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Exploring Insect Enzymes as Catalyst for Bioconversion of Value Added Products
Date
2012Type
ThesisDepartment
Biochemistry and Molecular Biology
Degree Level
Master's Degree
Abstract
Demand for fine chemicals and value-added products has increased annually
concomitantly with the rapid expansion of biocatalytic production of these chemicals.
With an increased mandate for chemicals in the pharmaceutical, agricultural, scents and
flavorants industry, it is essential to identify enzymes capable of performing specific,
complex chemical reactions in an efficient, economical and environmentally friendly
manner. Insects produce secondary metabolites that are potential value-added chemicals,
therefore insect enzymes provide a rich source of catalysts that can be added to the
collection of biocatalysts used to perform these intricate tasks.
This thesis explores the biochemical role of three insect enzymes, CYP9T3,
MPB-CPR, and CG4020, which are involved in lipid metabolism and could potentially be
used as biocatalysts for value-added products. All three enzymes were expressed with a
baculoviral system in Sf9 cells and used in functional assays.
Eastern Ips pini CYP9T3 is a cytochrome P450 with 94% sequence identity to
western Ips pini CYP9T2. CYP9T3 accepts myrcene, (+)- and (-)-α-pinene, (+)- and (-)-
limonene, and (+)-3-carene as substrates, a pattern similar to CYP9T2. However, the
enantiomeric ratio of (4R)-(-)-ipsdienol to (4S)-(+)-ipsdienol is significantly different
between CYP9T3 and CYP9T2. Additionally, the product from (-)-α-pinene is transverbenol with CYP9T3 and myrtenol with CYP9T2. Assays with β-pinene, terpinolene,
γ-terpinene, α-phellandrene did not yield detectable products by either CYP9T3 or
CYP9T2.
Cytochromes P450 require the transfer of electrons from a protein partner,
commonly cytochrome P450 reductases. Mountain pine beetle cytochrome P450
ii
reductase (MPB-CPR) is the first bark beetle cytochrome P450 reductase to be isolated
and characterized. MPB-CPR is 69% identical to house fly-CPR (HF-CPR), the
reductase used in previous bark beetle P450 functional assays. Recombinant MPB-CPR
microsomes reduce cytochrome c with apparent Km and vmax of 85.04 µM and 8.42
nmol·min-1·µg total protein-1, respectively. Initial kinetic assays with CYP9T3 indicate
that MPB-CPR reduces the P450 in the presence of myrcene with apparent Km and vmax
values of 15.8 uM and 11.3 nmol·min-1·U enzyme-1, respectively.
The final step in hydrocarbon biosynthesis in insects is the removal of one carbon
from a fatty aldehyde through the action of an oxidative decarbonylase, CYP4G2.
Experimental evidence from M. domestica microsomal preparations indicates that a fatty
acyl-CoA is reduced to a fatty aldehyde prior to the oxidative decarbonylation reaction
and the candidate enzyme is a fatty acyl-CoA reductase (FAR). CG4020, a Drosophila
melanogaster FAR, has an expression pattern similar to CYP4G1, the D. melanogaster
homologue of CYP4G2. Additionally, suppression of CG4020 produces flies with fifty
percent less total hydrocarbon than wild type flies. Preliminary assays with expressed
CG4020 did not confirm the biochemical function of this enzyme, however they did
provide relevant background information from which future experiments can be
designed.
Permanent link
http://hdl.handle.net/11714/3752Additional Information
Committee Member | Blomquist, Gary J.; Forister, Matthew |
---|---|
Rights | In Copyright(All Rights Reserved) |
Rights Holder | Author(s) |