Prospective Graduate Students / Postdocs
This faculty member is currently not actively recruiting graduate students or Postdoctoral Fellows, but might consider co-supervision together with another faculty member.
This faculty member is currently not actively recruiting graduate students or Postdoctoral Fellows, but might consider co-supervision together with another faculty member.
Processing α-glucosidase-I (Glu-I) is an endoplasmic reticulum inner membrane-bound enzyme that plays a critical role in N-glycosylation and quality-control of protein-folding. Despite its role, details of the catalytic mechanism of Glu-I function are still unknown. The objective of this research was to address the main obstacles in studying this enzyme, namely the lack of sufficient quantities of relatively pure enzyme and a substrate to test its function. Initially, a soluble form of yeast Glu-I was expressed in Escherichia coli with a yield of 6-8 mg of Glu-I per litre of culture. After single-step purification using immobilised metal affinity chromatography, this recombinant 6xHis-tagged Glu-I showed a Km of 1.27 mM with the synthetic trisaccharide substrate α-D-Glc1,2α-D-Glc1,3α-D-Glc-O-CH3. Since the catalytic domain of Glu-I is located at the C-terminus, expression of the C-terminal domain (Cwh41Δ1-525p) was attempted, but yielded insoluble bodies. Expression of Cwh41Δ1-525p with solubility-enhancing fusion tags or the co-expression of molecular chaperones did not improve solubility. Subsequently, based on a published tertiary structure of Glu-I, I identified that Cwh41Δ1-525p lacks two α-helices of the catalytic (α/α)6 toroid domain. Therefore, the N-terminus of Cwh41Δ1-525p was extended to include the missing helices and expression of the two new constructs (Cwh41Δ1-349p and Cwh41Δ1-314p) was attempted. However, these proteins also expressed as insoluble bodies. Co-expression of the N-terminal domain (Cwhnp) improved the expression of soluble Cwh41Δ1-525p, but the expressed protein was not functional. Catalytic domain released by trypsin hydrolysis from Glu-I was 2.2 times more active than the intact Glu-I. This catalytic domain was purified using size-exclusion chromatography. Since the enzymatic hydrolysis of a glycosidic bond typically occurs with general acid and general base assistance from two amino acid side chains, generally carboxylic amino acids, site-directed mutagenesis of all six conserved carboxylic residues of the catalytic domain was carried out. Glutamic acid 804 was identified as the catalytic base with the aid of nucleophile rescue. Further studies on the structure-function of yeast Glu-I will be helpful in establishing a model to study the inborn errors of metabolism involving this enzyme (CDG-IIb) in humans.
Phenylalanine ammonia lyase (PAL, EC 18.104.22.168) catalyzes the conversion of phenylalanine (Phe) to trans cinnamic acid and, in some instances tyrosine (Tyr) to para hydroxycinnamic acid (pHCA). The specificity of PAL for Phe relative to Tyr varies by over 10⁶ between biological sources. An understanding of the basis for this astounding range of substrate preference is required to rationally engineer a highly efficient Tyr-specific enzyme for use in synthesizing pHCA for health, pharmaceutical and flavour applications. Of the plant seedlings screened for PAL activity, Triticum aestivum displayed the highest level of activity with both substrates. A unique 103 kDa PAL polypeptide was detected by Western blot analysis, along with two others at 74 and 83 kDa. Dual substrate activity was identified for the first time in the dicot Lens culinaris. Microbial Trichosporon cutaneum PAL (TcPAL) possessed the highest level of activity with Tyr of the sources investigated. Induction with Tyr (2 mM) produced the highest ratio of Phe:Tyr activity (1.6 ± 0.3: 0.4 ± 0.1 µmol/h g wet weight). TcPAL displayed Michaelis Menten kinetics with Phe (Km 5.0 ± 0.7 mM) but allosteric kinetics with Tyr (K' 1.7 ± 0.8 mM, Hill coefficient 1.8 ± 0.2). A greater specificity for Phe was demonstrated by a Phe [Vmax/Km] / Tyr [Vmax/Km] ratio of 2. The enzyme had a pH optimum of 8 - 8.5, temperature optimum of 32°C, showed no metal dependence, and had a monomer molecular mass of 79 kDa.Of the three methods investigated for purifying TcPAL, anion exchange chromatography using a Hi-Trap Q-Sepharose column produced the highest yield (20%) and purification fold (50). The PAL gene from Trichosporon cutaneum was cloned into the pET30a vector and sequenced. Five amino acid residues different from a previous report were identified, namely Arg74Glu, Val274Ala, Ala298Val, Ser322Pro and Arg486Lys. The gene had an intron of 1062 base pairs, starting at position 121. A His-Gln motif, which appears to be characteristic of yeast PALs with dual substrate activity, was identified in the substrate selectivity region. Residues that could potentially enhance the tyrosine activity of the enzyme were identified for future mutagenesis studies.
