CN111575265A - Keratinase mutant with improved thermal stability - Google Patents
Keratinase mutant with improved thermal stability Download PDFInfo
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- CN111575265A CN111575265A CN202010439769.9A CN202010439769A CN111575265A CN 111575265 A CN111575265 A CN 111575265A CN 202010439769 A CN202010439769 A CN 202010439769A CN 111575265 A CN111575265 A CN 111575265A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
Abstract
The invention discloses a keratinase mutant with improved thermal stability, belonging to the technical field of genetic engineering and enzyme engineering. The invention adopts whole plasmid PCR to carry out site-directed saturation mutagenesis on the plasmid carrying the keratinase gene, constructs and screens the plasmid to obtain a heat-stable mutant recombinant bacterium, and obtains keratinase mutants T78C and T78E. Results of the thermostability studies showed that the half-lives of the keratinase mutants T78C and T78E at 60 ℃ were 2.25-fold and 2.1-fold that of the parent enzyme.
Description
Technical Field
The invention relates to a keratinase mutant with improved thermal stability, belonging to the technical field of genetic engineering and engineering.
Background
Keratinase is a specific keratinase capable of degrading keratin substrates (e.g., feather, wool, cow horn, etc.) and is produced by various microorganisms such as fungi, actinomycetes, and bacteria. The keratinase has wider substrate specificity and strong hydrolysis catalytic ability, is widely applied to the animal husbandry industry, the feed industry, the tanning industry and the medicine industry, and has great research and application values.
However, wild keratinase is generally poor in enzyme activity and thermostability. The requirement on the enzyme in industrial application is severe, for example, the enzyme activity of the keratinase can be greatly influenced in a high-temperature environment. In practice, heating is usually required to accelerate the reaction, which increases energy consumption, and the spray drying process for preparing the keratinase powder also requires heating. Therefore, thermostable keratinase is very important in practical applications. At present, methods for improving the thermal stability of enzymes mainly include immobilization, chemical modification, additive supplementation, post-translational modification of enzymes, protein engineering and the like. While the first two strategies can improve the thermostability of the enzyme, additional processing is required to treat the enzyme, increasing production costs. Therefore, the method of applying protein engineering to improve the thermal stability of enzyme from molecular level is a new research method.
With the promotion of green industry, the realization of keratinase industrialization is urgently needed, but at present, the thermal stability of the keratinase generally still does not meet the requirement of commercial application. The keratinase activity of the keratinase recombinant strain with high keratinase activity is higher than 426.60KU/mL (the keratinase is described in the Biotransformation of keratin water to amino acids and active peptide base on cell-free catalysis literature), and the keratinase recombinant strain is the highest level of recombinant keratinase expression reported in the literature at present. However, since this keratinase is not highly thermostable, it is necessary to further improve the thermostability of the keratinase in order to further improve the application performance of the enzyme.
Disclosure of Invention
In order to solve the technical problems, the invention provides a keratinase mutant, and a site-directed saturation mutation strategy is adopted to perform mutation transformation on the amino acid site of a parent enzyme so as to improve the thermal stability of keratinase.
The invention provides a keratinase mutant, which takes a keratinase with an amino acid sequence shown as SEQ ID NO.2 as a parent and carries out mutation on the 78 th site of the parent keratinase.
In one embodiment of the invention, the 78 th threonine of the parent keratinase is mutated to cysteine to obtain mutant T78C, wherein the amino acid sequence of the mutant T78C is shown in SEQ ID NO. 3.
In one embodiment of the invention, the 78 th threonine of the parent keratinase is mutated to glutamic acid to obtain mutant T78E, wherein the amino acid sequence of the mutant T78E is shown in SEQ ID NO. 4.
The invention also provides a gene for coding the keratinase mutant.
The invention also provides a recombinant plasmid carrying the gene encoding the mutant enzyme.
In one embodiment of the present invention, the vector of the recombinant plasmid is a pP43NMK plasmid.
The invention also provides a host cell carrying the gene encoding the mutant enzyme or the recombinant plasmid.
In one embodiment of the invention, the host cell is a bacterium or a fungus.
In one embodiment of the invention, the host cell is Bacillus subtilis WB 600.
In one embodiment of the present invention, the recombinant cell is constructed by transferring a recombinant expression vector carrying a gene encoding the mutant enzyme into a host cell by electroporation or chemical transformation.
The invention provides a method for degrading keratinase, which takes chicken feathers as substrates, and adds mutant T78C and/or T78E for reaction.
In one embodiment of the invention, the method is to take keratin-containing substances as substrates, add purified mutant T78C and/or T78E, and react for 4h at 37 ℃.
