Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

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     

Immunocytochemical characterisation of endophytic bacteriaAzospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria andGluconacetobacter diazotrophicus

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.     

Virus transmission to man from Phlebotomus flies: role of the Tuscany virus (Bunyaviridae, Phlebovirus) in the etiology of infections of the central nervous system

During field studies on the ecology of arboviruses in Italy, strains of a new virus were isolated from sand flies, mostly females, of a pure colony of Phlebotomus perniciosus collected in a central Italian region (Tuscany). The prototype was named Toscana (TOS) virus, assigned to the Phlebotomus fever serogroup of arboviruses (family Bunyaviridae, genus Phlebovirus) and registered in 1980 in the International Catalogue of Arboviruses. Neutralizing antibodies to TOS virus were detected in human sera. Serologic surveys were carried out to estimate the natural distribution of TOS virus in Italy and it was shown that antibodies to this virus could be detected in humans mainly of the Tuscany region. In particular, a high infection rate (24.8%) was observed among residents of the province of Florence. Furthermore, several patients with a diagnosis of aseptic meningitis presented a serologic response which reflected a recent infection with TOS virus in the Tuscany and Marche regions. Consequently, between 1980 and 1986 studies were conducted at selected sites of the Tuscany region to determine the possible occurrence of vectors and presence of foci of TOS virus and to assess its public health importance. As a result of these investigations, several virus strains were isolated from pools of wild caught Ph. perniciosus and Ph. perfiliewi. Of the virus isolates obtained, 37 were identified as TOS virus and 47 as a new serotype, member of the Phlebotomus fever group. For this virus we suggested the name of Arbia (ARB) virus, Arbia being the river flowing across both Florence and Siena provinces from which the isolates originated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypohidrotic ectodermal dysplasia

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.     

Acquisition of desiccation tolerance by isolated maize embryos exposed to different conditions: the questionable role of endogenous abscisic acid

The effect of exogenous ABA on acquisition of desiccation tolerance has been well documented for the embryos of several species. including maize ( Zea mays L.). It has also been suggested that endogenous ABA plays a role in regulating the same phenomena. To test this hypothesis, endogenous ABA was quantified by radioimmunoassay. Our results show that: (1) during embryogenesis in maize, endogenous ABA increase-concomitantly with the acquisition of desiccation tolerance: (2) ABA deficient embryos of the vp 5 mutant are desiccation intolerant, but tolerance can he induced by exogenous ABA: and (3) desiccation tolerance is acquired if desiccation sensitive embryos undergo a slow drying treatment, during which ABA increases. However, when embryos were preincubated in fluridone to prevent ABA accumulation during slow drying, desiccation tolerance was induced in spite of the low level of endogenous ABA in the embryo. Our results cast doubts on an exclusive role of ABA in the acquisition of desiccation tolerance in maize embryo.     

Chromosomal localization of zein genes by in situ hybridization in Zea mays

Summary In order to localize the genes coding for zein, the major storage protein of maize endosperm, zein ¹²⁵I-mRNA and ³H-cDNA labelled at high specific activity were used for in situ hybridization on heterozygous interchanges and paracentric inversions of the KYS strain of Zea mays. The analysis of the diplotene-metaphase I microsporocytes indicated the presence of zein structural genes on the long arm of chromosomes 4 and 5, the short arm of chromosome 7 and the distal segment of the long arm of chromosome 10. The two hybridization sites on chromosomes 7 and 10 are found near opaque-2 and opaque-7 loci which are known to regulate zein synthesis. The present data are discussed in relation to results obtained by other authors using genetical mapping of zein genes.     

Co-aggregation of penicillin g acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media

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.