Processing α-glucosidase I (Glu I) holds a key regulatory position in the N-glycosylation pathway. Glu I mutations in humans have severe or fatal outcomes. Despite the important role of human Glu I (MOGS), very little information is available regarding the function, mechanism and structure of the enzyme. The lack of methods to produce sufficient quantities of the enzyme is a major drawback in carrying out further structural and mechanistic studies. Therefore, this thesis was focused on establishing a Pichia pastoris expression system for MOGS. MOGS with an N-terminal histidine (6xHis) tag was overexpressed in P. pastoris and purified by FPLC using a His-trap column. The purified MOGS was N-linked glycosylated and was enzymatically active (specific activity of 735 U/mg) against the synthetic substrate α-D-Glc1,2α-D-Glc1,3α-D-Glc–OCH₃. However, the overexpression of MOGS in P. pastoris was not reproducible and it was overexpressed intracellularly in subsequent expression trials. Therefore, overexpression of MOGS in E. coli was attempted, but the expressed MOGS accumulated as inclusion bodies with either a MBP (maltose binding protein) tag or a 6xHis-tag. Two methods (urea denaturation-refolding and freeze-thawing) were used to purify MOGS from inclusion bodies. However, less than 0.5 mg from one litre culture was recovered and it was not enzymatically active. As the overexpression of active MOGS in either P. pastoris or E. coli was not successful, further characterization of Glu I was continued with the study of the N-terminal function of Saccharomyces cerevisiae Glu I (Cwh41p). The Cwh41p clones with N-terminal truncations were constructed and the proteins were overexpressed in E. coli. Among the six truncations, five truncations (Cwh41Δ1-167p, Cwh41Δ1-93p, Cwh41Δ1-74p, Cwh41Δ1-54p and Cwh41Δ1-45p) were expressed mainly in inclusion bodies and the eluted fractions from His-trap column were not enzymatically active. However, the eluted fractions of Cwh41Δ1-39p and the internal control (Cwh41Δ1-34p) were active with a specific activity of 266 U/mg and 2000 U/mg, respectively. This indicates that the residues 35-45 of Cwh41p may be essential for the proper folding of Cwh41p. I propose that the α-helix (NH1) of Cwh41p plays a positive role in protein folding by interacting with C-domain of the enzyme.
