Mining <Emphasis Type="Italic">Xanthomonas</Emphasis> and <Emphasis Type="Italic">Streptomyces</Emphasis> genomes for new pectinase-encoding sequences and their heterologous expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Zhizhuang Xiao Jason Boyd Stephan Grosse Manon Beauchemin Elizabeth Coupe Peter C K Lau |
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Institution: | (1) Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC, H4P 2R2, Canada |
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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. |
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Keywords: | Natural fibers processing Bioscouring Genome mining Pectinases Cold-active enzyme |
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