CN105200021A - High-activity Bacillus pumilus CotA laccase modified with site-directed mutagenesis - Google Patents

Abstract

The invention discloses a Bacillus pumilus CotA laccase mutant with improved catalytic performance and belongs to the field of genetic engineering and enzyme engineering. A composite mutant L386W/G417L CotA laccase gene constructed in the early stage in a laboratory is taken as a template, Asp at the 195th site of the composite mutant is mutated to Trp, Phe, Try and Ile respectively, then a wild type CotA laccase is taken as a control, and the composite mutant L386W/G417L/D195Y is discovered to have higher catalytic activity and specificity on the substrate ABTS (diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)), and the industrial application prospect of the Bacillus pumilus CotA laccase is promoted.

Description

A highly active Bacillus pumilus CotA laccase modified by site-directed mutagenesis

technical field

The invention relates to a bacillus pumilus CotA laccase mutant with improved catalytic efficiency, belonging to the fields of genetic engineering and enzyme engineering.

Background technique

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 will help the paper industry to achieve clean production; in the field of food processing, laccase can be used to remove turbidity caused by phenolic compounds in fruit juices, thereby improving the quality of fruit juices. In addition, laccase can also oxidize chlorophenols and their derivatives, reduce their toxicity, and reduce environmental pollution caused by using chlorophenols as industrial raw materials to produce dyes, preservatives, herbicides, insecticides and other chemical products.

Laccases can be divided into three categories according to different sources: plant laccases, fungal laccases and bacterial laccases. Bacterial laccases include the CotA protein of Bacillus, the PpoA protein of Halomonas, the CueO protein of Escherichia coli, the EpoA protein of Streptomyces griseus, etc., which are similar to fungal and plant laccase proteins in structure, and all have four copper ion binding location.

Our laboratory has cloned and recombinantly expressed CotA laccase from the self-screened Bacillus pumilus strain W3 (Bacillus pumilus W3) in the early stage. The study found that the CotA laccase has the following advantages compared with other types of laccases: high enzyme activity at alkaline pH, High temperature resistance, good thermal stability, high-concentration organic solvent and halogen ion environment, etc. These excellent characteristics are exactly what is urgently needed for the industrial application of laccase in the field of printing and dyeing wastewater treatment. However, the natural expression level of wild Bacillus pumilus CotA laccase is very low, and the substrate specificity is poor, and the catalytic activity is low, which has become a bottleneck for industrial application. Therefore, the present invention utilizes genetic engineering and enzyme engineering means to improve the catalytic activity of Bacillus pumilus CotA laccase, and further improve its industrial application prospect.

Contents of the invention

The present invention provides a Bacillus pumilus laccase mutant, which takes the double mutant L386W/G417L laccase gene obtained by transformation in the laboratory as a template, and further adds the 195th aspartic acid of the double mutant to Acid (Asp, D) was mutated into tryptophan (Trp, W), phenylalanine (Phe, F), tyrosine (Tyr, Y), isoleucine (Ile, I), respectively, and then wild Type CotA laccase was used as a control, and finally a mutation with higher specificity for the substrate 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) was selected body.

The parental amino acid sequence of the B. pumilusCotA laccase is consistent with the amino acid sequence of the B. pumilusCotA laccase in the NCBI database (submitted by our laboratory, GenBank accession number: KF040050).

The mutants are the 195th Asp in the compound mutant L386W/G417L/G57F laccase gene mutated into Trp, Phe, Tyr, and Ile, respectively, named L386W/G417L/D195W, L386W/G417L/D195F , L386W/G417L/D195Y, L386W/G417L/D195I.

Description of drawings

Figure 1: Three-dimensional simulation structure of wild Bacillus pumilus CotA laccase

Figure 2: Schematic diagram of the molecular operation principle of the process of constructing mutant plasmids

Detailed ways

The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

The present invention will be further described in detail below in conjunction with specific preparation examples and application examples, and with reference to data. It should be understood that these examples are only for illustration of the present invention, but not to limit the scope of the present invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1 Expression and purification of wild Bacillus pumilus CotA laccase.

The recombinant expression strain CotA/pColdII/BL21 (DE3) constructed from glycerol tube inoculation earlier stage was cultivated overnight in LB liquid medium (containing 100mg/L ampicillin), and the seeds were inserted into LB liquid fermentation medium (containing 100mg/L ampicillin) by 2% inoculum. 100mg/L). Escherichia coli was cultured at 37°C for 2 hours, then induced by adding 0.3mM IPTG at a final concentration, and continued to ferment and culture in a shaker at 15°C for 26 hours. The collected cells were resuspended in phosphate buffer, and after resuspension, the cells were crushed with an ultrasonic cell disruptor to release intracellular proteins. After the crushing was completed, the crushed liquid was centrifuged at 4°C and 8000 rpm for 10 min to collect the supernatant. The collected supernatant was used for CotA laccase protein purification.

