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Optimum pH (optimum + ph)
Selected AbstractsPurification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10FEMS MICROBIOLOGY LETTERS, Issue 2 2002Kinya Fujishiro Abstract A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55°C, respectively. The enzyme acted on 3,-hydroxysteroids such as cholesterol, pregnenolone, and ,-sitosterol at high rates, but on dehydro- epi -androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (Km= 30,M). [source] Some properties of polyphenol oxidase from lilyINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2008Ying Yang Summary A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first-order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the Vmax/Km ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l -cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning. [source] Characterization of ,- d -glucosidase extracted from soil fractionsEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 2 2000M. D. Busto Summary One way to study the state in which stabilized extracellular enzymes persist and are active in the soil is by extraction from the soil, with subsequent fractionation of enzyme,organomineral complexes and characterization of such complexes. In order to investigate the location and characteristics of soil ,-glucosidase, three soil fractions were obtained both from real (undisturbed) soil aggregates and from structural (dispersed in water and physically disrupted) aggregates using two different granulometric procedures. The ,-glucosidase activity of the fraction was then assayed. When the aggregates were dispersed, more than 73% of activity was in the soil microaggregates with diameters of less than 50 ,m (SF50). These aggregates were associated with strongly humified organic matter. Solutions of diluted pyrophosphate at neutral pH liberated active ,-glucosidase from all fractions, although the efficacy of extraction varied according to the type of fraction. The SF50 fraction and aggregates of 2000,100 ,m obtained by sieving (SF2000) showed the greatest ,-glucosidase activity (34.5 and 36.0%, respectively). Micro- and ultrafiltration of SF50 extracts increased the total ,-glucosidase activity, whereas these procedures, applied to the RF2000 fraction, decreased it. Humus,,-glucosidase complexes in the SF50 fraction, between 0.45 ,m and 105 nominal molecular weight limit ( nmwl) (SF50II) and < 105nmwl (SF50III) showed an optimum pH at 5.4, and in the SF50I fraction (> 0.45 ,m) the optimum was 4.0. The stability of ,-glucosidase in the aggregates of the smallest size SF50II and SF50III decreased at acid pHs. The presence of two enzymes (or two forms of the same enzyme) catalysing the same reaction with different values of Michaelis constant and maximum velocity was observed in all but one of the ,-glucosidase complexes extracted and partially purified from the SF50 aggregates. [source] Identification of yeast aspartyl aminopeptidase gene by purifying and characterizing its product from yeast cellsFEBS JOURNAL, Issue 1 2006Ryo Yokoyama Aspartyl aminopeptidase (EC 3.4.11.21) cleaves only unblocked N-terminal acidic amino-acid residues. To date, it has been found only in mammals. We report here that aspartyl aminopeptidase activity is present in yeast. Yeast aminopeptidase is encoded by an uncharacterized gene in chromosome VIII (YHR113W, Saccharomyces Genome Database). Yeast aspartyl aminopeptidase preferentially cleaved the unblocked N-terminal acidic amino-acid residue of peptides; the optimum pH for this activity was within the neutral range. The metalloproteases inhibitors EDTA and 1.10-phenanthroline both inhibited the activity of the enzyme, whereas bestatin, an inhibitor of most aminopeptidases, did not affect enzyme activity. Gel filtration chromatography revealed that the molecular mass of the native form of yeast aspartyl aminopeptidase is ,,680 000. SDS/PAGE of purified yeast aspartyl aminopeptidase produced a single 56-kDa band, indicating that this enzyme comprises 12 identical subunits. [source] Numerical calculations of the pH of maximal protein stabilityFEBS JOURNAL, Issue 1 2004The effect of the sequence composition, three-dimensional structure A large number of proteins, found experimentally to have different optimum pH of maximal stability, were studied to reveal the basic principles of their preferenence for a particular pH. The pH-dependent free energy of folding was modeled numerically as a function of pH as well as the net charge of the protein. The optimum pH was determined in the numerical calculations as the pH of the minimum free energy of folding. The experimental data for the pH of maximal stability (experimental optimum pH) was reproducible (rmsd = 0.73). It was shown that the optimum pH results from two factors , amino acid composition and the organization of the titratable