<strong>Table S2. List of all peptides detected by proteomics for AA9-X282 proteins.</strong>

The table contains all peptides detected by proteomic analysis (high-resolution tandem mass spectrometry) of three AA9-X282 proteins produced heterologously in Pichia pastoris and purified by IMAC, namely the AA9-X282-CBM1  from Coprinopsis cinerea (CciAA9-X282-CBM1), the AA9-X282-9res from Trametes ljubarskyi (TljAA9-X282-9res) and Pycnoporus coccineus (syn. Trametes coccinea) (PcoAA9-X282-9res). </p

MOESM2 of Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies

Additional file 2: Figure S2. F-values of principal coefficients and their interactions for each GH45 probe in FAX/DHP assemblies. A: probe type, B: FAX concentration, C: DHP concentration and their interactions AB, AC, BC, ABC

Vliv přídavku technického konopí Cannabis sativa na antioxidační aktivitu u laboratorních potkanů

This thesis studies an influence of hemp added to feed rations on the organism of tested animals. In this feeding experiment the effect of supplied hemp to weight increments and antioxidant activity was observed. The tested animals were rats of strain Wistar albino. The experiment was performed with 20 male rats which were divided into 4 groups. Two groups were fed with hemp mixture and other two with control mixture. During the experiment subjects were weighed once a week and weight gain and feed consumptions were recorded. Spectrofotometric method Folin-Ciocalteu for determinations of polyphenolic compounds was used. Antioxidant activity was measured for the individual samples of feed and liver tissues of tested animals. They were FRAP and TEAC method used. The results of measurement of antioxidant activity in liver tissue proved inconclusive for both methods. Demonstrable differences were found only in samples of feed. The processing was used ANOVA statistical method using the Scheffe test

List of primers used in the study.

<p>List of primers used in the study.</p

Molecular Engineering of Fungal GH5 and GH26 Beta-(1,4)-Mannanases toward Improvement of Enzyme Activity

<div><p>Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast <i>Yarrowia lipolytica</i> to improve the specific activity of two fungal endo-mannanases, <i>Pa</i>Man5A and <i>Pa</i>Man26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several <i>Pa</i>Man5A and <i>Pa</i>Man26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of <i>Pa</i>Man5A and <i>Pa</i>Man26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the <i>Pa</i>Man5A-G311S single mutant, which displayed an impressive 8.2-fold increase in <i>k_cat</i>/K_M due to a significant decrease of K_M, is located within the core of the enzyme. The <i>Pa</i>Man5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The <i>Pa</i>Man26A-P140L/D416G double mutant yielded a 30% increase in <i>k_cat</i>/K_M compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (k_cat/K_M).</p></div

Structural view of <i>Pa</i>Man5A (PDB 3ZIZ) exhibiting substituted amino-acids.

<p>A. Surface view of the catalytic cleft of <i>Pa</i>Man5A with mannotriose modelled in the −2 and −3 subsites and mannobiose modelled in the +1 and +2 subsites. The structures of GH5 from <i>T. reesei</i> and <i>T. fusca</i> in complex with mannobiose and mannotriose, respectively, were superimposed on the top of the structure of <i>Pa</i>Man5A to map the substrate-binding subsites. The two catalytic glutamate residues, E177 and E283, are coloured in red. The substituted amino-acids are labelled and coloured in yellow. B. Structural based sequence alignment of the region around position 311 (according to <i>Pa</i>Man5A numbering) from <i>Podospora anserina</i> (<i>Pa</i>Man5A), <i>Aplysia kurodai</i> (<i>Ak</i>Man, PDB 3VUP), <i>Mytilus edulis</i> (<i>Me</i>Man5A, PDB 2C0H), <i>Cellvibrio mixtus</i> (<i>Cm</i>Man5A, PDB 1UUQ), <i>Trichoderma reesei</i> (<i>Tr</i>Man5A, PDB 1QNR), <i>Lycopersicon esculentum</i> (<i>Le</i>Man4A, PDB 1RH9) and <i>Thermomonospora fusca</i> (<i>Tf</i>Man5, PDB 2MAN). Secondary structure elements, α-helix α7 and β-strand β8, are indicated below the sequences as a cylinder and an arrow, respectively. Strictly conserved residues, G311 and W315 (according to <i>Pa</i>Man5A numbering), are shown with a yellow and a grey background, respectively. C. Surface view of <i>Pa</i>Man5A rotated of about 90° along the horizontal axis. The front clipping plane has been moved in order to visualize the location of G311 inside the molecule. The zoom shows a compact hydrophobic core in the vicinity of G311.</p

Screening strategy and mutant selection.

<p>The number of variants screened at each step is indicated at the top (<i>Pa</i>Man5A) and at the bottom (<i>Pa</i>Man26A) of the diagram.</p

Error-prone PCR strategy used in the study.

<p>PCR1: error prone-PCR performed on <i>paman5a</i> (HM357135) and <i>paman26a</i> (HM357136); PCR2: PCR without mutation performed on Ura3d1 (selection marker), pPOX2 (inducible promotor of acyl-coA oxidase 2) and prepro Lip2 (secretion signal sequence); PCR3: overlapping PCR to reconstruct the entire sequence between zeta platforms. Primers used are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079800#pone-0079800-t003" target="_blank">Table 3</a>.</p

Kinetic constants of wild-type enzymes and selected variants toward galactomannan, mannohexaose (M₆) and mannopentaose (M₅).

<p>The kinetic parameters were determined at 40°C in sodium acetate buffer 50 mM, pH 5.2 as described in the Methods section. Paired t test was used to compare the kinetic parameters of mutants versus native enzyme. The difference was considered statistically significant when <i>p</i><0.05 (*). wt, wild type.ND: not determined.</p

Mannanase activity of selected <i>Y. lipolytica</i> variants.

<p>The mannanase activity was measured at 40°C in sodium acetate buffer 50 mM, pH 5.2 using 1% (w/v) galactomannan. The coefficient of variation (CV) was defined as the ratio of the standard deviation to the mean and was calculated for each of the wild-type enzymes. wt, wild type.</p