Since the recombinantly expressed CotA laccase protein has a polyhistidine tag (His ₆ .tag), the target protein was separated using nickel ion affinity chromatography. Nickel ion affinity chromatography purification steps: (1) balance: equilibrate HisTrapHP nickel ion column (1mL) with 20mM buffer solution (containing 5mM imidazole) of 10 times column volume; Load the sample at a flow rate of 1 mL/min; (3) Elution: Use a high concentration of imidazole for gradient elution, collect the tube number corresponding to the peak shape under the elution condition, and perform enzyme activity detection. Finally, the purified wild-type CotA laccase was obtained.

Embodiment 2 CotA laccase mutant construction and preparation

(1) Site-directed mutation

Using the B. pumilusCotA laccase gene sequence of the previously constructed double mutant L386W/G417L as a template, the aspartic acid (Asp, D) at position 195 in the laccase was mutated to tryptophan (Trp, W) respectively , phenylalanine (Phe, F), tyrosine (Tyr, Y), isoleucine (Ile, I), named L386W/G417L/D195W, L386W/G417L/D195F, L386W/G417L/D195Y, L386W/G417L/D195I.

The site-directed mutagenesis primers for introducing the D195W mutation are:

Forward primer 5'-CCTTTAATGATCATG TGG CGGACATTCCAG-3' (the mutation site is underlined)

Reverse primer 5'- CCA CATGATCATTAAAGGGATATCATATTC-3' (underlined is the mutation site)

The site-directed mutagenesis primers for introducing the D195F mutation are:

Forward primer 5'-CCTTTAATGATCATG TT CCGGACATTCCAG-3' (the mutation site is underlined)

Reverse primer 5'- AA CATGATCATTAAAGGGATATCATATTCA-3' (the mutation site is underlined)

The site-directed mutagenesis primers for introducing the D195Y mutation are:

Forward primer 5'-CCTTTAATGATCATG T ACCGGACATTCCAG-3' (the mutation site is underlined)

Reverse primer 5'- A CATGATCATTAAAGGGATATCATATTCAT-3' (the underline is the mutation site)

The site-directed mutagenesis primers for introducing the D195I mutation are:

Forward primer 5'-CCTTTAATGATCATG AT CGGACATTCCAG-3' (the mutation site is underlined)

Reverse primer 5'- AT CATGATCATTAAAGGGATATCATATTCA-3' (underlined is the mutation site)

Using the above primers, the double mutant plasmid pColdII-CotA (WL) was used as a template for PCR reaction. All reactions were carried out in a 50 μL system, and the reaction conditions were: 95°C pre-denaturation for 3 minutes, followed by 25 cycles (95°C for 20 s, 57°C for 20 s, 72°C for 6 min), after the cycle, 72°C extension for 7 minutes, and finally 4°C incubation. 10 μL of PCR products were detected by 1% agarose gel electrophoresis. After the target product is detected, add 1 μL DMT enzyme to the remaining PCR product, mix well, and incubate at 37°C for 1 hour. All the products after incubation were separated by 1% agarose gel electrophoresis, gel was cut, and the target fragment was recovered with a gel recovery kit. The recovered target fragments were transformed into DMT competent cells after assembly of mutant fragments, and the transformed products were spread on LB plates containing 100 mg/L ampicillin, cultured overnight at 37°C, and 10 single colonies were picked from the plates for colony PCR verification. Pick 3 single colonies from the successfully verified colonies and insert them into LB liquid medium. After 10 hours, store each bacterial solution in two glycerol tubes, one for storage at -20°C and one for sequencing. The mutants with correct sequencing were inoculated from glycerol tubes into LB liquid medium for overnight culture. After overnight, the glycerol tubes were preserved, and then the plasmids were extracted from the remaining bacterial liquid and transformed into BL21 (DE3) competent cells.

(2) Expression and purification of mutant enzymes

The mutant expression and purification process was as described in Example 1.

Example 3 Analysis of CotA Laccase Mutant Enzyme Activity.

(1) Definition of enzyme activity unit

When using the ABTS method to measure the enzyme activity of laccase, the amount of enzyme required to convert 1 μmol of substrate per minute is defined as an activity unit.

(2) Enzyme Activity Determination Steps

Preheating: Take 2.4 mL of citric acid buffer solution at pH 4.0 in a test tube, add 0.5 mL of ABTS solution (the final concentration of ABTS is 0.5 mM) into the test tube and place it in a 37°C water bath for 2 min.

Reaction: Add 0.1mL sample enzyme solution and shake evenly.

