Wenn das Licht ausgeht: Ein Kupfer- Biosensor wurde durch gentechnischen Ersatz von L-Tyrosin in grün fluoreszierendem Protein durch die chelatisierende nichtkanonische Aminosäure L-DOPA hergestellt (siehe Bild). Die spezifische Bindung von Cu2+ durch das modifizierte Protein war reversibel und führte zu einer mit dem Cu2+-Gehalt skalierenden Fluoreszenzlöschung. EDTA=Ethylendiamintetraessigsäure. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Folding up: Residue-specific incorporation of noncanonical amino acids (NCAAs) often results in loss of protein function either by misfolding or aggregation (left). However, engineering the protein sequence for enhanced folding increases the mutational robustness of the protein to accommodate novel side chains and generate tailor-made proteins with new properties (right). Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Abstract A current challenge in high‐throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid‐agar plate‐based HTS assay for screening ortho ‐specific hydroxylation of daidzein by sensing formaldehyde generated from the O‐dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde‐specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho ‐carbon position to COH correlates with a linear relationship to O‐dealkylation activity on chemically introduced methoxy group at the corresponding COH. As a model system, a 4′,7‐dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B‐ring, was examined for O‐dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O‐dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A‐ring inactivated in order to find enhanced 3′‐regioselectiviy). The whole‐cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L −1 production titer and greatly increased 3′‐regioselectiviy (3′/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions.
Abstract We report a highly atom‐efficient integrated cofactor/co‐product recycling cascade employing cycloalkylamines as multifaceted starting materials for the synthesis of nylon building blocks. Reactions using E. coli whole cells as well as purified enzymes produced excellent conversions ranging from >80 and 95 % into desired ω‐amino acids, respectively with varying substrate concentrations. The applicability of this tandem biocatalytic cascade was demonstrated to produce the corresponding lactams by employing engineered biocatalysts. For instance, ϵ‐caprolactam, a valuable polymer building block was synthesized with 75 % conversion from 10 mM cyclohexylamine by employing whole‐cell biocatalysts. This cascade could be an alternative for bio‐based production of ω‐amino acids and corresponding lactam compounds.
β-Transaminases (β-TAs) have shown considerable potential as biocatalysts for the synthesis of chiral β- and γ-amino acid. Herein, a (R)-β-TA was successfully created by the directed evolution of d-amino acid aminotransferase. The specific activities of created (R)-β-TA for β-phenylalanine (1.74 U/mg) and γ-phenylbutanoic acid (1.67 U/mg) were comparable with those of naturally occurring (S)-β-TAs. Moreover, the designed (R)-β-TA also showed potential for the biocatalytic synthesis of a δ-amino acid with specific activity (0.23 U/mg), which has not yet been explored. Enantiopure (S)- and (R)-forms of various β-, γ- and δ-amino acids (>99% ee) were synthesized using engineered (R)-β-TA-mediated kinetic resolution and asymmetric synthesis, respectively.
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