Foodborne Listeria monocytogenes (Lm) causes listeriosis, a rare but severe disease affecting at risk populations. Contamination of ready-to-eat (RTE) food has been linked with persistent Lm in the food processing environment (FPE), though mechanisms of persistence are not fully understood. Recent surveys in British Columbia (BC) detected Lm in seafood processing facilities and in RTE seafood products, yet little is known regarding Lm persistence and risk posed by strains from these sources. The objectives of this study were to determine the prevalence of Listeria spp., including Lm, in a RTE seafood processing facility and assess persistence capabilities and potential risk of Lm recovered. Environmental and RTE food samples were collected over 18 months from a BC seafood processor (n=2,959) and assessed for the presence of Listeria spp. Isolated Lm were subjected to phenotypic and genetic characterization and a subset of isolates (n=28) were characterized for attributes that may facilitate FPE persistence: surface adherence, resistance to quaternary ammonium-based sanitizers, and adaptation to salt and refrigerated temperatures. Ability of cold-smoked salmon from this processor to support Lm growth, evaluation of virulence gene (inlA) sequence, and antibiotic resistance in the subset were used to assess consumer risk. Non-Lm Listeria spp. and Lm were found in 2.6% and 1.5% of samples, respectively. Molecular characterization revealed raw materials as the primary contamination source and two recurrent subtypes. Lm typically possessed one attribute favorable to persistence in the FPE, though there was no association between these attributes and strain recurrence. Cold-smoked salmon supported the growth of Lm and most strains recovered in the FPE (n=14) belonged to serotypes linked to listeriosis (92%) and possessed full-length inlA (93%). These strains, however, did not show resistance to antibiotics commonly used to treat listeriosis. The results of this study highlight the importance of processor-level control strategies to minimize Lm FPE persistence, product contamination, and risk to consumers. While these results improve understanding of Lm in a BC seafood processing environment, more work is needed to determine whether these strains are truly persisting in this FPE and how these findings compare to similar facilities.
Listeria monocytogenes (Lm), a foodborne pathogen, causes the rare but severe disease listeriosis in at-risk populations. Resistance that increases environmental fitness in Lm, including resistance to heavy metals and/or food processing sanitizers, is of concern as it may increase survival in natural and food processing environments that leads to increased potential for product contamination. Lm (n=46) from the British Columbia food chain were evaluated for resistance to food processing sanitizers and heavy metals. All isolates were found to be sensitive to triclosan (16 µg/ml) and peroxyacetic acid (150 ppm exposure for 60 seconds). Seventeen isolates were resistant to quaternary ammonium compounds (QUATs; 10 µg/ml), with all positive for one known resistance determinant (bcrABC, n=16; emrE, n=1). Resistance to cadmium (Cd) and arsenic was found in 89% and 24% of the isolates, respectively. Under sub-lethal concentrations of Cd, all Cd sensitive isolates showed reduced growth. Decreasing pH and increasing Cd resulted in a minimum inhibitory concentration for a representative Cd sensitive isolate at 1.5 µg/ml CdCl₂ at pH 6 and 0.5 µg/ml CdCl₂ at pH 5.5. All bcrABC positive isolates were Cd resistant, and two were capable of co-transferring QUAT and Cd resistance to L. monocytogenes 08-5578. When bcrABC and emrE were combined through conjugation, there was no observable increase in resistance to QUATs. While L. monocytogenes from British Columbia remain sensitive to common sanitizers, a sub-population is resistant to low QUAT concentrations and co-resistant to Cd. Further work is needed to explore if co-selection between heavy metal and sanitizer resistance is a concern in the food chain. As Lm continue to cause disease, surveillance of antibiotic resistance within the population of Lm is necessary to aid in effective early treatment. A collection of sequence type 120 Lm (n=21) were all found to be sensitive to the primary drugs of choice for treatment of listeriosis - ampicillin, gentamicin, and co-trimoxazole. Four isolates were resistant to an ampicillin alternative, linezolid. This data supports the continued use of the primary drugs of choice in treating listeriosis in Canada from sequence type 120 Lm, but suggests the possibility of decreased effectiveness of alternative antibiotics.
