CN110540979B - Hydrogen peroxide tolerant lipase mutant and application thereof - Google Patents

Hydrogen peroxide tolerant lipase mutant and application thereof Download PDF

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CN110540979B
CN110540979B CN201910844697.3A CN201910844697A CN110540979B CN 110540979 B CN110540979 B CN 110540979B CN 201910844697 A CN201910844697 A CN 201910844697A CN 110540979 B CN110540979 B CN 110540979B
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hydrogen peroxide
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lipase
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CN110540979A (en
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王永华
蓝东明
李爽
赵泽鑫
杨博
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South China University of Technology SCUT
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/02—Working-up waste paper
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/02—Working-up waste paper
    • D21C5/025—De-inking
    • D21C5/027—Chemicals therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract

The invention discloses a hydrogen peroxide tolerant lipase mutant and application thereof, belonging to the technical field of biology, wherein an enzyme mutant library is designed and constructed based on enzyme protein structural analysis, a lipase mutant with enhanced hydrogen peroxide tolerance is obtained by screening, and pichia pastoris engineering bacteria for efficiently expressing the mutant are constructed, the hydrogen peroxide tolerant lipase mutant is obviously enhanced relative to the wild type hydrogen peroxide tolerant capability, the residual activity of the mutant is still 78 percent and is about 4 times of that of the wild type after the mutant is treated in 1M hydrogen peroxide for 24 hours, the mutant can be applied in the process of waste paper deinking, has good stability, can prolong the service life in the application process, save the production cost and improve the production efficiency.

