Home About us Contact
|
Home About us Contact | ||
|
|
||
U Mg (u + mg)
Selected AbstractsGlucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: expression of the g6pd gene and characterization of an extremely thermophilic enzymeFEMS MICROBIOLOGY LETTERS, Issue 2 2002Thomas Hansen Abstract The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80°C) on glucose-6-phosphate and NADP+ followed Michaelis,Menten kinetics with apparent Km values of 0.15 mM and 0.03 mM, respectively; apparent Vmax values were about 20 U mg,1. The enzyme also reduced NAD+ (apparent Km 12 mM, Vmax 12 U mg,1). The 1000-fold higher catalytic activity (kcat/Km) with NADP+ over NAD+ defines the G6PD as NADP+ specific in vivo. G6PD activity was competitively inhibited by NADPH with a Ki value of 0.11 mM. With a temperature optimum of 92°C the enzyme is the most thermoactive G6PD described. [source] Pigment and amylase production in Penicillium sp NIOM-02 and its radical scavenging activityINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 12 2009Mohan Appasaheb Dhale Abstract Penicillium sp NIOM-02 was isolated from the marine sediment, produced red pigment. The pigment extracted from this fungus scavenged 2, 2-diphenyl-1-pycrylhydrazyl (DPPH) radical. Penicillium sp NIOM-02 grown in media containing corn steep liquor scavenged 72,88% of DPPH radical. During solid-state fermentation on wheat (S1), the fungus produced more pigment (9.232 OD Units). Penicillium sp NIOM-02 grown on sugarcane bagasse scavenged 91% of DPPH radicals. It secreted more amylase (246 U mg,1) in culture medium No. 5 and the zymogram analysis revealed its molecular mass (53 kDa). The taka-amylase like character of amylase was determined by acarbose incorporated studies in the culture media. Production of pigment and radical scavenging activity of Penicillium sp NIOM-02, suggested its applications in food, pharmaceuticals and nutraceutical industries. [source] Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodiesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008D.E.C.S. Rao Abstract Aims:, To isolate, clone and express a novel phytase gene (phy) from Bacillus sp. in Escherichia coli; to recover the active enzyme from inclusion bodies; and to characterize the recombinant phytase. Methods and Results:, The molecular weight of phytase was estimated as 40 kDa on SDS-polyacrylamide gel electrophoresis. A requirement of Ca2+ ions was found essential both for refolding and activity of the enzyme. Bacillus phytase exhibited a specific activity of 16 U mg,1 protein; it also revealed broad pH and temperature ranges of 5·0 to 8·0 and 25 to 70°C, respectively. The Km value of phytase for hydrolysis of sodium phytate has been determined as 0·392 mmol l,1. The activity of enzyme has been inhibited by EDTA. The enzyme exhibited ample thermostability upon exposure to high temperatures from 75 to 95°C. After 9 h of cultivation of transformed E. coli in the bioreactor, the cell biomass reached 26·81 g wet weight (ww) per l accounting for 4289 U enzyme activity compared with 1·978 g ww per l producing 256 U activity in shake-flask cultures. In silico analysis revealed a ,-propeller structure of phytase. Conclusions:, This is the first report of its kind on the purification and successful in vitro refolding of Bacillus phytase from the inclusion bodies formed in the transformed E. coli. Significance and Impact of the Study:, Efficient and reproducible protocols for cloning, expression, purification and in vitro refolding of Bacillus phytase enzyme from the transformed E. coli have been developed. The novel phytase, with broad pH and temperature range, renaturation ability and substrate specificity, appears promising as an ideal feed supplement. Identification of site between 179th amino acid leucine and 180th amino acid asparagine offers scope for insertion of small peptides/domains for production of chimeric genes without altering enzyme activity. [source] Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applicationsJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2005S. Halaouli Abstract Aims:, Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. Methods and Results:, Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8,10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45·4 and 163·6 U g,1 protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35,38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4·5,5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6,7, in a large temperature range (30,70°C), and was stable below 60°C. The main kinetic constants were determined. The tyrosinase was able to convert p -tyrosol and p -coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. Conclusions:, Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg,1 protein for monophenolase and diphenolase respectively. Significance and Impact of the Study:, This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking. [source] pH Control of the production of recombinant glucose oxidase in Aspergillus nidulansJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2004R. Luque Abstract Aims:, Recombinant Aspergillus nidulans sVAL040, capable of synthesizing and secreting glucose oxidase derived from Aspergillus niger was used to study the influence of pH and carbon source on enzyme production. Methods and Results:, Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnB gene (encoding an acidic xylanase). A maximum specific glucose oxidase activity of approx. 10 U mg,1 protein and a maximum volumetric productivity of 29·9 U l,1 h,1 were obtained at pH 5·5, after 80 h of growth by using xylose as inducer. Enzyme volumetric productivity increased when xylans were used instead of xylose; however, specific glucose oxidase activity did not differ significantly. Conclusions:, Specific GOX activity obtained at pH 5·5 are two to three times more than those previously described for goxC multicopy transformants of A. nidulans. Xylans were a more powerful inducer than xylose although fungal growth was lower when the polymers were used. Significance and Impact of the Study:, The obtained results by using xlnB promoter in A. nidulans could be useful in improving heterologous enzyme production by using genetic- and process-engineering strategies. [source] Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesisJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004L. Wu Abstract Aims:, Isolation, identification and characterization of a highly efficient isomaltulose producer. Methods and Results:, After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis. An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity. Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90,100 U mg,1 DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41,66 U mg,1 DW) or a reference strain of Erwinia rhapontici (0·3,0·9 U mg,1 DW). Maximum yield of isomaltulose at 78,80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe. UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose. Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype. Significance and Impact of Study:, This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose. [source] Purification of Aspergillus carbonarius polygalacturonase using polymeric membranesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2008E. Nakkeeran Abstract BACKGROUND: Microfiltration (MF: 70,450 nm) and ultrafiltration (UF: 10,500 kDa) membranes were used to eliminate carbohydrates and other non-protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes. RESULTS: MF resulted in removal of 2,25% carbohydrates with an enzyme recovery of 69,82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10-fold while the purity improved from 4.6 to 49.4 U mg,1 of dry matter. CONCLUSIONS: The results of this study focused on the elimination of carbohydrates and other non-protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non-protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. Copyright © 2008 Society of Chemical Industry [source] Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2004Augustine C Ogbonna Abstract A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion-exchange chromatography on Q- and S-Sepharose (fast flow), gel filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The enzyme was purified 8.4-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg,1 protein. SDS,PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0,8.0 and retained over 60% activity at 70 °C after 30-min incubation. It was highly significantly (P < 0.001) inhibited by Hg2+, appreciably (P < 0.01) inhibited by Ag+, Ba2+ and Pb2+ but highly significantly (P < 0.001) activated by Co2+, Mn2+ and Sr2+. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p -chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: Km = 0.33 mg ml,1; Vmax = 0.08 µmol ml,1 min,1. Copyright © 2004 Society of Chemical Industry [source] Description of the chemical and pharmacological characteristics of a new hemisynthetic ultra-low-molecular-weight heparin, AVE5026JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2009C. VISKOV Summary.,Background and objectives: AVE5026 is a novel, hemisynthetic, ultra-low-molecular-weight heparin (ULMWH), which is in clinical development for prevention of venous thromboembolism. Its unique structural features result from the highly selective depolymerization of heparin by the phosphazene base that protects the antithrombin (AT)-binding site from destruction. In the present paper, we describe the chemical and biological characteristics of AVE5026, as well as its effects on experimental thrombosis as compared to those of the low-molecular-weight heparin (LMWH) enoxaparin after a single subcutaneous (s.c.) administration in certain animal models. Method and results: AVE5026 has a higher anti-factor Xa (anti-FXa) activity (,160 U mg,1) along with a catalytic anti-thrombin (anti-FIIa) activity (,2 U mg,1) as a result of its structure being strongly enriched in specific AT-binding oligosaccharides. In human plasma, potent inhibition of thrombin generation by AVE5026 was closely related to its anti-FXa activity. In a rat venous thrombosis model, AVE5026 showed a dose-dependent antithrombotic activity comparable to that of enoxaparin (ED50-AVE5026 = 1.6 mg kg,1, ED50-enoxaparin = 2.8 mg kg,1). Interestingly, non-occlusive venous thrombosis in rabbits was inhibited by an ED50 of 0.1 mg kg,1 AVE5026, whereas 0.316 mg kg,1 enoxaparin was not active. In a canine model, similarly to enoxaparin (ED50 = 1.3 mg kg,1), AVE5026 dose-dependently inhibited arterial thrombosis (ED50 = 2.0 mg kg,1). At equipotent doses, AVE5026 did not affect bleeding parameters, whereas enoxaparin showed increased hemorrhage in rats, rabbits and dogs. Conclusion: These unique structural attributes distinguish AVE5026 from the LMWH class. Based on these data in well-established arterial and venous thrombosis models, AVE5026 could represent a valuable alternative in thrombosis prevention with an improved benefit-risk profile as compared to that of enoxaparin. [source] Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiaeLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2007J. Hou Abstract Aims:, To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. Methods and Results:, The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP+ -dependent activity in both strains with mutated XDH, reaching 0·782 and 0·698 U mg,1. In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. Conclusions:, Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. Significance and Impact of the Study:, The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important. [source] Digestive enzyme activity at different developmental stages of blackspot seabream, Pagellus bogaraveo (Brunnich 1768)AQUACULTURE RESEARCH, Issue 4 2008Laura Ribeiro Abstract Blackspot seabream, Pagellus bogaraveo (Brunnich), has been identified as a potential species to diversify European aquaculture production. Although rearing aspects have been widely investigated, little information exists on the nutritional requirements for this species. The aim of this study was to build up information on the activity of digestive enzymes at certain developmental stages of blackspot seabream in order to understand the nutritional needs of larvae and post larvae. Fish larvae were reared from hatching to 55 days after hatching (dah), and the feeding plan consisted in rotifers (5,35 dah), Artemia naupli (30,35 dah) metanaupli (35,45) and Gemma microdiet (45,55 dah). At 7, 11, 21, 45 and 55 days after hatching (dah), pooled samples of fish larvae were collected for analysis of trypsin, amylase, lipase, alkaline phosphatase and leucine,alanine peptidase activity. Up to 21 dah, the whole larvae body was used for enzymatic analysis, whereas in older larvae only the dissected abdominal cavity was used. Blackspot seabream body dry weight growth was exponential, increasing from 60 ,g at 5 dah to 30±9.7 mg at 55 dah. Amylase specific activity decreased significantly during development, exhibiting at 11 dah (0.6 U mg,1 protein) an average value 2.7 times lower than at 7 dah, and remaining stable between 45 and 55 dah (0.7 U mg protein,1). Trypsin specific activity remained constant until 21 dah (between 38 and 44 mU mg protein,1), which could be related to the larvae feeding regime. At later stages of development, lipase-specific activity exhibited a significant increase (P<0.05), being three times higher at 55 dah (8 U mg protein,1) than at 45 dah. The total activity of the studied digestive enzymes increased significantly during larval development (until 21 dah), whereas afterwards only lipase and leucine,alanine peptidase increased significantly between 45 and 55 dah. The pattern of digestive enzymes activity was related to organogenesis and the type of food used at different developmental stages. [source] | |||||||||||||