Novel thermostable and alkalitolerant amylase production by Geobacillus stearothermophilus HP 3

This study reports the production and characterisation of a novel thermostable alkaline amylase from the newly isolated Geobacillus stearothermophilus HP 3. The optimum temperature and pH for enzyme production was 55°C and 9.0, respectively. The amylase powder obtained from the culture filtrate by pre-chilled acetone treatment was stable over a wide pH range and liquefied thick starch slurries at 75°C. Ca2? and Co2? were required for the thermostability of the enzyme preparation. The present purified amylase therefore could be defined as thermostable and alkalitolerant with new properties that make the enzyme applicable for many starch processing applications for use in the food industry.     

A uniform estimate for scalar curvature equation on manifolds of dimension 4

We consider the solutions to the prescribed scalar curvature equation on a four-dimensional Riemannian manifold M. We prove an upper bound for the supremum of all the solutions on every compact subset K of M, provided that all the solutions on M are bounded from below by a positive number.     

Crystal Structures of 1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene and 2,4,6-Trimethyl-1,3,5-tris(4-oxymethyl-1-formylphenyl)benzene

Abstract  

The synthesis and X-ray crystal structure of 2,4,6-trimethyl-1,3,5-tris(4-oxymethyl-1-formylphenyl)benzene (2) and the crystal structure of its core molecule, 1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene (1) are reported. The compounds 1 and 2 crystallize in the triclinic system with space group P ī. The lattice parameters of 1 and 2 are a = 9.454(2) ?, b = 9.478(4) ?, c = 9.573(2) ?, α = 116.53(2)°, β = 109.705(14)°, γ = 101.293(18)°, V = 658.5(4) and a = 15.7707(6) ?, b = 16.3646(5) ?, c = 18.2805(6) ?, α = 83.202(2)°, β = 66.0120(10)°, γ = 73.704(2)°, V = 4137.2(2), respectively. In the crystals of 1 and 2, there are one and three molecules in the asymmetric unit, respectively. The packing of the molecules in the solid is stabilized through van der Waals interactions in 1 and weak C–H···O and C–H···π interactions in 2.     

A mesoscopic model for (de)wetting

We present a mesoscopic model for simulating the dynamics of a non-volatile liquid on a solid substrate. The wetting properties of the solid can be tuned from complete wetting to total non-wetting. This model opens the way to study the dynamics of drops and liquid thin films at mesoscopic length scales of the order of the nanometer. As particular applications, we analyze the kinetics of spreading of a liquid drop wetting a solid substrate and the dewetting of a liquid film on a hydrophobic substrate. In all these cases, very good agreement is found between simulations and theoretical predictions.     

Molecular dynamics of amorphous/crystalline polymer blends studied by broadband dielectric spectroscopy

The molecular dynamics of poly(vinyl acetate), PVAc, and poly(hydroxy butyrate), PHB, as an amorphous/crystalline polymer blend has been investigated using broadband dielectric spectroscopy over wide ranges of frequency (10⁻² to 10⁵ Hz), temperature, and blend composition. Two dielectric relaxation processes were detected for pure PHB at high and low frequency ranges at a given constant temperature above the T_g. These two relaxation peaks are related to the α and α′ of the amorphous and rigid amorphous regions in the sample, respectively. The α′-relaxation process was found to be temperature and composition dependent and related to the constrained amorphous region located between adjacent lamellae inside the lamellar stacks. In addition, the α′-relaxation process behaves as a typical glass relaxation process, i.e., originated from the micro-Brownian cooperative reorientation of highly constraints polymeric segments. The α-relaxation process is related to the amorphous regions located between the lamellar crystals stacks. In the PHB/PVAc blends, only one α-relaxation process has been observed for all measured blends located in the temperature ranges between the T_g’s of the pure components. This last finding suggested that the relaxation processes of the two components are coupled together due to the small difference in the T_g’s (ΔT_g = 35 °C) and the favorable thermodynamics interaction between the two polymer components and consequently less dynamic heterogeneity in the blends. The T_g’s of the blends measured by DSC were followed a linear behavior with composition indicating that the two components are miscible over the entire range of composition. The α′-relaxation process was also observed in the blends of rich PHB content up to 30 wt% PHB. The molecular dynamics of α and α′-relaxation processes were found to be greatly influenced by blending, i.e., the dielectric strength, the peak broadness, and the dielectric loss peak maximum were found to be composition dependent. The dielectric measurements also confirmed the slowing down of the crystallization process of PHB in the blends.     

