Stephen Withers


Research Classification

Enzymes and Proteins
Blood Substitutes
Organic Molecules and Biomolecules
Biological and Biochemical Mechanisms

Research Interests

Drug development
enzyme inhibition
directed evolution

Relevant Degree Programs


Research Methodology

Organic synthesis, molecular biology, directed evolution, synthetic biology, metagenomics, Carbohydrate chemistry


Complete these steps before you reach out to a faculty member!

Check requirements
  • Familiarize yourself with program requirements. You want to learn as much as possible from the information available to you before you reach out to a faculty member. Be sure to visit the graduate degree program listing and program-specific websites.
  • Check whether the program requires you to seek commitment from a supervisor prior to submitting an application. For some programs this is an essential step while others match successful applicants with faculty members within the first year of study. This is either indicated in the program profile under "Requirements" or on the program website.
Focus your search
  • Identify specific faculty members who are conducting research in your specific area of interest.
  • Establish that your research interests align with the faculty member’s research interests.
    • Read up on the faculty members in the program and the research being conducted in the department.
    • Familiarize yourself with their work, read their recent publications and past theses/dissertations that they supervised. Be certain that their research is indeed what you are hoping to study.
Make a good impression
  • Compose an error-free and grammatically correct email addressed to your specifically targeted faculty member, and remember to use their correct titles.
    • Do not send non-specific, mass emails to everyone in the department hoping for a match.
    • Address the faculty members by name. Your contact should be genuine rather than generic.
  • Include a brief outline of your academic background, why you are interested in working with the faculty member, and what experience you could bring to the department. The supervision enquiry form guides you with targeted questions. Ensure to craft compelling answers to these questions.
  • Highlight your achievements and why you are a top student. Faculty members receive dozens of requests from prospective students and you may have less than 30 seconds to peek someone’s interest.
  • Demonstrate that you are familiar with their research:
    • Convey the specific ways you are a good fit for the program.
    • Convey the specific ways the program/lab/faculty member is a good fit for the research you are interested in/already conducting.
  • Be enthusiastic, but don’t overdo it.
Attend an information session

G+PS regularly provides virtual sessions that focus on admission requirements and procedures and tips how to improve your application.


Master's students
Doctoral students
Postdoctoral Fellows

Development of novel therapeutics for diabetes and lysosomal storage diseases
Discovery of enzymes for the enzymatic removal of cell surface antigens
In vitro and In vivo glycosylation of proteins

I support experiential learning experiences, such as internships and work placements, for my graduate students and Postdocs.
I am open to hosting Visiting International Research Students (non-degree, up to 12 months).

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Mar 2019)
Exploring novel human pancreatic alpha-amylase inhibitors : a departure from carbohydrate-based therapeutics (2018)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

View record

Harnessing natural diversity for the discovery of glycoside hydrolases and design of new glycosynthases (2018)

Plant biomass offers a sustainable source for energy and materials and an alternative to fossil fuels. However, the industrial scale production or biorefining of fermentable sugars from plant biomass is currently limited by the lack of cost effective and efficient biocatalysts. Microbes, the earth's master chemists - employing biocatalytic solutions to harvest energy, and transform this energy into useful molecules - offer a potential solution to this problem. However, a majority of microbes remain uncultured, limiting our access to the genetic potential encoded within their genomes. This has spurred the development of culture independent methods, termed metagenomics. In this thesis I harnessed high-throughput functional metagenomic screening to discover biomass deconstructing biocatalysts from uncultured microbial communities. Towards this goal, twenty-two clone libraries containing DNA sourced from diverse microbial communities inhabiting terrestrial and aquatic ecosystems were screened with 4-methylumbelliferyl cellobioside to detect glycoside hydrolase activity. This revealed 178 active clones containing glycoside hydrolases, often in gene clusters. This set of active clones was consolidated and further characterized through sequencing and rapid, plate-based, biochemical assays. Additionally, libraries sourced from beaver fecal and gut microbiomes were screened with four fluorogenic probes (6-chloro-4-methylumbelliferyl derivatives of cellobiose, xylobiose, xylose and mannose) for glycoside hydrolase activity. This revealed a total of 247 active fosmid-harbouring clones, that encoded many polysaccharide-degrading genes and gene cassettes. Specific candidate genes from the fecal library were sub-cloned, and the resulting purified enzymes were shown to be involved in synergistic degradation of arabinoxylan oligomers. The clone libraries that were generated through functional metagenomic screening were then employed to reveal the promiscuity of glycoside hydrolases towards unnatural azido- and aminoglycosides. Promiscuous enzymes identified from metagenomic and synthetic clone libraries were then used as a starting point for the generation of new glycosynthases capable of incorporating modified glucosides and galactosides. The resulting set of eight new glycosynthases are capable of synthesizing di- and trisaccharides, glycolipids and inhibitors such as 2,4-dinitrophenyl 4'-amino-2,4'-dideoxy-2-fluoro-cellobioside. Taken together this work has exploited the power of functional metagenomics to reveal new modes of biocatalysis and develop new synthetic tools.

