Acyltransferase for catalytic synthesis of simvastatin and mutant thereof
An acyltransferase and simvastatin technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of complicated product purification steps and low selectivity, and achieves the effects of short conversion time, high activity and friendly reaction environment.
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Embodiment 1
[0031] Example 1 Screening of acyltransferases
[0032] Using the reported acyltransferase LovD (derived from A. terreus) as a template, BLAST alignment was performed on NCBI, and the sequences with a similarity between 40-70% were selected for gene synthesis and induced expression, and used in the Enzymatic screening of vastatin. The screening system is as follows: Reaction system (substrate concentration 1g / L)
[0033] An enzyme that can be used for the enzymatic synthesis of simvastatin was successfully screened. The enzyme is an unknown protein derived from fungal sp. NO.14919. The encoded nucleotide sequence is shown in SEQ NO: 2, and the encoded amino acid sequence As shown in SEQ NO:1.
[0034] However, the reactivity of the enzyme is low, and directed evolution is required to meet the needs of industrial applications.
Embodiment 2
[0035] Embodiment 2, establishment of mutant library
[0036] Using the expression plasmid synthesized by the whole gene (synthesized by Changzhou Jiyu Biotechnology Co., Ltd., the amino acid sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2) was cloned on the plasmid, and the Primers F1 and R1 are forward and reverse primers respectively, and error-prone PCR is performed to construct a mutant library.
[0037] The primer sequences are as follows: F1: 5'-GGAATTCCATATGCAGGATATCGAAC-3'; R1: 5'-CCCAAGCTTTCAGTCGTTGCGCAGT-3', with Nde I and Hind III Restriction site.
[0038] The error-prone PCR reaction system is as follows: 10* PCR buffer 2.5μL, 10mM dGTP 0.5μL, 10mM dTTP 0.5μL, 10mM dCTP 2.5μL, 10mM dATP 2.5μL, 1mM MnCl 2 2.5 μL, 55 mM MgCl 2 2.5 μL, 10 μM F11 μL, 10 μM R1 1 μL, Template (10ng / μL) 1 μL, Taq DNA polymerase (5U / μL, takara) 0.2 μL, ddH 2 O to make up to 25 μL.
[0039] Error-prone PCR was performed on a Bio-Rad T100 thermal c...
Embodiment 3
[0041] Example 3 Activation and Induced Expression of Mutant Library
[0042]Pick a single clone from the plate cultured overnight to a 96-deep well plate filled with 1 mL of LB liquid medium with a final concentration of 50 μg / mL kanamycin sulfate, and culture overnight at 37°C and 220 rpm with shaking. On the next day, draw 100 μL of the culture solution from the 96 empty plates of the above overnight culture, and add it to a fresh 1 mL 96 deep-well plate containing LB liquid medium with a final concentration of 50 μg / mL kanamycin sulfate, at 37 ° C, 220 rpm After 4 hours of shaking culture, IPTG with a final concentration of 1 mM was added for induction, and then culture was continued at 30° C. for 20 hours. A total of 400 single clones were picked for activity screening.
[0043] Centrifuge the induced bacterial solution at 4°C and 4000 rpm for 15 min, and discard the supernatant. The cells were suspended in 100 μL of 50 mM sodium phosphate buffer (pH 7.0), added with ly...
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