Expression and high-level secretion of Trichoderma reesei endoglucanase I in Yarrowia lipolytica

The endoglucanase I (EGI) from fungus Trichoderma reesei was cloned, expressed, and secreted from Yarrowia lipolytica using the XPR2 promoter. The signal sequence of EGI transferred from T. reesei was efficiently processed in the Y. lipolytica secretory pathway and directed the secretion of active EGI into the culture medium. However, the recombinant EGI produced from YLCSIn strain was hyperglycosylated and significantly larger than the native enzyme produced by the parent strain. The expression of EGI using XPR2 preproregion has caused secretion of modified proteins that still retained cellulase activity. This resulted from imprecise processing of the N-terminus of recombinant protein. While the batch culture produced 5 mg EGI/L from YLCSIn strain, the EGI yield was increased approx 20-fold when the fed-batch fermentation process strategy in combination with the high-cell density cultivation technique was employed. These results showed that the Y. lipolytica is a useful host organism for production of a large amount of large size heterologous proteins, especially when used in combination with high-cell density and fed-batch culture techniques.     

Comparison of excited-state energy transfer in arrays of hydroporphyrins (chlorins, oxochlorins) versus porphyrins: rates, mechanisms, and design criteria

A set of chlorin-chlorin and oxochlorin-oxochlorin dyads has been prepared with components in the same or different metalation states. In each case a 4,4'-diphenylethyne linker spans the respective 10-position of each macrocycle. The dyads have been studied using static and time-resolved absorption and emission spectroscopy, resonance Raman spectroscopy, and electrochemical techniques. Excited-state energy transfer from a zinc chlorin to a free-base (Fb) chlorin occurs with a rate constant of (110 ps)(-1) and an efficiency of 93%; similar values of (140 ps)(-1) and 83% are found for the corresponding oxochlorin dyad. Energy transfer in both dyads is slower and less efficient than found previously for the analogous porphyrin dyad, which displays a rate of (24 ps)(-1) and a yield of 99%. The slower rates and diminished efficiencies in the ZnFb chlorin and oxochlorin dyads versus the ZnFb porphyrin dyad are attributed to substantially weaker linker-mediated through-bond (TB) electron-exchange coupling (as indicated by resonance Raman data). Although the through-space (TS, i.e., dipole-dipole) coupling in the ZnFb-chlorin and -oxochlorin dyads is enhanced relative to the ZnFb porphyrin dyad (as indicated by F?rster calculations), this enhancement is insufficient to compensate for the greatly diminished TB coupling. Taken together, the chlorin and oxochlorin dyads examined herein serve as benchmarks for elucidating the energy-transfer, electrochemical, and other properties of light-harvesting arrays containing multiple chlorins or oxochlorins.     

Solvent dependence of intramolecular electron transfer in a helical oligoproline assembly

The helical oligoproline assembly CH3-CO-Pro-Pro-Pro-Pra(Ptzpn)-Pro-Pro-Pra(RuIIb2m2+ -Pro-Pro-Pra(Anq)-Pro-Pro-Pro-NH2, having a spatially ordered array of functional sites protruding from the proline backbone, has been prepared. The 13-residue assembly formed a linear array containing a phenothiazine electron donor, a tris(bipyridine)ruthenium(II) chromophore, and an anthraquinone electron acceptor with the proline II secondary structure as shown by circular dichroism measurements. Following RuII --> b2m metal-to-ligand charge-transfer (MLCT) excitation at 457 nm, electron-transfer quenching occurs, ultimately to give a redox-separated (RS) state containing a phenothiazine (PTZ) radical cation at the Pra(Ptzpn) site and an anthraquinone (ANQ) radical anion at the Pra(Anq) site. The redox-separated state was formed with 33-96% efficiency depending on the solvent, and the transient stored energy varied from -1.46 to -1.71 eV at 22 +/- 2 degrees C. The dominant quenching mechanism is PTZ reductive quenching of the initial RuIII(b2m*-) MLCT excited state which is followed by m*- --> ANQ electron transfer to give the RS state. Back electron transfer is highly exergonic and occurs in the inverted region. The rate constant for back electron transfer is solvent dependent and varies from 5.2 x 10(6) to 7.7 x 10(6) s-1 at 22 +/- 2 degrees C. It is concluded that back electron transfer occurs by direct ANQ*- --> PTZ*+ electron transfer. Based on independently evaluated kinetic parameters, the electron-transfer matrix element is HDA approximately 0.13 cm-1.     

