Harry Brumer
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
The efficient depolymerization and/or utilization (valorization) of residual waste biomass is crucial for establishing a sustainable bio-based economy. Cellulose and chitin constitute the most abundant polymer components of biomass and represent renewable feedstocks for the production of biofuels, biomaterials, and other bio-based chemicals. The recalcitrance and low chemical reactivity of these polysaccharides limit their viability in numerous industrial applications. Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes secreted by saprotrophic organisms to assist in the depolymerization of cellulose and chitin fibers. The recent discovery of LPMOs has shifted the fundamental understanding of microbial biomass utilization while also potentiating many avenues for the commercial valorization of cellulose and chitin. This dissertation presents a thorough investigation of the production, functional analysis, and fiber-modifying application of bacterial LPMOs from Auxiliary Activity family 10 (AA10). Signal peptide-mediated periplasmic secretion through the general secretion pathway (Sec) in Escherichia coli was the most suitable approach for producing active AA10 LPMOs. Five AA10s encoded by the historically significant Cellulomonas flavigena and Cellulomonas fimi bacteria were purified and characterized. Four of these Cellulomonas AA10 homologs were cellulose-active and one was chitin-active. Thermostability studies confirmed only cellulose-active Cellulomonas AA10s could reversibly unfold. Three non-Cellulomonas AA10 catalytic domains were produced in E. coli using the Sec signal peptide derived from the lone AA10 encoded by C. fimi. Notably, Xylanimonas cellulosilytica LPMO10A demonstrated dual activity on cellulose and chitin, while Streptomyces pratensis LPMO10D and Legionella longbeachae LPMO10 were found to be strictly active on chitin or cellulose substrates, respectively. Time course kinetic assays show high initial LPMO-oxidizing activity commonly coincides with rapid inactivation from oxidation. These results highlight the importance of optimizing abiotic parameters such as pH and reductant type to maximize the catalytic potential of LPMOs. Lastly, as a proof-of-concept, a C1-oxidizing cellulose-active AA10 encoded by C. flavigena was used to chemo-enzymatically conjugate several fluorescent probes to cellulose fibers. The work presented here informs future studies on heterologous LPMO production, LPMO activity screening and characterization, as well as LPMO-mediated fiber defibrillation and chemical modification.
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The human gut microbiota (HGM) is a remarkably dense and dynamic community of microbes with incredible collective metabolic capacity. Complex glycans (“dietary fiber”) evade digestion by the human host and feed the HGM, driving its composition, and in turn influencing diverse facets of host health. In order to access these otherwise recalcitrant glycans, Bacteroidetes, a dominant bacterial phylum in the HGM, co-localize genes encoding a membrane-associated machinery that work in concert to bind, break down, and sequester target glycan into co-regulated Polysaccharide Utilization Loci (PULs). One important class of complex glycans with numerous documented health benefits is beta-glucans. In this thesis, I undertake holistic functional characterization, combining biochemistry, structural biology, microbiology, and (meta)genomics, of PULs targeting two distinct subclasses of beta-glucan: mixed-linkage beta-glucan (MLG), and beta(1,3)-glucan.The MLG utilization locus (MLGUL) from B. ovatus, necessary to enable growth on MLG, encodes an endo-beta-glucanase anchored to the cell surface to break down MLG, and a periplasmic exo-beta-glucosidase to completely saccharify the fragments imported by the TonB-dependent outer-membrane transporter. The process is aided by two cell surface glycan-binding proteins (SGBPs) which employ binding platforms shaped to complement that of target MLG to recruit and retain the glycan at cell surface. Growth analysis combined with comparative genomics reveal MLGULs serve as genetic markers for ability to grow on MLG. Metagenomic analysis further suggest MLGUL presence in, and consequent ability to utilize MLG by, the HGM of a majority of humans worldwide.A distinct set of syntenic beta(1,3)-glucan utilization loci (1,3GULs) from three prominent Bacteroides species (B. uniformis, B. thetaiotaomicron, and B. fluxus) were subject to similar holistic functional characterization. Differential ability to grow on beta-glucan congeners is driven by synergy between enzymes and SGBPs: a particular 1,3GUL can mediate utilization of a beta-glucan congener if it encodes both an enzyme that can hydrolyze the target, as well as an SGBP that can bind the target. Detailed structure-function analysis of glycoside hydrolases (GH), including a family-first structure of a GH158, and a suite of SGBPs reveal the molecular basis of catalytic and binding specificities that together give rise to species-differential specificity of 1,3GULs.
