CN105200020A

CN105200020A - High-substrate-specificity Bacillus pumilus CotA laccase modified with composite point mutation

Info

Publication number: CN105200020A
Application number: CN201510717404.7A
Authority: CN
Inventors: 管政兵; 陈宇; 廖祥儒; 蔡宇杰; 廖元元
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2015-10-29
Filing date: 2015-10-29
Publication date: 2015-12-30
Anticipated expiration: 2035-10-29
Also published as: CN105200020B

Abstract

The invention discloses a Bacillus pumilus CotA laccase mutant with improved substrate specificity and belongs to the field of genetic engineering and enzyme engineering. A single mutant L386W CotA laccase gene constructed in the early stage in a laboratory is taken as a template, Gly at the 417th site of a single mutant is further mutated to Leu, then a wild type CotA laccase is taken as a control, a composite mutant L386W/G417L is discovered to have higher specificity on a 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 kind of high substrate specificity bacillus pumilus CotA laccase by spot sudden change transformation

Technical field

The present invention relates to the bacillus pumilus CotA Laccase mutant that a kind of substrate specificity improves, belong to genetically engineered and enzyme engineering field.

Background technology

Laccase (laccase, E.C.1.10.3.2) is a kind of cupric polyphenoloxidase, can the redox reaction of phenol substance catalytic, plays a significant role in the biological degradation of xylogen and precursor analogue thereof.The oxidation substrates of laccase is very extensive, and comprise phenols and derivative, arylamine and derivative thereof, aromatic carboxylic acid and derivative thereof etc., therefore laccase application potential is huge.In wood processing field, laccase can replace chemical glue mixture, not only can improve the quality of products, and can alleviate the injury to HUMAN HEALTH and the pollution to environment; In paper industry, laccase is used for paper bio-bleaching and slurrying, can reduce the pollution of pulp and paper making, contribute to paper-making industry and finally realize cleaner production; At food processing field, laccase can be used for removing the muddiness that in fruit juice, phenolic compound causes, thus improves the quality of fruit juice.In addition, laccase is oxidable chlorophenol and derivative thereof also, reduces its toxicity, and reducing with chlorophenols is the environmental pollution that dyestuff produced by industrial raw material, sanitas, weedicide, agrochemical Chemicals cause.

Laccase by sources difference can be divided into three major types: plant laccase, fungal laccase and bacterial laccase.Bacterial laccase comprises the EpoA albumen etc. of the CotA albumen of Bacillus, the PpoA albumen of extra large Zymomonas mobilis, colibacillary CueO albumen, streptomyces griseus, with fungi and plant laccase protein structural similitude, all has 4 cupric ion binding sites.

This laboratory has in earlier stage been cloned and has been recombinated and have expressed CotA laccase from the bacillus pumilus bacterial strain W3 (BacilluspumilusW3) from row filter, research finds that this CotA laccase possesses following advantage relative to other kind laccase: high, the high temperature resistant and Heat stability is good of enzymic activity under alkaline pH, can tolerate high levels of organic solvents and halide-ions environment etc., and these good characteristics just current laccase carry out in treatment of dyeing wastewater field that industrial applications is badly in need of.But the natural expression amount of wild bacillus pumilus CotA laccase is very low, and Substratspezifitaet is poor, and enzymatic activity is on the low side, becomes the bottleneck of industrial applications.Therefore the present invention utilizes genetically engineered and enzyme engineering means, improves bacillus pumilus CotA laccase to the specificity of specific substrates, improves its industrial applications prospect further.

Summary of the invention

The invention provides a kind of bacillus pumilus Laccase mutant, this mutant has transformed the single mutant L386W laccase gene of acquisition for template with this laboratory early stage, further by the 417th of single mutant the glycine (Gly, G) leucine (Leu is sported, L), subsequently with wild-type CotA laccase for contrast, checking double-mutant joins nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) to substrate 2,2-and whether has higher specificity.

B.pumilusCotA laccase consensus amino acid sequence (being submitted to by this laboratory, GenBank accession number: KF040050) in the parent amino acid sequence of described B.pumilusCotA laccase and ncbi database.

Described mutant is that the Gly of the 417th in single mutant L386W laccase gene has been mutated into Leu, called after L386W/G417L.