groups with the 3D structure. It was demonstrated that the optimum pH and isoelectric point could be quite different. In many cases, the optimum pH was found at a pH corresponding to a large net charge of the protein. At the same time, there was a tendency for proteins having acidic optimum pHs to have a base/acid ratio smaller than one and vice versa. The correlation between the optimum pH and base/acid ratio is significant if only buried groups are taken into account. It was shown that a protein that provides a favorable electrostatic environment for acids and disfavors the bases tends to have high optimum pH and vice versa. [source] Partial purification and characterisation of banana [Musa (AAA Group) ,Gros Michel'] polyphenol oxidaseINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 4 2009Chitsuda Chaisakdanugull Summary Polyphenol oxidase (PPO) from pulp of banana [Musa (AAA Group) ,Gros Michel'] was extracted and precipitated with 80% saturated ammonium sulphate followed by conventional column chromatography on Sephacryl S-200 HR and fast protein liquid chromatography on Mono Q column. The lyophilised PPO obtained from Sephacryl S-200 HR column was used for characterisation and inhibition studies. The partially purified PPO obtained from the Mono Q column exhibited at least three isoenzymes. The banana PPO had optimum pH for activity at 7 and it was stable around the same pH. Only 48% of initial enzyme activity was lost after heating at 70 °C for 30 min. The enzyme was completely inhibited by 2 mm sodium metabisulphite, 2 mm l -cysteine, 4 mm ascorbic acid, and 100 mm 4-hexylresorcinol. The Km and Vmax of banana PPO for dopamine were 2.08 mm and 0.124 mm min,1 respectively. [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] Microbial production, immobilization and applications of ,- D -galactosidaseJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 4 2006Parmjit S Panesar Abstract ,- D -Galactosidase (,- D -galactoside galactohydrolase, E.C. 3.2.1.23), most commonly known as lactase, is one of the most important enzymes used in food processing, which catalyses the hydrolysis of lactose to its constituent monosaccharides, glucose and galactose. The enzyme has been isolated and purified from a wide range of microorganisms but most commonly used ,- D -galactosidases are derived from yeasts and fungal sources. The major difference between yeast and fungal enzyme is the optimum pH for lactose hydrolysis. The application of ,- D -galactosidase for lactose hydrolysis in milk and whey offers nutritional, technological and environmental applications to human life. In this review, the main emphasis has been given to elaborate the various techniques used in recent times for the production, purification, immobilization and applications of ,- D -galactosidase. Copyright © 2006 Society of Chemical Industry [source] Optimization of extraction of bulk enzymes from spent mushroom compostJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2003Avneesh D Singh Abstract The profiling of ligninase, hemicellulase and cellulase of Pleurotus sajor-caju after inoculation of spawn in bags containing sawdust was done at monthly intervals for a period of 6 months. Xylanase (EC 3.2.1.8) was produced throughout the 6 months studied with the productivity range from 5.60 to 7.51 U g,1. Cellulase (EC 3.2.1.4) and ,-glucosidase (EC 3.2.1.21) productivities were highest at 4 months, producing 3.31 U g,1 and 121.13 U g,1 respectively. Laccase (EC 1.10.3.2) productivity was highest at 2 months with a value of 7.59 U g,1. Lignin peroxidase (EC 1.11.1.14) productivity was highest at 5 months with a value of 206.20 U g,1. Total soluble proteins were highest at 4 months with a value of 0.139 mg cm,3. The profiling of lignin peroxidase in 5-month-old spent mushroom compost was monitored over a period of 10 months. It was observed that lignin peroxidase was produced throughout the period but productivity was variable. The average lignin peroxidase productivity ranged from 30 to 110 U g,1. The activities of the enzymes extracted in tap water at pH 8.4 were comparable to that extracted in 50 mmol sodium citrate buffer at pH 4.8 and distilled water at pH 5.2 at 4 °C using an incubator shaker at 200 rpm for 18 h. The optimum extraction time was 1 h using an incubator shaker at 4 °C. When an incubator shaker was used, there was no significant difference in the recovery of xylanase, cellulase and laccase at different pH values at 4 °C and 28 °C. No significant difference was observed in the recovery of ,-glucosidase using an incubator shaker at different pH values at 4 °C although the enzyme recovery was slightly higher at pH 8.12, with a value of 29.27 U g,1. The optimum extraction of ,-glucosidase was at pH 4 at room temperature using an incubator shaker. For the lignin peroxidase enzyme, the optimum pH for extraction was 6 at 4 °C