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Institute: search.filters.UBSInstitut.Institut für Technische BiochemieEnd date: 2001Author: search.filters.author.Lange, Stefan

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    Durch Überexpression in der Hefe Pichia pastoris zu erhöhter Enantioselektivität : ein neues Kapitel in der Anwendung von Schweineleberesterase
    (2001) Musidlowska, Anna; Lange, Stefan; Bornscheuer, Uwe Theo
    Lipases and Esterases can be used as efficient biocatalysts for the preparation of a wide variety of optically pure compounds. Whereas a range of lipases - especially of microbial origin - are commercially available, only a few esterases can be obtained for the kinetic resolution of racemates or desymmetrization. In the majority of publications, pig liver esterase (PLE) is used, which is isolated from pig liver by extraction. Although it could be demonstrated, that this preparation can convert a broad range of compounds at partially very high stereoselectivity, its application is encountered with a number of disadvantages.
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    High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris
    (2001) Lange, Stefan; Schmitt, Jutta; Schmid, Rolf D.
    In this report, we describe the high-yield secretory expression (~ 40 mg l-1) of pure, atrazine-specific Fab fragments (K411B) from P. pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of the alcohol oxidase promoter) into the genome of the yeast cells. Antibody-expressing clones were selected by SDS-PAGE and ELISA and fed-batch fermentations were carried out in a 5 l scale. Both chains of the Fab were successfully expressed upon methanol induction and almost no other proteins were secreted into the media. Approximately 30 % of the two chains formed the active Fab fragment containing the intermolecular disulphide bond, as determined by Western blot analysis under non-reducing conditions. Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC50 value for the detection of atrazine was determined from the standard curve as 3 µg l-1, which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in E. coli cells. In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E. coli and the parental mAb K4E7.