Description

Hydrogen peroxide tolerant lipase mutant and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a hydrogen peroxide tolerant lipase mutant and application thereof.
Background
Lipases, i.e., triacylglycerol acylhydrolases, catalyze the hydrolysis of natural substrate lipids to produce fatty acids, glycerol, and mono-or diglycerides. The basic building block of lipases is only amino acids, usually only one polypeptide chain. Its catalytic activity is determined solely by its protein structure (Schmid et al, 1998). The lipase has wide application value and has become the third industrial enzyme in the market. The lipase can catalyze reactions such as lipolysis, ester exchange, ester synthesis and the like, and is widely applied to industries such as feed additives, oil processing, foods, medicines, daily chemicals and the like.
The lipase can catalyze the hydrolysis of ester bonds in a water environment, and can catalyze the reactions such as ester exchange, esterification, alcoholysis, acidolysis and the like when being in an organic solvent environment. In the process of waste paper deinking, hydrogen peroxide is often added, but in the hydrogen peroxide environment, the activity of most enzymes is inhibited or even inactivated, so the application of the hydrogen peroxide in waste paper deinking is greatly limited. The difficulty in the actual biological catalysis process is to maintain higher lipase activity and the universality of lipase catalysis substrates in the hydrogen peroxide environment. Therefore, the development of hydrogen peroxide-tolerant lipase is of great significance in theoretical and industrial applications.
Disclosure of Invention
The invention aims to provide a hydrogen peroxide tolerant lipase mutant and application thereof, which are used for solving the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a hydrogen peroxide tolerant lipase mutant, which is characterized in that the 233 th amino acid of a lipase with an amino acid sequence of SEQ ID No.1 is changed from Val to Arg.
The amino acid sequence of the hydrogen peroxide tolerant lipase mutant is SEQ ID No. 2.
The nucleic acid sequence of the encoding gene of the hydrogen peroxide tolerant lipase mutant is SEQ ID NO. 3.
The invention also provides a recombinant plasmid for expressing the hydrogen peroxide tolerant lipase mutant with the amino acid sequence of SEQ ID NO.2 in the host cell.
Further, the recombinant plasmid contains a nucleic acid with the sequence of SEQ ID NO. 3.
The invention also provides an engineering strain, which contains recombinant plasmid of hydrogen peroxide tolerant lipase mutant with the amino acid sequence of SEQ ID NO.2 or recombinant plasmid with the nucleic acid sequence of SEQ ID NO. 3.
Further, the engineering strain is Pichia pastoris.
The invention also provides the hydrogen peroxide tolerant lipase mutant, the recombinant plasmid and the application of the engineering strain in the paper-making deinking process.
The invention discloses the following technical effects:
the lipase mutant with the enhanced hydrogen peroxide tolerance is obviously enhanced relative to the wild hydrogen peroxide tolerance based on the pichia pastoris engineering bacteria for efficiently expressing the mutant, the residual activity of the lipase mutant with the enhanced hydrogen peroxide tolerance is still 78 percent and is about 4 times of that of the wild type after being treated in 1M hydrogen peroxide for 24 hours, and the mutant can be applied to the processing process of waste paper deinking, has good stability, can prolong the service life in the application process, saves the production cost and improves the production efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a comparison graph of enzyme activity of MAS1 lipase and its mutant in hydrogen peroxide treatment process.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
EXAMPLE 1 construction of lipase mutants
And analyzing amino acids in the enzyme structure, which are contacted with hydrogen peroxide, wherein the amino acids have high interaction possibility with the hydrogen peroxide and can cause the structure change of the amino acids, so that the mutants N45Y, V233R, V202R and G40R/D are preferentially selected and designed.
Design of Lipase mutant primers Positive and reverse Primer sequences of the mutant were obtained using the website Primer X (http:// www.bioinformatics.org/Primer/cgi-bin/protein-3. cgi). Mutant primers were synthesized by Biotech. Construction of the mutants was accomplished by Site-directed Mutagenesis (Quik Change Site-directed Mutagenesis). Firstly, extracting plasmids from lipase wild type escherichia coli, taking the wild type plasmids as template plasmids, and carrying out PCR amplification by using mutant primers to obtain PCR amplification products, namely full-length plasmids of the mutants. The methylated template plasmid was then digested with Dpn I restriction enzyme. Finally, the PCR amplification product is transferred into an escherichia coli competent cell Top10 to obtain an enzyme gene mutant.
(1) The reaction system for PCR amplification of mutant plasmids is shown in Table 1:
TABLE 1
Figure DEST_PATH_IMAGE001
(2) PCR amplification procedure, as shown in Table 2
Figure DEST_PATH_IMAGE002
The PCR amplification product needs to be detected by nucleic acid electrophoresis to determine the full-length plasmid of the successfully amplified mutant.
