A kind of method for preparing (-)γ-lactam with high catalytic efficiency
A technology of lactam and lactamase, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve problems such as poor thermal stability and low catalytic efficiency, and achieve good solubility, high catalytic efficiency and excellent reaction conditions mild effect
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Embodiment 1
[0046] Example 1: Construction and culture of recombinant Escherichia coli BL21(DE3) / pET28a-TmLM
[0047] (1) Chemically synthesized (+) γ-lactamase TmLM whose nucleotide sequence is shown in SEQ ID NO.1.
[0048] (2) Construction of recombinant Escherichia coli:
[0049] The plasmids pET28a and TmLM were double digested with restriction enzymes BamH I and Xho I in a water bath at 37°C overnight, purified by agarose gel electrophoresis the next day, and the target fragments were recovered using an agarose recovery kit (eg. figure 1 shown). At 37°C, use T4 DNA ligase to connect the gene TmLM with the digested plasmid pET28a to obtain the recombinant expression vector pET28a-TmLM (such as figure 2 shown), the constructed recombinant expression vector pET28a-TmLM was thermally transferred into Escherichia coli BL21 (DE3) competent, coated with kanamycin-resistant LB solid medium, and the colony PCR was performed after overnight culture. The positive clone is recombinant Esche...
Embodiment 2
[0054] Example 2: Isolation and purification of recombinant Escherichia coli enzyme and (+)γ-lactamase
[0055] (1) The recombinant Escherichia coli BL21(DE3) / pET28a-TmLM obtained in Example 1 was suspended in liquid A (20 mmol·L -1 Sodium Phosphate, 500mmol·L -1 NaCl, 20mmol·L -1 imidazole, pH 7.4), the crude enzyme solution was obtained after ultrasonication and centrifugation.
[0056] (2) Purification of (+) γ-lactamase:
[0057] The column used for purification is HisTrap FF crude (nickel column), which is completed by affinity binding using the histidine tag on the recombinant protein. First, use solution A to equilibrate the nickel column, load the crude enzyme solution obtained in step (1), continue to use solution A to elute the breakthrough peak, and use solution B (20 mmol·L) after equilibration. -1 Sodium Phosphate, 500mmol·L -1 NaCl, 1000mmol·L -1 imidazole, pH 7.4) by gradient elution, the recombinant protein bound to the nickel column was eluted to obtain ...
Embodiment 3
[0059] Example 3: Properties of (+)γ-lactamase
[0060] (1) Under the conditions of 30°C and pH 7.0, according to the detection method of (+)γ-lactamase enzyme activity, the enzyme activity of (+)γ-lactamase on the substrate vinsolactone was determined after 30 minutes of reaction , the enzyme activity is: 2.0U·mg –1 .
[0061] (2) Prepare 100mmol·L -1 Buffers with different pH: Tris-HCl (8.0~9.0), sodium phosphate buffer (pH6.0~8.0), citric acid-sodium citrate buffer (pH5.0~6.0). Then racemic γ-lactam was used as the substrate to determine the relative enzymatic activity of TmLM in different pH buffers, as shown in Table 1. The optimum reaction pH of TmLM is 7.0-8.0, and the enzyme activity is 1.4-2.0 U·mg –1 .
[0062] Table 1 Relative enzyme activity of TmLM in different pH buffers
[0063]
[0064] (3) Racemic γ-lactam was used as the substrate, and the enzyme activities of TmLM at different temperatures (20-80°C) were measured respectively. The highest enzyme act...
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