and pH 7 at room temperature using an incubator shaker at 200 rpm for 1 h. Homogenization for 8 min at 8000 rpm using tap water at pH 4 had an advantage over the use of the incubator shaker for the extraction as high titers of enzymes were recovered. Copyright © 2003 Society of Chemical Industry [source] Production of L -methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactorJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 5 2002Ying-Jin Yuan Abstract Production of L -methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60,°C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (Km and Vm) of immobilized pellets were found to be 7.99,mmol,dm,3 and 1.38,mmol,dm,3 h,1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2,mol,dm,3. The L -methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4,mol,dm,3, respectively, at a flow rate of 7.5,cm3,h,1 using the small-scale packed bed reactor developed. The half-life of the L -aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L -methionine. © 2002 Society of Chemical Industry [source] CLONING AND SEQUENCING OF THE ,-AMYLASE GENE FROM BACILLUS SUBTILIS US116 STRAIN ENCODING AN ENZYME CLOSELY IDENTICAL TO THAT FROM BACILLUS AMYLOLIQUEFACIENS BUT DISTINCT IN THERMAL STABILITYJOURNAL OF FOOD BIOCHEMISTRY, Issue 2 2010EZZEDINE BEN MESSAOUD ABSTRACT The gene encoding for the ,-amylase AMYUS116 was cloned and sequenced. The amino acid sequence of AMYUS116 exhibited an almost perfect homology with the ,-amylase BACAAM, excluding the residues N205 and N217 of AMYUS116 that were changed to H205 and I217 into BACAAM. Three mutant derivatives from AMYUS116 (N205H, N217I and N205H/N217I) were created by site-directed mutagenesis and their physicochemical and kinetic properties were compared with those of the wild-type enzymes. Therefore, the undertaken amylases mainly generated maltohexaose from starch and had radically the same kinetic parameters and optimum pH and temperature. They, however, were significantly distinct in thermal stability; AMYUS116 was more thermosensible as its half-life time at 80C was 13 min, while those of BACAAM and the double mutant were likewise 38 min. The single-mutant amylases exhibited an identically intermediate thermal stability as their half-life times at 80C were roughly 22 min. PRACTICAL APPLICATIONS Of particular interest to the current search is that the different thermal stability between AMYUS116 and BACAAM can lead to novel findings pertaining to protein stability, which can bring about new strategies for protein engineering. Basically, the comparative study of closely related amylases and the protein engineering of already existing ones are certainly important because they offer opportunities to understand the structure,function relationships of these biocatalysts. [source] AN ESTEROLYTIC ACTIVITY FROM A WILD EDIBLE MUSHROOM, LYCOPERDON PERLATUMJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2009AHMET COLAK ABSTRACT Lycoperdon perlatum Pers. (Lycoperdaceae, Agaricales, Agaricomycetidae, Agaricomycetes, Basidiomycota, Fungi) was evaluated for its esterolytic potential. Native electrophoresis of the crude extracts showed four bands having Rf values of 0.34, 0.39, 0.52 and 0.59. The esterase showed the highest activity toward a short-chain substrate, p -nitrophenyl acetate. Optimum reaction conditions for L. perlatum crude extract were attained at pH 8.0 and 40C. Esterolytic activity of enzyme extract was stimulated in the presence of Mn2+, Fe2+, Ca2+ and Zn2+ in the reaction mixture. The enzyme activity was stimulated by incubation at pH 6.0 but retained 77% of its original activity at its optimum pH after 24 h. Thermal inactivation was displayed after incubation for 20 min at various temperatures above 30C. At 1 mM final concentration, 2-mercaptoethanol, dithiothreitol, ethylenediamine tetraacetic acid and p -methylphenyl sulfonylfluoride inhibited the esterolytic reaction. These results support that the crude L. perlatum extract possesses an esterolytic activity having properties similar to other esterases. PRACTICAL APPLICATIONS Esterases catalyzing the cleavage and formation of ester bonds are known ,/,-hydrolases (EC 3.1.1.X). Esterases are used for the synthesis of flavor esters for the food industry, modification of triglycerides for fat and oil industry and resolution of racemic mixtures used for the synthesis of fine chemicals for the pharmaceutical industry. Therefore, the search for new enzyme sources is important for the development of new enzymes and applications. [source] PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp.JOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2004ABSTRACT Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. ,-CD yield of CGTase from alkalophilic Bacillus species is usually much lower than ,-CD, while from alkalophilic Bacillus sp. 7-12. ,-CD yield is close to ,-CD. A CGTase from alkalophilic Bacillus sp. 7-12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and Sepharose CL-6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS-PAGE. The enzyme showed a Kmof 1.24 mg/mL and Vmax0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6,10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag+, Cu2+, Mg2+, Al3+, Co2+, Zn2+, Fe2+and slightly inhibited by Sn2+, Mn2+. The ions Ca2+and K+, EDTA and DTT had no influence on the enzyme activity. [source] STABILITY AND PROPERTIES OF A THERMOSTABLE ,-GALACTOSIDASE IMMOBILIZED ON CHTTINJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2000JADWIGA MACIU ABSTRACT Thermostable ,-galactosidase from an E. coli transformant containing the enzyme gene from P. woesei was immobilized at pH 4.0 and a glutaraldehyde concentration of 10 mM on chitin isolated from shrimp Crangon crangon shells. These preparations had a specific activity of 43,000 U/g of chitin at 85C using ONPG as substrate. The optimum pH and temperature for immobilized ,-galactosidase activity were 5.2 and 93C. Immobilization shifts the optimum pH for the activity of the enzyme by 0.2 units towards the acid side. The immobilized enzyme is stable at temperatures close to the optimal value, and the residual activity for ONPG hydrolysis of the preparations incubated 5 h in 0.1 M phosphate citrate buffer (pH 5.4) at 90C and 100C was 70% and 40% of the initial value, respectively. [source] Endopeptidase Isoenzyme Characteristics in Cucumis sativus Leaves During Dark-induced SenescenceJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 4 2007Peng Zhang Abstract The changes and characteristics of endopeptidase (EP) isoenzymes in cucumber (Cucumis sativus L.) leaves during dark-induced senescence were investigated by activity staining after gradient-polyacrylamide gel electrophoresis (G-PAGE) containing co-polymerized gelatin as substrate. The results showed that both the chlorophyll and the protein contents of leaves were decreased, and the protein degradation was correlated with the increase of proteolytic activity during the course of leaf senescence. Meanwhile, nine cucumber endopeptidases isoenzymes (CEP) with 140, 120, 106, 94, 76, 55, 46, 39 and 35 kDa molecular weights were detected. Four of these, CEP2, 3, 4 and CEP9 appeared all the time, but the changes of the activity were different during incubation. Another four CEPs (CEP5, 6, 7 and CEP8) whose activities increased with dark-induced time were only detected in senescent leaves. Furthermore, the biochemical properties of these nine CEP were also characterized. All the CEPs had high activities from 35 °C to 45 °C, and the optimum temperature was found to be 40 °C. However, the activities of CEPs were not detected below 25 °C or over 60 °C. The activity bands appeared at a wide range of pH from 5.0 to 9.0, but the optimum pH was found at 7.0. No CEPs were detected at pH 4 or pH 10. By inhibition analysis we concluded that CEP2, 3, 4 and CEP9 were serine endopeptidases and CEP6 was a kind of cysteine protease. It is suggested that serine endopeptidases might play a major role in cucumber leaf senescence, and for the first time, six senescence-related endopeptidases (CEP1, 5, 6, 7, 8 and 9) were found in cucumber leaves. [source] Structural Features of the NAD-Dependent In Situ Retinoic Acid Supply System in Esophageal MucosaALCOHOLISM, Issue 2010Hirokazu Yokoyama Background:, We previously reported that an NAD-dependent in situ retinoic acid supply system, which comprises some isoforms of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) and provides retinoic acid from retinol via a 2-step oxidation process, exists in the rat esophagus. Herein, their isoforms responsible for the pathway and its localization in the rat esophagus was examined. Methods:, The expressions of mRNAs of various isoforms of ADH and ALDH were examined in the fraction mainly comprising mucosal layer of the rat esophagus by RT-PCR. Expression levels of Class IV ADH and ALDH 1A1 were compared between the fractions and that mainly comprising muscle layer of the rat esophagus by quantitative PCR. The catalytic activities producing retinoic acid from retinal were compared between the 2 fractions and its optimum pH was also determined. Results:, Classes I, III, and IV ADHs and ALDHs 1A1 and 3A1 were predominant isoforms in the rat esophageal mucosa. The expression levels of mRNA of Class IV ADH and ALDH 3A1 were significantly higher in the mucosal than in the muscle layer. Consistently, the catalytic activities producing retinoic acid from retinal were significantly higher in the former than the latter. The optimum pH of the process was 9.0. Conclusions:, Considering the affinities for retinol and retinal of ADHs and ALDHs expressed in the rat esophagus, the NAD-dependent in situ retinoic acid supply system in the rat esophagus is thought