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1.
Marco Filice Marzia Marciello Lorena Betancor Alfonso V. Carrascosa Jose M. Guisan Gloria Fernandez‐Lorente 《Biotechnology progress》2011,27(4):961-968
Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 相似文献
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Antonella Canini Lorena Canuti Maria Grilli Caiola Maddalena Del Gallo 《Annals of microbiology》2006,56(4):393-398
In the present work an immunocytochemical characterisation of four endophytic bacterial species has been made by using polyclonal antiserum produced against each of the four bacterial strains previously heated at 60 °C. The aim of this researchsito identify common elements among bacteria associated with their endophytic behaviour. Analysis of extracts of each strain by immunoblotting and ELISA confirmed the presence of proteins from different bacterial strains made up of common epitopes. However, antisaproduced againstHerbaspirillum seropedicae andBurkholderia ambifaria show a high number of bands recognised on each extracts, while antisera againstAzospirillum brasilense andGluconacetobacter diazotrophicus show a low number of bands recognised on each extract. Immunogold labelling showed that epitopes are located both on the cell wall and in the cytoplasm; most likely they could be preursor cell wall proteins synthesized inside the cytoplasm and subsequently transported onto cell wall. Finally, the common bands amog bacterial strains revealed by immunoblotting could play a role as active hydrolases involved in host tissue penetration. 相似文献
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Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells. 总被引:3,自引:1,他引:2 下载免费PDF全文
P P Powell J W Kyle R D Miller J Pantano J H Grubb W S Sly 《The Biochemical journal》1988,250(2):547-555
A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells. 相似文献
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Simone Gilgenkrantz Claudine Blanchet-Bardon V. Nazzaro Lorena Formiga Patricia Mujica Y. Alembik 《Human genetics》1989,81(2):120-122
Summary A family carrying the X-linked gene for hypohidrotic ectodermal dysplasia (hereditary ectodermal polydysplasia or Christ-Siemens-Touraine syndrome) over three generations was monitored for more than 15 years. Two prenatal diagnoses were carried out by fetoscopy on skin biopsies. Polymorphic probes were used in the segregation analysis of the Xq11–21 region carried out on 30 members of the family. Current screening possiblitities for the carriers and prenatal diagnosis are discussed. 相似文献
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Co-aggregation of penicillin g acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media 总被引:1,自引:0,他引:1
Wilson L Illanes A Abián O Pessela BC Fernández-Lafuente R Guisán JM 《Biomacromolecules》2004,5(3):852-857
A novel type of biocatalyst that combines the good properties of cross-linked enzyme aggregates (CLEAs) and hydrophilic microenvironments has been developed. Dextran sulfate- and polyethyleneimine-coated CLEAs of penicillin acylase (CLEA-GDP) were prepared by adding the polymers of different sizes before the precipitation stage of the enzyme. This study presents the development and optimization of a protocol to produce such a biocatalyst using penicillin acylase as a model. Experiments show that CLEA-GDPs have a highly increased stability in organic media. The average half-life of the preparations was much higher than standard CLEA without a microenvironment (CLEA-G), (e.g., more than 25-fold) in the presence of dioxane. However, their thermal stability was not increased, which leads to the conclusion that the stability of CLEA-GDPs in organic media is due to the hydrophilic microenvironment that surrounds the protein enzyme more than to a conformational stiffening effect. This is further supported by solvation experiments that show a preferential hydration of CLEA when polymers are used to coat the enzyme. CLEA-GDPs are clearly better than other biocatalysts in terms of solvent stability. 相似文献
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Maria I. B. de Oliveira Lorena V. de Matos Lídia A. da Silva Edsandra C. Chagas Grazyelle S. da Silva Ana L. S. Gomes 《Journal of fish biology》2019,94(4):648-659
The objective of the present study was to describe the histology and histochemistry of the mucosal layer of the digestive tube of Piaractus brachypomus, and the histopathology associated with parasitism by Neoechinorhynchus sp. The digestive tube of P. brachypomus consists of three macroscopically distinct portions: short, rectilinear and elastic-walled ooesophagus, J-shaped siphon stomach and a long intestine with rectilinear and curved portions, defined by patterns of villi as foregut, midgut, and hindgut. Histological and histochemical differences were observed in the mucosal layers of the different digestive tube regions, such as intense production of neutral and acidic mucous substances in the pseudostratified mucosal epithelium of the oesophagus; positive periodic acid Schiff reagent (PAS)reactions at the apex of the columnar epithelial cells of the stomach and increased intensity of histochemical reactions in the hindgut region. Neoechinorhynchus sp. was present in 85.7% of specimens examined, with a mean intensity of 7.4 ± 6.2 (±) and abundance of 6.33. Good health of the fish indicated by high relative condition factor values ( Kn) and occurrence of only mild to moderate alteration in the mucosal layer indicated that Neoechinorhynchus sp. exhibits low pathogenicity towards P. brachypomus hosts in farming environments, with low levels of infection. 相似文献