scholarly journals A Phytase Characterized by Relatively High pH Tolerance and Thermostability from the Shiitake MushroomLentinus edodes

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Guo-Qing Zhang ◽  
Ying-Ying Wu ◽  
Tzi-Bun Ng ◽  
Qing-Jun Chen ◽  
He-Xiang Wang
Keyword(s):  
Sequence Similarity ◽  
Gel Filtration ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Maximal Activity ◽  
Phytase Activity ◽  
Ph Tolerance ◽  
Phosphorylated Compounds ◽  
Terminal Amino ◽  
Ph Range

A monomeric phytase with a molecular mass of 14 kDa was acquired from fresh fruiting bodies of the shiitake mushroomLentinus edodes. The isolation procedure involved chromatography on DEAE-cellulose, CM-cellulose, Q-Sepharose, Affi-gel blue gel, and a final fast protein liquid chromatography-gel filtration on Superdex 75. The purified phytase demonstrated the unique N-terminal amino acid sequence DPKRTDQVN, which exhibited no sequence similarity with those of other phytases previously reported. It expressed its maximal activity at pH 5.0 and 37°C. Phytase activity manifested less than 20% change in activity over the pH range of 3.0–9.0, considerable thermostability with more than 60% residual activity at 70°C, and about 40% residual activity at 95°C. It displayed a wide substrate specificity on a variety of phosphorylated compounds with the following ranking: ATP > fructose-6-phosphate > AMP > glucose-6-phosphate > ADP > sodium phytate >β-glycerophosphate. The phytase activity was moderately stimulated by Ca2+, but inhibited by Al3+, Mn2+, Zn2+, and Cu2+at a tested concentration of 5 mM.

scholarly journals Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

2015 ◽  
Vol 81 (19) ◽  
pp. 6938-6944 ◽  
Author(s):  
Tao Tu ◽  
Huiying Luo ◽  
Kun Meng ◽  
Yanli Cheng ◽  
Rui Ma ◽  
...  
Keyword(s):  
Rational Design ◽  
Residual Activity ◽  
Maximal Activity ◽  
Wild Type ◽  
Content Type ◽  
Highly Active ◽  
Wide Ph Range ◽  
Enzyme Thermal Stability ◽  
Ph Range ◽  
Charge Interactions

ABSTRACTImproving enzyme thermostability is of importance for widening the spectrum of application of enzymes. In this study, a structure-based rational design approach was used to improve the thermostability of a highly active, wide-pH-range-adaptable, and stable endopolygalacturonase (PG8fn) fromAchaetomiumsp. strain Xz8 via the optimization of charge-charge interactions. By using the enzyme thermal stability system (ETSS), two residues—D244 and D299—were inferred to be crucial contributors to thermostability. Single (D244A and D299R) and double (D244A/D299R) mutants were then generated and compared with the wild type. All mutants showed improved thermal properties, in the order D244A < D299R < D244A/D299R. In comparison with PG8fn, D244A/D299R showed the most pronounced shifts in temperature of maximum enzymatic activity (Tmax), temperature at which 50% of the maximal activity of an enzyme is retained (T50), and melting temperature (Tm), of about 10, 17, and 10.2°C upward, respectively, with the half-life (t1/2) extended by 8.4 h at 50°C and 45 min at 55°C. Another distinguishing characteristic of the D244A/D299R mutant was its catalytic activity, which was comparable to that of the wild type (23,000 ± 130 U/mg versus 28,000 ± 293 U/mg); on the other hand, it showed more residual activity (8,400 ± 83 U/mg versus 1,400 ± 57 U/mg) after the feed pelleting process (80°C and 30 min). Molecular dynamics (MD) simulation studies indicated that mutations at sites D244 and D299 lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability.

