Soluble expression increased bacillus pumilus W3 CotA laccase composite mutant

A technology of bacillus pumilus and mutants, applied in the field of bioengineering, can solve problems such as poor effect and secondary pollution

Inactive Publication Date: 2017-08-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the special chemical structure of the dye itself, physical or chemical methods (coagulati

Method used

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  • Soluble expression increased bacillus pumilus W3 CotA laccase composite mutant
  • Soluble expression increased bacillus pumilus W3 CotA laccase composite mutant
  • Soluble expression increased bacillus pumilus W3 CotA laccase composite mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of B. pumilus W3 laccase mutant

[0026] Principle of site-directed mutagenesis: The principle of point mutation uses primers to introduce mutation sites, and PCR SuperMix synthesizes mutant chains ( figure 2 ). Using the CotA laccase gene sequence of the previously constructed recombinant laccase mutant WLF as a template, primers were designed to mutate the 501st aspartic acid (Asp, D) in the laccase to glycine (Gly, D), and the mutation was successful Afterwards, position 317 was mutated with the D501G / WLF position template. The relevant forward primers and reverse primers designed are as follows:

[0027] 501 F5'-AACACGAGGATTAT GGC ATGATGCGGCC-3'

[0028] R5'- CC ATAATCCTCGTGTTCTAATATGTGAC-3'

[0029] 317 bit F5'-AACGATTGTTTTA AAC AATAAGGCAGGC-3'

[0030] R5'- GTT TAAAACAATCGTTTGGTTTTCGTAA-3'

[0031] The underlined parts represent the codons corresponding to the amino acids encoded by the mutant genes. The PCR amplification sys...

Embodiment 2

[0032] Example 2 Expression and purification of B.pumilus W3 laccase

[0033] Expression: Inoculate wild-type recombinant laccase expression strains and previously constructed WLF and K317N / D501G / WLF recombinant expression strains from glycerol tubes into LB medium for activation, 37°C, 200rpm overnight (10h). Insert the seeds into 50mL LB liquid fermentation medium (containing 100mg L -1 Ampicillin) 37 ℃ 200rpm shaker culture to OD 600 After reaching 0.5, the shaker temperature was adjusted to 15°C for static culture for 30 minutes, and then the final concentration of 0.4mM IPTG and 0.25mM CuSO was added 4 For induction, culture at 200 rpm at 15°C for 24 hours, centrifuge the fermentation broth at 8000 rpm at 4°C for 10 minutes to remove the supernatant, and collect the cells. Resuspend the collected bacteria with phosphate buffer, and after resuspension, use an ultrasonic cell disruptor to crush the bacteria to release intracellular proteins. The supernatant was heated in...

Embodiment 3

[0035] Example 3 Enzyme activity determination and expression analysis of B.pumilus W3 laccase

[0036] (1) Definition of enzyme activity unit: When using the ABTS method to determine the enzyme activity of laccase, define the amount of enzyme required to catalyze the conversion of 1 μmol of substrate into product per minute as an activity unit.

[0037] (2) Enzyme activity determination steps: 1 Preheating: Take 2.4mL of citric acid buffer solution with pH 4.0 in a test tube, add 0.5mL ABTS solution (the final concentration of ABTS is 0.5mM) into the test tube and place it in a water bath at 50°C for preheating. Heat for 5 minutes; 2 reactions: add diluted 0.1mL sample enzyme solution, shake evenly. 3 Measurement: Use a spectrophotometer to measure the kinetics of the uniformly oscillating sample, measure the change in OD value per minute within 30s at a wavelength of 420nm (the measurement reaction shows a straight line) and calculate the enzyme activity.

[0038] (3) Deter...

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Abstract

The invention discloses a soluble expression increased bacillus pumilus W3 Cota laccase composite mutant, and belongs to the field of biological engineering. The composite L386W/G417L/G57F(WLF)CotA laccase gene which is constructed at the early stage of the lab and has greatly improved catalyzing efficiency (ABTS is used as substrate) is used as a template to further mutate Asp on the 501 site of the composite mutant into Gly and Lys on the 317 site into Asn. The bacillus pumilus W3 CotA laccase mutant has a soluble expression which is 3.04 times that of WT-CotA and 3.63 times that of WLF, has a catalyzing efficiency whichis 4.6 times that of WT-CotA and 1.03 times that of WLF, and has excellent pH value and temperature stability. The CotA laccase mutant is more applicable to industrial application, and has a wide application prospect.

Description

technical field [0001] The invention relates to a compound mutant of Bacillus pumilus CotA laccase with increased soluble expression, and belongs to the technical field of bioengineering. Background technique [0002] Laccase (Laccase, E.C.1.10.3.2) is a copper-containing polyphenol oxidase that can catalyze the redox reaction of phenolic substances and plays an important role in the biodegradation of lignin and its precursor analogs. Laccase has a wide range of oxidation substrates, including phenols and their derivatives, aromatic amines and their derivatives, aromatic carboxylic acids and their derivatives, etc., so the application potential of laccase is huge. In the field of wood processing, laccase can replace chemical adhesives, which can not only improve product quality, but also reduce the harm to human health and environmental pollution; in the paper industry, laccase is used for paper biological bleaching and pulping, which can Reducing pollution in pulp and pape...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N1/21C12N15/53C02F3/34C12R1/07
CPCC12N9/0061C02F3/34C12Y110/03002
Inventor 管政兵罗权夏静王凯强廖祥儒
Owner JIANGNAN UNIV
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