51. Structure and biocatalytic properties of nitrilases from filamentous fungi
- Author
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Křenková, Alena, Martínková, Ludmila, Macek, Tomáš, and Klempier, Norbert
- Abstract
Filamentous fungi are alongside bacteria a rich source of nitrilases that can be used for biotechnological purposes. There is a large number of sequences of putative nitrilases in databases, but few of these enzymes have been expressed, purified and characterized. According to the sequences in GenBank database, synthetic genes of putative nitrilases from Aspergillus niger CBS 513.88 and Neurospora crassa OR74A were designed, expressed in Escherichia coli and the nitrilases produced were purified and characterized. The effect of chaperone-assisted nitrilase expression was also examined using a set of commercial chaperone plasmids. The structural properties of nitrilases were examined using various methods, such as homology modelling, sedimentation velocity analysis or electron microscopy. Recombinant nitrilase production was compared to their production by native producers - filamentous fungi. The cultivation of native producers was rather difficult and nitrilase production was very low, therefore, the advantage of using prokaryotic expression system was highlighted. Nitrilases with amino acids close to the catalytic cysteine replaced were prepared in order to study the effects of certain amino acids on the activity, chemoselectivity and enantioselectivity of nitrilases. The catalytic properties of...
- Published
- 2013