Showing papers by "Neil R. Gilkes published in 1999"

Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme.

Abstract: Several models have been proposed to explain the high temperatures required to denature enzymes from thermophilic organisms; some involve greater maximum thermodynamic stability for the thermophile, and others do not. To test these models, we reversibly melted two analogous protein domains in a two-state manner. E2cd is the isolated catalytic domain of cellulase E2 from the thermophile Thermomonospora fusca. CenAP30 is the analogous domain of the cellulase CenA from the mesophile Cellulomonas fimi. When reversibly denatured in a common buffer, the thermophilic enzyme E2cd had a temperature of melting (Tm) of 72.2 degrees C, a van't Hoff enthalpy of unfolding (DeltaHVH) of 190 kcal/mol, and an entropy of unfolding (DeltaSu) of 0.55 kcal/(mol*K); the mesophilic enzyme CenAP30 had a Tm of 56.4 degrees C, a DeltaHVH of 107 kcal/mol, and a DeltaSu of 0. 32 kcal/(mol*K). The higher DeltaHVH and DeltaSu values for E2cd suggest that its free energy of unfolding (DeltaGu) has a steeper dependence on temperature at the Tm than CenAP30. This result supports models that predict a greater maximum thermodynamic stability for thermophilic enzymes than for their mesophilic counterparts. This was further explored by urea denaturation. Under reducing conditions at 30 degrees C, E2cd had a concentration of melting (Cm) of 5.2 M and a DeltaGu of 11.2 kcal/mol; CenAP30 had a Cm of 2.6 M and a DeltaGu of 4.3 kcal/mol. Under nonreducing conditions, the Cm and DeltaGu of CenAP30 were increased to 4.5 M and 10.8 kcal/mol at 30 degrees C; the Cm for E2cd was increased to at least 7.4 M at 32 degrees C. We were unable to determine a DeltaGu value for E2cd under nonreducing conditions due to problems with reversibility. These data suggest that E2cd attains its greater thermal stability (DeltaTm = 15.8 degrees C) through a greater thermodynamic stability (DeltaDeltaGu = 6.9 kcal/mol) compared to its mesophilic analogue CenAP30.

Expression of a mannan binding domain to alter plant morphology

Abstract: Novel compositions and methods for their use are provided comprising recombinant nucleic acids coding for a mannan binding domain or functional portions thereof. The compositions can be synthesized or prepared by recombinant DNA technology. The compositions can be used to create transgenic plants expressing said mannan binding domain. The mannan binding domain can be expressed in a tissue specific manner and thereby modify plant properties including mannan-containing plant components.

C.fimi glycosyl hydrolase containing a mannan binding domain

Abstract: Novel polypeptide compositions and methods for their use are provided comprising fusion proteins in which the mannan binding domain or functional portion thereof is fused to a heterologous protein or is conjugated to a chemical moiety. The compositions can be synthesized or prepared by recombinant DNA technology. The compositions find use for separating and/or purifying biological compositions and living cells. In the method for purifying biological compositions, the biological composition is mixed with a phase-forming mannan to which the mannan binding peptide binds inducing phase separation. The phase containing the biological composition is then isolated. Phase separation is induced using the phase separation inducing agent or, when the oligosaccharide polymer is a thermo-separating polymer, by increasing the temperature above the cloud point temperature of the polymer.

Utilisation du domaine de fixation du mannane pour modifier la morphologie de plantes

Abstract: Cette invention se rapporte a de nouvelles compositions et a des procedes d'utilisation de ces compositions, qui comprennent des acides nucleiques recombines codant pour un domaine de fixation du mannane ou pour des parties fonctionnelles de ce domaine. Ces compositions peuvent etre synthetisees ou preparees par une technique d'ADN de recombinaison. Ces compositions peuvent servir a creer des plantes transgeniques exprimant ce domaine de fixation du mannane. Ce domaine de fixation du mannane peut etre exprime de facon specifique aux tissus et modifier par consequent les proprietes des plantes, y compris les composants vegetaux contenant du mannane.