(3) The cleavage system of the template plasmid is shown in Table 3:
TABLE 3
Figure DEST_PATH_IMAGE003
Reaction conditions are as follows: the mixture was incubated at 37 ℃ for 4 hours in a PCR apparatus.
The resulting enzyme-cleaved product was purified using a PCR product purification kit.
(4) Transformation of mutant plasmid into E.coli Top10
1. Coli competent cell Top10 (100. mu.L/tube) stored in a-80 ℃ refrigerator was taken out, and placed on ice to melt, 10. mu.L of the purified product was added, mixed well, and then left on ice for 30 min.
2. The resulting mixture was heat-shocked at 42 ℃ for 90 s and then rapidly moved to ice for cooling for 3 min.
3. In a sterile environment, the Escherichia coli containing the mutant plasmid obtained in the previous step is added into 1 mL LB liquid medium and mixed evenly, and is placed at 37 ℃ and cultured for 50 min in a shaking table at 200 rpm.
Centrifuging at 4.9000 rpm for 2 min, removing most supernatant, re-suspending thallus with 60-80 μ L of the remaining liquid, spreading on Amp-resistant LB solid plate, and culturing overnight at 37 deg.C in dark
5. Under sterile conditions, single colonies were picked from the plates and inoculated into 5 mL of LB liquid medium containing Amp resistance, and left to incubate overnight at 37 ℃. mu.L of the bacterial liquid is added to 500 mu.L of the 50% glycerol strain preservation liquid and preserved at the temperature of minus 20 ℃. And simultaneously extracting plasmids and carrying out sequence determination.
Example 2 Lipase mutant expression and purification
(1) Expression of target enzyme protein
The recombinant plasmid pET22b-MAS1 containing MAS1 lipase (amino acid sequence is SEQ ID NO: 1) and the mutant gene expression vector are transformed into an expression strain BL21 (DE 3), an Amp-resistant LB plate is coated, and a single clone grows out overnight. Single colonies were picked from the plate and cultured overnight in 5 mL LB medium for 12 h each time, and inoculated in 500 mL LB medium at an inoculum size of 1%. When the OD600 of the bacterial liquid is about 0.8, the bacterial liquid is cooled to 20 ℃ and induced by 2 mM IPTG with the final concentration for 20 h. After induction expression, the thalli is collected by using a refrigerated centrifuge to centrifuge (10000 r/min, 15 min) fermentation liquor, the thalli is resuspended by using 50 mM phosphate buffer solution (pH 7.4), and after ultrasonication, the thalli is centrifuged (10000 r/min, 25 min) to collect supernatant fluid to detect whether the target protein is expressed or not by SDS-PAGE electrophoresis.
(2) Purification of target enzyme protein
1. First, the nickel column was equilibrated with 20 mM PBS buffer (pH 7.4) to remove the ethanol stock solution contained in the column, and the flow rate was set to 4 mL/min, and the equilibration process was completed when the absorbance at A280, the salt ion concentration, and other parameters showed equilibration.
2. The concentrated protein solution was then pumped to a nickel column at a flow rate of 3 mL/min, and after loading was complete, the column was flushed with 20 mM PBS buffer (pH 7.4) until the absorbance curve was shown and the salt ion concentration curve appeared to be a horizontal line.
3. Then, gradient elution was started with 20 mM PBS buffer (0-500 mM imidazole, pH 7.4) containing different concentrations of imidazole, and samples of the eluate were collected in tubes and stored at 4 ℃.
4. And (4) carrying out protein electrophoresis determination on the penetrating fluid sample and the eluent sample, and storing the purified target protein sample according to an electrophoresis result.
Example 3 screening of Hydrogen peroxide tolerant lipase mutants
Example 3 the enzyme activities of the lipase MAS1 and its mutants were reflected by measuring the consumption of the substrate MCD. It is known that the substrate MCD has a maximum absorbance at a wavelength of 290 nm, whereas the MCD oxidized has no absorbance at this wavelength, and thus the enzyme activity can be measured therefrom. The enzyme activity is defined as the amount of enzyme required to oxidize 1. mu. mol MCD per minute under certain conditions, i.e., one unit of enzyme activity, expressed as U.
1. Determination of enzyme Activity
80 μ L of peroxy acid determination working solution and 10 μ L of 30% H were sequentially added2O2And 10 mu L of purified enzyme solution is added into a 96-hole enzyme label plate, the plate is placed into an enzyme label instrument to react for 5 min at 40 ℃, and the change of the light absorption value is measured under the wavelength of 290 nm. In the control experiment, the enzyme solution was replaced with 20 mM phosphate buffer, and the reaction conditions were otherwise the same. The experiment was repeated three more times.
MCD solutions with different concentrations are prepared, so that the concentrations of the MCD solutions in the peroxy acid determination working solution are 0, 36, 72, 108, 144 and 180 mu M respectively. H in the reaction system2O2The reaction conditions were the same except that ultrapure water was used instead. And (4) taking the MCD content in the system as an abscissa and the measured light absorption value as an ordinate to prepare a standard curve. The change in absorbance measured after the reaction can be further converted into enzyme activity by referring to a standard curve.
