Cellulase Thesis

Cellulase Thesis-79
The enzymes had optimal activity at p H 5.0-6.0 and temperature 50-60 ºC, and temperature stability up to 40 ºC on carboxymethyl cellulose (CMC).The enzymes were active on various substrates with β-1,4-glucosidic linkages in the backbone and on the lignocellulosic substrate switchgrass.The cluster contained both multi-domain enzymes and enzymes with a C-terminal secretion tag for the Type IX secretion system (T9SS).

Ideally, the isolation of a representative of the AGa phylotype would shed light on its true involvement in cellulose degradation.

Collectively, the work presented in this thesis contributes to the understanding of biomass degradation by anaerobic-bacteria, which can help improving the industrial conversion of biomass to valuable products in the future.

Combining the enzymes in cocktails gave no synergistic effects.

The weakly annotated CBM domain in one of the enzymes was shown to bind to the cellulosic substrate Avicel, indicating a correct annotation as a CBM.

Lignocellulosic biomass is generally regarded as a sustainable and environmentally friendly energy source.

Cellulase Thesis

However, the production of fuels from biomass is not sufficiently effective to compete fully with fossil fuels on economic terms, mostly due to the low efficiency of the enzymes needed to degrade the biomass.Denne oppgaven ble basert på en metagenomisk studie av en switchgrass prøve fra kumage, hvor videre undersøkelser fant et antatt cellulolytisk genkluster, fra en Bacteroidetes fylotypes genom (AGa), med fire antatt cellulolytiske glykosid-hydrolase familie 5 (GH5) enzymer.Klusteret inneholdt både multi-domene enzymer og enzymer med et C-terminalt sekresjonssignal for Type IX sekresjonssystemet (T9SS).Lignocellulose is the most abundant biomass on earth and has a great potential as a source for sustainable production of valuable chemicals and biofuels.Today’s depletion of fossil fuel reserves and pollution from their usage creates a need for a more sustainable source of energy.Some of the enzymes gave inclusion bodies upon expression and had to be expressed with fusion tags.Further, the enzymes were characterized, through enzymatic assays, in terms of p H and temperature optima, temperature stabilities, substrate specificities, product profiles and cellodextrin cleaving patterns.Product analysis showed release of mostly cellobiose from cellulosic substrates, and cellodextrin assays showed cleaving of cellopentaose to cellobiose and cellotriose, and cellohexaose to cellobiose, cellotriose and partly cellotetraose for the majority of the enzymes.One GH5 displayed a different cleaving pattern on cellohexaose, cleaving it to solely cellotriose.The cluster enzyme characterization gives insight into the use of the Type IX secreted multi-domain cellulases.Further work on the gene cluster can provide more insight into the degradation mechanism, and expression of the cluster in a bacterium harbouring the Type IX secretion system could be pursued to improve the expression of the multi-domain cellulases that were difficult to express.


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