Accompanying drawing explanation

Fig. 1: wild bacillus pumilus CotA laccase three-dimensional simulation structure

Fig. 2: the molecule manipulation principle schematic building mutant plasmid process

Embodiment

Term used in the present invention, unless otherwise specified, generally has the implication that those of ordinary skill in the art understand usually.

Below in conjunction with concrete preparation embodiment and Application Example, and comparable data describes the present invention in further detail.Should be understood that these embodiments just in order to demonstrate the invention, but not limit the scope of the invention by any way.

Below in an example, the various process do not described in detail and method are ordinary methods as known in the art.

The expression and purification of embodiment 1 wild bacillus pumilus CotA laccase.

From glycerine pipe preseed stage build recombinant strains CotA/pColdII/BL21 (DE3) in LB liquid nutrient medium (containing 100mg/L penbritin) incubated overnight, by 2% inoculum size, seed is accessed LB liquid fermentation medium (containing 100mg/L penbritin).After intestinal bacteria cultivate 2h at 37 DEG C, the IPTG adding 0.4mM final concentration induces, and after 15 DEG C of shaking tables continue fermentation culture 24h, by fermented liquid in 4 DEG C, 8000rpm centrifugal 10min removal supernatant, collects thalline.By resuspended for the thalline phosphate buffered saline buffer collected, bacterial cell disruption is discharged intracellular protein by resuspended rear ultrasonic cell disruption instrument, after fragmentation completes, by the liquid of fragmentation in 4 DEG C, 8000rpm centrifugal 10min collection supernatant.The supernatant collected is used for CotA laccase protein purifying.

Because recombinant expressed CotA laccase protein is with polyhistidine label (His ₆.tag), nickel ion affinity chromatograph method separate targets albumen is therefore used.Nickel ion affinity chromatograph purification step: (1) balances: balance HisTrapHP nickel ion post (1mL) with the 20mM damping fluid (imidazoles containing 5mM) of 10 times of column volumes; (2) loading: the sample of anticipating is with the flow velocity loading of 1mL/min; (3) wash-out: carry out gradient elution with high density imidazoles, the pipe number that under collection elution requirement, peak type is corresponding, and do Enzyme activity assay.The wild-type CotA laccase that final acquisition purifying is good.

Embodiment 2CotA Laccase mutant builds and prepares

(1) rite-directed mutagenesis

The glycine (Gly) of the 417th in laccase, for template, is mutated into leucine (Leu), called after L386W/G417L by the B.pumilusCotA laccase gene sequence of single mutant L386W successfully constructed with early stage.

The rite-directed mutagenesis primer introducing L386W sudden change is:

Forward primer 5 '-TATGGGCGCCCTATTT gGtTACTAGATAAC-3 ' (underscore is mutational site)

Reverse primer 5 '- cCaAATAGGGCGCCCATATTTATCTTGTGT-3 ' (underscore is mutational site)

The rite-directed mutagenesis primer introducing G417L sudden change is:

Forward primer 5 '-CGTTAACCCAACAAGG cTcACACACCCTAT-3 ' (underscore is mutational site)

Reverse primer 5 '- aGcCTTGTTGGGTTAACGATGTTCCATACC-3 ' (underscore is mutational site)

Utilize above-mentioned primer, with single mutant plasmid pColdII-CotA (W) for template, carry out PCR reaction.Reaction is all carried out in 50 μ L systems, and reaction conditions is: 95 DEG C of denaturation 3min, carries out 25 circulations (95 DEG C of 20s, 57 DEG C of 20s, 72 DEG C of 6min) subsequently, circulates latter 72 DEG C and extends 7min, last 4 DEG C of insulations.Get 10 μ LPCR products to detect through 1% agarose gel electrophoresis.Add 1 μ LDMT enzyme after object product having been detected in remaining PCR primer, mixing, hatches 1 hour for 37 DEG C.All be separated hatching the product after process, cut glue through 1% agarose gel electrophoresis, reclaim test kit with gel and reclaim object fragment.The object fragment reclaimed is transformed into DMT competent cell after mutant fragments assembling, the LB that converted product is coated containing 100mg/L penbritin is dull and stereotyped, through 37 DEG C of incubated overnight, choose 10 single bacterium colonies from flat board and carry out bacterium colony PCR checking, 3 single bacterium colony access LB liquid nutrient mediums are chosen from the bacterium colony be proved to be successful, be saved in two glycerine pipes by each bacterium liquid after 10h, a-20 DEG C of preservations, a being used for checks order.The correct mutant of order-checking is inoculated into incubated overnight LB liquid nutrient medium from glycerine pipe, after spending the night, first preserves glycerine pipe, then will remain bacterium liquid extracting plasmid and transform access BL21 (DE3) competent cell.