2. Tolerance to H2O2Determination of Capacity
Subjecting the purified enzyme solution to different H2O2The incubation was carried out at concentrations (5 mM, 50 mM, 500 mM and 1M) at room temperature, and the enzyme solutions were removed at different times for detection of peroxidase activity. Concrete implementThe test process is as follows: 80 mu L of 0.1M valeric acid as organic acid working solution, 10 mu L of 100 mM valeric acid as organic acid working solution and 10 mu L of enzyme solution are sequentially added into a 96-hole enzyme label plate, the reaction is carried out for 5 min, the light absorption value is measured at 290 nm, and the experiment is repeated for more than three times. To pass through H2O2The residual enzyme activity of the incubated enzyme solution was 100% as a reference.
The results are shown in FIG. 1, the designed lipase mutants have improved hydrogen peroxide tolerance to different degrees, wherein the residual enzyme activity of V233R (amino acid sequence SEQ ID NO: 2) is still 78% after incubation in 1M hydrogen peroxide solution for 24 hours, which is about 4 times that of the wild type.
Example 4 high Density fermentation preparation of Hydrogen peroxide tolerant Lipase mutants
Firstly, cloning MAS1-V233R gene (nucleotide sequence SEQ ID NO: 3) into a Pichia pastoris expression vector pPICZ alpha A vector to obtain the pPICZ alpha A-MAS1-V233R expression vector, electrically transforming the expression vector into a Pichia pastoris X-33 strain, and screening by using bleomycin to obtain a positive recombinant expression strain. Culturing the recombinant bacteria in a fermentation tank, and performing induction culture for 8 days under the conditions of 24 ℃ of temperature and 6.0 of pH value. After the fermentation process is finished, centrifuging to obtain a fermentation broth supernatant, concentrating by using a membrane package, measuring the hydrolase activity and the protein concentration of the fermentation broth, and storing in a refrigerator at 4 ℃.
Example 5 application of Hydrogen peroxide tolerant lipase mutants in deinking waste paper
The waste newspapers were manually shredded into pieces of about 3cm by 3cm size and then processed in a horizontal hydropulper. Specifically, the quality of waste paper is adjusted to 3% by deionized water, and is pulped for 8 min at 55 ℃, and then is wrung out and put into a refrigerator for standby. The appropriate amount of water was poured into a hydropulper, a wet pulp containing 140 g of oven dried pulp was added and the pH was adjusted with HCl or NaOH. Adding a certain amount of enzyme preparation or 3% hydrogen peroxide simultaneously, and performing enzyme deinking treatment at different times. Tap water was then added to adjust the pulp concentration of the enzyme deinked pulp to 0.5%, the pulp was uniformly dispersed by premixing in a flotation tank, and a sheet for paper whiteness determination was prepared using distilled water and a buchner funnel. The tests were grouped into 5 groups, as shown in table 3, group a: wild-type MAS1 lipase; group B MAS 1-mutants; group C: wild-type MAS1 lipase + hydrogen peroxide; group D: group B MAS 1-mutant + hydrogen peroxide, group F: hydrogen peroxide. The specific reaction conditions are as follows: the enzyme dosage is 20 U.g-1 (relative to oven dry pulp), the pH value of the treatment is 7.0-7.5, the temperature is 50-55 ℃, and the hydrogen peroxide is 3% (relative to the treatment volume).
TABLE 3 comparison of deinking efficiency of wastepaper from different treatment modes (whiteness/% ISO)
Figure DEST_PATH_IMAGE004
As can be seen from Table 3, after the lipase and the hydrogen peroxide are combined for 5 minutes, the whiteness can reach 72 percent, which is better than the effect of single treatment by the lipase and the hydrogen peroxide. The whiteness of the sample is not improved any more after the sample is treated for 10 minutes by combining MAS1 lipase with hydrogen peroxide, which indicates that the biological enzyme is influenced by the hydrogen peroxide, so that the activity is inhibited. The activity of the MAS1 lipase mutant is affected after the combination with hydrogen peroxide for 20 minutes, and the final whiteness of the sample reaches 74.56 percent, which is better than that of other combinations.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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<120> hydrogen peroxide-tolerant lipase mutant and application thereof
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tga 783

Claims (5)

1. A hydrogen peroxide tolerant lipase mutant is characterized in that the 233 th amino acid of the lipase with the amino acid sequence of SEQ ID No.1 is changed from Val to Arg.
2. A recombinant plasmid for expressing the hydrogen peroxide-tolerant lipase mutant according to claim 1 in a host cell.
3. An engineered strain comprising the recombinant plasmid of claim 2.
4. The engineered strain of claim 3, wherein the engineered strain is Pichia pastoris.
5. The hydrogen peroxide tolerant lipase mutant as defined in claim 1, the recombinant plasmid as defined in claim 2, or the engineered strain as defined in claim 3 or 4, for use in paper deinking.
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