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The utilization and valorization of biomass has been proposed as a sustainable alternative to petroleum-based energy, chemicals and materials. Microbial enzymes can be used as selective catalysts for the depolymerization and functionalization of plant material. In this regard, the copper radical oxidases (CROs) from Auxiliary Activity Family 5 subfamily 2 (AA5_2) are attractive targets since they oxidize primary alcohols in a chemo-selective manner to the corresponding aldehydes without utilizing complex and expensive organic cofactors. AA5_2 was first defined by the archetypal galactose oxidase from Fusarium graminearum, however, other fungal AA5_2 members have recently been shown to comprise a wide range of specificities for aromatic, aliphatic and furan-based alcohols. This suggests a broader substrate scope of CROs for biocatalytic applications. However, only 10% of the annotated AA5_2 members have been characterized to date.The work presented in this thesis describes the biochemical characterization of fourteen fungal copper radical oxidases, recombinantly produced in Pichia pastoris, encompassing galactose oxidases, raffinose oxidases, broad spectrum alcohol oxidases, non-carbohydrate alcohol oxidases and aryl alcohol oxidases. Detailed product analysis was conducted of carbohydrates, furans and glycerol oxidized by these novel enzymes to further explore their potential applications. Of note, we observed direct oxidation of 5-hydroxymethylfurfural (HMF), an important platform chemical derived from biomass, to 5-formyl-2-furoic acid (FFCA) by six enzymes. One enzyme facilitated the direct one-pot conversion of HMF to 2,5-furandicarboxylic acid (FDCA), a building block for bio-plastic production. Through docking of galactose into homology models of these newly characterized enzymes, and comparing it to the archetypal galactose oxidase, key amino acid residues important for substrate specificity have been proposed. Finally, the potential of enzyme immobilization using the cross-linked enzyme aggregate (CLEA) strategy has been investigated for a specific alcohol oxidase to increase its utilization in biocatalysis.This work expands our understanding of the catalytic diversity of copper radical oxidases from AA5_2 and doubles the number of characterized AA5_2 members. Detailed sequence and enzymatic analysis highlights their structure-function relationships while product analysis informs their possible biotechnological applications in bio-products manufacturing. Furthermore, immobilization was shown to be a promising avenue for increasing the biocatalytic potential of CROs.
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A defining feature of plants, the plant cell wall is an essential structure that provides mechanical support, cell shape, and protection from pathogens and the environment. Cell walls are dynamic structures that are composed of a network of crystalline cellulose microfibrils, amorphous polysaccharides, and structural proteins. The synthesis, maintenance, and modification of the plant cell wall polysaccharides are carried out by carbohydrate active-enzymes (CAZymes). Xyloglucan endotransglycosylase/hydrolase (XTH) are secreted cell wall enzymes that restructure the cell wall matrix glycan, xyloglucan, thereby facilitating cell wall remodelling and growth. The plant endo-glucanase 16 (EG16) enzymes were recently identified as a unique clade that is structurally and functionally intermediate between the bacterial mixed-linkage β(1,3)/(1,4)-glucanases and the plant XTHs in glycoside hydrolase family 16 (GH16). Correspondingly, EG16 members were broadly specific towards the cell wall polysaccharides mixed-linkage β-glucan (MLG) and xyloglucan, as well as soluble cellulosic substrates. Although EG16 activity and protein structure have been demonstrated, their biological role is unknown. This thesis employs a multi-disciplinary approach to the characterization of the newly designated EG16 group. A comprehensive census of the publicly available genome and transcriptomes showed that EG16 orthologs were found in all plant clades spanning nearly 500 million years of evolution. Notably, the presence of EG16 orthologs observed in the charophyte green algae species that are the most phylogenetically distant from angiosperms, support that EG16-like genes are ancestors to extant EG16 and XTH genes. EG16 substrate specificities, primary hydrolysis site, and digest products are consistent between vastly different plant species. Elucidation of the biological role of EG16 in the moss Physcomitrella patens and a hybrid poplar species, two different distantly related land plants, show gene expression in young tissues and leaderless cell wall localization. In P. patens, EG16 deletion yielded larger plants that senesced earlier than wild-type, whereas, in poplar, downregulation did not affect growth.This thesis serves as a foundation for the EG16 clade and informs their evolutionary history, conserved activity, cell wall localization, and involvement in growth. The research opens the door to investigate unexplored topics such as the EG16 specific secretion mechanism and their role in MLG-containing cell walls.
