Redox processes acidify and decarboxylate steam-pretreated lignocellulosic biomass and are modulated by LPMO and catalase.

BACKGROUND: The bioconversion of lignocellulosic feedstocks to ethanol is being commercialised, but further process development is required to improve their economic feasibility. Efficient saccharification of lignocellulose to fermentable sugars requires oxidative cleavage of glycosidic linkages by lytic polysaccharide monooxygenases (LPMOs). However, a proper understanding of the catalytic mechanism of this enzyme class and the interaction with other redox processes associated with the saccharification of lignocellulose is still lacking. The in-use stability of LPMO-containing enzyme cocktails is increased by the addition of catalase implying that hydrogen peroxide (H2O2) is generated in the slurry during incubation. Therefore, we sought to characterize the effects of enzymatic and abiotic sources of H2O2 on lignocellulose hydrolysis to identify parameters that could improve this process. Moreover, we studied the abiotic redox reactions of steam-pretreated wheat straw as a function of temperature and dry-matter (DM) content. RESULTS: Abiotic reactions in pretreated wheat straw consume oxygen, release carbon dioxide (CO2) to the slurry, and decrease the pH. The magnitude of these reactions increased with temperature and with DM content. The presence of LPMO during saccharification reduced the amount of CO2 liberated, while the effect on pH was insignificant. Catalase led to increased decarboxylation through an unknown mechanism. Both in situ-generated and added H2O2 caused a decrease in pH. CONCLUSIONS: Abiotic redox processes similar to those that occur in natural water-logged environments also affect the saccharification of pretreated lignocellulose. Heating of the lignocellulosic material and adjustment of pH trigger rapid oxygen consumption and acidification of the slurry. In industrial settings, it will be of utmost importance to control these processes. LPMOs interact with the surrounding redox compounds and redirect abiotic electron flow from decarboxylating reactions to fuel the oxidative cleavage of glycosidic bonds in cellulose.

Biomass conversion determined via fluorescent cellulose decay assay.

An example of a rapid microtiter plate assay (fluorescence cellulose decay, FCD) that determines the conversion of cellulose in a washed biomass substrate is reported. The conversion, as verified by HPLC, is shown to correlate to the monitored FCD in the assay. The FCD assay activity correlates to the performance of multicomponent enzyme mixtures and is thus useful for the biomass industry. The development of an optimized setup of the 96-well microtiter plate is described, and is used to test a model that shortens the assay incubation time from 72 to 24h. A step-by-step procedure of the final assay is described.

Effects of non-ionic surfactants on the interactions between cellulases and tannic acid: a model system for cellulase-poly-phenol interactions.

Addition of non-ionic surfactants (NIS) is known to accelerate enzymatic lignocellulose hydrolysis. The mechanism behind this accelerating effect is still not elucidated but has been hypothesized to originate from favorable NIS-lignin interactions which alleviate non-productive adsorption of cellulases to lignin. In the current work we address this hypothesis using tannic acid (TAN) as a general poly-phenolic model compound (for lignin and soluble phenolics) and measure the mutual interactions of cellulases (CBHI, CBHII, EGI, EGII and BG), TAN and NIS (Triton X-100) using isothermal titration calorimetry (ITC). The experimental results suggest rather strong enzyme-specific interactions with TAN in reasonable agreement with enzyme specific lignin inhibition found in the literature. Enzyme-TAN interactions were disrupted by the presence of NIS by a mechanism of strong TAN-NIS interaction. The presence of NIS also alleviated the inhibitory effect of TAN on cellulase activity. All together the current work provides strong indications that favorable NIS-poly-phenol interactions alleviate non-productive cellulase-poly-phenol interactions and hence may provide a mechanism for the accelerating effect of NIS on lignocellulose hydrolysis.

A kinetic model for the burst phase of processive cellulases.

