Bacillus pumilus W3 CotA laccase mutant with improved soluble expression

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

Inactive Publication Date: 2017-10-03
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, the use of physical or chemical methods (coagulation, ozone, activated carbon) is often ineffective, and it is easy to cause secondary pollution

Method used

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  • Bacillus pumilus W3 CotA laccase mutant with improved soluble expression
  • Bacillus pumilus W3 CotA laccase mutant with improved soluble expression
  • Bacillus pumilus W3 CotA laccase mutant with improved soluble expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of B. pumilus W3 laccase mutant

[0024] Principle of site-directed mutagenesis: The principle of point mutation uses primers to introduce mutation sites, and PCR SμperMix synthesizes mutant chains ( figure 2 ). Using the CotA laccase gene sequence of the recombinant laccase mutant WLF successfully constructed earlier as a template, primers were designed to mutate the 501st aspartic acid (Asp, D) in the laccase to glycine (Gly, D). The relevant forward primers and reverse primers designed are as follows:

[0025] Foreword F 5'-AACACGAGGATTAT GGC ATGATGCGGCC-3'

[0026] Back-quoted R 5'- CC ATAATCCTCGTGTTCTAATATGTGAC-3'

[0027]The underlined parts represent the codons corresponding to glycine at position 501 encoded by the mutant gene. The PCR amplification system is: plasmid DNA 3 μL, front primer (10 μM) 1 μL, back primer (10 μM) 1 μL, PCR SμperMix 25 μL, ddH 2 Make up to 50 μL with O, the PCR amplification conditions are denaturation ...

Embodiment 2

[0028] Example 2 Expression and purification of B.pumilus W3 laccase

[0029] Expression: Inoculate the wild-type recombinant laccase expression strain and previously constructed WLF and D501G / WLF recombinant expression strains from glycerol tubes into LB medium for activation, 37°C, 200rpm overnight (10h). The seeds were inserted 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 30min, then the final concentration of 0.4mM IPTG and 0.25mM CuSO4 were added to induce, and cultured at 15°C for 24h at 200rpm, the fermentation broth was centrifuged at 4°C at 8000rpm for 10min to remove the supernatant and collect bacteria. 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 a water bath...

Embodiment 3

[0031] Example 3 Enzyme activity determination and expression analysis of B.pumilus W3 laccase

[0032] (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.

[0033] (2) Enzyme activity assay steps: 1 Preheating: Take 2.4mL of pH4.0 citrate buffer solution 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 Preheat for 5 minutes; 2 reactions: Add 0.1mL of diluted sample enzyme solution and shake evenly. 3 Measurement: Use a spectrophotometer to measure the kinetics of the uniformly shaken 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.

[0034] (3) Determination of kinetic parameters...

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Abstract

The invention discloses a Bacillus pumilus W3 CotA laccase mutant with improved soluble expression, belonging to the field of bioengineering. According to the invention, a composite mutant, i.e., L386W / G417L / G57F (WLF) CotA laccase gene, constructed in our laboratory in an early period and having greatly improved catalytic efficiency (with ABTS as a substrate) is used as a template, and Asp at the site 501 of the composite mutant is further mutated into Gly. The soluble expression level of the Bacillus pumilus W3 CotA laccase mutant provided by the invention is 3.75 times the soluble expression level of WT-CotA and 4.48 times the soluble expression level of WLF; the catalysis efficiency of the Bacillus pumilus W3 CotA laccase mutant is 4.4 times the catalysis efficiency of WT-CotA and 0.993 time the catalysis efficiency of WLF; and the Bacillus pumilus W3 CotA laccase mutant maintains good pH and temperature stability. The CotA laccase mutant obtained in the invention is more suitable for industrial application and has better application prospects.

Description

technical field [0001] The invention relates to a Bacillus pumilus CotA laccase mutant with increased soluble expression, which belongs to the technical field of bioengineering. Background technique [0002] Laccase (Laccase, E.C.1.10.3.2) is a copper-containing polyphenol oxidase, which 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 paper mills...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N1/21C12N15/53C02F3/34C12R1/07
CPCC12N9/0061C02F3/34C02F2101/308C12Y110/03002
Inventor 管政兵罗权夏静王凯强廖祥儒
Owner JIANGNAN UNIV
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