An anomalous behavior of trypsin immobilized in alginate network

Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N _α-benzoyl-l-arginine-4-nitroanilide hydrochloride (l-BAPNA), and in the presence or absence of d-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 °C. It was found that at 37 and 42 °C a strong acceleration of the reaction was obtained, whereas at 10 °C and at room temperature, the presence of d-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with d-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus l-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.     

Direct characterization of Frankia and of close phyletic neighbors from an Alnus viridis rhizosphere

To analyze the population structure of Frankia soil populations, DNA was extracted from an Alnus viridis rhizosphere, PCR-amplified with a primer designed to be specific, and cloned. A PCR-RFLP analysis of the resulting clones showed that none had a pattern close to those expected from an analysis made on Frankia collection strains, on which Hha I, Hpa II and Hae III were found to be the most discriminant restriction enzymes. Sequencing was done on a subset of soil clones and these were compared to DNA data banks. Some sequences were typical of lowGC Firmicutes ( Bacillus and relatives), whereas others were typical of highGC Firmicutes (Actinomycetes and relatives). Two of these sequences appear to be in the same line of descent as Frankia and Geodermatophilus and represent as yet uncharacterized soil microorganisms. This shows that the design of specific or generic primers must be refined constantly to take into account the deposition of such new sequences in DNA data banks.