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Carbohydrates are ubiquitous in Nature and fundamental to the sustenance of organisms across all domains of life. Carbohydrates serve as sources and reserves of metabolic energy, participate in various cellular communication events, and provide structural support to plant and animal cells. Highly specific enzymes have evolved over several millennia to bind and manipulate carbohydrate substrates. Glycoside hydrolases (GHs) are a class of carbohydrate-active enzymes that cleave glycosidic linkages in complex carbohydrates. Organisms across all domains of life dedicate part of their genome to the production of GHs. New GHs are continually discovered through genome sequencing, while their structural and functional characterization, particularly in complex native environments, poses a persistent challenge to the dynamic field of GH characterization. One of the fundamental ways of ascribing protein function is the exploration of protein active sites, which can be used to deduce important details regarding substrate-enzyme interactions.The work presented in this thesis describes the development of six new probes targeting a variety of GHs by irreversible covalent inhibition. These probes, developed on oligosaccharide scaffolds, feature either an N-bromoacetylglycosylamine electrophilic warhead or 2', 4' dinitrophenyl 2-deoxy-2-fluoro substitutions, facilitating irreversible inhibition of the target GH. The analysis presented in this work reveals key information about enzyme-inhibitor interactions through enzyme kinetic analyses, intact-protein mass spectrometry, and inhibitor-bound protein X-ray crystallography. Enzymes of diverse GH families and substrate preferences including endo-xyloglucanases, mixed-linkage glucanases, and β-(1,3) glucanases are featured to demonstrate the potency of this library of inhibitors. This small-molecule inhibitor toolkit targeting specific GH enzymes has the potential to enhance our knowledge of the structural and functional characteristics of GHs and to provide a platform for activity-based enzyme profiling.
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Xyloglucan (XyG) is a ubiquitous plant heteropolysaccharide representing up to one-quarter of the total carbohydrate content of terrestrial plant cell walls. Given its structural complexity, XyG requires a consortium of backbone-cleaving endo-xyloglucanases and sidechain-cleaving exo-glycosidases for complete saccharification. The Gram-negative soil saprophyte Cellvibrio japonicus is a treasure trove for carbohydrate active enzymes (CAZymes) due to its robust capacity to degrade different plant polysaccharides. The XyG utilization machinery in C. japonicus is incompletely understood, despite recent characterization of associated sidechain-cleaving exo-glycosidases. I present here my attempts to identify and functionally characterize the endo-xyloglucanase(s) and the XyG-specific β-1,4 exo-glucosidase catalyzing the first and final steps, respectively, in the XyG saccharification pathway in C. japonicus. Bioinformatic analysis identified one Glycoside Hydrolase Family 74 (CjGH74), three GH5_4 (CjGH5D, CjGH5E and CjGH5F) and three GH9 (CjGH9A, CjGH9B and CjGH9C) candidates with putative endo-xyloglucanase activity. Biochemical and structural analyses that involved CjGH74 and the three CjGH5_4 enzymes clearly demonstrated the exquisite specificity of the four enzymes towards XyG. Scrutiny of the modular architecture of the CjGH5_4 enzyme, CjGH5F, identified the module of unknown function X181. Affinity gel electrophoresis and isothermal titration calorimetry identified the X181 module as a member of a new CBM family that exclusively binds galactose-containing polysaccharides including XyG and galactomannans, congruent with the displayed endo-xyloglucanase activity of the pendent catalytic domain. Surprisingly, reverse genetic analysis in C. japonicus displayed the lack of growth perturbation on XyG upon the deletion of the four specific endo-xyloglucanases, suggesting the presence of other enzyme(s) with potential endo-xyloglucanase activity. Biochemical characterization of the GH9 enzyme CjGH9B revealed its high catalytic efficiency towards mixed linkage β-glucan and its weak side-activity against xyloglucan, which might be sufficient to rescue the quadruple deletion mutant. Bioinformatic analysis identified the four CjGH3 enzymes Bgl3A, Bgl3B, Bgl3C, and Bgl3D as potential targets for the XyG-specific exo-β-glucosidase in C. japonicus. Comprehensive genetic and biochemical approaches interestingly revealed the fundamental contribution of Bgl3D in XyG utilization in C. japonicus. Together, these data shed light on the initial and final steps of xyloglucan saccharification in C. japonicus and identify useful enzymes for selective biomass deconstruction.