(2) expression and purification of mutant enzyme

Mutant Expression and purification process as described in Example 1.

Embodiment 3CotA Laccase mutant enzyme is lived and is analyzed.

(1) enzyme unit definition alive

When adopting ABTS method to measure laccase activity, enzyme amount required when definition per minute transforms 1 μm of ol substrate is as a unit of activity.

(2) enzyme activity determination step

Preheating: get the citrate buffer solution of 2.4mLpH4.0 in test tube, adds 0.5mLABTS solution (final concentration of ABTS is 0.5mM) and is placed in 37 DEG C of water-bath preheating 2min in test tube.

Reaction: add 0.1mL sample enzyme liquid, concussion evenly.

Measure: uniform for concussion sample spectrophotometer is carried out kinetic measurement, under 420nm wavelength, measure the variable quantity (speed of reaction is at the uniform velocity reaction) of OD value p.s. in 30s and calculate enzyme activity.

Embodiment 4 laccase Determination of Kinetic Parameters

The kinetic parameter of pure enzyme is measured using the ABTS of different concns as substrate.In the reaction system of 3mL, the final concentration scope of ABTS is 5-500 μM.The reaction system of 3mL comprises 2mL citrate buffer solution (0.1M; PH4.0), the ABTS solution (being diluted to 0.9mL with deionized water get the ABTS mother liquor of respective volume according to final concentration after) of the pure enzyme liquid of 0.1mL, 0.9mL.Reaction system is placed in 37 DEG C of water-baths reaction a moment, under 420nm wavelength, measures the variable quantity (speed of reaction is at the uniform velocity reaction) of OD value p.s. in 30s.Live according to enzyme activity formulae discovery enzyme.With mapping, draws a straight line.The slope of straight line is with the intersection point of the longitudinal axis (0, ), with the intersection point of transverse axis ( 0).Kinetic parameter V can be obtained like this _maxand K _m, after measuring protein content, just can obtain k _cat.

Enzyme activity formula: enzyme activity

In formula: the changing value V of Δ OD-absorbancy OD _alwaysthe volume of-reaction system

The n-enzyme liquid extension rate Δ t-reaction times

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

The Substratspezifitaet of embodiment 5 Laccase mutant

Take ABTS as the kinetic parameter V that substrate determines wild-type and mutant laccase _max, K _m, k _cat, k _cat/ K _m.

The kinetic parameter of table 1 wild-type (WT) and mutant laccase

K _mvalue can judge specificity and the natural substrate of enzyme, the maximum K of avidity of enzyme during suitable substrate _mminimum.

In certain enzyme concn, enzyme is to the V of specific substrates _maxalso be a constant.

K _catrepresent the molecule number of each enzyme molecule conversion p.s. substrate when enzyme is saturated by substrate, this constant is commonly referred to as catalytic constant, and the catalytic efficiency of its value larger expression enzyme is higher.

K _cat/ K _mthe apparent secondary rate constant that enzyme and substrate reactions are formed, sometimes also referred to as specificity constant.

Can calculate from table 1, mutant is compared with wild-type laccase, and specificity and the catalytic efficiency of L386W/G417L are greatly improved.The specificity of mutant L386W/G417L is 4.3 times of wild-type, and catalytic efficiency is 1.8 times of wild-type.

Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims

1. a mutant for bacillus pumilus CotA laccase, is characterized in that, is for its Gly of the 417th is sported Leu by template with the bacillus pumilus CotA laccase gene of L386W single mutation.

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

(1) with early stage build single mutant plasmid pColdII-CotA (W) for template, adopt the method for rolling ring PCR, in introducing mutational site, overlapping region during design primer, pcr amplification plasmid pColdII-CotA (W) 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 0.4mM induces, and during 24h, centrifugal acquisition precipitation, resuspended with damping fluid, is crude enzyme liquid.Complex mutation body L386W/G417L joins nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) to substrate 2,2-and has higher specificity, be 4.3 times of wild-type, and catalytic efficiency is 1.8 times of wild-type.