| 1. |
- Börjesson, Johan, et al.
(författare)
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Enhanced enzymatic conversion of softwood lignocellulose by poly(ethylene glycol) addition
- 2007
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 40:4, s. 754-762
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Tidskriftsartikel (refereegranskat)abstract
- Ethanol production from lignocellulose has great potential and is an important step in changing fuel consumption to a more environmentally friendly alternative. Lignocellulose is a large source of biomass. However, with lignocellulose and softwood lingocellulose in particular, high conversion of cellulose into fermentable sugars requires large amounts of enzymes. Addition of surfactants is known to increase the enzymatic conversion and decrease the amount of enzymes needed. Surfactants and polymers with various amount of ethylene oxide (EO) content were used to study the conversion of steam-pretreated spruce lignocellulose. Increasing conversion was obtained with longer EO chains on the non-ionic surfactants. Similar results were obtained by using only the hydrophilic part of the surfactant, i.e. by addition of ethylene oxide polymers such as poly(ethylene glycol) (PEG) to the hydrolysis mixture. Interactions of enzymes and PEG with substrate was monitored with C-14-labeled PEG 4000 and H-3-labeled Cel7A (CBH I), the dominating cellulase from Trichoderma reesei. Addition of PEG to enzyme hydrolysis of lignocellulose increased the conversion from 42% without addition to 78% in 16 h. Adsorption of Cel7A decreased from 81 to 59%. No effect of PEG was seen on a delignified substrate. By addition of PEG it was possible to perform hydrolysis at 50 degrees C leading to both high cellulose conversion (80%) and shorter process time (48 h). Two different interactions are proposed in PEG adsorption on lignocellulose, hydrogen bonding and hydrophobic interactions. Our conclusions from experiments on lignocellulose and delignified substrate are that EO containing surfactants and polymers, such as PEG, bind to lignin by hydrophobic interaction and hydrogen bonding and reduce the unproductive binding of enzymes. (c) 2006 Elsevier Inc. All rights reserved.
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| 2. |
- Everberg, Henrik, et al.
(författare)
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Aqueous two-phase partitioning for proteomic monitoring of cell surface biomarkers in human peripheral blood mononuclear cells
- 2006
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 5:5, s. 1168-75.
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Tidskriftsartikel (refereegranskat)abstract
- For proteomic monitoring of processes such as allergy or inflammation an efficient pre-fractionation strategy is required. We isolated plasma membranes from human peripheral blood mononuclear (PBM) cells by aqueous two-phase partitioning. After 1DE combined with LC-MS/MS, several cell surface marker proteins and in total 60 different plasma membrane proteins (out of 84 identified proteins, i.e., 72%) were detected. Plasma membranes obtained were from only one human donor, the procedure is therefore applicable for individual patient screening.
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| 3. |
- Gough, Simon, et al.
(författare)
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A new method for isolating physiologically active Mg-protoporphyrin
- 2007
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Ingår i: Plant Physiology and Biochemistry. - : Elsevier BV. - 1873-2690 .- 0981-9428. ; 45:12, s. 932-936
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Tidskriftsartikel (refereegranskat)abstract
- Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H2O/25% NH3. Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.
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| 4. |
- Gough, Simon P, et al.
(författare)
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A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway.
- 2007
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Ingår i: Plant Physiology and Biochemistry. - : Elsevier BV. - 0981-9428. ; 45:12, s. 932-6
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Tidskriftsartikel (refereegranskat)abstract
- Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H2O/25% NH3. Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.
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| 5. |
- Lundqvist, Joakim, et al.
(författare)
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ATP-Induced Conformational Dynamics in the AAA plus Motor Unit of Magnesium Chelatase
- 2010
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 18:3, s. 354-365
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Tidskriftsartikel (refereegranskat)abstract
- Mg-chelatase catalyzes the first committed step of the chlorophyll biosynthetic pathway, the ATP-dependent insertion of Mg2+ into protoporphyrin IX (PPIX). Here we report the reconstruction using single-particle cryo-electron microscopy of the complex between subunits BchD and BchI of Rhodobacter capsulatus Mg-chelatase in the presence of ADP, the nonhydrolyzable ATP analog AMPPNP, and ATP at 7.5 angstrom, 14 angstrom, and 13 angstrom resolution, respectively. We show that the two AAA+ modules of the subunits form a unique complex of 3 dimers related by a three-fold axis. The reconstructions demonstrate substantial differences between the conformations of the complex in the presence of ATIP and ADP, and suggest that the C-terminal integrin-I domains of the BchD subunits play a central role in transmitting conformational changes of BchI to BchD. Based on these data a model for the function of magnesium chelatase is proposed.