Isolation and some properties of extracellular heat-stable lipases fromPseudomonas fluorescensstrain AFT 36

1983 ◽  
Vol 50 (1) ◽  
pp. 77-89 ◽  
Author(s):  
Patrick F. Fox ◽  
Leszek Stepaniak
Keyword(s):  
Phosphate Buffer ◽  
Lipolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Lipase Production ◽  
Maximum Activity ◽  
Temperature Range ◽  
Milk Serum ◽  
Heat Stable ◽  
Ph Range

SummaryAeration increased the growth and lipase production in milk byPseudomonas fluorescensstrain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented ∼ 71 % of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8·0 and 35 °C; it had aKmon tributyrin of 3·65 mM. and was inhibited by concentrations of substrate > ∼ 17 mM. The enzyme was very stable over the pH range 6–9; it was relatively heat-labile in phosphate buffer in the temperature range 60–80 °C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100–150 °C: theDvalues at 150 °C were ∼ 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the correspondingZvalues in the temperature range 100–150 °C were ∼ 40 and ∼ 42 °C and theEafor inactivation were 7·65 × 104J mol-1and 6·97 × 104J mol-1respectively.

scholarly journals Some properties of cytochrome b5 from liver microsomes of man, monkey, pig and chicken

1969 ◽  
Vol 115 (4) ◽  
pp. 849-856 ◽  
Author(s):  
F. G. Nóbrega ◽  
P. S. Araujo ◽  
M. Pasetto ◽  
I. Raw
Keyword(s):  
Amino Acids ◽  
Spectral Properties ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gradient Elution ◽  
Closely Related Species ◽  
Ammonium Sulphate Fractionation ◽  
Terminal Amino

1. Cytochrome b5 was released from liver microsomes of man, monkey, pig and chicken by incubation with a crude lipase preparation. 2. By using DEAE-cellulose chromatography, ammonium sulphate fractionation, Sephadex-gel filtration and a final gradient elution on DEAE-Sephadex A-50, cytochromes b5 were obtained from the four species studied, all possessing similar spectral properties. 3. Stokes radii of the cytochromes were measured by gel filtration. 4. N-Terminal amino acids for the different cytochromes were serine for man and monkey, alanine for pig and glycine for chicken. 5. Amino acid analyses of the cytochromes are presented. 6. Peptide ‘fingerprint’ patterns of tryptic digests of the different cytochromes are discussed and clearly show increasing similarity for more closely related species.

Pectic enzymes produced by Pseudomonas fluorescens, an organism associated with “pink eye” disease of potato tubers

1972 ◽  
Vol 50 (12) ◽  
pp. 2479-2488 ◽  
Author(s):  
S. S. Hagar ◽  
G. A. McIntyre
Keyword(s):  
Pseudomonas Fluorescens ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pectin Methylesterase ◽  
Viscosity Reduction ◽  
Cellulose Acetate Electrophoresis ◽  
Pectic Enzymes ◽  
Ph Range ◽  
Two Peaks ◽  
Layer Chromatography

No pectin methylesterase (PME) activity was observed in crude or dialyzed extracts from macerated potato tuber tissue inoculated with Pseudomonas fluorescens; however, pectic lyase (syn. polygalacturonic acid transeliminase, PATE) activity was observed. Two PATE enzymes (peaks 1 and 2) were eluted from a pH 9.4 DEAE-cellulose column using a gradient of pH 7.6 Tris-HCl buffer (0.01–0.1 M). Enzyme in peak 1 was about 6 times more active than enzyme in peak 2 based on reducing group assays, and 10 times more active in viscosity reduction of 1% Na-polypectate (NaPP) at pH 8.5. No increase in absorbancy was observed at 515 nm of clarified reaction mixtures, indicating that saturated oligouronides did not accumulate. Other properties of the two peaks: optimum pH range was 8.5–9.5, substrate preference was NaPP vs. pectin, addition of Ca2+ (0.001 M) enhanced activity while EDTA (0.001 M) decreased activity to [Formula: see text], cellulose acetate electrophoresis revealed one band of protein per peak, and heat of inactivation was 51–60C. Thin-layer chromatography of hydrolytic products from NaPP revealed unsaturated uronides and pectic fragments after 2 h hydrolysis; after 96 h hydrolysis only unsaturated uronides were observed. Molecular weight estimations by Sephadex G-200 gel filtration were about 18 000 (peak 1) and 22 500 (peak 2). Enzyme in either peak macerated 400-μ sections of potato tissue.