Measurement: Use a spectrophotometer to measure the kinetics of the uniformly oscillating sample, measure the change in OD value per second within 30s at a wavelength of 420nm (the reaction rate is a uniform reaction) and calculate the enzyme activity.

Embodiment 4 Laccase Kinetic Parameters Determination

The kinetic parameters of pure enzyme were determined with different concentrations of ABTS as substrate. The final concentration range of ABTS in the 3 mL reaction system was 5-500 μM. The 3mL reaction system includes 2mL citric acid buffer (0.1M; pH4.0), 0.1mL pure enzyme solution, 0.9mL ABTS solution (according to the final concentration, take the corresponding volume of ABTS mother solution and dilute it to 0.9mL with deionized water) . The reaction system was placed in a water bath at 37°C to react for a while, and the change in OD value per second within 30 s was measured at a wavelength of 420 nm (the reaction rate was a uniform reaction). Enzyme activity was calculated according to the enzyme activity formula. by Draw a graph to get a straight line. The slope of the line is Intersection with the vertical axis Intersection with horizontal axis In this way, the kinetic parameters V _max and K _m can be obtained, and k _cat can be obtained after the protein content is measured.

Enzyme Activity Formula: Enzyme Activity

In the formula: ΔOD-the change value of absorbance OD V _total -the volume of the reaction system

n-enzyme solution dilution factor Δt-reaction time

V ₀ - the volume of the enzyme solution ε - the molar absorptivity of the substrate

The catalytic activity analysis of embodiment 5 laccase mutants

Kinetic parameters V _max , K _m , k _cat , k _cat /K _m of wild-type and mutant laccases were measured with ABTS as substrate.

Table 1 Kinetic parameters of wild type (WT) and mutant laccase

The K _m value can judge the specificity of the enzyme and the natural substrate. When the substrate is the most suitable, the affinity of the enzyme is the largest and the K _m is the smallest.

At a certain enzyme concentration, the V _max of an enzyme for a specific substrate is also a constant.

k _cat represents the number of molecules converted per enzyme molecule per second when the enzyme is saturated with the substrate. This constant is commonly known as the catalytic constant. The larger the value, the higher the catalytic efficiency of the enzyme.

k _cat /K _m is the apparent second-order rate constant formed by the reaction between the enzyme and the substrate, sometimes also called the specificity constant.

It can be calculated from Table 1 that compared with the wild-type laccase, the catalytic efficiency and specificity of the mutant L386W/G417L/D195Y have the greatest improvement. The catalytic efficiency of the mutant L386W/G417L/D195Y was 2.3 times that of the wild type, and the substrate specificity of ABTS was 3.7 times that of the wild type.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (4)

1. a mutant for bacillus pumilus CotA laccase, is characterized in that, is for its Asp of the 195th is sported Trp, Phe, Try, Ile by template respectively with the bacillus pumilus CotA laccase gene of the two sudden change of L386W/G417L.

2. the preparation method of mutant according to claim 1, comprises the steps:

(1) with early stage build double-mutant plasmid pColdII-CotA (WL) for template, adopt the method for rolling ring PCR, in introducing mutational site, overlapping region during design primer, pcr amplification plasmid pColdII-CotA (WL) is utilized to obtain the open loop recombinant vectors of the gene order containing coding CotA Laccase mutant;

(2) in PCR primer, add DMT enzymic digestion PCR primer, then carry out 1% agarose gel electrophoresis and reclaim fragment;

(3) utilize special recombinase and homologous recombination principle, by seamless spliced for open loop recombinant vectors, form ring texture;

(4) be transformed into by cyclic plasmid in DMT competent cell, slat chain conveyor picking list bacterium colony checks order, in order that extract correct mutant plasmid;

(5) by correct recombinant plasmid transformed e. coli bl21 (DE3), abduction delivering.

3. the method according to right 2, is characterized in that, also comprises further and uses AVANT protein purification instrument and HisTrapHP1mL nickel post, desalting column, anion-exchange column to carry out purifying to laccase.

4. the method according to right 2, is characterized in that, recombinant bacterium step (5) obtained 37 DEG C of liquid culture in the LB substratum containing penbritin are spent the night, and the LB fermentation broth 37 DEG C that rear access contains penbritin is cultured to OD ₆₀₀≈ 0.5, be cooled to rapidly 15 DEG C and leave standstill at least 30min, the inductor IPTG adding ultimate density 0.3mM induces, and during 26h, centrifugal acquisition precipitation, resuspended with damping fluid, is crude enzyme liquid.Complex mutation body L386W/G417L/D195Y joins nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) to substrate 2,2-and has higher catalytic activity, be 2.3 times of wild-type, and specificity is 3.7 times of wild-type.