View record

Discovery and engineering of carbohydrate-modifying enzymes using targeted high-throughput approaches (2017)

Carbohydrate-modifying enzymes can be used for the enzymatic synthesis or cleavage of complex glycans. In this study, various high-throughput assays were assessed as methods for the discovery of glycoside hydrolases (GHs) and glycosyltransferases (GTs) within environmental samples. Fluorescence-activated cell sorting (FACS) was evaluated as an approach for the functional enrichment of sialyltransferase (ST) genes in environmental samples. A model ST from Campylobacter jejuni, CstI, was successfully enriched from a mixture of genomic DNA (proof-of-principle), but active STs could not be isolated from a set of metagenomic libraries. The same FACS screen was applied to the directed evolution of multifunctional sialyltransferase from the Pasteurella multocida, PmST1, in order to isolate mutants with reduced sialidase activity and improved synthetic efficiency over the wild-type enzyme. Sialidase activity (kcat/KM) of the best mutants was reduced approximately 2-fold, with an approximately 2.5-fold increase in the sialyltransferase activity (kcat/KM). Despite these improvements, the maximum product yield of the mutants did not increase appreciably. While engineering PmST1, a study of the sialidase and trans-sialidase mechanisms of PmST1 and other STs from the glycosyltransferase family 80 was also undertaken. The mechanisms of both these activities were found to follow a reversible sialylation path, varying from that previously proposed in literature. A high-throughput plate-based assay was also evaluated as a functional screen for the identification of blood antigen-cleaving enzymes within the human gut microbiome. One GH enzyme from Bacteroides vulgatus (BvGH109) was found to be capable of converting the blood type A antigen into blood type O, offering a new enzyme for the engineering of universal donor blood. Two enzymes with α-N-acetylgalactosaminidase activity were also isolated and determined to represent a new sub-family of the GH family 31.

View record

Binding of substrates and inhibitors to human pancreatic alpha amylase (2016)

Human pancreatic alpha-amylase (HPA) is the enzyme responsible for hydrolyzing starch within the gut into shorter oligosaccharides. Selective inhibition targeted at only HPA could be used to modulate blood glucose levels for the treatment of diabetes and obesity.Montbretin A (MbA) is a potent (Ki = 8.1 nM) and specific inhibitor of HPA. Controlled degradation studies on MbA, coupled with inhibition analysis, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide, mini-MbA. X-ray structural analyses of the complex of MbA-HPA confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal pi-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the catalytic residues of HPA. The simplified analogue mini-MbA therefore offers potential for new strategies for glycosidase inhibition and therapeutic development.As part of a search for selective, mechanism-based covalent inhibitors of HPA, chemo-enzymatic syntheses of oligoglycosyl epi-cyclophellitols are described. Alpha-1,4 glucosyl epi-cyclophellitol, synthesized from epi-cyclophellitol by coupling of a glucosyl moiety using maltose phosphorylase, inactivated HPA stoichiometrically. X-ray crystallographic analysis of the covalent derivative so formed confirmed its reaction at the active site with the catalytic nucleophile Asp197. Another trisaccharide analogue 4’-O-methyl-alpha-maltosyl epi-cyclophellitol was synthesized enzymatically or by in situ elongation by HPA. Both of the inhibitors showed time-dependent inactivation of HPA, with the trisaccharide version being a better inactivator. This new class of mechanism-based inhibitors will be useful as activity-based probes for amylases. Several potential starch binding sites have been identified on the surface of HPA by crystallography, but their role, if any, in starch degradation is unknown. Through analysis of the binding of HPA mutants, modified individually at each site, to soluble and granule starch, two of these surface binding sites (SBSs) were shown to play a role in starch granule binding. A quite separate site was shown to be important for binding to, and cleavage of, soluble starch. Binding at SBSs was distinguished from binding to the active site by blocking the active site with the glycosyl epi-cyclophellitol mechanism-based inactivators.

