3) ◽  
pp. 231-241
Author(s):  
Kuang-Yu Hu ◽  
Jia-An Wuu ◽  
Ming-Ching Kao ◽  
Yu-Tien Liu ◽  
Shou-Hsiung Pai
Keyword(s):  
Restriction Endonuclease ◽  
Type Ii ◽  
Streptomyces Sp ◽  
Isolation And Characterization

Serum phenoloxidase activity in the hemolymph of the anomuran crab Albunea symmysta (Linnaeus, 1758) (Decapoda: Anomura: Albuneidae)

2021 ◽  
Vol 41 (1) ◽  
Author(s):  
Shanthi Sivakumar ◽  
Mullaivanam R Sivakumar ◽  
Rayvathy Balasubramanian
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl Sulfate ◽  
Functional Activity ◽  
Sodium Dodecyl ◽  
Substrate Affinity ◽  
Optimal Conditions ◽  
Phenoloxidase Activity ◽  
In Vitro Inhibition ◽  
Pronase E

Abstract We characterized the optimal conditions for measuring serum phenoloxidase activity and its functional activity and susceptibility to an inhibitor and various activators in an anomuran crab, Albunea symmysta (Linnaeus, 1758). The substrate affinity of the phenoloxidase (PO) enzyme was determined using different phenolic substrates in which only diphenols were found to be oxidized. The enzyme was characterized as a catecholoxidase-type of PO and 3,4-dihydroxy-DL-phenylalanine (DL-Dopa), the enzyme showing the highest substrate affinity to the serum. The optimal enzyme activity was observed at 5 mM DL-Dopa in 10 mM Tris-HCl buffer at a pH of 7.5 at 25 °C for 10 min, and absorbance at 470 nm. Serum-PO activity was inhibited by 7 mM phenylthiourea (PTU), and activated by activators such as trypsin, chymotrypsin, pronase-E, and detergent-like sodium dodecyl sulfate (SDS). We also identified the chemicals causing in vitro inhibition or activation of the enzyme as a serum of the crab having a potent PO activity.

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