In one embodiment of the invention, the keratin-containing material comprises fur, elastin, scales, fibers.
In one embodiment of the invention, the keratin-containing material comprises skin, casein, hair, nails, feathers.
The invention provides an application of the mutant, the gene, the expression vector or the host cell in animal husbandry, feed industry, leather industry and medical industry.
The invention also claims the use of said mutant, gene, expression vector or host cell for degrading skin, hair, casein, elastin, hair, nails, scales, fibers, hair keratin.
The invention has the beneficial effects that: the invention adopts whole plasmid PCR construction to obtain keratinase mutants T78C and T78E, and the results of thermal stability researches show that the half-life periods of the keratinase mutants T78C and T78E at 60 ℃ are 2.25 times and 2.1 times of that of parent enzymes, so that the invention has more application value and potential.
Drawings
FIG. 1 is a SDS-PAGE gel of Bacillus subtilis supernatants from keratinase mutants T78C, T78E; where M represents the protein molecular weight standard, the different lanes represent the different muteins, and the arrow indicates the position of the protein band of interest.
FIG. 2 shows the comparison of the thermal stability at 60 ℃ of different mutants with that of a control strain.
FIG. 3 is a graph of mutant degraded feathers; a is a reaction system of 0 h; b is the reaction system after 4 h.
Detailed Description
Coli JM109 referred to in the examples below was purchased from North Naphthora; the pP43NMK plasmids referred to in the examples below were purchased from a haloghic organism; bacillus subtilis WB600 mentioned in the examples below is described in patent application publication No. CN 102492645A.
The culture medium involved in the first embodiment is:
LB liquid medium: yeast powder 5.0 g.L-1Tryptone 10.0 g.L-1、NaCl 10.0g·L-1。
LB solid medium: yeast powder 5.0 g.L-1Tryptone 10.0 g.L-1、NaCl 10.0g·L-115g/L of agar powder.
Fermentation medium: peptone 20 g.L-1Yeast powder 10 g.L-120 g.L of sucrose-1、KH2PO43g·L-1、Na2HPO46g·L-1、MgSO40.3g·L-1。
The detection method involved in the second embodiment is as follows:
measurement of the enzyme activity of keratinase: taking 50 μ L of the fermentation supernatant diluted properly, adding 150 μ L of 50mM Gly/NaOH solution as buffer and 100 μ L of 2.5% water-soluble keratin (purchased from Taishiai (Shanghai) chemical industry development Co., Ltd., product code: K0043) as substrate, mixing, and reacting at 40 deg.C for 20 min; the reaction was stopped by adding 200. mu.L of 4% (w/v) trichloroacetic acid (TCA) and centrifuged at 8000r/min at room temperature for 3 min. The supernatant was taken to 200. mu.L, and 1mL of 4% (w/v) Na was added2CO3Mixing with 200 μ L of Folin phenol reagent, mixing, developing at 50 deg.C for 10min, and measuring clear solution light absorption value at 660nm with 0.5cm quartz cuvette; 3 experimental groups are paralleled, the blank control is that the reaction terminator TCA is added before the substrate is added, and the rest operations are the same as above;
definition of enzyme activity: OD under this condition6600.001 per literThe enzyme demand is one enzyme activity unit (1U).
And (3) purifying the enzyme: and purifying the recombinant protein by adopting an AKTAavant protein purifier. Because different keratinase mutants contain histidine tags, the method can be used for separation and purification by a nickel ion affinity chromatography purification column, and comprises the following specific steps: (1) balancing: equilibrating the column with 5 volumes of 20mmol/LpH 7.4.4 Tris-HCl buffer; (2) loading: loading the pretreated sample at a flow rate of 0.5ml/min, wherein the loading volume is generally not more than 5 times of the column volume; (3) and (3) elution: comprises eluting unadsorbed substances, hybrid proteins and target proteins at the flow rate of 2.0ml/min, carrying out gradient elution on an eluent which is 20mmol/LpH 7.2.2 Tris-HCl buffer solution containing 50mM imidazole, wherein the detection wavelength is 280nm, and collecting the eluent containing keratinase activity in batches; only one target protein elution peak appears in the elution process, and subsequent enzyme activity measurement and SDS-PAGE protein electrophoresis find that the enzyme solution collected at the peak top is the purest part no matter the original enzyme or the mutant enzyme.
Example 1: construction of site-directed mutants of keratinase
In this study, the amino acid sites (alanine at position 1, threonine at position 78, glycine at position 131, serine at position 159, asparagine at position 240, and glutamine at position 275) of keratinase having the amino acid sequence of SEQ ID NO.2 were selected for mutation transformation studies.