Chilean tinamou (Nothoprocta perdicaria) egg white was characterized and compared to chicken (Gallus gallus) and emu (Dromaius novaehollandiae) egg whites for composition and antimicrobial components. Tinamou and chicken appeared more similar in terms of protein, sialic acid, ash, and iron content than tinamou and emu, even though phylogenetic analysis places tinamous within the ratites. Egg white proteins were separated by anion–exchange Fast Protein Liquid Chromatography, followed by SDS–PAGE. Tinamou ovotransferrin, ovomucoid, and lysozyme C were identified by peptide mass fingerprinting of SDS-PAGE bands. Similar sized ovotransferrins were present in all egg whites species, however higher quantities were observed for ratites. It is possible that ovotransferrin has an essential antimicrobial function and therefore its presence is conserved among distinct species. The antimicrobial activity of tinamou and chicken ovotransferrins against two food related pathogens, Escherichia coli O157:H7 and Staphylococcus aureus COL was bicarbonate, concentration, and avian species dependent, as evaluated by turbidly and viability assays. Native ovotransferrins were the most effective against E. coli O157:H7, followed by apo and holo forms. Native ovotransferrins exhibited a significant bactericidal activity at a concentration of 10 mg/ml with bicarbonate. In the presence of bicarbonate, chicken apo and holo ovotransferrins were more bacteriostatic than tinamou ovotransferrins. Additionally, there was no significant difference in the antimicrobial activity of apo and holo ovotransferrins applied at 5 and 10 mg/ml. Holo ovotransferrins exhibited moderate antimicrobial activity, only in the presence of bicarbonate; therefore it is possible that bicarbonate contributes to the antimicrobial activity of ovotransferrin by a mechanism other than a bridging ligand between ovotransferrin and iron. Native chicken and tinamou ovotransferrins at 10 mg/ml were bactericidal against S. aureus COL, whereas tinamou ovotransferrin was more bacteriostatic. In conclusion, tinamou ovotransferrin combined with bicarbonate was found to be bactericidal against two foodborne pathogens. In the absence of bicarbonate, tinamou ovotransferrins exhibited minor bacteriostatic activity, while chicken ovotransferrin was not effective. It is possible that tinamou ovotransferrin possesses different amino acid sequences from the chicken protein that form unique antimicrobial motifs; therefore it should be further investigated as a natural antimicrobial agent for use in food matrices or food preparation surfaces.
The objectives of this research were to use molecularly imprinted polymers (MIP) and microfluidic chips as an approach to a rapid and low cost analytical method for niacinamide analysis. Lab-on-a-chip (microfluidics) devices are becoming increasingly popular due to their relatively low cost, sensitivity, and speed. MIPs may be able to serve as solid-phase extraction packing material in microfluidic chips. To reach the objectives, it was necessary to identify the mechanisms by which binding of analyte to polymer occur, determine the optimal functional monomer to cross-linker ratio, and gain an understanding of the polymeric structure and characteristic bonds. An MIP was created using niacinamide (NAM) as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, azobisisobutyronitrile (AIBN) as the free-radical initiator, and chloroform as the porogen. It was hypothesized that rebinding occurs via hydrogen-bonding of the carbonyl and amide groups of NAM to the oxygen atoms in the carboxyl group of MAA. Rebinding studies were conducted using compounds with similar functional groups to NAM to determine binding mechanism to the polymer. Both the pyridyl nitrogen and the amide group were found to be important in hydrogen bonding interactions with the polymer. Polymers were optimized for rebinding by using different ratios of functional monomer:cross-linker (MAA:EGDMA) and determining imprint factor of NAM to each polymer. The 1:4 polymer yielded the highest imprinting factor, indicating that the polymer is most selective for NAM.FTIR was conducted to determine the structure of polymers created and whether NAM detection and quantification was possible. There was a peak at 1725 cm⁻¹, which was a shift of the C=O stretching band from 1694 cm⁻¹ in MAA and 1717 cm⁻¹ in EGDMA, indicating a chemical interaction between the two compounds. The disappearance of a peak at 1633 cm⁻¹ showed a loss of conjugation in the carboxylic acid in the polymeric structure. Through this research, knowledge was gained about the polymer optimization and structure. However, more studies need to be conducted to determine the feasibility of an MIP application for a lab-on-a-chip device.
Doctor of Philosophy in Food Science (PhD) 
Plant screening for tyrosine/ phenylalanine ammonia lyase and biochemical characterisation, purification and cloning of the tyrosine/phenylalanine ammonia lyase enzyme from Trichosporon cutaneum.
Lecturer and Programme Coordinator
University of the West Indies