Cell-Free Biosystems Comprised of Synthetic Enzymatic Pathways: Development of Building Blocks, Immobilization of Enzymes, Stabilization of Cascade Enzymes, and Generation of Hydrogen
|dc.description.abstract||The production of hydrogen from low-cost abundant renewable biomass would be vital to sustainable development. Cell-free (in vitro) biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it is essential to produce (purified) enzymes at low costs and stabilize them for long periods to decrease biocatalyst costs.|
Thermophilic recombinant enzymes as building blocks were discovered and developed: fructose 1,6-bisphosphatase (FBP) from Thermotoga maritime, phosphoglucose isomerase (PGI) from Clostridium thermocellum, triose phosphate isomerase (TIM) from Thermus thermophiles and fructose bisphosphate aldolase (ALD) from T. maritima and T. thermophilus. The recombinant proteins were over-expressed in E. coli, purified and characterized.
For purification and stabilization of enzymes, one-step, simple, low-cost purification and immobilization methods were developed based on simple adsorption of cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose. Also, a simple, low-cost purification method of thermophilic enzymes was developed utilizing a combination of heat and ammonium sulfate precipitation.
For development of cascade enzymes as building modules (biocatalyst modules), it was discovered that the presence of other enzymes/proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme (e.g., PGI) due to the in vitro macromolecular crowding effect. And substrate channeling among CBM-tagged self-assembled three-enzyme complex (synthetic matabolon) immobilized on the easily-recycled cellulose-containing magnetic nanoparticles can not only increase cascade reaction rates greatly, but also decrease enzyme cost in cell-free biosystems.
The high product yield and fast reaction rate of dihydrogen from sucrose was validated in a batch reaction containing fifteen enzymes comprising a non-natural synthetic pathway. The yield of dihydrogen production from 2 mM of sucrose was 96.7 % compared to theoretical yield at 37 oC. The maximum rate was increased 3.1 fold when the substrate concentration was increased from 2 to 50 mM in a fed-batch reaction.
The research and development of cell-free biosystems for biomanufacturing require more efforts, especially in low-cost recombinant thermostable enzymes as building blocks, efficient cofactor recycling, enzyme and cofactor stabilization, and fast reaction rates.
|dc.rights||This Item is protected by copyright and/or related rights. Some uses of this Item may be deemed fair and permitted by law even without permission from the rights holder(s), or the rights holder(s) may have licensed the work for use under certain conditions. For other uses you need to obtain permission from the rights holder(s).||en_US|
|dc.subject||synthetic enzymatic pathway||en_US|
|dc.title||Cell-Free Biosystems Comprised of Synthetic Enzymatic Pathways: Development of Building Blocks, Immobilization of Enzymes, Stabilization of Cascade Enzymes, and Generation of Hydrogen||en_US|
|dc.contributor.department||Biological Systems Engineering||en_US|
|thesis.degree.grantor||Virginia Polytechnic Institute and State University||en_US|
|thesis.degree.discipline||Biological Systems Engineering||en_US|
|dc.contributor.committeechair||Zhang, Yi Heng Percival||en_US|
|dc.contributor.committeemember||Barone, Justin Robert||en_US|
|dc.contributor.committeemember||Winkel, Brenda Sophia J.||en_US|
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