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| 6. |
- Peterson Wulff, Ragna
(författare)
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Protein complexes in chlorophyll biosynthetic enzymes
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are found on the inside, in the membrane, on the surface and on the outside of cells. They form complicated structures and they interact with other molecules and proteins. Protein complexes and protein-protein interactions are challenging to investigate and in the beginning of protein research most studies were done with single proteins, often in water. Although, in vivo proteins rarely function alone.To study protein complexes, two enzymes in the chlorophyll biosynthetic pathway were selected, Mg-chelatase in Rhodobacter capsulatus (bacteria) and the MPE cyclase complex in Hordeum vulgare (barley) and Arabidopsis thaliana (mouse-ear cress). Chlorophyll is a pigment formed through a complicated reaction path. Chlorophyll biosynthesis takes place in chlorophyll-producing organisms. The firstcommitted step towards chlorophyll biosynthesis is performed by the enzyme complex Mg-chelatase. Mg-chelatase inserts a Mg2+ ion into the porphyrin substrate. The pathway is continued by a methyltransferase and thereafter the MPE cyclase complex which performs a complicated ring-closure in the porphyrin.Mg-chelatase is composed of three proteins, BchI (40 kDa), BchD (60 kDa) and BchH (130 kDa). A cryo-electron microscopy model of the BchID complex (7.5 Å) revealed a two-tired hexameric ring structure with an arrangement of the subunits as a trimer of dimers. The transient full complex of Mg-chelatase, BchIDH, was chemically cross-linked and BchH was found to interact with the Dside of the BchID complex.The MPE cyclase complex was more difficult to study and two of the three core components of the complex are still unknown. An interesting enzyme, NADPH-dependent thioredoxin reductase C (NTRC), was found to stimulate the MPE cyclase reaction together with a 2-Cys peroxiredoxin. NTRC was characterised further with regards to function and structure. The enzyme consists of a fusion between a NADPH-dependent thioredoxin reductase polypeptide and a thioredoxin polypeptide in the C-terminal. The three-dimensional structure ofNTRC was determined with cryo-electron microscopy (10.0 Å) and revealed a tetramer.
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| 7. |
- Peterson Wulff, Ragna, et al.
(författare)
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The activity of barley NADPH-dependent thioredoxin reductase C is independent of the oligomeric state of the protein: tetrameric structure determined by cryo-electron microscopy
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 50:18, s. 3713-3723
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Tidskriftsartikel (refereegranskat)abstract
- Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H(2)O(2). These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hordeum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.
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| 8. |
- Rosengren, Anna, et al.
(författare)
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The role of subsite +2 of the Trichoderma reesei beta-mannanase TrMan5A in hydrolysis and transglycosylation
- 2012
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 30:3, s. 338-352
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Tidskriftsartikel (refereegranskat)abstract
- The N-terminal catalytic module of beta-mannanase TrMan5A from the filamentous fungus Trichoderma reesei is classified into family 5 of glycoside hydrolases. It is further classified in clan A with a (beta/alpha)(8) barrel configuration and has two catalytic glutamates (E169 and E276). It has at least five other residues conserved in family 5. Sequence alignment revealed that an arginine (R171 in TrMan5A) is semi-conserved among beta-mannanases in family 5. In a previously published mannobiose complex structure, this residue is positioned in hydrogen bonding distance from the C2 hydroxyl group of the mannose residue bound at the +2 subsite. To study the function of R171, mutants of this residue were constructed. The results show that arginine 171 is important for substrate binding and transglycosylation. A mutant of TrMan5A with the substitution R171K displayed retained activity on polymeric galactomannan but reduced activity on oligosaccharides due to an increase of K-m. While the wild-type enzyme produces mannobiose as dominant product from mannotetraose the R171K mutant shows an altered product profile, producing mannotriose and mannose. The cleavage pattern of mannotetraose was analysed with a method using isotope labelled water ((H2O)-O-18) and mass spectrometry which showed that the preferred productive binding mode of mannotetraose was shifted from subsite -2 to +2 in the wild-type to subsite -3 to +1 in the R171K mutant. Significant differences in product formation after manno-oligosaccharide incubation showed that the wild-type enzyme can perform transglycosylation on to saccharide acceptors while the R171K mutant cannot, likely due to loss of acceptor affinity. Interestingly, both enzymes show the ability to perform alcoholysis reactions with methanol and butanol, forming new beta-linked glyco-conjugates. Furthermore, it appears that the wild-type enzyme produces mainly mannobiose conjugates using M-4 as substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding.
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| 9. |
- Stenbaek, Anne, et al.
(författare)
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NADPH-dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection of Mg–protoporphyrin monomethyl ester cyclase
- 2008
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 582:18, s. 2773-2778
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Tidskriftsartikel (refereegranskat)abstract
- Abstract The chloroplast-localized NADPH-dependent thioredoxin reductase (NTRC) has been found to be able to reduce hydrogen peroxide scavenging 2-Cys peroxiredoxins. We show that the Arabidopsis ntrc mutant is perturbed in chlorophyll biosynthesis and accumulate intermediates preceding protochlorophyllide formation. A specific involvement of NTRC during biosynthesis of protochlorophyllide is indicated from in vitro aerobic cyclase assays in which the conversion of Mg–protoporhyrin monomethyl ester into protochlorophyllide is stimulated by addition of the NTRC/2-Cys peroxiredoxin system. These findings support the hypothesis that this NADPH-dependent hydrogen peroxide scavenging system is particularly important during periods with limited reducing power from photosynthesis, e.g. under chloroplast biogenesis.
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| 10. |
- Söderberg, Christopher, et al.
(författare)
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Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation.
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:6
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Tidskriftsartikel (refereegranskat)abstract
- Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using (14)N/(15)N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers.
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