scholarly journals Characterization of the Family I inorganic pyrophosphatase fromPyrococcus horikoshiiOT3

Archaea ◽  
2005 ◽  
Vol 1 (6) ◽  
pp. 385-389 ◽  
Author(s):  
Sung-Jong Jeon ◽  
Kazuhiko Ishikawa
Keyword(s):  
Maximum Velocity ◽  
Heat Stability ◽  
Hydrolytic Activity ◽  
Heat Inactivation ◽  
Inorganic Pyrophosphatase ◽  
Gene Encoding ◽  
Sds Page ◽  
The Family ◽  
Hydrolysis Of

A gene encoding for a putative Family inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeonPyrococcus horikoshiiOT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) fromP. horikoshiishowed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase fromP. horikoshii(PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability ofPhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+being most effective; Zn2+, Co2+and Mn2+efficiently supported hydrolytic activity in a narrow range of concentrations (0.05– 0.5 mM). The Kmfor pyrophosphate and Mg2+were 113 and 303 µM, respectively; and maximum velocity,Vmax, was estimated at 930 U mg–1.

scholarly journals Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
Author(s):  
Takane Katayama ◽  
Akiko Sakuma ◽  
Takatoshi Kimura ◽  
Yutaka Makimura ◽  
Jun Hiratake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Sugar Chain ◽  
Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Isolation, purification and characterization of an ascorbate peroxidase from celery and overexpression of the AgAPX1 gene enhanced ascorbate content and drought tolerance in Arabidopsis

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Jie-Xia Liu ◽  
Kai Feng ◽  
Ao-Qi Duan ◽  
Hui Li ◽  
Qing-Qing Yang ◽  
...  
Keyword(s):  
Drought Stress ◽  
Ascorbate Peroxidase ◽  
Drought Resistance ◽  
Stomatal Closure ◽  
High Survival ◽  
Gene Encoding ◽  
Sds Page ◽  
Relative Water ◽  
New Evidence

Abstract Background Celery is a widely cultivated vegetable abundant in ascorbate (AsA), a natural plant antioxidant capable of scavenging free radicals generated by abiotic stress in plants. Ascorbate peroxidase (APX) is a plant antioxidant enzyme that is important in the synthesis of AsA and scavenging of excess hydrogen peroxide. However, the characteristics and functions of APX in celery remain unclear to date. Results In this study, a gene encoding APX was cloned from celery and named AgAPX1. The transcription level of the AgAPX1 gene was significantly upregulated under drought stress. AgAPX1 was expressed in Escherichia coli BL21 (DE3) and purified. The predicted molecular mass of rAgAPX1 was 33.16 kDa, which was verified by SDS-PAGE assay. The optimum pH and temperature for rAgAPX1 were 7.0 and 55 °C, respectively. Transgenic Arabidopsis hosting the AgAPX1 gene showed elevated AsA content, antioxidant capacity and drought resistance. Less decrease in net photosynthetic rate, chlorophyll content, and relative water content contributed to the high survival rate of transgenic Arabidopsis lines after drought. Conclusions The characteristics of APX in celery were different from that in other species. The enhanced drought resistance of overexpressing AgAPX1 in Arabidopsis may be achieved by increasing the accumulation of AsA, enhancing the activities of various antioxidant enzymes, and promoting stomatal closure. Our work provides new evidence to understand APX and its response mechanisms to drought stress in celery.

scholarly journals Functional Diversity among Metallo-β-Lactamases: Characterization of the CAR-1 Enzyme of Erwinia carotovora

2008 ◽  
Vol 52 (7) ◽  
pp. 2473-2479 ◽  
Author(s):  
Magdalena Stoczko ◽  
Jean-Marie Frère ◽  
Gian Maria Rossolini ◽  
Jean-Denis Docquier
Keyword(s):  
Functional Diversity ◽  
Clinical Importance ◽  
Erwinia Carotovora ◽  
Exchange Chromatography ◽  
Genome Database ◽  
Reading Frame ◽  
The Family ◽  
Functional Features ◽  
Hydrolysis Of

