A kind of tyrosine decarboxylase mutant and its gene and application
An amino acid and serine technology, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as unclear key sites, research reports on molecular transformation of tyrosine decarboxylase, etc., and achieve the effect of improving application potential
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Embodiment 1
[0029] The preparation of embodiment 1 Lactobacillus brevis tyrosine decarboxylase mutant
[0030] Site-directed mutagenesis using II Site-Directed Mutagenesis Kit (Stratagene, Catalog #200522) described the protocol to operate. First design mutation primers containing mutation points, as shown in Table 1, including the 98th, 99th, 100th, 101st, 102nd, 103rd, 294th in the sequence shown in SEQ ID NO: 1 in the sequence table , 295, 296, 297, 298, 299, 331, 391, 395, 396, 397, 398, 505, 586 and 588 for introducing mutations, and for 586 A primer for saturation mutation (mutating the serine at position 586 to any one of the remaining 19 amino acids). The sequences of the above primers are shown in the sequence table SEQ ID No.2-81:
[0031] Mutation primers of table 1 tyrosine decarboxylase
[0032]
[0033]
[0034]
[0035] Using the above primers, the pET-24a plasmid containing the LbTDC gene was used as a template for the whole plasmid PCR reaction. The reactio...
Embodiment 2
[0038] Example 2 Expression and purification of Lactobacillus brevis tyrosine decarboxylase mutant
[0039] 1. Expression of Tyrosine Decarboxylase Mutants
[0040] The expression bacterial strain of the mutant that sequencing succeeds among the embodiment 1 is inoculated in 100mL the LBG liquid medium (10g / L peptone, 5g / L yeast extract, 10g / L NaCl and 20g / L Glucose), cultured at 37°C, 180rpm to OD 600 After cooling down to 25°C, and adding IPTG with a final concentration of 0.2mmol / L, after 6 hours of induction, the bacteria were collected by centrifugation at 8000×g for 7 minutes. The cells were suspended in solution A (25mmol / L Tris-HCl, pH 7.4, 300mmol / L NaCl, 20mmol / L imidazole, 5mmol / L β-mercaptoethanol) and then sonicated (power 45%, ultrasonic 1s, intermittent 3s, total time 15min), and then centrifuged at 8000×g for 30min at 4°C, and the supernatant was the crude enzyme solution of the tyrosine decarboxylase mutant. The obtained crude enzyme solution is freeze-drie...
Embodiment 3
[0048] The enzyme activity analysis of embodiment 3 tyrosine decarboxylase mutants
[0049] The activity of tyrosine decarboxylase was measured by reverse phase HPLC (C18 column), and the activity of tyrosine decarboxylase was determined by measuring the increase of tyramine before and after the reaction. Enzyme activity is defined as 1 U of enzyme required to convert 1 μmol of substrate into product in 1 min under the assay conditions. Specific activity is defined as the number of units of activity per milligram of protein.
[0050] The assay system for tyrosine decarboxylase (1 mL) is: 950 μL of 2.75 mmol / L substrate preheated at 40°C (dissolved in 0.2 mol / L, pH 5.0 sodium acetate buffer), 5 mmol / L coenzyme PLP 40 μL , 10 μL of enzyme solution of appropriate concentration. The reaction was carried out in a 2mL EP tube. After reacting at 40°C for 10 minutes, it was inactivated with a metal bath at 100°C for 10 minutes to terminate the reaction. After the reaction solution ...
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