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Plant biomass is both the most abundant organic carbon source and the most abundant organic carbon sink on our planet. This carbon is stored primarily in the cell wall, where carbohydrates, proteins and polyphenols are interwoven to form complex purpose-built composite materials. Within plants, diverse polysaccharides are built up and broken down as part of their natural life cycle. Within the guts of multicellular organisms, a diverse and adaptable collection of bacteria anaerobically ferments complex plant polysaccharides. In this thesis, the structure and function of enzymes involved in these two processes are described. The xyloglucan endo-transglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodelling. Investigations into the ancestry of the XTH family revealed a subfamily of endo-glucanases which share a common ancestor with the XTHs. Based on product analysis, kinetics, and X-ray crystallography these EG16s have been identified as a family of β(1,4)-specific endo-glucanases with an uncommon mode of substrate recognition. Although the biological role(s) of EG16 orthologues remains to be fully resolved, the presented biochemical and tertiary structural characterisation provide insight into plant glycoside hydrolase evolution, and will continue to inform studies of plant cell walls.Within the gut, Prevotella are an important genus of Gram-negative bacteria associated with carbohydrate-rich diets. Recent genome sequencing has shown that they possess many undescribed polysaccharide utilisation loci (PULs). A revisited broad-specificity cross-linking glycan-degrading endo-glucanase (PbGH5A) is associated with a PUL of unknown function within Prevotella bryantii, an obligate anaerobe originally isolated from the bovine gut. Based on X-ray crystallography, product identification, binding assays, and kinetic measurements, the structures and functions of a variety of proteins involved in the recognition and breakdown of complex β-mannans have been determined. These include a broad-specificity endo-β-glucanase, an endo-β-mannanase, two β-mannan-binding proteins, two β-mannan acetylesterases, a mannobiose-2-epimerase, and a mannosylglucose phosphorylase. Characterisation of the two β-mannan acetylesterases provides a basis for the expansion of CAZy family CE7 and the formation of a new CE family. Furthermore, the presented model of the Prevotella β-mannan utilisation locus provides a genetic template for identifying systems which degrade complex galactomannans and glucomannans across the Bacteroidetes phylum.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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Chronic wounds, which fail to heal or heal only very slowly, remain a major challenge in medical treatment and a burden to healthcare systems. Chronic wounds are exacerbated by bacterial infection and endogenous proteases. In particular, increased levels of human neutrophil elastase (HNE) have been observed in chronic wound fluid, and has been utilized as a severity indicator via clinical assays. To facilitate chronic wound treatment, a facile, in situ, detection method for HNE would be advantageous. Here, cellulose-based analytical devices have been designed and produced as a proof-of-concept for chronic wound point-of-care diagnostics. Specifically, two distinct fluorogenic HNE substrates amenable to click-chemistry were synthesized based on tetrapeptide (Ala4) conjugates to the rhodamine derivatives, carboxyrhodamine110-PEG3-azide (cRho110-PEG₃-N₃) and rhodamine 110 (Rho110). Michaelis-Menten kinetics were used to demonstrate activity of HNE toward these compounds that was comparable to known chromogenic substrates. Following attachment under mild aqueous conditions to alkyne-functionalized Whatman No. 1 filter paper, a pure cellulosic substrate and model for cotton gauze, HNE detection on a solid surface was demonstrated visually under specific illumination and was quantified with a fluorescence scanner. These results validate the concept of in situ protease detection using modified cellulose surfaces to monitor chronic wounds toward improved treatment outcomes. Furthermore, the modular design of the cellulose-based analytical devices presented here suggests a broader potential for the detection of specific protease activity in diverse applications.
