A kind of recombinant lipase mutant, coding gene, recombinant engineering bacteria and application
A technology of genetically engineered bacteria and encoded genes is applied in recombinant lipase mutants, encoded genes, recombinant engineered bacteria and application fields, and can solve the problems of low concentration of split reaction substrates, low catalytic activity of lipase, and long reaction time. , to achieve the effect of less catalyst dosage, high stereoselectivity and short reaction time
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Embodiment 1
[0031] Embodiment 1: Construction of recombinant lipase genetically engineered bacteria E.coli Rosetta(DE3) / pET22b-SRL
[0032] The gene sequence of Sporisorium reilianum SRZ2 lipase (SRL) was optimized by yeast codons, and the pGEM-T-SRL plasmid was obtained by total gene synthesis. Design homologous recombination primer 1 (GCGATGGCCACTCCATTGGTTAAGAGA), primer 2 (GTGGTGGTGCAAGATAACACCAGAACA), primer 3 (GTTATCTTGCACCACCACCACCACCAC), primer 4 (CAATGGAGTGGCCATCGCCGGCTGGGC), using Max Super-Fidelity DNA polymerase is amplified with pGEM-T-SRL and pET22b plasmid as template, obtains the lipase gene sequence of 966bp (nucleotide sequence as shown in SEQ ID NO.1, aminoacid sequence SEQ ID NO.2 Shown) and the pET22b expression vector gene sequence of 5427bp. A one-step cloning kit was used to connect the lipase gene fragment with the pET22b expression vector gene fragment to construct the expression vector pET22b-SRL. The constructed recombinant expression vector was transformed in...
Embodiment 2
[0033] Example 2: Rational design and construction of recombinant lipase mutant mut-Ile194Lys
[0034] Using the recombinant bacteria (E.coli Rosetta(DE3) / pET22b-SRL) containing the expression vector pET22b-SRL as the starting strain, a mutation was introduced at the 194th position of the amino acid sequence of SRL by site-directed mutagenesis (the amino acid shown in SEQ IN NO.2 The 194-position Ile of the sequence is mutated to Lys), which improves the catalytic activity of lipase to the substrate racemic N-acetyl-piperidine-2,3-dicarboxylic acid dimethyl ester. Design primers for site-directed mutagenesis as follows:
[0035] Ile194Lys:
[0036] Upstream primer 5: 5'-CTCTGCTACTGACGACAAGGTTCAACCACAAAAC-3'
[0037] Downstream primer 6: 5'-GTTTGTGGTTGAACCTTGTCGTCAGTAGCAGAG-3'
[0038] Using the pET22b-SRL plasmid as a template, mutations were introduced by PCR. The PCR reaction procedure was as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 6 min, repeating...
Embodiment 3
[0040] Embodiment 3: Continued transformation and screening of recombinant lipase mutant mut-Ile194Lys
[0041] Using the mutant mut-Ile194Lys as the starting strain, the catalytic activity of lipase to the substrate racemic N-acetyl-piperidine-2,3-dicarboxylate dimethyl was further improved by site-directed saturation mutagenesis. Primers were designed as follows:
[0042] Leu(L)145:
[0043] Upstream primer 7:
[0044] 5'-ACTACAAGGGTACTGTTNNKGCTGCTTTCTTGACTAC-3'
[0045] Downstream primer 8:
[0046] 5'-GTAGTCAAGAAAGCAGCMNNAACAGTACCCTTGTAGT-3'
[0047] Ala(A)146:
[0048] Upstream primer 9:
[0049] 5'-ACAAGGGTACTGTTTTGNNKGCTTTCTTGACTACTCC-3'
[0050] Downstream primer 10:
[0051] 5'-GGAGTAGTCAAAGAAAGCMNNCAAAACAGTACCCTTGT-3'
[0052] Leu(L)149:
[0053] Upstream primer 11:
[0054] 5'-CTGTTTTGGCTGCTTTCNNKACTACTCCAGGTTTGGC-3'
[0055] Downstream primer 12:
[0056] 5'-GCCAAACCTGGAGTAGTMNNGAAAGCAGCCAAAACAG-3'
[0057] Leu / Ser(L / S)154 / 156 combination:
[0058] U...
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