View record

Mechanistic investigations of glycosaminoglycan degrading enzymes (2013)

Glycosaminoglycans are the main structural polysaccharides of vertebrates, and represent a major barrier to the spread of both bacterial infection and tumours. The enzymes by which mammals and pathogens degrade these polysaccharides use very different mechanisms, and may represent suitable therapeutic targets. In this thesis, work is presented towards an understanding of the mechanisms of Clostridium perfringens unsaturated glucuronyl hydrolase (UGL), the second enzyme in the bacterial pathway for degradation of glycosaminoglycans, and human heparanase, the enzyme by which the abundant glycosaminoglycan heparan sulfate is remodelled.For UGL, evidence was presented for a hydration reaction scheme that had previously been proposed on the basis of crystallographic evidence. This was shown by characterisation of products formed by reaction in D₂O and 10 % methanol, and by demonstrating hydrolysis of three compounds that are only expected to be turned over by the enzyme if this reaction is correct. Investigation of the effects of substituents on the transition state stability, by measurement of a linear free-energy relationship for a series of aryl glycosides, kinetic isotope effects, and rate determination for heteroatom-substituted substrates, led to the proposal of alternate mechanisms. Attempts to verify these mechanisms were made by testing of potential inhibitors, rescue of a catalytic-residue mutant, trapping of a covalent glycosyl-enzyme intermediate, or synthesis of a potential intermediate, but without success. The mechanism that appears most likely proceeds through protonation of the substrate C4-C5 double bond, with the resulting C5 positive charge being quenched by opening of the pyranose ring to give a C5 ketone and a C1-C2 epoxide. Subsequent hydration of the ketone and opening of this epoxide reforms the pyranose ring and gives the same product as direct hydration, but through a lower energy path.For mammalian heparanase, several new potential substrates and a potential inactivator were synthesized and tested. While this work was largely unsuccessful, it indicated that optimisation of sulfation patterns without modification of the aglycone is likely a futile strategy. A redesigned aglycone was proposed, representing a new path towards the goal of studying this enzyme for its eventual use as a therapeutic target in cancer therapy.

View record

Investigating the enzymatic mechanisms of the inverting and retaining glycosyltransferases by NMR spectroscopy (2012)

The overall goal of this thesis was to investigate the structures and enzymatic mechanisms of glycosyltransferases using NMR spectroscopy and enzyme kinetic measurements. The bifunctional sialyltransferase CstII from Campylobacter jejuni and the α-1,4-galactosyltransferase LgtC from Neisseria meningitidis were chosen to be the model inverting and retaining enzymes, respectively. By systematically introducing point mutations at the subunit interfaces of CstII, two active monomeric variants were obtained and characterized. In contrast to the wild-type tetramer, the monomeric CstII variants yielded good quality amide ¹H/¹⁵N-HSQC and methyl-TROSY NMR spectra. However, the absence of signals from approximately one half of the amides in the ¹H/¹⁵N-HSQC spectra of both monomeric forms suggests that the enzyme undergoes substantial conformational exchange on a msec-µsec time-scale. The histidine pKa values of CstII-F121D in its apo form were measured by monitoring the pH-dependent chemical shifts of biosynthetically incorporated [¹³Cε¹]-histidine. Consistent with its proposed catalytic role, the site-specific pKa value ~ 6.6 for His188 matches the apparent pKa value ~ 6.5 governing the pH-dependence of kcat/Km for CstII towards CMP-Neu5Ac. The enzymatic mechanism of the retaining glycosyltransferase LgtC appears to involve a “front-side attack” SNi or SNi-like mechanism with a short-lived oxocarbenium-phosphate ion pair intermediate. Furthermore, based upon X-ray crystallographic studies, two flexible loops were proposed to become ordered over the active site of LgtC upon sugar donor binding. Accordingly, NMR spectroscopy was used to investigate the dynamic properties of the enzyme with an emphasis on delineating the possible roles of these motions. The amide ¹H/¹⁵N-TROSY-HSQC and methyl-TROSY spectra of LgtC were partially assigned using a variety of NMR spectroscopic approaches, combined with mutagenesis of all the isoleucine residues. More than the expected number of methyl signals was observed, indicating that LgtC adopts multiple conformational states in equilibrium on a seconds time-scale, and that their relative populations change upon mutation and substrate binding. Furthermore, relaxation dispersion studies indicated substantial msec-µsec time-scale motions of methyl groups both within and distal to the active site in apo and substrate-bound forms of LgtC. Thus LgtC exhibits a range of dynamic behaviours potentially linked to its catalytic function. They were studies in this thesis using NMR spectroscopy and kinetic studies.

