Novel xylanases from metagenomic libraries

GRADU ON SALAINEN JOULUKUUHUN 2012 SAAKKA

Novel xylanases from metagenomic libraries

GRADU ON SALAINEN JOULUKUUHUN 2012 SAAKKA

Cloning of novel bacterial xylanases from lignocellulose-enriched compost metagenomic libraries

Xylanases are in important class of industrial enzymes that are essential for the complete hydrolysis of lignocellulosic biomass into fermentable sugars. In the present study, we report the cloning of novel xylanases with interesting properties from compost metagenomics libraries. Controlled composting of lignocellulosic materials was used to enrich the microbial population in lignocellulolytic organisms. DNA extracted from the compost samples was used to construct metagenomics libraries, which were screened for xylanase activity. In total, 40 clones exhibiting xylanase activity were identified and the thermostability of the discovered xylanases was assayed directly from the library clones. Five genes, including one belonging to the more rare family GH8, were selected for subcloning and the enzymes were expressed in recombinant form in E. coli. Preliminary characterization of the metagenome-derived xylanases revealed interesting properties of the novel enzymes, such as high thermostability and specific activity, and differences in hydrolysis profiles. One enzyme was found to perform better than a standard Trichoderma reesei xylanase in the hydrolysis of lignocellulose at elevated temperatures.Peer reviewe

Cost-efficient sugar-based cellulase production

Enzymes remain a significant operational cost in biochemical biorefining processes. Inexpensive streams of sugar are available at many potential biorefining sites, and these could be an optimal raw material for the on-site production of cellulase enzymes. However, simple sugars do not support cellulase production using conventional cellulase-producing strains, necessitating either process or strain improvements. Here we describe improved glucose-based enzyme production processes enabling industrially relevant cellulase production kinetics. Extracellular protein titers of up to 53 g/L could be reached in under 93 hours, representing an up to 260% improvement in productivity over previously reported results. Glucose-produced enzyme is also shown to perform well on pre-treated corn stover. Using the NREL biorefinery model, enzyme production costs are estimated to be $2,8/kg protein or 6/L produced ethanol. Additionally, we briefly discuss how organic nitrogen could be cycled in enzyme production, and how the described processes could fit into existing and emerging biorefining operations.</p

Comparison of pretreatments and cost-optimization of enzymatic hydrolysis for production of single cell protein from grass silage fibre

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Enzymatic reduction of galactooligosaccharide content of faba bean and yellow pea ingredients and food products

Intake of Fermentable Oligo,- Di,- Monosaccharides, And Polyols, abbreviated as FODMAP, is known to worsen gastrointestinal symptoms in people with functional bowel disorders. Legume-based foods are climate-friendly and healthy alternatives to meat, but also high in fermentable oligosaccharides, galactooligosaccharides in particular. In this study, we have screened commercial and produced, new α-galactosidases for hydrolysis of galactooligosaccharides in pea and faba bean based materials. Commercial DS30 and recombinantly produced Neosartorya fisherii α-galactosidase were further applied to enzymatic removal of galactooligosaccharides from leguminous ingredients as well as moist (spoonable), semi-moist (wet-extrudate) and dry (crackers) food prototypes. Galactooligosaccharide levels were reduced by both enzymes by over 90% during production of a pea protein-based spoonable product and formulations prepared for extrusion cooking. Extrusion cooking increased the analyzable galactooligosaccharide content to some extent, presumably because heating and shearing released more galactooligosaccharides from the material. Cracker dough galactooligosaccharide concentration was decreased less during α-galactosidase treatment, most likely because of the slightly alkaline pH of the material, which was not optimal for α-galactosidase action

A tentative model for mTTF function.

<p>A. Normally, replication pauses at mTTF binding sites, where orderly passage of transcription and replication is mediated. mTTF binding sites may additionally be the places where an RNA bootlace is supplied <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003800#pgen.1003800-Reyes1" target="_blank">[14]</a>, via termination of a nascent transcript produced by an arriving transcription complex. Note that, under this model, as the fork advances further, RNA/DNA hybrid is laid down behind the fork, whilst the replicative helicase unwinds the parental duplex ahead of the fork. The lagging-strand RNA can then be processed to generate primers for lagging-strand DNA synthesis, as the fork proceeds. Letters mark corresponding positions on RNA and DNA strands. B. In case of mTTF depletion by RNAi, uncontrolled collisions between the replication and transcription machineries take place outside of the mTTF binding site, leading to fork reversal and a failure of normal lagging-strand synthesis. C. Depletion of mTerf5 by RNAi is proposed to enhance the binding or inhibit the dissociation of mTTF, resulting in stronger pausing, a block to onward fork progression, and early completion of the lagging strand in the rRNA gene region.</p

Replication pauses at mTTF binding sites.

<p>A. Schematic map of <i>D. melanogaster</i> mtDNA with positions of probes, mTTF binding sites (bs1, bs2), gene clusters (bold), tRNA genes (open circles), non-coding region (NCR, grey) origin and direction of replication (open arrow) and restriction endonuclease sites for Hind III, Cla I, Nde I and Bsp 1407I. Positions of genes for which expression was analyzed are shown in blue. B. 2DNAGE of ClaI- or HindIII-digested mtDNA. Red arrows indicate discrete spots on standard Y-arcs, representing major pause sites (replication fork barriers), analogous with those documented previously in other systems <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003800#pgen.1003800-Brewer2" target="_blank">[59]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003800#pgen.1003800-Greenfeder1" target="_blank">[88]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003800#pgen.1003800-Shinomiya1" target="_blank">[89]</a>: (see also relevant reviews cited in text, explaining the species seen by 2DNAGE). Blue arrows denote broader replication slow-zone in the HindIII fragment detected by probe 3. For more accurate mapping of pause sites by multiple digests, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003800#pgen.1003800.s001" target="_blank">Fig. S1</a>.</p