26 research outputs found
Statistical Mechanics of Glass Formation in Molecular Liquids with OTP as an Example
We extend our statistical mechanical theory of the glass transition from
examples consisting of point particles to molecular liquids with internal
degrees of freedom. As before, the fundamental assertion is that super-cooled
liquids are ergodic, although becoming very viscous at lower temperatures, and
are therefore describable in principle by statistical mechanics. The theory is
based on analyzing the local neighborhoods of each molecule, and a statistical
mechanical weight is assigned to every possible local organization. This
results in an approximate theory that is in very good agreement with
simulations regarding both thermodynamical and dynamical properties
Altering the Activation Mechanism in <i>Thermomyces lanuginosus</i> Lipase
It
is shown by rational site-directed mutagenesis of the lid region
in <i>Thermomyces lanuginosus</i> lipase that it is possible
to generate lipase variants with attractive features, e.g., high lipase
activity, fast activation at the lipid interface, ability to act on
water-soluble substrates, and enhanced calcium independence. The rational
design was based on the lid residue composition in <i>Aspergillus
niger</i> ferulic acid esterase (FAEA). Five constructs included
lipase variants containing the full FAEA lid, a FAEA-like lid, an
intermediate lid of FAEA and TlL character, and the entire lid region
from <i>Aspergillus terreus</i> lipase (AtL). To investigate
an altered activation mechanism for each variant compared to that
of TlL, a combination of activity- and spectroscopic-based measurements
were applied. The engineered variant with a lid from AtL displayed
interfacial activation comparable to that of TlL, whereas variants
with FAEA lid character showed interfacial activation independence
with pronounced activity toward pNP-acetate and pNP-butyrate below
the critical micelle concentration. For variants with lipase and esterase
character, lipase activity measurements further indicated a faster
activation at the lipid interface. Relative to their activity toward
pNP-ester substrates in calcium-rich buffer, all lid variants retained
between 15 and 100% activity in buffer containing 5 mM EDTA whereas
TlL activity was reduced to less than 2%, demonstrating the lid’s
central role in governing calcium dependency. For FAEA-like lid variants,
accessible hydrophobic surface area measurements showed an approximate
10-fold increase in the level of binding of extrinsic fluorophores
to the protein surface relative to that of TlL accompanied by a blue
shift in emission indicative of an open lid in aqueous solution. Together,
these studies report on the successful alteration of the activation
mechanism in TlL by rational design creating novel lipases with new,
intriguing functionalities
The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study
Triacylglycerol
hydrolases (EC 3.1.1.3) are thought to become activated
when they encounter the water–lipid interface causing a “lid”
region to move and expose the catalytic site. Here, we tested this
idea by looking for lid movements in <i>Thermomyces lanuginosus</i> lipase (TL lipase), and in variants with a mutated lid region of
esterase (Esterase) and esterase/lipase (Hybrid) character. To measure
lid movements, we employed the tryptophan-induced quenching (TrIQ)
fluorescence method to measure how effectively a Trp residue on the
lid of these mutants (at position 87 or 89) could quench a fluorescent
probe (bimane) placed at nearby site 255 on the protein. To test if
lid movement is induced when the enzyme detects a lower-polarity environment
(such as at the water–lipid interface), we performed these
studies in solvents with different dielectric constants (ε).
The results show that lid movement is highly dependent on the particular
lid residue composition and solvent polarity. The data suggest that
in aqueous solution (ε = 80), the Esterase lid is in an “open”
conformation, whereas for the TL lipase and Hybrid, the lid remains
“closed”. At lower solvent polarities (ε <
46), the lid region for all of the mutants is more “open”.
Interestingly, these behaviors mirror the structural changes thought
to take place upon activation of the enzyme at the water–lipid
interface. Together, these results support the idea that lipases are
more active in low-polarity solvents because the lid adopts an “open”
conformation and indicate that relatively small conformational changes
in the lid region play a key role in the activation mechanism of these
enzymes
Density, Viscosity, and Surface and Interfacial Tensions of Mixtures of Water + n -Butyl Acetate + 1-Propanol at 303.15 K and Atmospheric Pressure
Abstract Experimental densities, viscosities, and surface and interfacial tensions have been measured at 303.15 K for liquid mixtures of water + n-butyl acetate + 1-propanol. The excess molar volume, V E, viscosity, ?, and surface tension, ?, were calculated and rational functions due to Myers and Scott, and Pando et al. were used to describe the composition dependence of these properties. The viscosity, ?, of the mixtures was correlated using a theoretically based method developed from the Eyring theory using the above-mentioned rational functions to express the excess Gibbs energy of activation for viscous flow, G ?E. The UNIMOD model based on the Eyring theory was used to correlate the viscosity of the binaries and to predict the same property for ternary mixtures. To describe the above-mentioned properties of the ternary system, binary pair additivity and some empirical models were considered. The methods of Fu et al. and Li et al. were used to correlate the binary surface tension and also to predict the ternary behavior. The interfacial tension was correlated by the Li and Fu method