According to the sequence of keratinase (the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2), mutation primers are respectively designed, and the site-directed saturation mutation is carried out on the plasmid pP43NMK carrying the keratinase gene.
Wherein, the primers used for the alanine at the 1 st position are as follows:
a forward primer: 5 '-CCATGCCTTGNNKCAAACCGTTCCTTACGGCATTCC-3' (SEQ ID NO. 5);
reverse primer: 5 '-CGGTTTGMNNCAAGGCATGGGCCACATGATC-3' (SEQ ID NO. 6);
the primers for threonine 78 were as follows:
a forward primer: 5'-GCTTGACAATNNKACGGGTGTATTAGGCGTTGC-3' (SEQ ID NO. 7);
reverse primer: 5'-CACCCGTMNNATTGTCAAGCGCAGCTACTGTAC-3' (SEQ ID NO. 8);
the primers for the 131 th glycine were as follows:
a forward primer: 5'-GGGAGCATCANNKTCGACAGCGATGAAACAGGCAG-3' (SEQ ID NO. 9);
reverse primer: 5'-CTGTCGAMNNTGATGCTCCCCCAAGGCTCAT-3' (SEQ ID NO. 10);
the primers used for serine 159 were as follows:
a forward primer: 5'-CAGCGGATCTNNKGGAAACACGAATACAATTGGCTATCCTG-3' (SEQ ID NO. 11);
reverse primer: 5'-TGTTTCCMNNAGATCCGCTGTTCCCTGCT-3' (SEQ ID NO. 12);
the primers used for asparagine at position 240 are as follows:
a forward primer: 5'-AAAACATCCGNNKCTTTCAGCTTCACAAGTCCGCAAC-3' (SEQ ID NO. 13);
reverse primer: 5'-CTGAAAGMNNCGGATGTTTTGACAAGATCAAAGCTG-3' (SEQ ID NO. 14);
the primers used for glutamine 275 were as follows:
a forward primer: 5'-AGCTGCCGCTNNKTAATGATGAAAGCTTGGCGTAATCATGGT-3' (SEQ ID NO. 15);
reverse primer: 5'-ATCATTAMNNAGCGGCAGCTTCGACAT-3' (SEQ ID NO. 16).
The PCR reaction systems are as follows: PrimeSTAR Max Premix (2X) 25. mu.L, 2.5mM dNTPs 4. mu.L, 10. mu.M forward primer 1. mu.L, 10. mu.M reverse primer 1. mu.L, template DNA 1. mu.L, 2.5U/. mu.LPrime STARTaqHS 0.5. mu.L, double distilled water was added to 50. mu.L;
the PCR product amplification conditions were all: pre-denaturation at 98 ℃ for 3 min; then carrying out 30 cycles of 10s at 98 ℃, 5s at 55 ℃ and 2min at 72 ℃; finally, keeping the temperature at 72 ℃ for 10 min;
detecting the PCR amplification product by using 1% agarose gel electrophoresis, after the detection is finished, adding 0.5 mu L of methylated template digestive enzyme (DpnI) into 10 mu L of the amplification product, blowing and sucking a gun head to uniformly mix, reacting for 1.5h at 37 ℃, converting the amplification product processed by the Dpn I into escherichia coli JM109, coating the conversion product on an LB solid culture medium, culturing for 8-10 h at 37 ℃, transferring single colonies on the LB solid to a bacteria shaking tube by using an LB liquid culture medium, culturing for 2-3 h at 37 ℃ and 220rpm, extracting plasmids, and sending to a company for sequencing to obtain the recombinant plasmid containing the mutant gene.
Example 2: construction of recombinant bacterium
Transforming recombinant plasmids into Bacillus subtilis WB600, coating a transformed product on an LB solid culture medium, culturing at 37 ℃ for 8h, inoculating a large number of obtained single colonies into a 96 deep-hole plate containing a fermentation culture medium, and culturing at 37 ℃ and 220rpm for 24h by taking an original strain as a control to obtain a fermentation liquid;
the fermentation broth was centrifuged at 4000rpm for 10min at 4 ℃ to obtain the fermentation supernatant.
And detecting the enzyme activity in the fermentation supernatant, and retaining the mutant recombinant bacteria with the enzyme activity similar to that of the original strain.