Cellobiohydrolases (exocellulases) hydrolyze cellulose processively, i.e. by sequential cleaving of soluble sugars from one end of a cellulose strand. Their activity generally shows an initial burst, followed by a pronounced slowdown, even when substrate is abundant and product accumulation is negligible. Here, we propose an explicit kinetic model for this behavior, which uses classical burst phase theory as the starting point. The model is tested against calorimetric measurements of the activity of the cellobiohydrolase Cel7A from Trichoderma reesei on amorphous cellulose. A simple version of the model, which can be solved analytically, shows that the burst and slowdown can be explained by the relative rates of the sequential reactions in the hydrolysis process and the occurrence of obstacles for the processive movement along the cellulose strand. More specifically, the maximum enzyme activity reflects a balance between a rapid processive movement, on the one hand, and a slow release of enzyme which is stalled by obstacles, on the other. This model only partially accounts for the experimental data, and we therefore also test a modified version that takes into account random enzyme inactivation. This approach generally accounts well for the initial time course (approximately 1 h) of the hydrolysis. We suggest that the models will be useful in attempts to rationalize the initial kinetics of processive cellulases, and demonstrate their application to some open questions, including the effect of repeated enzyme dosages and the 'double exponential decay' in the rate of cellulolysis.

Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

Plant paralog to viral movement protein that potentiates transport of mRNA into the phloem.

CmPP16 from Cucurbita maxima was cloned and the protein was shown to possess properties similar to those of viral movement proteins. CmPP16 messenger RNA (mRNA) is present in phloem tissue, whereas protein appears confined to sieve elements (SE). Microinjection and grafting studies revealed that CmPP16 moves from cell to cell, mediates the transport of sense and antisense RNA, and moves together with its mRNA into the SE of scion tissue. CmPP16 possesses the characteristics that are likely required to mediate RNA delivery into the long-distance translocation stream. Thus, RNA may move within the phloem as a component of a plant information superhighway.

Rice phloem thioredoxin h has the capacity to mediate its own cell-to-cell transport through plasmodesmata.

Rice (Oryza sativa L.) phloem sieve tubes contain RPP13-1, a thioredoxin h protein that moves around the plant via the translocation stream. Such phloem-mobile proteins are thought to be synthesized in the companion cells prior to being transferred, through plasmodesmata, to the enucleate sieve-tube members. In this study, in-situ hybridization experiments confirmed that expression of RPP13-1 is restricted to companion cells within the mature phloem. To test the hypothesis that RPP13-1 enters the sieve tube, via plasmodesmata, recombinant RPP13-1 was expressed in Escherichia coli, extracted, purified and fluorescently labeled with fluorescein isothiocyanate (FITC) for use in microinjection experiments into tobacco (Nicotiana tabacum L.) mesophyll cells. The FITC-RPP13-1 moved from the injected cell into surrounding cells, whereas the E. coli thioredoxin, an evolutionary homolog of RPP13-1, when similarly labeled and injected, failed to move in this same experimental system. In addition, co-injection of RPP13-1 and FITC-dextrans established that RPP13-1 can induce an increase in plasmodesmal size exclusion limit to a value greater than 9.4 but less than 20 kDa. Nine mutant forms of RPP13-1 were constructed and tested for their capacity to move from cell to cell; two such mutants were found to be incapable of movement. Crystal-structure prediction studies were performed on wild-type and mutant RPP13-1 to identify the location of structural motifs required for protein trafficking through plasmodesmata. These studies are discussed with respect to plasmodesmal-mediated transport of macromolecules within the companion cell-sieve tube complex.

Molecular cloning and expression of a novel adhesion molecule, SC1.

SC1, an integral membrane glycoprotein of 100 kd, is uniquely and transiently expressed on spinal cord motoneurons early in development and appears in peripheral neurons and several other tissues during development. SC1 has been purified by immunoaffinity techniques, and SC1 cDNA clones have been obtained by screening an E4 chick embryo phage expression library with a rabbit polyclonal antibody produced against purified SC1. The deduced protein sequence of 588 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The sequence is most similar to MUC18, reported as a tumor progression marker in human melanoma. Transfection of SC1 cDNA into mammalian cells leads to cell surface expression of SC1 antigen and a subsequent increase in cell-cell adhesion. SC1 molecules bind to each other via a homophilic adhesion mechanism, independently of calcium or magnesium ions. SC1 may have a role in lateral motor column formation or neurite growth or fasciculation.

Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family.

