9A-G and 10A-K, respectively. AAO76671). In particular examples, a nitrile oxido-reductase includes a QueF motif sequence (SEQ ID NO: 5). Please suggest some good methods? Other methods for expressing a nitrile oxido-reductase amino acid sequence from an exogenous nucleic acid molecule include, but are not limited to, constructing a nucleic acid molecule such that a regulatory element promotes the expression of a nucleic acid sequence that encodes a nitrile oxido-reductase. Nitriles can be reduced to primary amines when treated with LiAlH4 or to aldehydes when a milder reducing agent such as DIBAL is used. Alternatively catalytic hydrogenation to reduce the nitrile groups could work depending on the rest of your molecule. This example describes methods that can be used to reduce nitrites to the corresponding amine in vivo. In particular examples, reduction of a nitrile containing compound can be performed at 20-40° C. (such as 25-37° C., 25-30° C., such as 30° C.), at a pH of 6-8.5 (such as 6-8, 6-7.5, or 7-7.5, such as pH 7.2 or 7.5), for 30 seconds to several hours (such as 30 seconds to 6 hours, 1 minute to 3 hours, or 30 minutes to 2 hours). One potential enzymatic activity included the conversion of preQ0 to preQ1, which involves the conversion of a nitrile to an amine. Therefore, using the symmetry of the FolE multimer, a homodimer of the B. subtilis QueF monomeric model was generated and energy minimized in CNS. The presence of the N-terminal His6 fusion did not affect catalytic activity; both the fusion and cleaved recombinant enzymes exhibited identical molar specific activities. However, one skilled in the art will appreciate that the choice of nitrile oxido-reductase will depend on the nitrile to be reduced (or the amine to be produced). This example describes methods that can be used to alter the Glu98 residue in B. subtilis QueF to another amino acid, thereby changing the substrate specificity of the nitrile oxido-reductase. A nucleic acid molecule encoding a peptide having nitrile oxido-reductase activity can be identified and obtained using any method known in the art. One skilled in the art will appreciate that the culture medium will depend on the cell used. What's the finest readily-available way to write on paper? J. Very often, you may end up reducing these derivatives to alcohols and oxidizing them to aldehydes since DIBAL will do a complete reduction like LiAlH4 does. YP—217894). ZP—00039169). 4:947-52, 1997), and dihydroneopterin adolase (n=4) (Hennig et al. Any method can be used to introduce an exogenous nucleic acid molecule into a cell. 313:373-6, 1996 and Matsuura et al. After $\pu{30 min}$ the starting material has disappeared and the solution is allowed to cool down and then poured into ice. Transfer hydrogenation of unsaturated bonds in the absence of base additives catalyzed by a cobalt-based heterogeneous catalyst. For example, the methods can be used to reduce a nitrile to its corresponding primary amine. FIG. NP—662521). For example, the exogenous nitrile oxido-reductase nucleic acid molecule can be operably linked to a secretory sequence, thereby producing a nitrile oxido-reductase fusion protein that is secreted from the cell. The conserved QueF motif is in green, and the side-chains of the Glu and Cys residues conserved in both QueF and FolE are in red. Nat. Thus, changing this residue can be used to create a nitrile oxido-reductase enzyme with altered substrate specificity with regards to functional groups located at positions equivalent to C2. The method includes incubating one or more nitrites with one or more nitrile oxido-reductases under conditions that permit substantial reduction of the nitrites to the appropriate amine. site design / logo © 2020 Stack Exchange Inc; user contributions licensed under cc by-sa. Privacy Policy SEQ ID NO: 69 is an exemplary nitrile oxido-reductase amino acid sequence GenBank Accession no. Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity. 15. A naturally-occurring peptide is any peptide having an amino acid sequence as found in nature, including wild-type and polymorphic polypeptides. 9A-G and 10 A-K). In particular examples, the sample includes a recombinant nitrile oxido-reductase. Particular types of promoters that can be used to practice the methods disclosed herein include, but are not limited to, constitutive promoters and inducible promoters (such as a promoter responsive or unresponsive to a particular stimulus, for example such as light, oxygen, or chemical concentration, such as an IPTG or tetracycline inducible promoter). Engin. CAE06978). The present disclosure also provides methods for producing an amine, such as a primary amine. This example describes methods used to analyze the structure of the QueF Class I (YkvM) and Class II (YqcD) families. Jaime Z. Saavedra, Angel Resendez, Alexander Rovira, Scott Eagon, Dustin Haddenham, and Bakthan Singaram . 82:25-30, 2004). It is recommended that BH3-THF is used below 35 C (for safety). All rights reserved. 388:134-45, 2004). 9A-G and 10A-K). For example substitution of Glu98 as follows: E98A, E98L, E98I, E98M, E98V, E98Q, or E98N (wherein the Glu98 refers to SEQ ID NO: 2, but that one skilled in the art will recognize that the corresponding Glu can be identified in other QueF sequences, for example using the alignment provided in FIGS.

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