ABSTRACT Metallo-β-lactamases (MBLs) are zinc-dependent bacterial enzymes characterized by an efficient hydrolysis of carbapenems and a lack of sensitivity to commercially available β-lactamase inactivators. Apart from the acquired subclass B1 enzymes, which exhibit increasing clinical importance and whose evolutionary origin remains unclear, most MBLs are encoded by resident genes found in the genomes of organisms belonging to at least three distinct phyla. Using genome database mining, we identified an open reading frame (ORF) (ECA2849) encoding an MBL-like protein in the sequenced genome of Erwinia carotovora, an important plant pathogen. Although no detectable β-lactamase activity could be found in E. carotovora, a recombinant Escherichia coli strain in which the ECA2849 ORF was cloned showed decreased susceptibility to several β-lactams, while carbapenem MICs were surprisingly poorly affected. The enzyme, named CAR-1, was purified by means of ion-exchange chromatography steps, and its characterization revealed unique structural and functional features. This new MBL was able to efficiently hydrolyze cephalothin, cefuroxime, and cefotaxime and, to a lesser extent, penicillins and the other cephalosporins but only poorly hydrolyzed meropenem, while imipenem was not recognized. CAR-1 is the first example of a functional naturally occurring MBL in the family Enterobacteriaceae (order Enterobacteriales) and highlights the extraordinary structural and functional diversity exhibited by MBLs.

scholarly journals Characterization of a Feruloyl Esterase from Lactobacillus plantarum

2013 ◽  
Vol 79 (17) ◽  
pp. 5130-5136 ◽  
Author(s):  
María Esteban-Torres ◽  
Inés Reverón ◽  
José Miguel Mancheño ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Bacterial Species ◽  
Hydrolytic Activity ◽  
Feruloyl Esterase ◽  
Gene Encoding ◽  
Content Type ◽  
Cell Extracts ◽  
Methyl Ferulate ◽  
Methyl Caffeate

ABSTRACTLactobacillus plantarumis frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant.L. plantarumWCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methylp-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methylp-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present amongL. plantarumstrains. The present study constitutes the description of feruloyl esterase activity inL. plantarumand provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species.

A novel endo-β-1,4-xylanase GH30 from Dickeya dadantii DCE-01: Clone, expression, characterization, and ramie biological degumming function

2017 ◽  
Vol 89 (4) ◽  
pp. 463-472 ◽  
Author(s):  
Ruijun Wang ◽  
Zhengchu Liu ◽  
Lifeng Cheng ◽  
Shengwen Duan ◽  
Xiangyuan Feng ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Ph Value ◽  
Hydrolytic Activity ◽  
Glycoside Hydrolase Family ◽  
Dickeya Dadantii ◽  
Beechwood Xylan ◽  
Expression Characterization ◽  
Hydrolysis Of ◽  
Hydrolase Family

Xylanase plays an important role in the hydrolysis of hemicellulose and has gained much attention in the field of biological degumming. The research for xylanases with cellulase-free and high activity for biological degumming has intensified in recent years. In the present research, heterologous expression of a novel endo-β-1,4-xylanase (GH30) from Dickeya dadantii DCE-01 in Escherichia coli BL21 (DE3) was reported. Biochemical characterization of the enzyme and a potential application in ramie biological degumming was discussed. The results showed that the xylanase gene consists of 1251 nucleotides, belonging to glycoside hydrolase family 30 (GH30). The optimal activity of the xylanase was observed at 50℃ and a pH value of 6.4. The Km and Vmax values for beechwood xylan were 14.25 mg/mL and 296.6 μmol/mg, respectively. The catalytic activity was enhanced by addition of 1 mM Cu2+, Ca2+, Mg2+, and K+. The recombinant enzyme was specific for xylan substrates. The enzyme exhibited hydrolytic activity toward ramie hemicellulose. The recombinant xylanase could be effectively applied to ramie degumming.

