Method for producing imidazole dipeptide
A technology of imidazole bis and carnosine, which is used in biochemical equipment and methods, peptides, enzymes, etc.
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[0043] 1. The preparation method of imidazole dipeptide
[0044] One of the embodiments of the present invention is a method for preparing imidazole dipeptide using a microorganism having imidazole dipeptide synthesis activity.
[0045] More specifically, the present invention is a method for preparing imidazole dipeptide using a microorganism having imidazole dipeptide synthesis activity. The aforementioned preparation method does not include the process of adding ATP, or includes adding The process of calculating the amount of ATP less than the amount of imidazole dipeptide to be prepared in conversion.
[0046] In the present specification, the sequence of a protein is described by a three-letter or one-letter notation for commonly used amino acids well known to those skilled in the art. The amino acids in this specification are L-forms unless otherwise specified. In addition, when expressing a mutant protein, the one-letter label of the mutated amino acid to be introduce...
Embodiment 1
[0127] In the following examples, polynucleotide (DNA, mRNA) preparation, PCR, nucleotide sequence determination, transformation, HPLC analysis, etc. can be carried out using methods well known to those skilled in the art. Please refer to, for example, Sambrook, J. and Russell, D.W., Molecular Cloning, A Laboratory Manual 3rd Edition, Cold Spring Harbor Laboratory Press (2012).
[0128] Introduction of site-specific mutations to YwfE
[0129] Target mutations were introduced using QuickChange Site-Directed Mutagenesis (Strategene, USA) according to the manufacturer's instructions using the pET vector assembled with the YwfE gene (SEQ ID NO: 1) as a template. The PCR reaction was carried out under the reaction conditions shown in Table 1 (composition) and Table 2 (PCR cycle). In the PCR reaction, KOD-Plus Neo DNA polymerase (Toyobo Co., Ltd., Osaka) was used. Using primers (SEQ ID NOS: 2 to 5), a vector into which a site-specific mutation of N108E or I112V was introduced wa...
Embodiment 2
[0183] Synthesis of Anserine Based on Cell Reaction Method
[0184] Escherichia coli BL21 (DE3) (wild type) and the PepD-deficient strain (APepD) of the Escherichia coli were used as hosts to synthesize the anserine of the transformant expressing the double mutant YwfE (I112V / H378K), using the composition shown in Table 7 The reaction solution was carried out at a temperature of 30° C. and a pH of 7.0 to 8.0 for 20 hours. After completion of the reaction, the anserine concentration was quantified by HPLC in the same manner as in Example 1, and the anserine synthesis activity was evaluated. It should be noted that the standard product of anserine was prepared using commercially available anserine (Wako Pure Chemical Industries, Ltd., Osaka) with a wet cell mass of 10 mg / ml.
[0185] [Table 8]
[0186]
[0187] result
[0188] Compared with the glucose non-added group (G-), the transformation of E. coli BL21 (DE3) in the glucose added group (G+) and the PepD-deficient st...
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