Molecular dynamics at the root of expansion of function in the M69L inhibitor-resistant TEM beta-lactamase from Escherichia coli

Clavulanate, an inhibitor for beta-lactamases, was the very first inhibitor for an antibiotic resistance enzyme that found clinical utility in 1985. The clinical use of clavulanate and that of sulbactam and tazobactam, which were introduced to the clinic subsequently, has facilitated evolution of a set of beta-lactamases that not only retain their original function as resistance enzymes but also are refractory to inhibition by the inhibitors. This article characterizes the properties of the clinically identified M69L mutant variant of the TEM-1 beta-lactamase from Escherichia coli, an inhibitor-resistant beta-lactamase, and compares it to the wild-type enzyme. The enzyme is as active as the wild-type in turnover of typical beta-lactam antibiotics. Furthermore, many of the parameters for interactions of the inhibitors with the mutant enzyme are largely unaffected. The significant effect of the inhibitor-resistant trait was a relatively modest elevation of the dissociation constant for the formation of the pre-acylation complex. The high-resolution X-ray crystal structure for the M69L mutant variant revealed essentially no alteration of the three-dimensional structure, both for the protein backbone and for the positions of the side chains of the amino acids. It was surmised that the difference in the two enzymes must reside with the dynamic motions of the two proteins. Molecular dynamics simulations of the mutant and wild-type proteins were carried out for 2 ns each. Dynamic cross-correlated maps revealed the collective motions of the two proteins to be very similar, yet the two proteins did not behave identically. Differences in behavior of the two proteins existed in the regions between residues 145-179 and 155-162. Additional calculations revealed that kinetic effects measured experimentally for the dissociation constant for the pre-acylation complex could be mostly attributed to the electrostatic and van der Waals components of the binding free energy. The effects of the mutation on the behavior of the beta-lactamase were subtle, including the differences in the measured dissociation constants that account for the inhibitor-resistant trait. It would appear that nature has selected for incorporation of the most benign alteration in the structure of the wild-type TEM-1 beta-lactamase that is sufficient to give the inhibitor-resistant trait.     

Computational investigation of irreversible inactivation of the zinc-dependent protease carboxypeptidase A

Zinc proteases are ubiquitous and the zinc ion plays a central function in the catalytic mechanism of these enzymes. A novel class of mechanism-based inhibitors takes advantage of the zinc ion chemistry in carboxypeptidase A (CPA) to promote covalent attachment of an inhibitor to the carboxylate of Glu-270, resulting in irreversible inhibition of the enzyme. The effect of the active site zinc ion on irreversible inactivation of CPA was probed by molecular orbital (MO) calculations on a series of active site models and the Cl(-) + CH(3)Cl S(N)2 reaction fragment. Point charge models representing the active site reproduced energetics from full MO calculations at 12.0 A separation between the zinc and the central carbon of the S(N)2 reaction, but at 5.0 A polarization played an important role in moderating barrier suppression. ONIOM MO/MO calculations that included the residues within 10 A of the active site zinc suggest that about 75% of the barrier suppression arises from the zinc ion and its ligands. A model of the pre-reactive complex of the 2-benzyl-3-iodopropanoate inactivator with CPA was constructed from the X-ray structure of l-phenyl lactate bound in the active site of the enzyme. The model was fully solvated and minimized by using the AMBER force field to generate the starting structure for the ONIOM QM/MM calculations. Optimization of this structure led to the barrierless S(N)2 displacement of the iodide of the inhibitor by Glu-270, assisted by interaction of the zinc ion with the leaving group. The resulting product is in good agreement with the X-ray structure of the covalently modified enzyme obtained by irreversible inhibition of CPA by 2-benzyl-3-iodopropanoate.