Cold Adaptation of a Mesophilic Subtilisin-like Protease by Laboratory Evolution

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (kcat) at 10 °C 6.6 times and a catalytic efficiency (kcat/KM) 9.6 times that of wild type. Its half-life at 70 °C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in kcat is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77.4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation

Isotropic photonic band gap and anisotropic structures in transmission spectra of two-dimensional 5-fold and 8-fold symmetric quasiperiodic photonic crystals

We measured and calculated transmission spectra of two-dimensional quasiperiodic photonic crystals (PCs) based on a 5-fold (Penrose) or 8-fold (octagonal) symmetric quasiperiodic pattern. The photonic crystal consisted of dielectric cylindrical rods in air placed normal to the basal plane on vertices of tiles composing the quasiperiodic pattern. An isotropic photonic band gap (PBG) appeared in the TM mode, where electric fields were parallel to the rods, even when the real part of a dielectric constant of the rod was as small as 2.4. An isotropic PBG-like dip was seen in tiny Penrose and octagonal PCs with only 6 and 9 rods, respectively. These results indicate that local multiple light scattering within the tiny PC plays an important role in the PBG formation. Besides the isotropic PBG, we found dips depending on the incident angle of the light. This is the first report of anisotropic structures clearly observed in transmission spectra of quasiperiodic PCs. Based on rod-number and rod-arrangement dependence, it is thought that the shapes and positions of the anisotropic dips are determined by global multiple light scattering covering the whole system. In contrast to the isotropic PBG due to local light scattering, we could not find any PBGs due to global light scattering even though we studied transmission spectra of a huge Penrose PC with 466 rods.Comment: One tex file for manuscript and 12 PNG files for figures consisting of Fig.1a-d, 2,3, ...

Equivalence between Schwinger and Dirac schemes of quantization

This paper introduces the modified version of Schwinger's quantization method, in which the information on constraints and the choice of gauge conditions are included implicitly in the choice of variations used in quantization scheme. A proof of equivalence between Schwinger- and Dirac-methods for constraint systems is given.Comment: 12pages, No figures, Latex, The proof is improved and one reference is adde

Novel phase diagram for antiferromagnetism and superconductivity in pressure-induced heavy-fermion superconductor Ce2_2RhIn8_8 probed by In-NQR

We present a novel phase diagram for the antiferromagnetism and superconductivity in Ce$_2$RhIn$_8$ probed by In-NQR studies under pressure ($P$). The quasi-2D character of antiferromagnetic spin fluctuations in the paramagnetic state at $P$ = 0 evolves into a 3D character because of the suppression of antiferromagnetic order for $P > P_{\rm QCP}\sim$ 1.36 GPa (QCP: antiferromagnetic quantum critical point). Nuclear-spin-lattice-relaxation rate $1/T_1$ measurements revealed that the superconducting order occurs in the $P$ range 1.36 -- 1.84 GPa, with maximum $T_c\sim$ 0.9 K around $P_{\rm QCP}\sim$ 1.36 GPa.Comment: 5 pages, 5 figures, submitted to PR

Crystal-field-induced magnetostrictions in the spin reorientation process of Nd2_2Fe14_{14}B-type compounds

Volume expansion $\Delta V / V$ associated with the spin reorientation process of Nd$_2$Fe$_{14}$B-type compounds has been investigated in terms of simple crystalline-electric-field (CEF) model. In this system, $\Delta V / V$ is shown to be a direct measure of second order CEF energy. Calculated anomalies in $\Delta V / V$ associated with the first-order magnetization process of Nd$_2$Fe$_{14}$B are presented, which well reproduced the observations.Comment: 2 pages, 2 figures, to appear in J. Magn. Magn. Mate

Magnetic ground state of pyrochlore oxides close to metal-insulator boundary probed by muon spin rotation

Magnetism of ruthernium pyrochlore oxides A2Ru2O7 (A = Hg, Cd, Ca), whose electronic properties within a localized ion picture are characterized by non-degenerate t2g orbitals (Ru5+, 4d3) and thereby subject to geometrical frustration, has been investigated by muon spin rotation/relaxation (muSR) technique. The A cation (mostly divalent) was varied to examine the effect of covalency (Hg > Cd > Ca) on their electronic property. In a sample with A = Hg that exhibits a clear metal-insulator (MI) transition below >> 100 K (which is associated with a weak structural transition), a nearly commensurate magnetic order is observed to develop in accordance with the MI transition. Meanwhile, in the case of A = Cd where the MI transition is suppressed to the level of small anomaly in the resistivity, the local field distribution probed by muon indicates emergence of a certain magnetic inhomogeneity below {\guillemotright} 30 K. Moreover, in Ca2Ru2O7 that remains metallic, we find a highly inhomogeneous local magnetism below >>25 K that comes from randomly oriented Ru moments and thus described as a "frozen spin liquid" state. The systematic trend of increasing randomness and itinerant character with decreasing covalency suggests close relationship between these two characters. As a reference for the effect of orbital degeneracy and associated Jahn-Teller instability, we examine a tetravalent ruthernium pyrochlore, Tl2Ru2O7 (Ru4+, 4d4), where the result of muSR indicates a non-magnetic ground state that is consistent with the formation of the Haldane chains suggested by neutron diffraction experiment.Comment: 12 pages, 13 figure