scholarly journals Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

1988 ◽  
Vol 170 (12) ◽  
pp. 5901-5907 ◽  
Author(s):  
R Lahti ◽  
T Pitkäranta ◽  
E Valve ◽  
I Ilta ◽  
E Kukko-Kalske ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inorganic Pyrophosphatase ◽  
Gene Encoding ◽  
K 12

scholarly journals Characterization of Gelatin Profile of Chicken Broiler (Gallus domestica) Bone Using SDS-PAGE Electrophoresis

ALCHEMY ◽  
2019 ◽  
Vol 7 (1) ◽  
pp. 7 ◽  
Author(s):  
Dewi Yuliani ◽  
Dhienda Risa Awalsasi ◽  
Akyunul Jannah
Keyword(s):  
Ammonium Sulfate ◽  
Protein Concentration ◽  
Peptide Fragments ◽  
Strong Base ◽  
Chicken Bone ◽  
Sds Page ◽  
Hydrolysis Of ◽  
Β Chain ◽  
Page Electrophoresis

<p>Gelatin, a proteinaceous additive, is obtained from hydrolysis of collagen in the bone, hide and skin of animals. As natural product, gelatin has been applied in many industries with various functions. This study attempt to characterize gelatin profile of broiler chicken (<em>Gallus domestica</em>) using SDS-PAGE electrophoresis. The chicken bone was pretreated using a strong base, sodium hydroxide, producing type B gelatin. The gelatin was purified through precipitation using the variation of ammonium sulfate concentrations (40-70%) and dialysis using cellophane membrane. The purified gelatin was characterized through SDS-PAGE electrophoresis. Based on electrophoresis visualization, reduction of band intensity by ammonium sulfate 40% showed removal of small peptide fragments. The remained gelatin showed two major bands, α-chains and a β-chain with the respective molecular weight of ~135 and ~245 kDa. The protein content of the unpurified gelatin (E1) was 71.65±0.60 mg/L.  The purified E1 gelatins by 40-70% of ammonium sulfate addition contained 61.42±3.90, 60.45±1.36, 59.89±0.24, and 55.32±1.05 mg/L of protein concentration, respectively.</p><p> </p><p>Keywords: chicken bone, gelatin profile, protein electrophoresis</p>

scholarly journals Identification and Characterization of Bacterial Lipase from Plateu Soil in West Java

2017 ◽  
Vol 18 (02) ◽  
pp. 103-108
Author(s):  
Vivitri Dewi Prasasty ◽  
Vinella Winata ◽  
Muhammad Hanafi
Keyword(s):  
Heat Stability ◽  
Industrial Applications ◽  
West Java ◽  
Ph Optimum ◽  
Good Efficiency ◽  
Optimum Ph ◽  
Glycerol Ester ◽  
Lipase Screening ◽  
Hydrolysis Of

Lipases are known as glycerol ester hydrolases that catalyze the hydrolysis of triglycerides into free fatty acids and glycerol. Lipases are found in human, animal, plant, and microorganisms. The aim of this research is to identify lipase producers and characterize bacterial lipase from West Java plateau soil. Plateau soil bacteria samples were isolated on lipase screening medium containing Rhodamine B. Olive oil was used as a substrate in screening and production medium bacterial lipases. From 16 bacterial isolate of lipase producers, 14 were identified as Bacillus sp. and the others were identified as Pseudomonas alcaligenes. All isolates were taken into production step to determine their lipase activities. Moreover, top 3 lipase activities out of 16 lipase activities were chosen to find the optimum pH and temperature. Both characterizations showed pH optimum and temperature optimum from each lipase. These optimum condition were used in heat stability characterization for each lipase samples. The result showed that lipase from isolate COK 2 in optimum pH 4 and temperature 50oC was the most stable lipase due to this sample has good and stable activity for 1 to 5 hours incubation time. Lipase sample from isolate COK 2 has good efficiency for lipase productivity in acid condition and high temperature. Results of this investigation could encourage utilization of these activity enhancers for various industrial applications.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

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