Purification and regulation of an AMP-specific cytosolic 5'-nucleotidase from dog heart

1993 ◽  
Vol 264 (5) ◽  
pp. H1528-H1534 ◽  
Author(s):  
A. Darvish ◽  
P. J. Metting
Keyword(s):  
Adenine Nucleotides ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximal Activity ◽  
Maximal Velocity ◽  
Sucrose Density Gradient Centrifugation ◽  
Dog Heart ◽  
Myocardial Hypoxia

The major enzyme responsible for adenosine production during myocardial hypoxia or ischemia is 5'-nucleotidase. We purified an AMP-specific 5'-nucleotidase to homogeneity from the 150,000-g supernatant of dog heart homogenate using phosphocellulose, DEAE-cellulose, and ADP-agarose affinity chromatography. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of the purified enzyme yielded a single protein band of 43 kDa. The molecular mass of the holoenzyme, determined by gel filtration and sucrose density-gradient centrifugation, was approximately 166 kDa, suggesting a tetrameric structure. Dog heart cytosolic 5'-nucleotidase was active at physiological pH (6.8-7.8) and demonstrated a preference for AMP over IMP as substrate. The enzyme exhibited sigmoidal saturation kinetics, with half-maximal activity at 2.6 mM AMP in the absence of ADP. ADP (0-250 microM) activated cytosolic 5'-nucleotidase by increasing maximal velocity and affinity for AMP. The enzyme was inhibited by 4 mM ATP, but 5'-nucleotidase activity increased as [ATP] was reduced. Mg2+ was required for activity, with maximal activation at approximately 3.5 mM free Mg2+. These data suggest that the regulation of AMP-specific cytosolic 5'-nucleotidase by adenine nucleotides and free Mg2+ may be important in the production of adenosine during conditions promoting ATP hydrolysis, such as myocardial hypoxia or ischemia.

scholarly journals Characterization of a Thermostable l-Arabinose (d-Galactose) Isomerase from the Hyperthermophilic Eubacterium Thermotoga maritima

2004 ◽  
Vol 70 (3) ◽  
pp. 1397-1404 ◽  
Author(s):  
Dong-Woo Lee ◽  
Hyeung-Jin Jang ◽  
Eun-Ah Choe ◽  
Byoung-Chan Kim ◽  
Sang-Jae Lee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Elevated Temperatures ◽  
Thermotoga Maritima ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Recombinant Enzyme ◽  
Amino Acid Sequence Similarity ◽  
Calculated Molecular Weight

ABSTRACT The araA gene encoding l-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni2+ affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90�C and pH 7.5 under the assay conditions used. Its apparent Km values for l-arabinose and d-galactose were 31 and 60 mM, respectively; the apparent V max values (at 90�C) were 41.3 U/mg (l-arabinose) and 8.9 U/mg (d-galactose), and the catalytic efficiencies (k cat/Km ) of the enzyme were 74.8 mM−1 � min−1 (l-arabinose) and 8.5 mM−1 � min−1 (d-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn2+ and/or Co2+ than in the absence of these ions. The enzyme carried out the isomerization of d-galactose to d-tagatose with a conversion yield of 56% for 6 h at 80�C.

scholarly journals Production, purification and characterization of an exo-polygalacturonase from Penicillium janthinellum sw09

2016 ◽  
Vol 88 (suppl 1) ◽  
pp. 479-487 ◽  
Author(s):  
YUPING MA ◽  
SIWEN SUN ◽  
HUI HAO ◽  
CHUNPING XU
Keyword(s):  
Gel Filtration ◽  
Maximal Activity ◽  
Growth Conditions ◽  
Purification And Characterization ◽  
Penicillium Janthinellum ◽  
Soil Isolate ◽  
Sds Page ◽  
The Stability ◽  
Ph Range

ABSTRACT A soil isolate, Penicillium janthinellum sw09 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as exo-polygalacturonase (exo-PG). By optimizing growth conditions, P. janthinellum sw09 produced high amount of exo-PG (16.54 units/mL). The crude enzyme was purified by gel filtration chromatography and two exo-PG activity peaks (designated as PGI and PGII) were revealed. On SDS-PAGE analysis, purified PGII using DEAE-Sepharose FF column, was found to be a single band with a molecular mass of 66.2 kDa. The purified PGII exhibited maximal activity at the temperature of 45 oC and pH 5.0. The stability profiles show that PGII is more stable in the pH range of 4.0-8.0 and below 60 oC. The Km and Vmax for the enzyme was 1.74 mg/mL and 18.08 μmol/ (mL•min), respectively. Due to this enzymatic characterization, this pectinase is an attractive candidate for applications in degradation of pectin.

