scholarly journals Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus: Effects of Thioredoxin Fusion Tag and Triton X-100

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Sylvia Maria Campbell Alquéres ◽  
Roberta Vieira Branco ◽  
Denise Maria Guimarães Freire ◽  
Tito Lívio Moitinho Alves ◽  
Orlando Bonifácio Martins ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Optimum Temperature ◽  
Optimal Temperature ◽  
Thermostable Lipase ◽  
Fusion Tag ◽  
Triton X

In this work, the lipase from Pyrococcus furiosus encoded by ORF PF2001 was expressed with a fusion protein (thioredoxin) in Escherichia coli. The purified enzymes with the thioredoxin tag (TRX−PF2001Δ60) and without the thioredoxin tag (PF2001Δ60) were characterized, and various influences of Triton X-100 were determined. The optimal temperature for both enzymes was 80°C. Although the thioredoxin presence did not influence the optimum temperature, the TRX−PF2001Δ60 presented specific activity twice lower than the enzyme PF2001Δ60. The enzyme PF2001Δ60 was assayed using MUF-acetate, MUF-heptanoate, and MUF-palmitate. MUF-heptanoate was the preferred substrate of this enzyme. The chelators EDTA and EGTA increased the enzyme activity by 97 and 70%, respectively. The surfactant Triton X-100 reduced the enzyme activity by 50% and lowered the optimum temperature to 60°C. However, the thermostability of the enzyme PF2001Δ60 was enhanced with Triton X-100.

Molecular characterization of an endo-type chitosanase from the fish pathogenRenibacteriumsp. QD1

2014 ◽  
Vol 94 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Peichuan Xing ◽  
Dan Liu ◽  
Wen-Gong Yu ◽  
Xinzhi Lu
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Maximal Activity ◽  
Optimum Temperature ◽  
Sds Page ◽  
Ph Range ◽  
Tlc Analysis

Renibacteriumsp. QD1, a bacteria strain capable of hydrolysing chitosan, was isolated from the homogenate of small crabs. An extracellular chitosanase, Csn-A, was purified from the QD1 fermentation broth. The enzyme was purified to homogeneity, with a yield of eight-fold, 67% recovery and a specific activity of 1575 U/mg proteins. The molecular weight of Csn-A was estimated to be 26.1 kDa by SDS-PAGE. Unlike other chitosanases, the purified Csn-A displayed maximal activity at a pH range of 5.3–6.5, and it was stable in a broad pH range of 5.0–10.0. The optimum temperature for chitosanlytic activity was 55°C. The enzyme activity was strongly stimulated by Mn2+but inhibited by Fe3+, Cu2+, Al3+, Zn2+and SDS. TLC analysis demonstrated that Csn-A hydrolysed N-deacetylated polymeric glucosamines into chito-biose and -triose in an endo-type manner. The amino acid seuquence of Csn-A showed close identity with an uncharacterized chitosanase of strain ATCC33209.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

scholarly journals Escherichia coli S-adenosylhomocysteine/5′-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action

1985 ◽  
Vol 232 (2) ◽  
pp. 335-341 ◽  
Author(s):  
F Della Ragione ◽  
M Porcelli ◽  
M Cartenì-Farina ◽  
V Zappia ◽  
A E Pegg
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Mechanism Of Action ◽  
Specific Activity ◽  
Maximal Rate ◽  
Purification Procedure ◽  
Protein Affinity ◽  
Naturally Occurring ◽  
Purine Ring ◽  
Ribose Moiety

S-Adenosylhomocysteine/5′-methylthioadenosine nucleosidase (EC 3.2.2.9) was purified to homogeneity from Escherichia coli to a final specific activity of 373 mumol of 5′-methylthioadenosine cleaved/min per mg of protein. Affinity chromatography on S-formycinylhomocysteine-Sepharose is the key step of the purification procedure. The enzyme, responsible for the cleavage of the glycosidic bond of both S-adenosylhomocysteine and 5′-methylthioadenosine, was partially characterized. The apparent Km for 5′-methylthioadenosine is 0.4 microM, and that for S-adenosylhomocysteine is 4.3 microM. The maximal rate of cleavage of S-adenosylhomocysteine is approx. 40% of that of 5′-methylthioadenosine. Some 25 analogues of the two naturally occurring thioethers were studied as potential substrates or inhibitors of the enzyme. Except for the analogues modified in the 5′-position of the ribose moiety or the 2-position of the purine ring, none of the compounds tested was effective as a substrate. Moreover, 5′-methylthioformycin, 5′-chloroformycin, S-formycinylhomocysteine, 5′-methylthiotubercidin and S-tubercidinylhomocysteine were powerful inhibitors of the enzyme activity. The results obtained allow the hypothesis of a mechanism of enzymic catalysis requiring as a key step the protonation of N-7 of the purine ring.

A β-Mannanase from Bacillus subtilis B36: Purification, Properties, Sequencing, Gene Cloning and Expression in Escherichia coli

2006 ◽  
Vol 61 (11-12) ◽  
pp. 840-846 ◽  
Author(s):  
Ya Nan Li ◽  
Kun Meng ◽  
Ya Ru Wang ◽  
Bin Yao
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Specific Activity ◽  
Ph Value ◽  
High Specificity ◽  
Apparent Molecular Weight ◽  
Cloning And Expression ◽  
Gene Cloning And Expression ◽  
Reading Frame

Abstract MANB36, a secrete endo-β-1,4-D-mannanase produced by Bacillus subtilis B36, was puri­fied to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 °C. The enzyme activity of MANB36 is remarkably thermostable at 60 °C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoetha- nol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted mo­lecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.

scholarly journals Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

scholarly journals Molecular Characterization of the PceA Reductive Dehalogenase of Desulfitobacterium sp. Strain Y51

2002 ◽  
Vol 184 (13) ◽  
pp. 3419-3425 ◽  
Author(s):  
Akiko Suyama ◽  
Masaki Yamashita ◽  
Sadazo Yoshino ◽  
Kensuke Furukawa
Keyword(s):  
Escherichia Coli ◽  
Molecular Characterization ◽  
Specific Activity ◽  
Chlorinated Ethenes ◽  
Reductive Dehalogenation ◽  
Reductive Dehalogenase ◽  
Dna Fragment

ABSTRACT The tetrachloroethene (PCE) reductive dehalogenase (encoded by the pceA gene and designated PceA dehalogenase) of Desulfitobacterium sp. strain Y51 was purified and characterized. The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE). The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to cis-1,2-dichloroethene at a specific activity of 113.6 nmol · min−1 · mg of protein−1. The apparent Km values for PCE and TCE were 105.7 and 535.3 μM, respectively. Chlorinated ethenes other than PCE and TCE were not dehalogenated. However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane. The pceA gene of Desulfitobacterium sp. strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in Escherichia coli for the preparation of antibodies. Immunoblot analyses located PceA in the periplasm of the cell.

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.

scholarly journals The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1970 ◽  
Vol 117 (2) ◽  
pp. 319-324 ◽  
Author(s):  
G. J. Mulder
Keyword(s):  
Enzyme Activity ◽  
Density Gradient ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Male Rats ◽  
Uridine Diphosphate ◽  
Triton X

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The Km of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

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