VTechWorks staff will be away for the Independence Day holiday from July 4-7. We will respond to email inquiries on Monday, July 8. Thank you for your patience.
 

Development of Building Blocks - Thermostable Enzymes for Synthetic Pathway Biotransformation (SyPaB)

Simple item page
dc.contributor.authorSun, Fangfangen
dc.contributor.committeechairZhang, Y. H. Percivalen
dc.contributor.committeememberZhang, Chenming Mikeen
dc.contributor.committeememberCapelluto, Daniel G. S.en
dc.contributor.departmentBiological Systems Engineeringen
dc.date.accessioned2017-04-04T19:50:56Zen
dc.date.adate2012-06-05en
dc.date.available2017-04-04T19:50:56Zen
dc.date.issued2012-04-25en
dc.date.rdate2016-09-17en
dc.date.sdate2012-05-10en
dc.description.abstractHydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes that microbes cannot do. One of the largest challenges is the high cost and instability of enzymes and cofactors. To overcome this obstacle, strong motivations have driven intensive efforts in discovering, engineering, and producing thermostable enzymes. In this project, ribose-5-phosphate isomerase (RpiB), one of the most important enzymes in the pentose phosphate pathway, was cloned from a thermophile Thermotoga maritima, and heterologously expressed in Escherichia coli, purified and characterized. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 80°C and pH 6.5-8.0. It had a half lifetime of 71 h at 60°C, resulting in its turn-over number of more than 2 x108 mol of product per mol of enzyme. Another two thermostable enzymes glucose-6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) and their fusion proteins G6PDH-DI and DI-G6PDH were cloned from Geobacillus stearothermophilus, heterologouely expressed in E. coli and purified through its His-tag. The individual proteins G6PDH and DI have good thermostability and reactivity. However, the presence of DI in fusion proteins drastically decreased G6DPH activity. However, a mixture of G6PDH and a fusion protein G6PDH-DI not only restored G6PDH activity through the formation of heteromultimeric network but also facilitated substrate channeling between DI and G6PDH, especially at low enzyme concentrations. My researches would provide important building blocks for the on-going projects: high-yield hydrogen production through cell-free enzymatic pathways and electrical energy production through enzymatic fuel cells.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-05102012-110527en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-05102012-110527/en
dc.identifier.urihttp://hdl.handle.net/10919/77009en
dc.language.isoen_USen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectsubstrate channelingen
dc.subjectsynthetic pathway biotranformation (SyPaB)en
dc.subjectdiaphoraseen
dc.subjectglucose-6-phosphate dehydrogenaseen
dc.subjectRpiBen
dc.subjectribose-5-phosphate isomeraseen
dc.subjectthermostable enzymesen
dc.subjectbiofuelen
dc.titleDevelopment of Building Blocks - Thermostable Enzymes for Synthetic Pathway Biotransformation (SyPaB)en
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiological Systems Engineeringen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
etd-05102012-110527_Sun_F_T_2012.pdf
Size:
1.24 MB
Format:
Adobe Portable Document Format
Download

Collections

Masters Theses