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A technology of transaminase and mutant, applied in the field of enzyme engineering, can solve the problems of limited application, poor tolerance of transaminase activity, etc.
Active Publication Date: 2018-08-10
ASYMCHEM LIFE SCI TIANJIN
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[0011] The main purpose of the present invention is to provide a transaminase mutant and its application, so as to solve the pro
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[0134] The crude enzyme was treated at 30° C., pH 9.5, and DMSO concentration was 50% for 1 h, then in a 10 mL reaction flask, 0.1 g of substrate 1 was added, and 4 eq of isopropylamine hydrochloride and 0.6-1 mg of PLP ( 5'-pyridoxal phosphate), and then add 5 mg of the above-mentioned treated enzyme, and stir for 16 hours at 30° C., pH 9.5, and 50% DMSO concentration. The conversion rate of the system was detected by HPLC, and the mutant reaction data are shown in Table 12.
[0135] Table 12:
[0136]
Embodiment 2
[0137] Example 2: Mutant 45°C, pH 10, 50% DMSO tolerance test
[0138] Crude enzyme was treated at 45°C, pH 10, DMSO concentration was 50% environment for 1h, then in a 10mL reaction flask, 0.1g substrate 1 was added, and 4eq isopropylamine hydrochloride and 0.6-1mgPLP (5'- pyridoxal phosphate), and then add 5 mg of the enzyme after the above treatment, and stir for 16 hours at 45° C., pH 10, and DMSO concentration of 50% in an environment with constant temperature. The conversion rate of the system was detected by HPLC, and the mutant reaction data are shown in Table 13.
[0139] Table 13:
[0140]
[0141]
Embodiment 3
[0142] Example 3: Mutant 30°C, pH8, 35% Methanol Tolerance Detection
[0143] Treat the crude enzyme at 30°C, pH8, 35% MeOH concentration environment for 1h, then add 0.1g substrate 1, 4eq isopropylamine hydrochloride and 0.6-1mgPLP (5'- pyridoxal phosphate), and then add 5 mg of the enzyme after the above treatment, and continue stirring at 30° C., pH 8, and 35% MeOH concentration environment for 16 hours. The conversion rate of the system was detected by HPLC, and the mutant reaction data are shown in Table 14.
[0144] Table 14:
[0145]
[0146]
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Abstract
The invention provides a transaminase mutant and applications of the transaminase mutant. The amino acid sequence of the transaminase mutant is the amino acid sequence with mutation as shown in SEQ IDNO:1, the amino acid sites with mutation comprise the T7C+S47C site. For the transaminase mutant with the mutation sites, the enzyme activity and/or stability are/is greatly improved, and the transaminase mutant can be applied in the relatively extreme environment. In addition, the transaminase mutant with the mutation sites can be further prepared into immobilized enzyme through the immobilization technology, the activity and the stability of the immobilized enzyme are relatively high, and the immobilized enzyme can be recycled for a plurality of times, and is suitable for being applied to continuous flow reaction of a packed bed.
Description
technical field [0001] The invention relates to the field of enzyme engineering, in particular to a transaminase mutant and its application. Background technique [0002] ω-Aminotransferase (ω-TA) belongs to the class of transferases, and like other transaminases, it catalyzes a process of exchanging an amino group with a keto group. In most cases, ω-transaminase refers to a class of enzymes. As long as the substrate or product of the transamination reaction catalyzed by an enzyme does not contain α-amino acids, the enzyme can be called ω-transaminase. ω-transaminase uses ketones as raw materials to efficiently produce chiral amines through stereoselective transamination. Enantiomeric chiral amines are key intermediates of many pharmaceutical compounds with broad biological activities (ChemBioChem.9, 2008, 363-365, Chem.Commun.46, 2010, 5569-5571, Biotechnol. 1479-1493). Because of its relatively cheap substrates and high product purity, it has attracted more and more att...
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