The association and activities of pteridines in photosynthetic systems

Abstract— Unconjugated pteridines are associated with the photosynthetic systems of several organisms. Inhibition of the development of the photosynthetic system of Rhodospirillum rubrum with the pteridine inhibitor 4-phenoxy-2,6-diamino pyridine (PDAP) is reversed by biopterin, a natural pteridine. Evidence is presented which indicates an electron transport function for pteridines. Specific interaction of reduced pteridines with isolated pigment protein complexes, leading to red-shifted absorption bands, has suggested a mechanism for photochemical energy trapping.     

Functional analysis of the lipoglycodepsipeptide antibiotic ramoplanin

The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.     

ELECTRON TRANSFER FROM PHOTOEXCITED SINGLET AND TRIPLET BACTERIOPHEOPHYTIN—II. THEORETICAL

Abstract— A previous paper showed that collision of the first excited singlet state of bacteriopheophytin (Bph*) and p-benzoquinone (Q) returns Bph* to the ground state; however, excited triplet (Bph⁺) and quinone on collision produce the radical ions, (Bph⁺) and (Q^?). This paer rationalizes these findings by first estimating the half cell potentials Bph⁺/Bph* and Bph⁺/Bph^T, the energy for the various collision complexes, and the energy of the charge separated ions Bph⁺+ Q? and then estimating the rates for conversion among these various states. Thus it is estimated that the complexes [Bph*Q] or [Bph^TQ], live ?5 ps before dissociating. This is long enough for electron transfer to occur, producing the singlet and triplet charge transfer complexes, [Bph⁺Q?]^S or [Bph⁺Q?]^T, either of which could separate to Bph⁺+ Q? in ?230ps. In the singlet case, quenching by reverse charge transfer [Bph⁺Q?]^S→[Bph Q] occurs more rapidly than ion separation; however, the analogous triplet process, [Bph⁺Q?]^T→ [Bph Q], is spin forbidden, so that ion separation competes successfully with quenching. Spin scrambling, [Bph⁺Q?]^S? [Bph⁺Q?]^T, is estimated to be slow, as this explanation requires. In the bacterial photosynthetic reaction center, the initial electron transfer from an excited singlet state of the bacteriochlorophyll dimer complex (BB)* to bacteriopheophytin, giving [(BB⁺)(Bph?)]^S, successfully leads to ion separated species (i) because reverse charge transfer [(BB⁺)(Bph?)]^S→ [(BB)(Bph)] is slowed by a fairly large Franck-Condon energy, ΔE? lev, which is difficult to convert from electronic to vibrational degrees of freedom and (ii) because of the rapid subsequent electron transfer from (Bph⁺) to another acceptor X.     

World Year of Physics: a direct test of E=mc2

One of the most striking predictions of Einstein's special theory of relativity is also perhaps the best known formula in all of science: E=mc(2). If this equation were found to be even slightly incorrect, the impact would be enormous--given the degree to which special relativity is woven into the theoretical fabric of modern physics and into everyday applications such as global positioning systems. Here we test this mass-energy relationship directly by combining very accurate measurements of atomic-mass difference, Delta(m), and of gamma-ray wavelengths to determine E, the nuclear binding energy, for isotopes of silicon and sulphur. Einstein's relationship is separately confirmed in two tests, which yield a combined result of 1-Delta(mc2)/E=(-1.4+/-4.4)x10(-7), indicating that it holds to a level of at least 0.00004%. To our knowledge, this is the most precise direct test of the famous equation yet described.