View record

Towards universal blood : mechanistic studies on blood group cleaving glycosidases (2012)

The ABO blood groups are vitally important in blood transfusion and organ transplantation. Transfusion with an incorrect blood type results in destruction of the incompatible blood cells, which can result in death. In my thesis, the catalytic mechanisms of three enzymes, two of which can directly be used on red blood cells (RBCs), were investigated in detail as follows.YesZ, a family GH 42 β-galactosidase (retaining), was used as a model system for the identification of catalytic residues. The mechanism-based inhibitor, 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-galactopyranoside was synthesized and used to inactivate YesZ via trapping of a reaction intermediate. Subsequent proteolytic digestion and comparative MS analysis identified the labeled peptide which, combined with, sequence alignments identified the catalytic nucleophile, a glutamate in the sequence ETSPSYAASL. Use of the acid/base mutant for trapping experiments provided support for its role thereby providing experimental verification of the identities of the catalytic residues in Family GH42.EABase, a family GH98 endo-β-galactosidase, cleaves blood group A and B trisaccharides from glycoconjugates and RBCs. The mechanism of Family 98 glycosidases was unknown but inferred to be retaining. The DNP-β-A-trisaccharide substrate was synthesized by in vivo enzymatic and subsequent chemical methods and direct 1H NMR analysis of its hydrolysis by EABase revealed that EABase is an inverting glycosidase. Both activated and nonactivated substrates were used to kinetically characterize EABase and its mutants, revealing that D453 and/or E506 act as the base catalyst and that E354 is the acid catalyst. EABase was used, in collaboration with Dr. Kizhakkedathu’s lab, to generate “universal blood cells” from type-B blood. Several α-L-fucosidases from family GH29 (retaining), which cleave α(1,2)-fucose from glycoconjugates were kinetically characterized in the hope of identifying the acid/base residue which is not conserved by sequence. A combination of modeling, sequence comparisons and phylogenetic tree analysis was used to identify candidate acid/base residues and further subgroup GH29 fucosidases based on these comparisons. The identity of the acid/base residue in four fucosidases is supported by kinetic characterization of a series of mutants of candidate residues and can now be predicted for all Family GH29 fucosidases.

View record

Mechanistic studies of retaining alpha-glycosidases (2010)

No abstract available.

Synthesis and enzymatic evaluation of activated fluorosugars as inactivators of lysosomal enzymes (2009)

Activated fluorosugars are covalent inactivators for a number of glycosidases, functioning through accumulation of stable glycosyl-enzyme intermediates. Two approaches were taken in designing new inactivators: more highly fluorinated sugars that could form more stable intermediates, and fluorosugars bearing novel aglycones that could be manipulated to improve selectivity and efficiency. Six novel difluorosugar fluorides were synthesized and evaluated as covalent inactivators. Four of the compounds were tested as time-dependent inactivators of the β-glucosidase from Agrobacterium sp. (Abg) and, while they were shown to behave as reversible competitive inhibitors, the only time-dependent inactivation was traced to the presence of an extremely small amount (
View record

Master's Student Supervision (2010-2017)
A metagenomic search for glycoside phosphorylases using a phosphate dependent 2,4-dinitrophenyl glycoside colorimetric assay (2015)

Carbohydrate active enzymes (CAZymes) comprise a large class of enzymes responsible for the assembly and degradation of glycans in biological systems. CAZymes are widely employed enzymes in industry, being used in brewing and food processing, animal feed preparation, industrial pulp and paper applications and increasingly in biofuel development. While the use of CAZymes is cost-effective in glycan degradation, glycan assembly generally requires the use of expensive nucleotide sugar phosphates as a starting material. The high cost of these materials makes an assembly approach towards industrial-scale glycan synthesis difficult and usually non-viable. One class of CAZyme that has received little attention from industry is that of the glycoside phosphorylases (GPases), which offer a potential solution to the high-costs associated with glycan synthesis. These enzymes bypass the need for expensive nucleotide sugar phosphates, and thus, we believe that approaches employing GPases will be of high market value. The bottleneck in this approach to glycan synthesis currently is the very limited range of GPases available, limiting the classes of glycan that can be assembled. To help increase the spectrum of known GPases available, we have turned to metagenomics as means to discover new enzymes belonging to this family. This will involve high-throughput (HT) screening of bacterial genome fragments recovered directly from the environment for expression of novel GPases. Here, we report the development of a novel HT screening methodology that allows the screening of large libraries constructed from metagenomic DNA. A dual approach is described using functional screening and bioinformatic techniques. By using a synthetic substrate analogue that produces a colorimetric response when processed by a GPase we are able to identify functional candidates from libraries containing upwards of 25 000 clones at a time. Likewise, utilising previous sequence data accumulated by our collaborators (Hallam Lab), we were able to identify GPases based on sequence homology. To date, through this screening methodology we have discovered 5 new GPases and a new class of CAZymes: the stereochemistry-retaining β-glycoside phosphorylases.