Human hemopexin. Preparation of the heme-rich protein

1982 ◽  
Vol 47 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Ladislav Morávek ◽  
Josef Borvák ◽  
Karel Grüner ◽  
Bedřich Meloun ◽  
Petr Štrop ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Analysis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Simplified Procedure ◽  
Pooled Samples ◽  
Terminal Amino Acid Sequence

A simplified procedure was developed for the preparation of hemopexin from Cohn fraction IV obtained from partially hemolyzed pooled samples of serum. The method is based on precipitation with rivanol, chromatography on DEAE-cellulose, and gel filtration; it permits large quantities of the material to be treated on a laboratory scale. The preparation of heme-rich hemopexin obtained was characterized by amino acid analysis and the following N-terminal amino acid sequence: Thr-Pro-Leu-Pro-Arg-Gly-Ser-Ala-His-Gly-Asn-Val-Ala-Glu-Gly-Glu-Thr(Thr)Thr-Asn-Pro-Asp-Val-(Gly)(Leu).

A proteolytic pseudomonad from skin lesions of rainbow trout (Salmo gairdnerii). II. Some properties of the proteinase

1968 ◽  
Vol 14 (8) ◽  
pp. 875-880 ◽  
Author(s):  
M. F. Li ◽  
Carol Jordan
Keyword(s):  
Rainbow Trout ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Skin Lesions ◽  
Complexing Agents ◽  
Bivalent Cations ◽  
Wide Ph Range ◽  
Ph Range ◽  
Trout Muscle ◽  
Cellulose Column

An extracellular proteinase from a pseudomonad pathogenic to rainbow trout was purified 33-fold by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G 75 gel filtration. The purified enzyme was active over a wide pH range, from pH 5.0 to 10.0. Heating at 98 °C for 1 h did not completely inactivate the enzyme. Its observed temperature optimum was 45 °C. Michaelis–Menten constants were found to be 0.625% for casein and 0.263% for rainbow trout muscle albumin. Activation energies calculated for these substrates were 8.1 kcal and 11.4 kcal per mole, respectively. The involvement of bivalent cations and free sulfhydryl groups in the enzymatic activity was demonstrated by the inhibition caused by metal-complexing agents and p-chloromercuribenzoate, respectively.

scholarly journals Vitamin A and carotenoids. The enzymic conversion of β-carotene into retinal in hog intestinal mucosa

1969 ◽  
Vol 114 (4) ◽  
pp. 689-694 ◽  
Author(s):  
Noel H. Fidge ◽  
Frank Rees Smith ◽  
DeWitt S. Goodman
Keyword(s):  
Intestinal Mucosa ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Enzyme Preparations ◽  
Optimum Ph ◽  
Tween 40 ◽  
Ph Range ◽  
Optimum Ph Range ◽  
Β Carotene

The conversion of β-carotene into retinal was studied in vitro with enzyme preparations from homogenates of hog intestinal mucosa. The hog mucosal enzyme was purified about 27-fold by precipitation with ammonium sulphate, chromatography on DEAE-Sephadex and gel filtration on Sephadex G-200. The reaction displayed a narrow optimum pH range (approx. 7·8–8·2). The enzyme was stimulated strongly by the addition of thiols, and was inhibited by thiol inhibitors and by the chelating agents αα′-bipyridyl and o-phenanthroline. The reaction required the addition of an appropriate detergent (or bile salt); maximal activity was obtained by addition of an appropriate combination of detergents and lipid (specifically Tween 40, sodium glycocholate and sphingomyelin). The reaction displayed Michaelis kinetics with Km1·3×10−6m and Vmax.1·1nmole of retinal formed/hr. (for 0·7mg. of enzyme protein). The properties of the hog enzyme are similar to those previously reported for a less purified rat enzyme preparation.

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