Zachary Armstrong

University of British Columbia - Vancouver, Vancouver, British Columbia, Canada

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Publications (6)20.17 Total impact

  • Hong-Ming Chen · Zachary Armstrong · Steven J Hallam · Stephen G Withers
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    ABSTRACT: Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa < 7, such as halogenated coumarins, allows direct screening at neutral pH. The synthesis and characterisation of a set of nine different glycosides of 6-chloro-4-methylumbelliferone are described. The use of these substrates in a pooled format for screening of expressed metagenomic libraries yielded a “hit rate” of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process.
    No preview · Article · Feb 2016 · Carbohydrate research
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    Zachary Armstrong · Keith Mewis · Cameron Strachan · Steven J Hallam
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    ABSTRACT: Plant biomass offers a sustainable alternative to the energy and materials produced from fossil fuels. The industrial scale production or biorefining of fermentable sugars and aromatics from plant biomass is currently limited by the lack of cost effective and efficient biocatalysts. One potential solution to this problem is the discovery of biomass deconstructing biocatalysts from uncultivated microbial communities. Here we review recent progress in recovering such biological devices from environmental genomes and consider how this information can be used to build better biorefining ecosystems. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Jul 2015 · Current opinion in chemical biology
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    Zachary Armstrong · Stephen G Withers
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    ABSTRACT: The synthesis of defined glycans enables us to further understand their roles in a biological context. While useful chemical methods have been developed for the synthesis of glycans, these typically require complex protection and deprotection steps along with challenging control of anomeric stereochemistry. Enzymatic methods offer an attractive alternative to chemical synthesis. In particular, the use of glycosynthases and thioglycoligases, classes of engineered glycoside hydrolases, offer an enticing approach to the stereo- and regioselective synthesis of glycans without the need for protecting groups. Herein we describe recent progress in the use of glycosynthases and thioglycoligases for the synthesis of glycans and glycopolymers.
    Full-text · Article · Oct 2013 · Biopolymers
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    ABSTRACT: Functional metagenomics has emerged as a powerful method for gene model validation and enzyme discovery from natural and human engineered ecosystems. Here we report development of a high-throughput functional metagenomic screen incorporating bioinformatic and biochemical analyses features. A fosmid library containing 6,144 clones sourced from a mining bioremediation system was screened for cellulase activity using 2,4-dinitrophenyl β-cellobioside, a previously proven cellulose model substrate. Fifteen active clones were recovered and fully sequenced revealing 9 unique clones with the ability to hydrolyze 1,4-β-D-glucosidic linkages. Transposon mutagenesis identified genes belonging to glycoside hydrolase (GH) 1, 3, or 5 as necessary for mediating this activity. Reference trees for GH 1, 3, and 5 families were generated from sequences in the CAZy Database for automated phylogenetic analysis of fosmid end and active clone sequences revealing known and novel cellulase encoding genes. Active cellulase genes recovered in functional screens were subcloned into inducible high copy plasmids, expressed and purified to determine enzymatic properties including thermostability, pH optima, and substrate specificity. The workflow described here provides a general paradigm for recovery and characterization of microbially-derived genes and gene products based on genetic logic and contemporary screening technologies developed for model organismal systems.
    Full-text · Article · Jul 2013 · Journal of Biotechnology
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    Full-text · Article · Mar 2010 · ChemBioChem
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    ABSTRACT: Thioglycoligases are engineered enzymes for the synthesis of thioglycosides that are derived from retaining glycosidases by replacing the acid/base catalyst. The optimal choice of substitution for the acid/base mutant is currently unknown, so to investigate this question a complete acid/base library of the model glycosidase Bacillus circulans xylanase (Bcx) was generated by using site-saturation mutagenesis. A novel screening approach combining active site titration with semiquantitative product analysis by thin layer chromatography was established and used to evaluate specific activities of each mutant enzyme within crude cell lysates. The six most active Bcx variants were analyzed in more detail, a pH optimum of 8.5 was established and the identity of reaction products was confirmed. Optimal choices for substitution were small, preferably polar amino acids such as threonine, cysteine, and serine. We discuss the resultant data in the context of previously published studies on thioglycoligases.
    No preview · Article · Mar 2010 · ChemBioChem