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Xiao Z.,National Research Council Canada | Grosse S.,National Research Council Canada | Bergeron H.,National Research Council Canada | Lau P.C.K.,National Research Council Canada | And 2 more authors.
Applied Microbiology and Biotechnology | Year: 2014

The only available genome sequence for Rhizopus oryzae strain 99-880 was annotated to not encode any β-1,4-endoxylanase encoding genes of the glycoside hydrolase (GH) family 10 or 11. Here, we report the identification and cloning of two such members in R. oryzae strain NRRL 29086. Strain 29086 was one of several selected fungi grown on wheat or triticale bran and screened for xylanase activity among other hydrolytic actions. Its high activity (138 U/ml) in the culture supernatant led to the identification of two activity-stained proteins, designated Xyn-1 and Xyn-2 of respective molecular masses 32,000 and 22,000. These proteins were purified to electrophoretic homogeneity and characterized. The specific activities of Xyn-1 and Xyn-2 towards birchwood xylan were 605 and 7,710 U/mg, respectively. Kinetic data showed that the lower molecular weight Xyn-2 had a higher affinity (Km = 3.2 ± 0.2 g/l) towards birchwood xylan than Xyn-1 by about 4-fold. The melting temperature (Tm) of the two proteins, estimated to be in the range of 49.5–53.7 °C indicated that they are rather thermostable proteins. N-terminal and internal peptide sequences were obtained by chemical digestion of the purified xylanases to facilitate cloning, expression in Escherichia coli, and sequencing of the respective gene. The cloned Rhizopus xylanases were used to demonstrate release of xylose from flax shives-derived hemicellulose as model feedstock. Overall, this study expands the catalytic toolbox of GH10 and 11 family proteins that have applications in various industrial and bioproducts settings. © 2014, Her Majesty the Queen in Right of Canada.


Leisch H.,NRC Biotechnology Research Institute | Grosse S.,NRC Biotechnology Research Institute | Iwaki H.,Kansai University | Hasegawa Y.,Kansai University | And 2 more authors.
Canadian Journal of Chemistry | Year: 2012

The biocatalytic performance of a cloned cyclohexylamine oxidase derived from Brevibacterium oxydans IH-35A towards structurally different amines was investigated. Cycloalkyl primary amines, alkyl aryl amines, and α-carbon-substituted aliphatic amines were identified as suitable substrates for the biocatalyst based on an activity assay. Kinetic resolutions of several amines by either recombinant whole cells or crude enzyme extracts prepared therefrom gave enantiomerically pure (R)-amines besides the corresponding ketones. When cyclohexylamine oxidase in combination with a borane-ammonia complex as reducing agent was applied to the deracemization of several substrates, excellent enantiomeric ratios (>99:1) and good isolated yields (62%-75%) of the corresponding (R)-amines were obtained. © 2012 Published by NRC Research Press.


Hexagonally well-organized ZnCl2-OMA materials have been successfully synthesized by a one-pot approach using a sol-gel method at ambient temperature and pressure. These materials were prepared through simultaneous self-assembly process with F127 as directing agent combined with in situ impregnation of ZnCl2. The new approach was found to be compatible with various common aluminium precursors and carboxylic acids used as self-assembly interfacial protectors. The obtained ZnCl2-OMA materials were fully characterized using XRD, N2 adsorption-desorption, TEM, SEM, EDX, XPS and 1H and 27Al MAS-NMR techniques. These materials were compared with the zinc chloride-doped organized mesoporous alumina (ZnCl2-OMA) supports prepared through a conventional two steps process that includes OMA synthesis and then post-synthesis incorporation of ZnCl2. Thus, the one-pot synthesized materials were found to exhibit improved properties compared to the conventional ones, particularly larger BET surface area. The synthesized ZnCl2-OMA materials were then used as catalytic supports for methyltrioxorhenium (MTO), showing enhanced catalytic performance for methyl oleate self-metathesis, demonstrating better activity and selectivity towards desired metathesis products. These features are highly beneficial for large-scale materials synthesis through fast, simple, easy and low-cost introduction of functionalities on mesoporous materials surface without multi-step procedures. © The Royal Society of Chemistry.


Morley K.L.,National Research Council Canada | Grosse S.,National Research Council Canada | Leisch H.,National Research Council Canada | Lau P.C.K.,National Research Council Canada | And 2 more authors.
Green Chemistry | Year: 2013

Canolol (4-vinylsyringol, VS), a potent antioxidant and an alkylperoxyl radical scavenger originally discovered in crude canola oil (rapeseed), is produced by decarboxylation of sinapic acid (SA) during canola seed roasting. Chemical syntheses of VS from SA require thermal or microwave induced decarboxylation in the presence of a base. A laboratory-evolved enzyme, designated SA decarboxylase (SAD), was developed in this study. In a biphasic bioreactor system, SAD was shown to produce VS from SA extracts prepared from canola meal with an overall yield of 3.0 mg VS per g of canola meal. In addition, we investigated the application of VS in polymerization to produce polyvinylsyringol (PVS) as a potential biodegradable polymer. The characteristics of PVS determined by thermogravimetric analysis, differential scanning calorimetry and nanoindentation tests are described. © 2013 The Royal Society of Chemistry.


Iwaki H.,Kansai University | Grosse S.,National Research Council Canada | Bergeron H.,National Research Council Canada | Leisch H.,National Research Council Canada | And 5 more authors.
Applied and Environmental Microbiology | Year: 2013

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km(32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km=3.6 μM; kcat=283 s-1) in preference to flavin adenine dinucleotide (FAD) (Km(19 μM; kcat(128 s-1). Sequence determination of~40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25-1 for 2,5-DKCMO-1 and camE25-2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and~533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates. © 2013, American Society for Microbiology.

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