Penicillin acylase and coding genes, producing bacteria and application thereof
A technology of penicillin acylase and coding gene, which is applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effects of wide substrate range, good pH stability, and strong tolerance to organic solvents
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Embodiment 1
[0027] This example illustrates the screening procedure for penicillin acylase-producing strains.
[0028] The following method was used for the initial screening: use high-concentration penicillin G as the screening pressure to obtain high-concentration penicillin-resistant microorganisms from samples such as the soil around the pharmaceutical factory. The specific medium formula is: yeast extract 5 g / L, peptone 5 g / L, NaCl 2 g / L, phenylacetic acid 2 g / L, penicillin 10 6 U / ml, pH 7.0. The culture temperature was 30°C, the culture time was 24-48 h, and the shaker speed was 180 rpm. This method can screen a large number of high-concentration penicillin-resistant microorganisms.
[0029] Re-screen the candidate strains of primary screening, adopt the following method: use 50mM phosphate buffer (pH7.5) to prepare 2mg / ml substrate analogue 2-nitro-5-phenylacetamide benzoic acid 2-nitro-5- phenylacetamidobenzoic acid (NIPAB), and added to a transparent 48-well plate, pick the cand...
Embodiment 2
[0032] This experiment illustrates the isolation and cloning procedure of the gene encoding penicillin acylase PGAPX02.
[0033] Total DNA was extracted by the phenol-chloroform method. The whole gene sequencing results of Achromobacter xylosoxidans A8 (NCBI Reference Sequence: NC_014640.1) were selected for analysis, and the gene encoding penicillin acylase derived from Achromobacter was obtained, and primers: PX02-F1 and PX02-R1 were designed according to the gene sequence.
[0034] PX02-F1 (SEQ ID: 3): 5'-ATGAAGCAGCAATGGTTGTCGGC-3';
[0035] PX02-R1 (SEQ ID: 4): 5'-TCAGTAGCGCAACGTCTCTACCG-3'.
[0036] Using the total DNA of Achromobacter xylosoxidans PX02 as a template, the CDS coding sequence of penicillin acylase PGAPX02 was amplified with primers PX02-F1 and PX02-R1. The amplified 2500 bp PCR fragment was connected to the pMD18-T vector for sequence determination and analysis. The gene sequence of penicillin acylase is shown in SEQ ID NO: 1, and the amino acid sequence...
Embodiment 3
[0038] This experiment illustrates the construction of the expression vector of penicillin acylase PGAPX02 and its expression in E.coli BL21 (DE3).
[0039] Using PGAPX02 total DNA as a template, design primers as follows:
[0040] PX02-F2 (SEQ ID: 5): 5′-ATA GCTAGC ATGAAGCAGCAATGGTTGTCGGC-3' (the underlined part is the NheI restriction site);
[0041] PX02-R2 (SEQ ID: 6): 5′-ATA CTCGAG TCAGTAGCGCAACGTCTCTACCG-3′ (the underlined part is the XhoI restriction site)
[0042] The penicillin acylase PGAPX02 gene was amplified with PX02-F2 and PX02-F2, and after purification, it was double-digested with restriction endonucleases Nhe I and Xho I, and the vector pET28a (purchased (from Novagen), the digested product was subjected to agarose gel electrophoresis, the gene fragment and the vector were recovered, and the gene fragment and the vector were ligated with T4 DNA ligase. The positive clone identified by enzyme digestion and PCR screening was named pET28a-pgapx02 , thus co...
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