Towards the microbial production of a switchable solvent by using the enzyme indoleamine N-methyltransferase

• Author / Creator

  Soon, Emily F

• Switchable solvents are a novel class of chemicals that are characterized with the ability to reversibly switch specific properties with the addition/removal of a trigger. For instance, a solution of low ionic strength can become one of high ionic strength, and vice versa. Switchable solvents have potential application in industrial processes as a greener alternative for product extraction. The ability to reversibly switch properties allows these solvents to be recycled, which reduces material usage and waste generation. Moreover, the switchable reaction occurs at low temperature and pressure, which reduces energy input and operation costs. Currently, application of switchable solvents is limited by difficult chemical synthesis and/or commercial unavailability. It is therefore necessary to find alternative, cheaper means to produce switchable solvents. Microorganisms have a long history of being used and modified to produce numerous chemicals of value. With growing concerns regarding the sustainability and environmental impact of the petroleum-based industry, there has been increased interest in converting biomass using microorganisms for the production of materials and chemicals. The main goal of this research study was to investigate the microbial production of the switchable solvent N,N,N’,N’-tetramethyl-1,4-diaminobutane through enzymatic conversion of putrescine using the enzyme indolethylamine N-methyltransferase (INMT). In the first study, the growth characteristics of two common microbial hosts, Escherichia coli and Saccharomyces cerevisiae were assessed for switchable solvent tolerance and potential consumption of the switchable solvent as a nutrition source. Both microorganisms grew well in the presence of N,N,N’,N’-tetramethyl-1,4-diaminobutane, as determined by microbial growth curves and cell viability assays. HPLC-ELSD analysis demonstrated that neither microorganism was found to consume N,N,N’,N’-tetramethyl-1,4-diaminobutane as a nutrition source during cultivation. The objective of the second study was to obtain a pure isolate of INMT for characterization purposes. DNA encoding rabbit lung INMT was cloned into E.coli and the enzyme was successfully expressed and purified. DNA sequencing and peptide sequencing experiments demonstrate that the isolated protein is INMT. In the third study, the activity of the purified enzyme was evaluated on tryptamine, a model compound, and putrescine using a microplate assay kit, high performance liquid chromatography (HPLC) and gas chromatography (GC) analysis. The enzyme assays showed that the purified enzyme from E. coli has activity on both tryptamine and putrescine. In summary, this thesis has laid down the ground work for the microbial-based production of switchable solvents. This thesis presents the first study characterizing the switchable solvent tolerance of both E. coli and S. cerevisiae as well as demonstrating that the switchable solvent is not consumed by either microorganism during growth. Additionally, this thesis has shown that the enzyme INMT was successfully cloned into E. coli, expressed and purified to obtain a pure isolate. Enzyme assay studies with INMT demonstrated that it has activity on tryptamine, the model compound, and putrescine, the switchable solvent precursor.

• Subjects / Keywords

  • N-methyltransferase
  • Switchable solvent
  • Escherichia coli
  • Enzyme characterization

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3BZ61D8J

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.