Manon Beauchemin

National Research Council Canada, Ottawa, Ontario, Canada

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Publications (6)9.7 Total impact

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    ABSTRACT: Mining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD600 > 200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ∼8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71-87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively.
    Applied biochemistry and biotechnology 09/2013; · 1.94 Impact Factor
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    ABSTRACT: The production of lactic acid from triticale starch was demonstrated for the first time. Direct fermentation by Rhizopus oryzae in batch mode led to a conversion yield of 0.74 g lactic acid/g starch. Kinetics analysis of the fermentation suggested a multiphase process which consisted of quick hydrolysis of starch, rapid glucose accumulation followed by fast production of lactic acid. In batch fermentation mode, correct timing and proper dosage of a neutralizing agent (calcium carbonate) were found to be critical factors that affect the organic acid yield. Addition of CaCO3 at the time point when glucose accumulation had reached its peak (24 h) resulted in the highest lactic acid yield. The best ratio (by weight) of triticale starch to CaCO3 for lactic acid production was 1:1. It was important to maintain a pH of about 5 during the whole fermentation process. Fermentation carried out in an optimized, commercially available small-scale parallel bioreactor (DASGIP) yielded up to 0.87 g lactic acid/g triticale starch when R. oryzae spores were directly added to the fermentation medium. Our fermentation results indicated that triticale starch from this nonfood crop is a promising renewable feedstock for production of lactic acid.
    Industrial Biotechnology 04/2011; 7(2):129-134.
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    ABSTRACT: Microbial genome sequencing has left a legacy of annotated yet uncharacterized genes or open reading frames, activities that may have useful applications in health and/or the environment. We are interested in the discovery and characterization of potentially new pectinolytic activities for the enzymatic retting of natural bast fibers such as hemp and flax. A highlight in this study is the discovery of a cold-active pectate lyase among five pectate-lyase-encoding sequences and two polygalacturonase-encoding sequences that we have cloned from the genomes of Xanthomonas campestris pv. campestris and Streptomyces coelicolor A3(2). Heterologous expression of these sequences as active pectate lyases and polygalacturonases required their subcloning in Escherichia coli Rosetta cells. The most active recombinant pectate lyase (XcPL NP_638163), a cold-active pectate lyase (XcPL NP_636037), and a polygalacturonase (XcPG NP_638805) were purified to near homogeneity and their kinetic parameters were determined. A significant amount of pectin degradation products was shown to be released by the two pectate lyases but not the polygalacturonase when hemp fiber pectin was used as substrate. Results of this study showed that genome data mining, besides an economical approach to new gene acquisition, may uncover new findings such as the discovery of a cold-active pectate-lyase-encoding sequence from X. campestris, a mesophilic microorganism.
    Applied Microbiology and Biotechnology 05/2008; 78(6):973-81. · 3.81 Impact Factor
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    ABSTRACT: In the vast number of random mutagenesis experiments that have targeted protein thermostability, single amino acid substitutions that increase the apparent melting temperature (Tm) of the enzyme more than 1 to 2 degrees C are rare and often require the creation of a large library of mutated genes. Here we present a case where a single beneficial mutation (R236F) of a hemp fiber-processing pectate lyase of Xanthomonas campestris origin (PL(Xc)) produced a 6 degrees C increase in Tm and a 23-fold increase in the half-life at 45 degrees C without compromising the enzyme's catalytic efficiency. This success was based on a variation of sequence alignment strategy where a mesophilic amino acid sequence is matched with the sequences of its thermophilic counterparts that have established Tm values. Altogether, two-thirds of the nine targeted single amino acid substitutions were found to have effects either on the thermostability or on the catalytic activity of the enzyme, evidence of a high success rate of mutation without the creation of a large gene library and subsequent screening of clones. Combination of R236F with another beneficial mutation (A31G) resulted in at least a twofold increase in specific activity while preserving the improved Tm value. To understand the structural basis for the increased thermal stability or activity, the variant R236F and A31G R236F proteins and wild-type PL(Xc) were purified and crystallized. By structure analysis and computational methods, hydrophobic desolvation was found to be the driving force for the increased stability with R236F.
    Applied and Environmental Microbiology 03/2008; 74(4):1183-9. · 3.95 Impact Factor
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    ABSTRACT: Yes L'environnement doit faire les corrections, dans ce cas ci soit Sector Santé ou Bioproc. qui doivent faire cette entrée en Crossector. Je ne peux faire aucun changement vu tant que l'env. n'enleve pas le # affecté sur RM. Katy
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    ABSTRACT: Yes SVP demandez à l'environnement de faire la modification sur cette pub. Il faut annuler cette entrée à NPArC. C'est une Pub. Health,Crossector,Bio. et non Env,Crossector,Bio!! Merci Katy