Modelling of enzyme hydrolysis of maltose in a single pellet of immobilized biocatalyst

The reversible hydrolysis of maltose to glucose by immobilized glucoamylase entrapped in spherical solid particles is studied theoretically. For this purpose a known kinetic model taking into account these reversible reactions and the competitive synthesis of iso-maltose was adopted. The mass transfer limitations in the bulk liquid and in the pores of the particles containing the enzyme are considered, using Fick's law. On the basis of mathematical modelling the optimum conditions for biocatalyst performance are established. An appropriate combination of particle size and initial substrate concentration may lead to reduction of undesirable mass transfer resistance and therefore product inhibition and to an improved selectivity of the biocatalyst with respect of glucose formation.List of Symbols C _i kmoles/m³ current concentration ofi-th component along the radius - C _oi kmoles/m³ bulk concentration ofi-th component - C _i ^* kmoles/m³ concentrations ofi-th component on the pellet surface - D _si ,D _i m²/s internal and molecular diffusion coefficient ofi-th component - W _M kmoles/m³·s reaction rate of maltose hydrolysis - W _IM kmoles/m³·s reaction rate of iso-maltose formation - W _G kmoles/m³·s reaction rate of glucose production - R ₀ m pellet radius - r m current radius of the pellet - t s time coordinate - r ₀ ratio of the time step to the square of the radial coordinate - Re Reynolds number =w·R/v - Sc Schmidt number =v/D - Bi Biot number = R/D - A _j ,B, C _j coefficients in the system of linear equations, Eq. (8) - X _i dimensionless degree of transformation - NR number of independent reactions - N number of division sections of the pellet radius - G kmoles/m³ concentration of glucose - M kmoles/m³ concentration of maltose - IM kmoles/m³ concentration of isomaltose - K _m kmoles/m³ Michaelis constant - V _max kmoles/m³·s maximum reaction rate in Eq. (6) - K _i kmoles/m³ inhibition constant - K _1eq ,K _2eq equilibrium constants in Eq. (6) - , h steps along the time and radial coordinate in the pellet - m/s mass transfer coefficient - dimensionless radius of the pellet - computation accuracy Indices i number of reaction component - j index along the radius of the pellet - k index along the time coordinate This work was accomplished with thanks to the financial support of the Bulgarian National Fund for Scientific Investigations —Grant No. MU-1-BE/93.     

Mutations affecting pigmentation and shape of the adult zebrafish

 Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish. Received: 17 June 1996 / Accepted: 15 July 1996     

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear β-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyrano-syl residues of which 80–85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N-acetylglucosaminyltransferase activity in the formation     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.     

Insertion elements in Staphylococcus intermedius

Staphylococcus intermedius cultures from dogs, pigeons, horses and mink were investigated for the prevalence of the insertion elements IS 256 and IS 257 in relation to their antibiotic resistance. Copies of IS 256 could not be detected in any of the Staph. intermedius isolates tested whereas single copies of IS 257 occurred in the isolates from dogs and horses. The mink strains did not harbour IS 257 elements, whereas Staph. intermedius isolates from pigeons carried multiple copies of IS 257 as predicted from the hybridization patterns obtained with a gene probe derived from the internal part of the IS 257 -encoded transposase gene. Independently of the origin of the Staph. intermedius isolates, all IS 257 copies were found to be located in the chromosomal DNA. The large number of chromosomal IS 257 copies in the pigeon strains might help to explain chromosomal multiresistance in many of those strains.     

Targeted development of microsatellite markers from the defined region of bovine Chromosome 6q21-31

A methodical strategy for the isolation of microsatellite markers specific for targeted regions of bovine chromosomes is presented. The procedure involves directed microdissection of one defined subchromosomal area, its DOP-PCR-amplification and cloning. With this approach, a library specific to the BTA 6q21-31 chromosomal region was constructed. Eleven unique microsatellite-containing sequences were isolated, converted into sequence-tagged microsatellite sites, and characterized concerning their species-specific origin. Seven primer pairs generated bovine-specific PCR products and provided a set of microsatellite markers that generally revealed high informativity in the HF breed. Linkage analysis assigned six of them to their predefined subchromosomal origin on BTA 6 corresponding to the specific rehybridization signal of the DOP-PCR product generated from the microdissected chromosome area 6q21-31. The results underline the usefulness of the BTA 6q21-31 library for targeted isolation of unique sequences that are specific for the dissected chromosomal region as demonstrated here by the isolation of microsatellite markers. Received: 27 March 1997 / Accepted: 14 July 1997     

Host-cell cyclophilin is important for the intracellular replication of Leishmania major

The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host–parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.     

Nutritional, growth, and reproductive responses of maize (Zea mays L.) to arbuscular mycorrhizal inoculation during and after drought stress at tasselling

The effects of root colonization by the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith on nutritional, growth, and reproductive attributes of two tropical maize cultivars with different sensitivities to drought were studied. Freshly regenerated seeds of selection cycles 0 (cv. C0, drought-sensitive) and 8 (cv. C8, drought-resistant) of the lowland tropical maize population "Tuxpeño sequía" were used in this greenhouse experiment. Maize plants were subjected to drought stress for 3 weeks following tasselling (75–95 days after sowing) and rewatered for the subsequent 5 weeks until harvest. Mycorrhizal (M+) plants had significantly higher uptake of N, P, K, Mg, Mn, and Zn into grain than non-mycorrhizal (M–) plants under drought conditions. AM inoculation also produced significantly greater shoot masses in C0 and C8 regardless of the drought-stress treatment. In the sensitive cultivar C0, drought stress reduced the shoot mass and grain yield by 23% and 55%, respectively, when roots were not colonized, while the reductions were only 12% and 31%, respectively, with mycorrhizal association. In addition, the emergence of tassels and silks was earlier in M+ plants than in M– plants under drought conditions. Mycorrhizal response was more pronounced under both well-watered and drought conditions in C0 than in the C8 cultivar. The overall results suggest that AM inoculation affects host plant nutritional status and growth and thereby alters the reproductive behaviour of maize under drought conditions.