Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes

Flavourzyme is sold as a peptidase preparation from Aspergillus oryzae. The enzyme preparation is widely and diversely used for protein hydrolysis in industrial and research applications. However, detailed information about the composition of this mixture is still missing due to the complexity. The present study identified eight key enzymes by mass spectrometry and partially by activity staining on native polyacrylamide gels or gel zymography. The eight enzymes identified were two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one α-amylase from the <i>A. oryzae</i> strain ATCC 42149/RIB 40 (yellow koji mold). Various specific marker substrates for these Flavourzyme enzymes were ascertained. An automated, time-saving nine-step protocol for the purification of all eight enzymes within 7 h was designed. Finally, the purified Flavourzyme enzymes were biochemically characterized with regard to pH and temperature profiles and molecular sizes

Unraveling the Degradation Process of LiNi_0.8Co_0.15Al_0.05O₂ Electrodes in Commercial Lithium Ion Batteries by Electronic Structure Investigations

The degradation of LiNi_0.8Co_0.15Al_0.05O₂ (LNCAO) is reflected by the electrochemical performance in the fatigued state and correlated with the redox behavior of these cathodes. The detailed electrochemical performance of these samples is investigated by galvanostatic and voltammetric cycling as well as with the galvanostatic intermittent titration technique (GITT). Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy was used to investigate the oxidation state of all three materials at the Ni L_2,3, O K, and Co L_2,3 edges at five different states of charge. Surface and more bulklike properties are distinguished by total electron yield (TEY) and fluorescence yield (FY) measurements. The electrochemical investigations revealed that the changes in the cell performance of the differently aged materials can be explained by considering the reaction kinetics of the intercalation/deintercalation process. The failure of the redox process of oxygen and nickel at low voltages leads to a significant decrease of the reaction rates in the fatigued cathodes. The accompanied cyclic voltammogram (CV) peaks appear as two peaks because of the local minimum of the reaction rate, although it is one peak in the CV of the calendarically aged LNCAO. The absence of the oxidation/reduction process at low voltages can be traced back to changes in the surface morphology (formation of a NiO-like structure). Further consequences of these material changes are overpotentials, which lead to capacity losses of up to 30% (cycled with a C/3 rate)

Transition-Metal Phthalocyanines on Transition-Metal Oxides: Iron and Cobalt Phthalocyanine on Epitaxial MnO and TiO<i>_x</i> Films

The interaction at interfaces between transition-metal phthalocyanines and ultrathin transition-metal oxide films is studied by means of photoemission (PES) and X-ray absorption spectroscopy (XAS). Our results are compared to the recently investigated system CoPc on MnO. A flat-lying adsorption geometry of iron and cobalt phthalocyanines (FePc and CoPc) on the different oxide substrates was observed: ultrathin epitaxially grown MnO films and ultrathin TiO<i>_x</i> films. A charge transfer from the MnO, in particular, to the Fe atom of the FePc molecule is observed by both PES and XAS. X-ray absorption spectra of the N K-edge of FePc do not hint at a nitrogen involvement in the interaction process. As a consequence of the charge transfer, a shift of the Fermi level of the semiconducting MnO films is observed, which is visible as a shift of MnO related core levels to lower binding energy. In contrast, CoPc and FePc deposited on TiO<i>_x</i> show no hints for a charge transfer, although the flat-lying adsorption geometry allows in principle a maximum interaction between the π-system and the substrate. Increased surface roughness (compared to MnO) and an oxygen termination of the surface of the TiO<i>_x</i> films is considered to suppress a possible strong interaction between the organic molecules and the substrate

Apparent kinetic parameters of reactivated apo-<i>Lb</i>-PepA using H-Asp-<i>p</i>NA or H-Glu-<i>p</i>NA as a substrate.

<p>The calculation of the kinetic parameters were performed by nonlinear regression fitting using SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA).</p

Relative PepA activity¹^,² in the presence of potential product inhibitors.

<p>Relative PepA activity<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152139#t002fn001" target="_blank">¹</a>^,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152139#t002fn002" target="_blank">²</a> in the presence of potential product inhibitors.</p

Apparent kinetic parameters of <i>Lb</i>-PepA and <i>Lc</i>-PepA using H-Asp-<i>p</i>NA or H-Glu-<i>p</i>NA as a substrate.

<p>The calculation of the kinetic parameters were performed by nonlinear regression fitting using SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA).</p

Summary of selected characteristics of PepA from different microorganisms.

<p>Summary of selected characteristics of PepA from different microorganisms.</p

Comparision of the <i>pepA</i> gene and PepA protein from different microorganisms.

<p>Comparision of the <i>pepA</i> gene and PepA protein from different microorganisms.</p

A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from <i>Lactobacillus delbrueckii</i> with Promising Properties for Application - Fig 1

<p><b>Course of the bioreactor cultivation (working volume: 600 mL) of recombinant <i>E</i>. <i>coli</i> BL21(DE3) for the production of <i>Lb</i>-PepA (A) and <i>Lc</i>-PepA (B).</b> Cultivations began at 37°C and shifted to 30°C with simultaneous induction using IPTG (see arrow). The means ± standard deviation of three independent measurements are presented.</p

Spin State in Perfluorinated FePc Films on Cu(111) and Ag(111) in Dependence on Film Thickness

The electronic structure of the central iron ion of perfluorinated iron phthalocyanine (FePcF₁₆) in thin films has been studied on Cu(111) and Ag(111) using polarization dependent X-ray absorption spectroscopy (XAS). The data are compared to FePc on Ag(111). Ligand field parameters have been computed, and multiplet calculations (CTM4XAS) were carried out to simulate XAS spectra. The planar molecules are preferentially oriented lying flat on the substrate surface during the growth of the 1–4 nm thick films. A clear polarization dependence of the Fe L edge absorption spectra is observed, arising from transitions into orbitals with in-plane and out-of-plane character. The shape of the spectra for three to four monolayers of FePcF₁₆ on Cu(111) is comparable to that of the thin films of FePc on Ag(111). However, a drastic change of the XAS peak shape is observed for thicker FePcF₁₆ films on both Ag(111) and Cu(111), although the molecular orientation is very similar to coverages consisting of a few monolayers. Since in both cases the film thickness is distinctly beyond the monolayer regime, interface interactions can be ruled out as a possible origin of this behavior. Rather, the different XAS peak shapes seem to indicate that the multiplicity may depend on the detailed arrangement of the FePcF₁₆ molecules. The large flexibility of the ground state of Fe could be of high interest for spintronic applications