Cellulase immobilized on poly-L-glutamic acid

Cellulase (Cellulosin AC-8) was immobilized on poly-L-glutamic acid. This immobilized cellulase (IC) is water soluble in the neutral and alkaline solutions, where IC has the activity, while IC can be made insoluble by lowering the pH so that it can be recovered from the reaction mixture with its activity. The optimum pH and temperature were determined to be 5.5 and 55 degrees C, respectively. The stability of IC against change in the pH and temperature was improved by the immobilization. Solvolysis of 3N-NaOH-treated cellulose, with IC under the optimum conditions found here, led to the production of low-molecular-weight compounds.     

Flow-induced conformational changes and phase behavior of aqueous poly-L-lysine solutions

Poly-L -lysine exists as an α-helix at high pH and a random coil at neutral pH. When the α-helix is heated above 27°C, the macromolecule undergoes a conformational transition to a β-sheet. In this study, the stability of the secondary structure of poly-L -lysine in solutions subjected to shear flow, at temperatures below the α-helix to β-sheet transition temperature, were examined using Raman spectroscopy and CD. Solutions initially in the α-helical state showed time-dependent increases in viscosity with shearing, rising as much as an order of magnitude. Visual observation and turbidity measurements showed the formation of a gel-like phase under flow. Laser Raman measurements demonstrated the presence of small amounts of β-sheet structure evidenced by the amide I band at 1666 cm⁻¹. CD measurements indicated that solutions of predominantly α-helical conformation at 20°C transformed into 85% α-helix and 15% β-sheet after being sheared for 20 min. However, on continued shearing the content of β-sheet conformation decreased. The observed phenomena were explained in terms of a “zipping-up” molecular model based on flow enhanced hydrophobic interactions similar to that observed in gel-forming flexible polymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 239–246, 1998     

Dielectric properties of poly-L-glutamic acid in salt-free aqueous solutions

The electric permittivity of poly-L-glutamic acid (PGA) in salt-free aqueous solutions was measured in the frequency range 2.5 kHz – 100 MHz at different concentrations and degrees of ionization. Two samples of different molecular weight were investigated. The experimental results could under most circumstances be described by a superposition of two dispersion curves of the Cole-Cole type. The low-frequency dielectric parameters were strongly molecular weight dependent, the high-frequency ones not. Strong concentration effects were observed resulting in increasing specific dielectric increments and relaxation times with decreasing concentration. Using the theory proposed by Van der Touw and Mandel to interpret the experimental results these concentration effects could be ascribed to the influence of the polyion interactions on the average dimensions and the rigidity of the polyelectrolyte chains. The change in the total dielectric increment and low-frequency relaxation time with degree of ionization correctly reflects the helix-coil transition of PGA occurring in ths region α = 0.3–0.5. The effect of counterion size and charge on the dielectric behaviour was also found to be consistent with the theoretical model.     

Raman studies of the helix-to-coil transition in poly-L-glutamic acid and poly-L-ornithine

The conformational transitions in water and in the solid state of poly-L -glutamic acid (PGA) and poly-L -ornithine (PO) have been studied by Raman spectroscopy. The Raman spectra of PGA, PO, and the monomer, dimer, and trimer of PGA in aqueous solutions and solid state are presented. The Raman spectral changes of PGA and PO were followed through the helix-to-coil transition induced by p^H, temperature, and solvent composition. A hyperchromic shift in the intensity of the amide III line accompanying the helix-to-coil transition was observed. This hyperchromic intensity shift occurs abruptly as a function of p^H but more slowly with heat denaturation of the alpha helix indicating that the Raman spectrum is sensitive to the transition mechanism. The high-temperature coil and the charged coil may have different conformations as evidenced by different amide III frequencies but similar intensities in these two conformations.     

The precipitation of poly-L-glutamic acid. I. Alpha-precipitation.

Studies have been made of the precipitation of poly-L -glutamic acid from dilute aqueous solutions, over the pH range where the α-helical conformation is maintained. The major purpose is to establish the molecular conformation in the precipitate and to outline the mechanism of the precipitation. Utilizing molecular-weight fractions, it is shown that there are two distinctly different precipitation regions, which depend on temperature, concentration, and pH. In one of these regions, the α-region, a variety of physical properties demonstrate that precipitation occurs without any conformational change. The temperature coefficient of the precipitation process in this region indicates that it is nucleation-controlled.     

Unfolding of poly-L-glutamic acid by microbubbling of supercritical carbon dioxide

The conformational changes in alpha-helical poly-L-glutamic acid caused by microbubbling supercritical CO2 were investigated with circular dichroism spectra. After microbubbling using a micropore filter at 35 and 30 MPa for 30 min, alpha-helix content decreased to 37%, while without the filter it was 68%. The alpha-helix structure was significantly decomposed by a high density of CO2. No important changes were observed in heating, autoclaving, or pH-lowering.     

The precipitation of poly-L-glutamic acid. II. beta-Precipitation.

Infrared and X-ray diffraction studies have established that in the β-precipitation region of poly-L -glutamic acid the chains are in the β-conformation. Therefore, a major molecular conformational change has taken place upon precipitation. It is shown that the size of the α-helical aggregates remains constant with time in the β-region. Strong evidence can be offered to indicate that the transformation involves a transitory random-coil intermediate. Reasons are advanced, in view of the stability of the β-form, as to why two distinct precipitation regions exist.     

Synthesis of potent inhibitors of anthrax toxin based on poly-L-glutamic acid

We report the synthesis of biodegradable polyvalent inhibitors of anthrax toxin based on poly-L-glutamic acid (PLGA). These biocompatible polyvalent inhibitors are at least 4 orders of magnitude more potent than the corresponding monovalent peptides in vitro and are comparable in potency to polyacrylamide-based inhibitors of anthrax toxin assembly. We have elucidated the influence of peptide density on inhibitory potency and demonstrated that these inhibitory potencies are limited by kinetics, with even higher activities seen when the inhibitors are preincubated with the heptameric receptor-binding subunit of anthrax toxin prior to exposure to cells. These polyvalent inhibitors are also effective at neutralizing anthrax toxin in vivo and represent attractive leads for designing biocompatible anthrax therapeutics.