A universal assay for screening expression libraries for carbohydrases

Although many assays are available for the screening of expression libraries for carbohydrases, some enzymes cannot be detected because their substrates are incompatible with the existing assays. One thing that all carbohydrases have in common is that they increase the number of reducing ends when d...

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Bibliographic Details
Published in:Journal of bioscience and bioengineering 2000, Vol.89 (1), p.107-109
Main Authors: Meeuwsen, Patrick J.A., Vincken, Jean-Paul, Beldman, Gerrit, Voragen, Alphons G.J.
Format: Article
Language:English
Subjects:
bicinchoninic acid
Bioassay
Biodegradation
Biological and medical sciences
Biology of microorganisms of confirmed or potential industrial interest
Biotechnology
CARBOHIDRATOS
CARBOHYDRATES
DNA
ENSAYO
Enzymes
expression cloning
Food Chemistry
Food Chemistry Group
Fundamental and applied biological sciences. Psychology
GLUCIDE
HIDROLASAS
HYDROLASE
HYDROLASES
Leerstoelgroep Levensmiddelenchemie
Levensmiddelenchemie
Miscellaneous
Mission oriented research
Organic acids
Polysaccharides
screening assay
TESTAGE
TESTING
VLAG
xylogalacturonan hydrolase
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Summary:Although many assays are available for the screening of expression libraries for carbohydrases, some enzymes cannot be detected because their substrates are incompatible with the existing assays. One thing that all carbohydrases have in common is that they increase the number of reducing ends when degrading their substrates. In this paper we explore the possibility of detecting this increase with the highly sensitive bicinchoninic acid (BCA) reducing value assay. This assay can be used for the detection of all carbohydrases degrading any polysaccharide; enzymes with either an exo- or an endo-type of mechanism can be detected at the same time. A cDNA library of Aspergillus tubigensis expressed in Kluyveromyces lactis clones, was screened with this assay for the presence of xylogalacturonan degrading enzyme(s). High background absorbances caused by culture medium, by proteins produced by the clones and by substrate could be dealt with by using the precautions described in this note. Three xylogalacturonase producing clones were found using this procedure.
ISSN:1389-1723
1347-4421
DOI:10.1016/S1389-1723(00)88062-7