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The hydrolysis of xanthan is a commercially relevant reaction. This is due to the widespread use of this polysaccharide as a rheology modifier in many industries. Therefore, investigations into the structure and function of enzymes capable of altering xanthan properties, including endo-xanthanases, is warranted.This thesis describes investigations into the structure-function relationship of PspXan9, a bacterial xanthanase from glycoside hydrolase family 9 (GH9). To do this, enzyme kinetic assays were performed to determine preferred biochemical conditions, substrate specificity and Michaelis-Menten kinetics. These revealed that PspXan9 was highly specific for xanthan following a pretreatment with xanthan lyase, and notably, required calcium for increased activity and stability. The appearance of only two products over the course of a reaction, as monitored by high-performance size exclusion chromatography coupled to a UV-detector (HPSEC-UV), demonstrated that hydrolysis of lyase-treated xanthan with PspXan9 occurred in a processive fashion. However, the identity of these products as lyase-treated xanthan tetrasaccharides and octasaccharides was only realized following analysis with liquid chromatography coupled to mass spectrometry (LC-MS), tandem mass spectrometry (MS/MS) and 1D- and 2D-Nuclear Magnetic Resonance (NMR). Analysis of the PspXan9 x-ray crystal structure was used to reveal structural insights that guided targeted mutations to perform subsite mapping. Changes resulting from each mutation were tested for activity, and degree of processivity through viscometric and HPSEC-UV analysis. Lastly, protein similarity networks were utilized to find a putative GH9 xanthanase subgroup as well as a possible progenitor GH9 whose protein expression was attempted.
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Auxiliary Activity Family 5 (AA5) carbohydrate-active enzymes are mononuclear copper radical oxidases (CROs) capable of oxidizing a variety of alcohols to their corresponding aldehydes without organic cofactors. Classically, AA5 characterization is performed using a horseradish peroxidase (HRP) coupled assay using ABTS (2.2’-azino-bis(3-ehtylbenthiazoline-6-sulphonic acid) as a colorimetric indicator. However, this HRP-ABTS coupled assay indirectly monitors reaction progress – it measures a small portion of reactant conversion and provides no information about the extent of oxidation (percentage conversion) or potential product inhibition. Therefore, we developed an alternative approach to assess alcohol oxidation by AA5 enzymes that allows direct monitoring of reaction progress by incorporating tandem reaction progress analysis. This new approach collects time-resolved information about active chemical species and facilitates the interrogation and optimization of the system. CgrAlcOx, an AA5 oxidase from the phytopathogenic fungal species Colletotrichum graminicola, was previously characterized and displayed activity on a wide variety of aromatic and aliphatic alcohols. Reaction monitoring using the HRP-ABTS assay in this previous study was used as a benchmark to develop our new approach. We tested different instruments and identified high performance liquid chromatography coupled with an ultraviolet (UV) detector as the best way to directly monitor, on-line, the depletion of alcohols and the formation of aldehydes in alcohol oxidation catalyzed by CgrAlcOx. Reaction conditions were optimized, and the kinetics of aromatic alcohol oxidation were determined to support the applicability of this approach for monitoring enzymatic reactions. This new approach also allowed the detailed study of CgrAlcOx, including the screening of new substrates and the detection of product inhibition; previous assays did not permit the latter. Lastly, this new approach was combined with the HRP-ABTS coupled assay in a complementary way to overcome the inability of the previous assay to characterize reaction inhibitors. This allowed the discovery of new inhibitors of CgrAlcOx, such as benzylamine and benzyl mercaptan. In addition, using this combinatorial approach, butanethiol was identified as both an inhibitor and a substrate towards CgrAlcOx.
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