Protein identification was performed using the open-source software MaxQuant 1. Alginate lyase activity was assayed on 1. Degradation of substrates was monitored by measuring A LR was identified in P. The gene sequence was truncated to exclude the 22 amino acids long predicted signal peptide. The resulting construct was transformed into E. Then, a methanol fed-batch phase was initiated to induce expression and secretion of the enzyme. Protein concentrations were determined by A using the theoretically obtained molar extinction coefficients.
Reactions on 0. Substrate concentrations ranging from 0. The draft genome of P. In order to elucidate P. The first hit was the terrestrial plant pathogen Stemphylium lycopersici at E-value 0.
The result correlated with the reported close taxonomic relationship of P. The protein sequences from the S.
The P. Of the predicted P. The CAZyme annotations were binned according to broad substrate specificities and compared. The two fungi showed similar substrate profiles, however, P.
Chondroitin AC lyase chondroitin sulfate and alginate lyase were unique to P. Comparative distribution of CAZymes in P. The CAZyme profiles showed high similarity with respect to families and paralogue copies within them Fig. Compared to the three terrestrial fungal plant pathogens, P. Venn diagram of shared CAZyme domain families between P. The three unique families in P. Unique to P. Three brown algae species A. To assess the content and variation of sugars in the selected species, we performed monomer composition analysis by acid hydrolysis with subsequent HPAEC-PAD of products.
Fucose content, assumed to mainly stem from FCSPs, varied significantly across the three species, with A. The majority of the glucose is assumed to come from laminarin and cellulose of which F.
Mannitol content was relatively the same for all the species at approximately seven percent. The samples were analyzed in triplicates. Based on the combination of assumed substrate specificities of the CAZymes and the monomer analysis of the brown algae, it was possible to identify enzymes in the P.
The predicted PL7 genes were found on three different contigs and interestingly none of them contained introns. To expand on this, the sequences were aligned with PL7 domain sequences derived from CAZy and analyzed in a maximum likelihood phylogenetic analysis Fig. The three P. Recently three putative PL7 proteins were annotated in a marine Calcarisporium sp. KF Basidiomycota 57 and it would have been interesting to include these sequences in the phylogenetic analysis, however no sequence information from this genome was available at the time of this study.
Maximum likelihood phylogenetic analysis of selected CAZy-listed PL7 protein sequences, including the three sequences from P. The species names are indicated along with accession numbers of corresponding PL7 sequences.
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Branch numbers indicate bootstrap values. The tree scale bar indicates substitution changes per site. Sequences belonging to PL7 subfamily 4 are indicated in the same column. The tree was pruned from a larger phylogenetic tree containing all CAZy listed sequences Supplementary Fig. We found putative genes for the complete degradation of cellulose in P. The AA9 LPMOs have been reported to act mainly on cellulose which is the broad category chosen for them in this study, but there have also been reports of specificity towards hemicellulose 58 and xyloglucan Five sulfate transporter proteins PF, PF were also found: one sulfate permease 1 2.
We found several enzymes belonging to the mannitol II degradation pathway. A mannitol dehydrogenase EC 1. Two such putative isomerases and multiple hexokinases were found. The presence of one mannitolphosphate 5-dehydrogenase EC 1. Three putative trehalase genes EC 3.
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Nine sequences were found which were subsequently aligned with the sequences used by Fournier and colleagues 62 and analyzed with maximum likelihood analysis. The phylogenetic tree showed that one PAP2 sequence g We tested the fermentation supernatants on a variety of substrates found in plant, fungal and brown algal cell walls.
In the carbon limited fermentation, amylase activity was observed very soon after day two together with protease activity Supplementary Fig. Enzyme activities in the fermentation supernatants. The supernatants of P.
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Five biological replicates for each type of fermentation were analyzed. To confirm the in silico prediction of the mannuronic acid specificity EC 4. The recombinant PsAlg7A had an approx. S5 , lane 2 , which corresponds with the predicted molecular mass. Michaelis-Menten plots of PsAlg7A. Evolution of initial velocities measured as the formation of double bonds for increasing substrate concentrations of a alginate, b polymannuronic acid and c polyguluronic acid.
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Maxquant identified proteins from P. The relative quantity analysis of the secretomes of the four fermentations revealed different profiles for each of the four types Fig. As expected, based on the enzyme activities, CAZymes were represented two to three times higher in the fermentations with algae, both in relative distribution Fig.
Relative composition of the secreted proteomes of P. Starch-like polysaccharides or the genes necessary for their synthesis have not been found in brown algae. All three algal P. The essential activities for gluconic acid production in Aspergillus niger were found to consist of an AA3 glucose-oxidase EC 1. This enzyme was most likely responsible for the activity observed on arabinoxylan and xylan Fig.
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Additionally, two putative pectate lyases EC 4. One of the most abundant single proteins in all the P.
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The annotation analysis of the P. However, in spite of the lower numbers of CAZymes, P. The proteomic and genomic analysis of the CAZymes of P. The absence of alginate lyase families in the terrestrial relatives, the absence of introns in the genes and the phylogenetic placement among bacterial sequences could indicate possible horizontal gene transfers from marine bacteria, granting P.
This hypothesis however, is difficult to confirm and the GC content analysis of the genes and the low sequence identity to all other PL7 proteins provided no further evidence. The gene loss of the PL7 genes in the terrestrial fungi is also a possibility, but even more difficult to confirm.