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Heat Stable (heat + stable)
Selected AbstractsProbing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutantsFEBS JOURNAL, Issue 14 2001Bernard Clantin The Pyrococcus furiosus ornithine carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 °C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization. [source] Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from KimchiJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2008M.S. Shin Abstract Aims:, Screening and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi, a traditional Korean fermented vegetable. Methods and Results:, A total of 1000 lactic acid bacteria were isolated from various Kimchi samples and screened for the production of bacteriocin. Pediocin K23-2, a bacteriocin produced by the Pediococcus pentosaceus K23-2 strain, showed strong inhibitory activity against Listeria monocytogenes. The bacteriocin activity remained unchanged after 15 min of heat treatment at 121°C or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The bacteriocin was maximally produced at 37°C, when the pH of the culture broth was maintained at 5·0 during the fermentation, although the optimum pH for growth was 7·0. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis. Conclusions:,Pediococcus pentosaceus K23-2 isolated from Kimchi produces a bacteriocin, which shares similar characteristics to the Class IIa bacteriocins. The bacteriocin is heat stable and shows wide antimicrobial activity against Gram-positive bacteria, especially L. monocytogenes. Significance and Impact of the Study:, Pediocin K23-2 and pediocin K23-2-producing P. pentosaceus K23-2 could potentially be used in the food and feed industries as natural biopreservatives, and for probiotic application to humans or livestock. [source] Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airagJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2006B. Batdorj Abstract Aims:, The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. Methods and Results:, Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2,10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and , -chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N -termini were blocked hampering straightforward Edman degradation. Conclusions:, Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. Significance and Impact of the Study:, Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998). [source] Isolation and characterization of a protease from Pseudomonas fluorescens RO98JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2000R. Koka Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 °C for 48 h to allow production of protease. A zinc-metalloacid protease was purified from the cell-free concentrate by anion exchange and gel filtration chromatography. The purified protease was active between 15 and 55 °C, and pH 4·5 and 9·0, and was stable to pasteurization. The enzyme had pH and temperature optima for activity of 5·0 and 35 °C, respectively. It was heat stable with a D55 of 41 min and a D62·5 of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for kM of 144·28, 18·73, 110·20 and 35·23 µmol were obtained for whole, ,-, ,- and ,-casein, with a Vmax of 8·26, 0·09, 0·42 and 0·70 µmol mg,1 min,1, respectively. The enzyme hydrolysed ,-casein preferentially when incubated with artificial casein micelles. [source] Sequencing, expression, and characterization of cDNA expressed flavin-containing monooxygenase 2 from mouseJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2001Edward D. Karoly Abstract The cDNA clone of mouse flavin-containing monooxygenase 2 (FMO2) was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 1432164). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2, which contains 112 bases of 5, flanking region, 1607 bases of coding region, and 309 bases of 3, flanking region. This FMO2 sequence encodes a protein of 535 amino acids including two putative pyrophosphate binding sequences (GxGxxG/A) beginning at positions 9 and 191. Additionally, this mouse FMO protein sequence shows 87 and 86% homology to rabbit and human FMO2 respectively. The mouse FMO2 sequence was subcloned into the expression vector pJL-2, a derivative of pKK233-2 and used to transform XL1-Blue Escherichia coli. FMO activity in particulate fractions isolated from isopropyl-,-D-thiogalactopyanoside (IPTG) induced cells was heat stable (45°C for 5 min) and demonstrated optimal activity at a relatively high pH of 10.5. The expressed FMO2 enzyme showed catalytic activity towards the FMO substrate methimazole and further analysis of E. coli fractions utilizing NADPH oxidation demonstrated that the mouse FMO2 enzyme also exhibits catalytic activity towards thiourea, trimethylamine, and the insecticide phorate. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:300,308, 2001 [source] ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITORS FROM SOME THAI LEGUME SEEDSJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2000SOOTTAWAT BENJAKUL ABSTRACT Trypsin inhibitors from cultivars of cowpea (Vigna unguiculata (L.) Wasp.), pigeon pea (Cajanus cajan (L.) Millsp.) and bambara groundnuts (Voandzeia subterranea (L.) Thou) grown in Thailand were isolated and characterized. Extraction of seeds with NaCl rendered a higher recovery of trypsin inhibitor than other solvents tested (P<0.05). The extraction time affected the inhibitor recovery (P<0.05). The extraction time of 3 h was optimum for the recovery of trypsin inhibitor from pigeon and bambara groundnuts, whereas 1 h was optimum for cowpea. Based cn inhibitor activity of zones separated by electrophoresis, the molecular mass of the inhibitor from bambara groundnuts was 13 kDa. Two inhibitory bands were observed for cowpea (10 and 18 kDa) and pigeon pea (15 and 25 kDa). Partial purification of inhibitors was achieved by heat-treatment at 90C for 10 min, followed by ammonium sulfate precipitation with 30,65% saturation. The partially purified inhibitors from four seeds were heat stable up to 30 min at 90C at pH 7.0. The activities were also retained over a wide pH range at 25C but were lost when samples were treated with ,-mercaptoethanol prior to electrophoresis. [source] 163 Identification of Euglenoids That Produce Ichthyotoxin(S) (Euglenophyta)JOURNAL OF PHYCOLOGY, Issue 2003R. E. Triemer Diatoms, dinoflagellates, pelagiophytes, prymnesiophytes, and cyanobacteria are the only divisions of microalgae known to produce toxins. We now report toxin production by freshwater members of the genus Euglena. Fish mortalities (sheepshead minnows, catfish, striped bass, and tilapia) have been observed following exposure in the field to Euglena blooms and in the laboratory when exposed to unialgal isolates of two species of Euglena (E. sanguinea Ehrenberg and E. granulata (Klebs) Lemm.). Three toxic fractions have been isolated from unialgal isolates of both species, and include both water soluble and lipophilic compounds having ichthyotoxic activity. The toxins are stable at ,80°C for at least 60 days and are heat stable to 30°C. Erratic swimming behavior of fish suggests a neurological toxin. This is the first report of fish kills by any freshwater algal taxa from both field and laboratory studies. [source] | |||||||||||||