Methods for the immobilization of lipases and their use for ester synthesis

The lipase from Pseudomonas fluorescens was immobilized onto five different carriers: celite, octyl-silica, aminopropyl-silica, gluterdialdehyde-activated silica and Eupergit C250L. Activities and operational stabilities of the prepared catalysts were compared using the enantioselective acylation of (R,S)-1-phenylethanol by vinyl acetate as acyl donor and t-butylmethyl ether with variable water content (0.038-0.97% v/v) as reaction medium. The above carriers provide catalysts with widely different specific activities ranging from excellent 25 mmol/h mg protein (celite) to 0.07 mmol/h mg protein (glutardialdehyde-activated silica) on the lower end. The lipase immobilized onto Eupergit C250L exhibited the best operational stability among the catalysts studied. It retained 30% of its initial activity after 11 cycles of application, each with a duration between 2 and 6 h.     

Survival of genetically engineered Pseudomonas fluorescens in soil in competition with the parent strain

Abstract The population dynamics of two genetically engineered Pseudomonas fluorescens strains, D5 and C5t, introduced into a loamy sand soil, in competition with a spontaneous antibiotic-resistant mutant of the corresponding wildtype strain was studied. Strain D5 contained an insertion of transposon Tn5 in its genome, whereas strain C5t was obtained by insertion of Tn 5 :: tox , a Tn 5 -derivative containing a Bacillus thuringiensis var. morrisoni δ-endotoxin gene, into the chromosome using a suicide vector system. Southern hybridization analysis demonstrated the absence of vector sequences, and the presence of single copies of either Tn 5 or Tn 5 :: tox in the respective strains. Western blotting and a bio-assay on larvae of Anopheles stephensi suggested the tox gene was functional in clone C5t. Both D5 and C5t were prototrophic and their generation times in minimal medium were slightly below that of the corresponding wild-type strain. Tn 5 and Tn 5 :: tox were stable in both clones during growth in minimal medium for 16 generations. During growth in competition with the wild-type strain, D5 competed well, however C5t was outcompeted from 50 to below 3% of the population in 40 generations. During growth in competition in the sterile loamy sand, both strains were outcompeted by the parent strain; strain C5t was less competitive than D5. In non-sterile loamy sand, the introduced mixed populations showed a slow decline; both C5t and D5 were outcompeted by the parent strain. The decreased fitness of both modified strains, although significant, was considered to be small in ecological terms. Further, the addition of 10% bentonite clay to the loamy sand resulted in a significant enhancement of survival of the mixed populations, and a stabilization of the proportions between the modified strains and the parent. Finally, there was a trend towards a decrease in the proportion modified strain/parent strain in both mixes in the rhizosphere of wheat.     

Exocellular phosphatidylethanolamine production and multiple-metal tolerance in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens appeared to circumvent the challenge imposed by millimolar amounts of metals (5 mM Al³⁺, 5 mM Fe³⁺, 2 mM Ca²⁺, 1 mM Ga³⁺ and 3 mM Zn²⁺) by the formation of phosphatidylethanolamine. This lipid moiety constituted an important organic component of an insoluble gelatinous residue in which most of the test metals were immobilized at stationary phase of growth. Ultracentrifugation and dialysis experiments showed that the metals were associated with phosphatidylethanolamine from early stages of growth. Transmission electron microscopy revealed metal rich bodies in the cytoplasm prior to their secretion in the spent fluid. These results demonstrate a role of phosphatidylethanolamine in multiple-metal homeostasis.     

Some properties of polysaccharides of microorganisms from degraded straw

Extracellular polysachcarides from bacteria and yeasts isolated from decomposed straw contained various proportions of d-galactose, d-glucose, d-mannose, uronic acid, d-xylose, l-fucose and l-rhamnose. Molecular weights of the polymers determined by viscometry and gel filtration were in the range 40 000–1800 000. All the polysaccharides stabilized aggregates of volcanic ash and most were more effective than the polysaccharide from Lipomyces starkeyi. Effectiveness seemed to be more related to molecular weight than to chemical composition.     

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

Abstract An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( npt II) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.     

Purification and characterisation of a novel alkalophilic α-D-mannosidase from Pseudomonas fluorescens

An extracellular alkaline α-D-mannosidase in the cell culture of a marine bacterium Pseudomonas fluorescens JK-02 was purified to homogeneity with a 30.7-fold by ammonium sulphate fractionation, anion-exchange chromatography and gel-filtration chromatography. The molecular weight of the purified enzyme was estimated to be 50.5 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme were 8.5 and 30°C. The K_m and V_max values of the purified enzyme towards p-nitrophenyl-α-D-mannopyranoside were determined to be 77 µM and 0.23 µM min^?1mg^?1 of protein, respectively. The α-D-mannosidase showed higher substrate specificity to α-1,3-mannobiose than other isomeric substrates such as α-1,2- and α-1,6-mannobiose. In addition, molecular characterisation of this enzyme reveals that it belongs to a class II α-mannosidase from the glycosyl hydrolase family 38. To the best of our knowledge, this is the first report on the alkalophilic α-1,3 D-mannosidase of Pseudomonas species, which has selective algal-lytic activity against Alexandrium tamarense, Akashiwo sanguine, Gymnodinium catenatum, Gymnodinium mikimotoi and Prorocentrum dentatum.     

Degradation of mandelate and 4-hydroxymandelate by Rhizobium leguminosarum biovar trifolii TA1

Rhizobium leguminosarum biovar trifolii TA1 grows on 4-hydroxymandelate and enzymes involved in its catabolism are inducible. Strain TA1 does not grown on mandelate or cis, cis-muconate, but spontaneous mutants capable of growth on these substrates were isolated. Enzymes involved in mandelate degradation were also inducible. The presence of intermediates of the mandelate and hydroxymandelate pathways resulted in a significant decrease in some of the enzymes involved in their degradation. Succinate and acetate, end products of the pathways, and glucose caused reductions in the levels of enzymes in the mandelate and hydroxymandelate pathways.     

Cloning, sequence analysis, and expression of ansB from Pseudomonas fluorescens, encoding periplasmic glutaminase/asparaginase

A gene (ansB) encoding a class II glutaminase/asparaginase has been cloned from Pseudomonas fluorescens and characterized by DNA sequencing, promoter analysis and heterologous expression in Escherichia coli. We show that ansB is monocistronic and depends on the alternate sigma factor sigma 54 for expression. A second open reading frame located downstream of ansB is highly homologous to a number of bacterial genes that encode secreted endonucleases of unknown function.