View record

Synthesis of cellulosic glycolipids using engineered enzymes (2014)

Cellulose, a linear polymer of D-glucose units connected by β-1,4 glycosidic bonds, adopts a highly-ordered crystalline structure in solution. In cellulose I, the dominant form of cellulose in nature, the polymeric chains are aligned in the same direction. Previous attempts to synthesize cellulose I in vitro have resulted in the synthesis of cellulose II, which has the thermodynamically favored anti-parallel orientation of chains. The synthesis of soluble fragments or defined surfaces of cellulose I would enable more detailed study of carbohydrate binding domains and other proteins that interact with cellulose in nature.The objective of this thesis is to prepare a crystalline surface of cellulose I in a controlled manner through the alignment of cellulolipids. A major focus of this thesis is the synthesis of cellulolipids with a cellohexaosyl head group. Cellohexaose is the shortest cello-oligosaccharide with cellulosic properties, but is consequently insoluble in aqueous solution. To improve the solubility of cellohexaose, the addition of a removable charged functionality was explored: either a terminal sialic acid or a phosphate group at the 6 position of the non-reducing terminal glucose. Abg2F6 glycosynthase from Agrobacterium sp. was used to synthesize β-1,4 linked cello-oligosaccharide fluorides from DP = 2 to DP = 4. These cello-oligosaccharides were modified with a removable charged functionality and utilized as donor substrates by CelB glycosynthase, a mutant of a β-1,4 endoglucanase from Caldicellulosiruptor saccharolyticus. Through the combination of glycosynthase enzymes and charged functionalities, a variety of soluble cellohexaosyl analogs were synthesized.Lyso-glycosphingolipids were prepared by transferring cello-oligosaccharyl fluorides to D-erythro-C18-sphingosine using EGCase glycosynthase. CelB glycosynthase used charged glycosyl fluoride donors to extend the lyso-glycosphingolipids, yielding soluble cellulolipids. The soluble cellulolipids were aligned along an aqueous:organic interface and the charged functionality was removed. Thus, a surface was prepared that appeared to interact with a carbohydrate binding module functionalized with a fluorescent tag. The soluble cellulolipids were successfully incorporated into a nanodisc, as shown through the incorporation of phosphorylated cellohexaosyl sphingosine. Cleavage of the phosphate using alkaline phosphatase yielded a nanodisc containing cellulolipids.

View record

Fluoro-glycosyl acridinones as sensitive active site titrating agents for glycosidases (2013)

9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) is a fluorescent and chromophoric molecule with a relatively low pKa (5.3) and moderate water solubility. DDAO was evaluated as a leaving group for mechanism-based inhibitors and a suitable reporter molecule for active site titrations of glycosidases.The Koenigs-Knorr coupling of DDAO to a protected α-glucosyl bromide resulted in two connectivities of DDAO to the glycone – DDAOY β-D-glucopyranoside (DDAOY-Glc) with chlorines close to the glycone and DDAOR β-D-glucopyranoside (DDAOR-Glc) with the chlorines distal. Kinetic evaluation of DDAOY-Glc and DDAOR-Glc revealed that both compounds were excellent substrates for a model β-glucosidase from an Agrobacterium sp. (Abg), reacting at or near diffusion-controlled rates.Several novel fluorogenic DDAO 2-deoxy-2-fluoro-β-D-glycopyranosides were synthesized and kinetic parameters for inactivation of a variety of retaining glycosidases were determined. DDAOY 2-deoxy-2-fluoro-β-D-glucopyranoside (DDAOY-2FGlc) was found to be the most effective inactivator of Abg reported to date (ki/Ki = 10⁶ mM-¹ min-¹), also reacting at, or near to, diffusion controlled rates and approximately 10⁶-fold faster than the DDAOR analogue (DDAOR-2FGlc). Differences in reactivity are partially due to the different inherent reactivities of DDAOY and DDAOR aglycones since the rate constant for spontaneous hydrolysis of DDAOY-Glc was found to be 700 times greater than that for DDAOR-Glc. DDAOY 2-deoxy-2-fluoro-β-D-galactopyranosides, D-xylopyranosides and cellobiosides were also shown to be effective inactivators of a variety of cognate glycosidases, including Escherichia coli β-galactosidase (LacZ), Trichoderma reesei endo-glucanase I (EG I), Cellulomonas fimi endo-xylanase (Cex), and Bacillus halodurans β-xylosidase (β-Xyl).Finally, DDAOY-2FGlc was shown to be a valuable tool for determining the concentration of Abg by active site titration. Through fluorescence detection, enzyme concentrations down to 3 nM could be reliably measured. DDAOY fluorosugar inactivators and DDAOR glycosides may serve as excellent substrates and probes in metagenomic and directed evolution screens.