A complementary DNA (cDNA) for the rat luteal lutropin-choriogonadotropin receptor (LH-CG-R) was isolated with the use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein. As would be predicted from the cDNA sequence, the LH-CG-R consists of a 26-residue signal peptide, a 341-residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein (LRG) family, and a 333-residue region containing seven transmembrane segments. This membrane-spanning region displays sequence similarity with all members of the G protein-coupled receptor family. Hence, the LH-CG-R gene may have evolved by recombination of LRG and G protein-coupled receptor genes. Cells engineered to express LH-CG-R cDNA bind human choriogonadotropin with high affinity and show an increase in cyclic adenosine monophosphate when exposed to hormone. As revealed by RNA blot analysis and in situ hybridization, the 4.4-kilobase cognate messenger RNA is prominently localized in the rat ovary.

Peptide-based radioimmunoassay for insulin receptor. Detection of insulin-stimulated downregulation in IM-9 lymphocytes.

To overcome the difficulties encountered in quantifying the insulin receptor number by Scatchard analysis, a radioimmunoassay (RIA) for the human insulin receptor (hIR) has been developed that uses an antibody raised against a synthetic peptide (Gly-Lys-Lys-Asn-Gly-Arg-Ile-Leu-Thr-Leu-Pro-Arg-Ser-Asn-Pro-Ser) corresponding to the carboxyl terminal of the hIR. A second peptide (Tyr-Gly-Arg-Ile-Leu-Thr-Leu-Pro-Arg-Ser-Asn-Pro-Ser) was used as a standard and allowed preparation of monoiodinated derivative of theoretical specific activity for use as the radioactive ligand. The assay is specific, highly reproducible, and sensitive, with a detection limit of 10 fmol of receptor. One mole of purified receptor, measured by Scatchard analysis or amino acid analysis, is read as one mole of receptor in the RIA with peptide being the standard. The assay is effective with receptor from multiple sources and could determine the decrease in number of insulin receptors seen in IM-9 lymphocytes after treatment with insulin (downregulation).

Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact.

A purified opioid-binding protein has been characterized by cDNA cloning. The cDNA sequence predicts an extracellularly located glycoprotein of 345 amino acids. This protein does not possess a membrane-spanning domain but contains a C-terminal hydrophobic sequence characteristic of membrane attachment by a phosphatidylinositol linkage. It displays homology to the immunoglobulin protein superfamily, featuring three domains that resemble disulfide-bonded constant regions. More specifically, the protein is most homologous to a subfamily of proteins which includes the neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG) and one subgroup of the tyrosine kinase growth factor receptors comprising the platelet-derived growth factor receptor (PDGF R), the colony-stimulating factor 1 receptor (CSF-1 R) and the c-kit protooncogene. These sequence homologies suggest that the protein could be involved in either cell recognition and adhesion, peptidergic ligand binding or both.

Identification of the intact insulin receptor using a sequence-specific antibody directed against the C-terminus of the beta-subunit.

An antibody was raised against a synthetic peptide corresponding to the carboxyl-terminal amino acids of the human insulin receptor (Anti-R beta C). Immunoprecipitation of the human insulin receptor and immunoblotting to the beta-subunit by Anti-R beta C could be inhibited by competition with the corresponding peptide. However, even at saturating concentrations, anti-R beta C could not completely immunoprecipitate or immunodeplete insulin receptors compared to a human autoantibody (anti-R B2). Using receptor labeled directly by 125I, evidence of multiple forms of the beta-subunit was found. When the receptor could be immunoprecipitated by anti-R beta C, the beta-subunit migrated with an apparent mol wt (MW) of 96,000 (at or above the phosphorylase b MW marker). However, in preparations where anti-R beta C was not able to immunoprecipitate the insulin receptor, the beta-subunit migrated at a significantly lower MW of 91,000 (below phosphorylase b), as detected by immunoprecipitation with Anti-R B2. Intermediate forms could also be detected. Phosphorylation of partially purified insulin receptor did not affect is ability to be immunoprecipitated by anti-R beta C, although insulin-stimulated phosphorylation increased the apparent MW of the beta-subunit. However, insulin receptor that was phosphorylated in solubilized extracts of whole cells had a beta-subunit that migrated at lower MW and was not immunoprecipitated by anti-R beta C. One possible explanation for this is that the beta-subunit may be degraded during preparation. When the MW of insulin receptor that has been purified to homogeneity from human placenta is compared to our data, it is clear that many of these insulin receptor preparations contain lower MW beta-subunits. These results must be taken into account when the sites of phosphorylation and kinase activity of purified insulin receptor preparations are studied.