to comprise Class IV ADH and ALDH 1A1. In the rat esophagus, the system exists predominantly in the mucosal layer. [source] Kinetic behaviour and stability of Escherichia coli ATCC27257 alkaline phosphatase immobilised in soil humatesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 3 2003María C Pilar Abstract Alkaline phosphatase (EC 3.1.3.1) extracted from Escherichia coli ATCC27257 was immobilised by co-flocculation with soil humates in the presence of Ca2+. The effects of time, temperature, pH and concentration of enzyme and support on immobilisation were studied. Between 58 and 92% of the added phosphatase was strongly bound to the humates, depending on the conditions of immobilisation used. Some characteristics of the humate,phosphatase complexes and of the free enzyme were compared. The enzymatic complexes showed values of Km (2.22,mM) and activation energy (33.4,kJ,mol,1) similar to those of the free enzyme (2.00,mM and 27.6,kJ,mol,1). The pH/activity profiles revealed no change in terms of shape or optimum pH (10.5) upon immobilisation of alkaline phosphatase. However, the immobilised enzyme showed maximal activity in the range of 80,100,°C, while the free enzyme had its highest activity at 60,°C. The thermal stability of alkaline phosphatase was enhanced by complexation to the soil humates. © 2003 Society of Chemical Industry [source] Structure and enzyme properties of Zabrotes subfasciatus ,-amylaseARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2006Patrícia B. Pelegrini Abstract Digestive ,-amylases play an essential role in insect carbohydrate metabolism. These enzymes belong to an endo-type group. They catalyse starch hydrolysis, and are involved in energy production. Larvae of Zabrotes subfasciatus, the Mexican bean weevil, are able to infest stored common beans Phaseolus vulgaris, causing severe crop losses in Latin America and Africa. Their ,-amylase (ZSA) is a well-studied but not completely understood enzyme, having specific characteristics when compared to other insect ,-amylases. This report provides more knowledge about its chemical nature, including a description of its optimum pH (6.0 to 7.0) and temperature (20,30°C). Furthermore, ion effects on ZSA activity were also determined, showing that three divalent ions (Mn2+, Ca2+, and Ba2+) were able to enhance starch hydrolysis. Fe2+ appeared to decrease ,-amylase activity by half. ZSA kinetic parameters were also determined and compared to other insect ,-amylases. A three-dimensional model is proposed in order to indicate probable residues involved in catalysis (Asp204, Glu240, and Asp305) as well other important residues related to starch binding (His118, Ala206, Lys207, and His304). Arch. Insect Biochem. Physiol. 61:77,86, 2006. © 2006 Wiley-Liss, Inc. [source] Purification and Characterization of Thermostable ,-Amylase from a Newly Isolated Thermophilic Bacillus stearothermophilus GRE 1ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 5-6 2005S.M. Zakir Hossain A thermophilic bacterium, Bacillus stearothermophilus GRE 1, isolated from an Ethiopian hyperthermal spring produced a thermostable ,-amylase. Enzyme production in shake flask experiments while using optimum nutrient supplements and environmental conditions was 2.35 U/ml. Under optimized conditions in a bioreactor, 5-6 fold higher enzyme activity was obtained than that of a shake flask. Gel filtration chromatography yielded a purification factor of 33.62-fold and a recovery of 46.52%. The optimum temperature for activity was determined to be 60-70d,C and optimum pH was in the range of 5.5-6.0. The enzyme maintained 50% of its original activity after 45 minutes of incubation at 80d,C, and is stable at pH values of 5.0-9.0. Enzyme activity was strongly inhibited by Cu2+, Zn2+ and Fe2+. The enzyme is calcium independent and 94% and 86% relative activities were displayed with low concentrations of Co2+ and Mg2+, respectively. [source] First report of an antifungal amidase from Peltophorum ptercoarpumBIOMEDICAL CHROMATOGRAPHY, Issue 5 2010Sze Kwan Lam Abstract A 60,kDa antifungal amidase was purified from Peltophorum ptercoarpum seeds using an isolation procedure that entailed ion-exchange chromatography on Q-Sepharose, ion-exchange chromatography on DEAE-cellulose and FPLC,gel filtration on Superdex 75. Unlike most other antifungal proteins isolated previously, it was adsorbed on Q-Sepharose and DEAE-cellulose. The isolated protein, designated as peltopterin, exhibited an N -terminal amino acid sequence closely resembling those of amidases. It exhibited amidase activity and digested iodoacetamide with an optimum pH and temperature at pH 9 and 50°C, respectively. It also hydrolyzed acrylamide and urea. It impeded mycelial growth in Rhizotonia solani with an IC50 of 0.65,,m. Chitin deposition at hyphal tips in R. solani was observed by staining with Congo red after