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Cephalosporinase mutant with improved enzyme activity and construction method thereof

A cephalosporin enzyme and mutant technology, applied in the field of genetic engineering, can solve the problems of low cephalosporin enzyme activity, low protein expression, low-level expression of enzyme, etc., and achieve the effect of improving enzyme activity

Pending Publication Date: 2019-04-05
广州白云山拜迪生物医药有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

①Induced high-yielding type Most of the genes naturally exist on the chromosome of Enterobacter cloacae, but only a small amount of enzymes are produced under normal conditions, and when there are β-lactam antibiotics such as penicillins and cephalosporins that induce the effect, the enzyme production increases significantly double; ② There are also genes on the chromosomes of strains with continuous high-yield partial enzyme production, and through the mutation of the weak promoter of the regulatory gene and the attenuator of the hairpin structure or the mutation of the regulatory gene, it can be used regardless of whether there is β-inner In the presence of amide antibacterial drugs, they can produce high-yield enzymes, also known as de-repressed high-expression; ③ Very few enzyme-producing strains of the persistent low-yield type do not produce or produce defective proteins due to lack of regulatory genes or gene mutations, and cannot be produced in the presence of β -It acts as an activator in the presence of lactam antibacterial drugs, and cannot act as an inhibitor in the absence of β-lactam antibacterial drugs, so that the enzyme continues to express at a low level, which can be seen in the large intestine bacteria
[0006] The outstanding problems of heterologous expression of cephalosporinase are low protein expression and low activity of cephalosporinase

Method used

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  • Cephalosporinase mutant with improved enzyme activity and construction method thereof
  • Cephalosporinase mutant with improved enzyme activity and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Site-directed mutagenesis

[0021] Using the recombinant plasmid containing SEQ ID NO: 4 as a template plasmid, refer to TaKaRa biological products and operation manuals, use TaKaRa MuTanBEST mutation kit, and use the designed corresponding mutation primer pair to amplify the site-directed mutation sequence of BmPGA by PCR. The specific operation steps as follows:

[0022] (1) Mutation primers

[0023] Forward primer BLAB-154-F is 5'-GACCCTGAACACCGCTATC RHT GGTGACAAACGTGACAC-3',

[0024] The reverse primer BLAB-154-R is 5'-GTGTCACGTTTGTCACCADYGATAGCGGTGTTCAGGGTC-3'.

[0025] (2) Reaction system (10ul)

[0026] 10ul of 10xPfu polymerase buffer, 2ul of 10mmol / L dNTP solution, 25pmoles of forward primer, 25pmoles of reverse primer, 5U of Pfu DNA polymerase, 100-200pg of plasmid DNA template, add water to 100ul.

[0027] (3) PCR conditions: 94°C for 3 minutes, 94°C for 30s, 55°C for 30s, 72°C for 300s, 30 cycles, 72°C for 10 minutes.

[0028] Refer to the o...

Embodiment 2

[0030] The acquisition of embodiment 2 engineering bacteria

[0031] Referring to the method of "Molecular Cloning Experiment Guide", transform the mutated plasmid into Escherichia coli competent cells or yeast competent cells to obtain genetically engineered strains expressing mutant enzymes, and finally apply the engineered strains to kanamycin sulfate On the LB selective plate of antibiotics, incubate upside down at 37°C for about 12-18 hours, and those that can grow on the LB plate with kanamycin sulfate antibiotic are transformants transformed with recombinant plasmids.

Embodiment 3

[0032] Embodiment 3 Engineering bacteria fermentation culture

[0033] Pick a single colony from the LB selective culture plate, inoculate it into 3 mL of LB liquid medium, add kanamycin to a final concentration of 100 μg / mL, and culture at 250 rpm at 37°C overnight; take 2 mL and culture overnight Inoculated into 200mL of LB liquid medium, cultured at 250r / min at 37°C for 4-6h, and the OD600 reached between 1.0-1.6, to obtain the seed bacterial liquid. Insert the inoculation amount of 1:15 into a 5-liter fermenter containing 3 liters of TB medium. Under the fermentation conditions of 37°C, 400rpm, and 1.3vvm, ferment until the OD600 reaches 25, and adjust the temperature to 30°C. Induce with IPTG with a final concentration of 1%, continue culturing for 2 hours, then add IPTG with a final concentration of 0.5% for induction, continue culturing for about 14-16 hours, and the fermentation ends.

[0034] The fermentation curve of engineering bacteria is as follows: figure 1 As ...

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Abstract

The invention relates to a cephalosporinase mutant with improved enzyme activity and a construction method thereof, and belongs to the technical field of genetic engineering. The amino acid sequence of the cephalosporinase mutant with improved enzyme activity is shown as SEQ ID NO:1. According to the invention, with adoption of a single point mutation technique and a homology modeling based method, the molecular structure of cephalosporinase is optimized and the enzyme activity is improved by selecting specific amino acids, and the cephalosporinase mutant with improved enzyme activity and theconstruction method thereof are significant for satisfying the industrialization production demand of the cephalosporinase and reducing the production cost.

Description

technical field [0001] The invention relates to a cephalosporinase mutant with improved enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] The enzyme system is mainly produced by the chromosome or plasmid encoding of Gram-negative bacilli such as Enterobacter, which can hydrolyze and inactivate cephalosporin antibiotics. Most cephalosporinases (ampc enzymes) can hydrolyze oxyimino β-lactam antibiotics and are not inhibited by clavulanic acid. Oxyimino β-lactam antibiotics include third-generation (such as ceftazidime, cefotaxime) and monocyclic β-lactam antibiotics (such as aztreonam), all of which contain an oxyimino group in their molecular structure group. Cefoxitin and cefmetera are extremely unstable to them, and bacteria producing such enzymes can be highly resistant to them, but the fourth-generation cephalosporins such as cefoxitin, cefpirome, imipenem, meropenem, etc. Carbapene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/86C12N15/55C12N1/21C12N15/70C12R1/19
CPCC12N9/86C12N15/70C12Y305/02006
Inventor 李润明罗春郝宇丁姝元邱壮伟王金刚陈舒明粱岩
Owner 广州白云山拜迪生物医药有限公司