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Biosynthesis method for obtaining high-purity and high-efficiency microcystins (MCs) degrading enzyme (MlrA)

A algae toxin-degrading enzyme and biosynthetic technology, applied in the field of biotechnology applications or environmental protection, can solve problems such as time-consuming and material-consuming, affecting the application of algae-toxin-degrading enzymes, and harmful to health

Active Publication Date: 2014-02-05
湖北华大瑞尔科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although coagulation, activated carbon absorption, and membrane treatment technologies can partially reduce the content of algal toxins in water, they can only transfer algal toxins to other places and cannot effectively degrade algal toxins, and consume time and materials
Although the method of oxidative degradation of algae toxins has high degradation efficiency and low price, because of the strong biocidal ability of oxidants, the specificity is poor, and it is easy to react with organic pollutants to produce substances harmful to human health.
(2) Bacterial degradation method: This method uses fine sand filtration and biofilm containing selective biodegradable bacteria to treat water samples. This method is economical, but the operation is cumbersome, and the biofilm needs to be replaced frequently, and the biodegradable bacteria metabolize themselves Need to consume nutrients, easily affect the stability of the ecosystem, and may produce unknown secondary metabolic toxic substances, causing secondary pollution
However, these experiments are currently carried out under the condition of mixed enzyme solution, no pure enzyme has been obtained, and the expression level and expression activity of the enzyme are not high, which affects the further application of the algae toxin degrading enzyme

Method used

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  • Biosynthesis method for obtaining high-purity and high-efficiency microcystins (MCs) degrading enzyme (MlrA)
  • Biosynthesis method for obtaining high-purity and high-efficiency microcystins (MCs) degrading enzyme (MlrA)
  • Biosynthesis method for obtaining high-purity and high-efficiency microcystins (MCs) degrading enzyme (MlrA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of algal toxin degrading enzyme expression vector pMAL-C2X-MlrA

[0034] MlrA (nucleotide sequence shown in SEQ ID NO.1) was synthetically loaded into the cloning vector pUC19 to obtain pUC19-MlrA.

[0035] (1) Extraction of plasmid

[0036] Take 2 Erlenmeyer flasks containing 20mL LB (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, ampicillin 30mg / L) medium, and put the plasmids containing pUC19-MlrA and pMAL-C2X Single colonies were added to the culture medium, cultivated overnight at 37°C and 220rpm, and the plasmid was extracted according to the SDS lysis method. After the plasmid extraction is completed, mark it and put it in a 4°C refrigerator for use in the next step.

[0037] (2) PCR amplification of MlrA, MlrA and pMAL-C2X double enzyme digestion

[0038] PCR amplification reaction system: plasmid (pUC19-MlrA) 1 μL, 10×buffer 5 μL, dNTPs 4 μL, MlrA upstream primer 1 μL, MlrA downstream primer 1 μL, Taq DNA polymerase 1 μL, add ddH 2...

Embodiment 2

[0048] Example 2 Expression and Purification of Algae Toxin Degrading Enzymes

[0049] (1) Optimization of induction conditions

[0050] The concentration of the bacterial solution before induction, the concentration of the inducer IPTG, the induction expression temperature, and the induction time were optimized

[0051] The pMAL-C2X-MlrA plasmid in Escherichia coli DH5α was extracted, and the extracted plasmid was transformed into TB1 competent cells. Activate the expressing bacteria containing the target gene in a 20 mL LB medium flask containing 30 mg / L ampicillin at 37 °C and 220 rpm for 16 hours, take the activated bacterial liquid and inoculate it in 500 mL LB medium at an inoculation amount of 2.5%. Choose different induction conditions to induce expression. After induction of expression, the bacteria were collected, and the expression of the target protein was detected by SDS-PAGE to determine the optimal induction conditions. The result is as Figure 3-6 As show...

Embodiment 3

[0062] Example 3 Activity Detection of Algae Toxin Degrading Enzyme

[0063] (1) Cleaning balance of the chromatographic column

[0064] First turn on the high performance liquid chromatograph (Agilent 1200 LC) and elute the chromatographic column twice with methanol-containing aqueous solution from 100% to 2% gradient, and finally wash with 60% methanol to the baseline, and prepare the sample after the baseline leveled off for 20 minutes.

[0065] (2) Detection of samples

[0066] Sample information

[0067] The sample loading buffer is 0.05mol / L phosphate buffer, adjusted to pH=7.0; algae toxin MC-LR, MC-RR standard concentration is 10μg / mL, diluted with methanol to 5.0μg / mL, 1.0μg / mL , 0.5μg / mL, 0.25μg / mL; Methanol is HPLC chromatographically pure Fisher laboratory analytical reagent 4L; water is double distilled water, all samples need to be filtered with a 0.22μm filter membrane, and the bubbles dissolved in it are removed by ultrasonic waves.

[0068] Detection cond...

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Abstract

The invention discloses a biosynthesis method for obtaining a high-purity and high-efficiency microcystins (MCs) degrading enzyme (MlrA), belonging to the field of biotechnology application or environmental protection. The method comprises the following steps: constructing the sequences shown in SEQ ID NO.1 on pMAL-C2X to obtain pMAL-MlrA; transferring pMAL-MlrA to escherichia coli TB1 to obtain an expression bacterium of MlrA; carrying out induced expression on the expression bacterium of MlrA with isopropyl beta-D-1-thiogalactopyranoside (IPTG), collecting thalli after induced expression, crushing and purifying by using a maltose-binding protein (MBP) tag, thus obtaining the high-purity and high-efficiency MlrA. By optimizing an expression vector and expression conditions, the purity of the obtained MBP-MlrA is above 90% and the purity of MlrA after cutting off the MBP tag can be above 98%. The obtained MlrA has wide range of degradation and high degradation efficiency.

Description

[0001] technical field [0002] The invention relates to the field of biotechnology application or environmental protection, in particular to a biosynthesis method for obtaining high-purity and high-efficiency algal toxin degrading enzyme. Background technique [0003] Microcystins (MCs) are a serious class of toxins released by cyanobacteria during growth and after death (Duy T.N. et al. Toxicology and risk assessment of fresh water eyanobacteria (blue-green algae) toxic in water. Rev. Environ. Contam. Toxicaol . 2000, 163, 113-186), its structure diagram is as follows figure 1 shown. MCs is a cyclic heptapeptide compound, which divides the entire cyclic polypeptide into seven parts: 1, D-alanine; 2, R1; 3, D-β-methylisoaspartic acid; 4, R2; 5, Adda; 6, D-isoglutamic acid; 7, N-methyl dehydroalanine. At present, 67 algae toxins have been discovered, most of which are caused by the differences in amino acid residues at positions 2, 3, 4 and 7 in the ring structure. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/52C12R1/19
CPCC12N9/50C12N15/70
Inventor 冯玲玲万坚李俊吴迪苏佳丽任彦亮肖闪
Owner 湖北华大瑞尔科技有限公司
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