TABLE 1 enzyme Activity of fermentation supernatants of original and mutant enzymes
Example 3: thermostability of keratinase mutants
Separating a single colony from the mutant recombinant bacteria constructed and retained in the example 2 by plate streaking, selecting the single colony, inoculating the single colony into an LB liquid culture medium (100 mu g/mL kanamycin), and culturing at 37 ℃ and 220rpm for 14h to obtain a seed solution; inoculating the seed solution into a fermentation medium according to the inoculation amount of 5% (v/v), and culturing at 37 ℃ and 220rpm for 28h to obtain a fermentation solution; after the fermentation broth was centrifuged at 12000rpm for 20min at 4 ℃, the centrifuged fermentation supernatant was collected and analyzed by SDS-PAGE, and the molecular weights of T78E and T78C were both 28kDa (FIG. 1). The supernatant was purified.
Evaluation of recombinase Pre-and post-mutation half-Life t1/2In the experiment, the recombinant enzyme with poor stability is selected for detection at 60 ℃. Respectively carrying out heat treatment on the original enzyme without mutation and each mutant enzyme at 60 ℃, taking out a part every 15min to detect residual enzyme activity, and taking the enzyme activity without heat treatment as 100%. And selecting mutants with better thermal stability for sequencing to respectively obtain mutants with 78 th mutation of parent enzyme into cysteine and glutamic acid, which are respectively named as T78C and T78E.
Compared with the initial control, the T78C and T78E mutant enzymes have improved heat stability at 60 ℃, and the half-life T of T78C at 60 DEG C1/2The half-life T of T78E at 60 ℃ is increased from 20min before mutation to 45min after mutation and is 2.5 times of that of a control1/2The time is increased from 20min before mutation to 45min after mutation, which is 2.5 times of that of the control. FIG. 2 shows that the enzyme activity of the mutant enzyme T78C was 70.25KU/mL at 30min, T78E was 66.45KU/mL and WT was 36KU/mL, the enzyme activity of the mutant enzyme T78C was 41.25KU/mL at 60min, T78E was 39.80KU/mL and WT was 15.10 KU/mL.
TABLE 2 half-lives of original and mutant enzymes
Example 4: use of mutants for degrading keratin
Preparing the purified mutant T78C and/or T78E into 2000U/mL feather degradation enzyme solution, then adding 50mL feather degradation solution into a 50mL centrifuge tube containing 0.1g chicken feather, placing the reaction system at 37 ℃ and 220rpm for reacting for 4h, and observing the degradation condition of the feather. After the reaction was complete, the feathers were found to be completely degraded, as shown in figure 3.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
WUHAN INDUSTRIAL CONTROL INDUSTRIAL TECHNOLOGY INSTITUTE Co.,Ltd.
<120> keratinase mutant having improved thermostability
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<210>14
<211>36
<212>DNA
<213> Artificial sequence
<220>
<221>misc_feature
<222>(9)..(10)
<223>n is a, c, g, or t
<400>14
ctgaaagmnn cggatgtttt gacaagatca aagctg 36
<210>15
<211>42
<212>DNA
<213> Artificial sequence
<220>
<221>misc_feature
<222>(11)..(12)
<223>n is a, c, g, or t
<400>15
agctgccgct nnktaatgat gaaagcttgg cgtaatcatg gt 42
<210>16
<211>27
<212>DNA
<213> Artificial sequence
<220>
<221>misc_feature
<222>(9)..(10)
<223>n is a, c, g, or t
<400>16
atcattamnn agcggcagct tcgacat 27
Claims (10)
1. A keratinase mutant characterized in that a keratinase having an amino acid sequence shown in SEQ ID NO.2 is used as a parent, and the 78 th position of the parent is mutated.
2. The keratinase mutant according to claim 1, wherein the mutant is any one of:
(a) mutating threonine 78 to cysteine of parent keratinase;
(b) the threonine 78 th of the parent keratinase was mutated to glutamic acid.
3. A gene encoding the mutant of claim 1 or 2.
4. An expression vector carrying the gene of claim 3.
5. The expression vector according to claim 4, wherein the expression vector is pP43 NMK.
6. A host cell expressing the mutant of claim 1 or 2 or containing the gene of claim 3.
7. The host cell of claim 6, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
8. A method for degrading keratin, which comprises using the mutant according to claim 1 or 2, using a keratin-containing material as a substrate.
9. The method of claim 8, wherein the keratin-containing material comprises fur, elastin, scales, fibers.
10. Use of the mutant according to claim 1 or 2, or the gene according to claim 3, or the expression vector according to claim 4 or 5, or the host cell according to claim 6 or 7, or the method according to claim 8 or 9 for the degradation of keratin in the fields of animal husbandry, feed, tanning, and medicine.
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| CN113528493A (en) * | 2021-05-28 | 2021-10-22 | 江南大学 | Keratinase mutant with improved thermal stability and application thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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