Biocatalysis preparation method of D-tert-leucine
A technology of tertiary leucine and biocatalyst, which is applied in the direction of fermentation, etc., and can solve problems such as the inability to synthesize D-tertiary leucine
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Embodiment 1
[0022] The StDapdh wild-type gene Genbank number is AP006840.1. First, the gene is fully synthesized and connected to the pET32 vector to obtain a plasmid expressing the target gene: pET 32 -StDapdh, and soluble expression of the wild-type gene in E. coli BL21(DE3). The N-terminus of the expressed protein has a 6*his tag, which will facilitate the purification of the target protein using Ni-NTA. According to the site to be mutated, refer to the instructions of the Quick Change Mutagenesis Kit kit to synthesize the PCR mutation primers used in Table 1 below. PCR product amplification, Dpn1 digestion and subsequent nucleic acid recovery were all carried out according to the kit instructions.
[0023] Table 1: Primers used for mutation
[0024] Primer number
[0025] in pET 32 -The StDapdh plasmid was used as a template, and primers 1 and 2 were used to introduce the W121L mutation into the plasmid, and transformed into E. coli TOP10 competent, and the plasmid was ext...
Embodiment 2
[0026] Example 2: Expression and purification of mutant enzymes
[0027] Escherichia coli BL21(DE3) containing the triple mutant plasmid was cultured in 2LLB liquid medium, and cultivated to OD at 37°C 600 After about 0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM to induce expression, and the induction temperature is 25° C. for 20 hours. After induction of expression, the cells were collected by centrifugation at 5000×rpm for 5 minutes, resuspended and washed with buffer A (20 mM Tris-Cl pH 8.0, 500 mM sodium chloride, 5% glycerol). All subsequent purification experiments were performed at 4°C, and all buffers were pre-cooled to 4°C. First resuspend the bacteria with buffer A, high-pressure homogenate, and centrifuge at 14000×rpm for 30 minutes to remove the broken precipitate. The supernatant is pre-balanced with buffer A Ni-NTA chromatography column (GE health care), and buffered Solution B (20mM Tris-Cl pH8.0, 50mM imidazole, 500mM N...
Embodiment 3
[0028] Example 3: Viability assay of mutants
[0029] The activity of the mutants against 3,3-dimethyl-2-oxobutanoic acid was measured using a SPECTRAMAXM2e (MD, USA) microplate reader using a 96-well plate. The activity measurement system is as follows: the final concentrations of each component are: 20mM substrate 3,3-dimethyl-2-carbonylbutanoic acid, 200mM substrate ammonium chloride, 1mM coenzyme NADPH, appropriate amount of StDAPDH mutant pure enzyme, activity measurement The buffer is 100 mM sodium carbonate / sodium bicarbonate buffer solution pH 9.0, the final volume is 200 μL. The substrate and protein samples used in the activity assay were first added to a 96-well plate and equilibrated at 30°C for 10 minutes, and then an appropriate amount of coenzyme NADPH was added to it to initiate the reaction. By measuring the OD 340 The enzyme activity was determined by the reduction of NADPH (NADPH molar extinction coefficient at 340nm is 6.22mM -1 cm -1 ), the enzyme activ...
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