View record

Towards specific inactivators of Trypanosoma cruzi trans-sialidase (2013)

The Trypanosoma cruzi (T. cruzi) trans-sialidase is thought to be an essential enzyme for infection by T. cruzi since sialic acid is needed for its nutrition and pathogenesis yet T. cruzi cannot biosynthesize sialic acid. The overall goal of this project is to identify a tight binding and specific inactivator of the T. cruzi trans-sialidase (TcTS). A library of mechanism-based inactivators of TcTS was synthesized based upon the known inactivator 9-azido-3-fluorosialyl fluoride (parent inactivator) by click chemistry with a series of alkynes in yields of 40-81%. Kinetic parameters for the clicked inactivators and its parent were determined using 5-acetamido-3,5-dideoxy-2-(4-trifluoromethylumbelliferyl)-D-glycero-α-D-galacto-non-2-ulopyranosonic acid as substrate. Inactivation parameters for the clicked inactivators were generally inferior to those of parent compound 9-azido-3-fluorosialyl fluoride. The substrate binding pocket of TcTS prefers non-polar moieties near the 9-position, especially aliphatic rings. It was also found that TcTS can tolerate negative charges better than positive charges at this position. These results suggest that improved reagents might incorporate moieties attached to C-9 through an amide or an ether linker rather than through a triazole.

View record

Activity-based proteomics profiling for identification and quantification of Trichoderma reesei cellulases (2012)

Cellulosic ethanol holds great promise as a renewable fuel to supplement gasoline.The complete conversion of cellulose involves multiple steps, one of which is the enzymaticdegradation of cellulose to glucose. Trichoderma reesei (recently renamed as Hypocreajecorina) secretes a large variety of cellulases that work synergistically in the hydrolysis ofcellulose into glucose. However, enzymes tend to “die off” during biomass conversion,decreasing the efficiency of degradation, yet it is difficult to determine which enzyme in themixture loses activity. Furthermore, significant diversity in the composition of plant cellwalls requires optimization of enzyme mixture used for hydrolysis of each feedstock. Inorder to address the problems, tools to identify and quantitate the active enzyme speciespresent in the enzyme mixtures are required, and would characterize the composition of theactive enzymes in the hydrolysis mixtures.We proposed an activity-based protein profiling (ABPP) approach. It involves a set ofchemical probes, each containing a cellulase-specific inactivating functionality to label theactive enzymes in the hydrolysis mixture, and a reporter group to quantitate the modifiedenzymes using such reagents, the concentration of one specific enzyme could be determinedbased on the amount of labeled enzymes detected using the reporter group. A cellulasespecificinactivating functionality is either an affinity label or a mechanism-based inactivatorincorporating a specificity-determining chemical group. To evaluate the potential inactivatingfunctionalities, six cellobiose-based affinity labels based on reactive groups that have proveduseful for other glycosidases, and two mechanism-based inactivators have been tested withthe 4 principal T. reesei cellulases (provided by our collaborator) for their efficiencies. Seven of them inactivated at least one of the cellulases; among these, C-epoxypentyl cellobiosidehad the best overall performance. It had relatively good binding affinity and efficiencytoward three cellulases, and it will be included in a second generation of ABPP probe. Anenlarged inactivator library will be required to target other cellulases. Ultimately, at least oneABPP for each of the T. reesei cellulase will be generated and hopefully will prove useful tothe biofuel industries.

View record

News Releases

This list shows a selection of news releases by UBC Media Relations over the last 5 years.

Current Students & Alumni

This is a small sample of students and/or alumni that have been supervised by this researcher. It is not meant as a comprehensive list.

If this is your researcher profile you can log in to the Faculty & Staff portal to update your details and provide recruitment preferences.