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Long-chain aliphatic amines such as (S,Z)-hepta- dec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engi- neered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octyl- nonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer– Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.
Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades
(2019)
Plant-derived carbohydrates are an abundant renewable re- source. Transformation of carbohydrates into new products, in- cluding amine-functionalized building blocks for biomaterials applications, can lower reliance on fossil resources. Herein, bio- catalytic production routes to amino carbohydrates, including oligosaccharides, are demonstrated. In each case, two-step bio- catalysis was performed to functionalize d-galactose-contain- ing carbohydrates by employing the galactose oxidase from Fusarium graminearum or a pyranose dehydrogenase from
Agaricus bisporus followed by the w-transaminase from Chro- mobacterium violaceum (Cvi-w-TA). Formation of 6-amino-6- deoxy-d-galactose, 2-amino-2-deoxy-d-galactose, and 2-amino- 2-deoxy-6-aldo-d-galactose was confirmed by mass spectrome- try. The activity of Cvi-w-TA was highest towards 6-aldo-d-gal- actose, for which the highest yield of 6-amino-6-deoxy-d-galac- tose (67%) was achieved in reactions permitting simultaneous oxidation of d-galactose and transamination of the resulting 6- aldo-d-galactose.
In 2010, the identification of 17 novel (R)-ATAs represented a breakthrough for the biocatalytic asymmetric synthesis of chiral amines, because only one (R)-ATA was described before. These novel ATAs were identified in a bioinformatic approach by studying the substrate acceptance of BCATs and DATAs to deduce the unknown substrate coordination of (R)-ATAs. Article I describes an alternative approach for the identification of (R)-ATA activity by reengineering the substrate- recognition site of α-AATs. While the engineering of the eBCAT led to the formation of an initial (R)-amine acceptance only, the (R)-ATA activity was successfully introduced in the DATA scaffold. These results demonstrate the transformation of an α-AAT in a moderately active (R)-ATA for the first time and highlight the evolutionary relationship between α-AATs and ATAs. Despite the availability of different ATAs nowadays, their substrate spectrum is limited due to the natural composition of their active sites. Several protein-engineering studies showed the widening of the substrate spectrum and the acceptance of bulky substrates by screening large mutant libraries to identify beneficial variants. In Article II, we developed an in silico engineering approach for amine transaminases to improve the conversion of bulky substrates and to reduce the number of variants to be tested in the laboratory. The resulting double-mutants of the (S)-ATA from C. violaceum displayed a >200-fold improved activity towards the bulky benchmark substrate. These variants expand the available biocatalytic toolbox for the synthesis of bulky amines, and the developed framework paves the way for rational protein-engineering protocols.
By studying unconventional transaminase substrates, we explored the potential of the available in- house transaminase toolbox in Articles III, IV, V, and VI. In Article III, we showed the transamination of a β-keto ester, leading to the synthesis of β-phenylalanine. The described cascade in Article IV enables the synthesis of amino carbohydrates. In addition, Article V describes an enzymatic cascade for the synthesis of amino fatty acids, which was extended in Article VI to obtain fatty amines.
The findings of this thesis clearly contribute to the understanding of the substrate scope and specificity of amine transaminases and expand the application of this versatile biocatalyst beyond classical ketone substrates.