incubation with peltopterin. Its antifungal activity was stable throughout pH 0,14 and 25,100°C. It potently inhibited HIV-1 reverse transcriptase with an IC50 of 27,nm. Copyright © 2009 John Wiley & Sons, Ltd. [source] Structure of d -3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate d -3-hydroxybutyrate and NAD+ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Md Mominul Hoque d -3-Hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between d -3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate d -3-hydroxybutyrate and the cofactor NAD+ at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD+ and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of d -3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure. [source] Mung bean lipoxygenase in the production of a C6-aldehyde.BIOTECHNOLOGY JOURNAL, Issue 11 2007Natural green-note flavor generation via biotransformation Abstract Mung bean was investigated as a novel source of lipoxygenase in the natural production of the green-note aroma compound hexanal. Lipoxygenase extracted from mung bean catalyzed the oxidative reaction of linoleic acid, after which the intermediate hydroperoxide compound was split via green bell pepper hydroperoxide lyase to produce hexanal. In comparison to soybean lipoxygenase, mung bean lipoxygenase was found to be a good substitute as it produced 15.4 mM (76% yield) hexanal while soybean gave 60% yield. The mung bean pH profile comprised a wide peak (optimum pH 6.5) representing lipoxygenase-2 and lipoxygenase-3 isozymes, whereas two narrower peaks representing lipoxygenase-1 and lipoxygenase-2/3 isozymes were observed for soybean (optimum pH 10). Extraction at pH 4.5 was preferred, at which specific lipoxygenase activity was also the highest. [source] ,-Glucosidase-Catalyzed Hydrolysis of Indican from Leaves of Polygonum tinctoriumBIOTECHNOLOGY PROGRESS, Issue 5 2002Thierry Maugard In this article, a HPLC method to identify and quantify the dyes and the indigo precursors produced in Polygonum tinctorium is described. Using this technique, indican has been positively identified in extracts of P. tinctorium. Our work with two cultivars of P. tinctorium has confirmed that the quantity of indican is dependent on the cultivars, harvest period, and age of the leaves. Two enzymes, Novozym 188 (cellobiase) and Novarom G ( , -glucosidase), are compared on the basis of their activities to hydrolyze the indican at several pH values. We observed that Novarom G is more active than Novozym 188 whatever the pH and that optimum pH of both enzymes for indican hydrolysis is 3. Liberated indoxyl can be oxidized in alkaline media and transformed into indigo and indirubin. [source] Dispersant-free dyeing of polyester with temporarily solubilised azo disperse dyes from 1-substituted-2-hydroxypyrid-6-one derivativesCOLORATION TECHNOLOGY, Issue 4 2002J J Lee Four temporarily solubilised azo disperse dyes based on 1-substituted-2-hydroxypyrid-6-one were synthesised and characterised. The dyes showed high extinction coefficients and had a yellow shade on polyester fabric. They were successfully applied to polyester without the use of dispersants and the optimum pH was found to be 5. It was found that the dyes could be alkali-cleared due to ionisation of the dye under mild alkaline conditions. The dyes exhibited good to excellent fastness properties on the polyester fabric. [source] Numerical calculations of the pH of maximal protein stabilityFEBS JOURNAL, Issue 1 2004The effect of the sequence composition, three-dimensional structure A large number of proteins, found experimentally to have different optimum pH of maximal stability, were studied to reveal the basic principles of their preferenence for a particular pH. The pH-dependent free energy of folding was modeled numerically as a function of pH as well as the net charge of the protein. The optimum pH was determined in the numerical calculations as the pH of the minimum free energy of folding. The experimental data for the pH of maximal stability (experimental optimum pH) was reproducible (rmsd = 0.73). It was shown that the optimum pH results from two factors , amino acid composition and the organization of the titratable groups with the 3D structure. It was demonstrated that the optimum pH and isoelectric point could be quite different. In many cases, the optimum pH was found at a pH corresponding to a large net charge of the protein. At the same time, there was a tendency for proteins having acidic optimum pHs to have a base/acid ratio smaller than one and vice versa. The correlation between the optimum pH and base/acid ratio is significant if only buried groups are taken into account. It was shown that a protein that provides a favorable electrostatic environment for acids and disfavors the bases tends to have high optimum pH and vice versa. [source] | ||||||||||||||||||||||||||||