Publications by authors named "Kathrin Bönsch"

2 Publications

  • Page 1 of 1

Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering.

Protein Eng Des Sel 2011 May 27;24(5):439-46. Epub 2011 Jan 27.

Institute of Biochemistry, University of Leipzig, Deutscher Platz 5b, 04103 Leipzig, Germany.

A focused library comprising nine residues of the active site of P450cam monooxygenase resulting in ∼ 300,000 protein variants was screened for activity on diphenylmethane (DPM). The assay was based on the depletion of NADH by an in vitro reconstituted P450cam system in a 96-well scale. The throughput was increased by the parallel cultivation, purification and analysis of 20 variants per well (cluster screening). Thus ∼ 20,000 protein variants could be screened in summary of which five were found to transform DPM with a specific activity of up to 75% of the wild-type activity on d-camphor and a coupling rate of 7-18%. One variant converting DPM to 4-hydroxydiphenylmethane (4HDPM) was subjected to site-directed mutagenesis and saturation mutagenesis, which revealed the particular importance of positions F87, Y96 and L244 for substrate selectivity and the possibility for further improvements of this variant. Moreover, a reduction in size of the amino acid at position 396 decreased specific activity dramatically but increased coupling and switched the main product formation from 4HDPM towards diphenylmethanol.
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http://dx.doi.org/10.1093/protein/gzq119DOI Listing
May 2011

Characterization and engineering of a novel pyrroloquinoline quinone dependent glucose dehydrogenase from Sorangium cellulosum So ce56.

Mol Biotechnol 2011 Mar;47(3):253-61

Institute of Biochemistry, University of Leipzig, Deutscher Platz 5b, 04103 Leipzig, Germany.

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized. The recombinant protein has a relative molecular mass of 63 kDa and shows 43% homology to PQQ GDH-B from Acinetobacter calcoaceticus. In the presence of PQQ and CaCl₂ the enzyme has dehydrogenase activity with the substrate glucose as well as with other mono- and disaccharides. The thermal stability and its pH activity profile mark the enzyme as a potential glucose biosensor enzyme. In order to decrease the activity on maltose, which is unwanted for a potential application in biosensors, the protein was rationally modified at three specified positions. The best variant showed a 59% reduction in activity on maltose compared to the wild type enzyme. The catalytic efficiency (k(cat)/K(M)) was reduced fivefold but the specific activity still amounted to 63% of the wild type activity.
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http://dx.doi.org/10.1007/s12033-010-9339-5DOI Listing
March 2011