CN113528493B

CN113528493B - Keratinase mutant with improved thermal stability and application thereof

Info

Publication number: CN113528493B
Application number: CN202110592639.3A
Authority: CN
Inventors: 史劲松; 苏畅; 郑可欣; 许正宏; 龚劲松; 钱建瑛; 江佳宇; 秦安琪; 何纪萌
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2022-11-25
Anticipated expiration: 2041-05-28
Also published as: CN113528493A

Abstract

The invention discloses a keratinase mutant with improved thermal stability and application thereof, belonging to the technical field of genetic engineering and enzyme engineering. The mutants of the invention are A165E, Q170T, N292F, L190Q, H92N and S171G, and the mutants can improve the thermal stability to different degrees on the basis of better maintaining the catalytic activity, wherein the Q170T mutant has the optimal thermal stability, and the half-life period at 60 ℃ is improved from 17.3min of the wild type to 38.5min. The half-life of the six-site combined mutant is obviously improved to 66.1min, and the optimal reaction temperature is increased from 40 ℃ to 60 ℃. The mutant has better catalytic activity and thermal stability, and has higher application value and potential compared with wild type keratinase. And the protein thermal stability improving method based on computer-aided analysis can become a general way for improving the thermal stability of the industrial enzyme.

Description

Keratinase mutant with improved thermal stability and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a keratinase mutant with improved thermal stability and application thereof.

Background

The keratinase has the function of specifically degrading natural keratin and is a protease with strong biological activity. Most are extracellular serine proteases and a small number are conjugated or intracellular proteases. The keratinase-producing microorganisms reported in the literature are mainly derived from bacteria, fungi or actinomycetes, and are mostly isolated from the accumulation of feathers, bird feathers or hair waste. Keratinase can treat keratin-containing materials more mildly and efficiently than other proteases, and at the same time, can improve the nutritional value of the treated keratin. At present, keratinase is widely applied to industries such as medicine, chemical industry, feed, textile, tanning and the like, and has important application values in the aspects of preparation of biological agents, utilization of waste biological resources and the like, so research on keratinase is concerned.

High temperatures favor the rapid hydrolysis of keratin fibers such as wool, feathers, hair, and the like, and the hydrolysis process is usually carried out for a prolonged period of time, which requires that the keratinase have good thermal stability. Most of the proteases reported to date are generally unstable in industrial environments. In previous studies, the inactivation half-life of KerBp keratinase at 60 ℃ is only about 17min, and insufficient stability is a bottleneck problem limiting the application of KerBp keratinase. Improving the stability of proteases, especially proteases of unknown structure, is a challenging task. Traditional modification methods, such as directed evolution, are time-consuming and labor-consuming; the complete rational design has limitations, and although the amount of protein structure data information which can be applied at present is increased rapidly and the computer technology level is enhanced, the structure-activity relationship of the protein is not completely resolved. Currently, cross-coupling of computational biology and directed evolution further accelerates the rational engineering process of enzymes. Protein engineering with the aid of computer-aided design opens up a new area for efficient production of enzymes with stability based on precise and comprehensive structural analysis, advanced genetic manipulation and new methods combined with multidisciplinary techniques.

Disclosure of Invention

In order to solve the technical problems, the invention modifies bacillus pumilus keratinase molecules by means of computer-aided calculation and analysis and respectively carrying out site-directed mutation and multi-site combined mutation on a high-flexibility Loop region and a calcium ion binding region of keratinase and low-frequency amino acids in a sequence, so as to construct a recombinant keratinase engineering strain with improved thermal stability, and simultaneously provide an effective enzyme protein thermal stability modification strategy.

The first purpose of the invention is to provide a keratinase mutant, which takes the keratinase with an amino acid sequence shown as SEQ ID NO.2 as a parent, and mutates at least one of alanine at position 165, asparagine at position 292, glutamine at position 170, leucine at position 190, histidine at position 92 and serine at position 171 of the parent keratinase into other amino acids.

In one embodiment of the invention, the parent keratinase is at least one of mutated alanine at position 165 to glutamic acid, mutated asparagine at position 292 to phenylalanine, mutated glutamine at position 170 to threonine, mutated leucine at position 190 to glutamine, mutated histidine at position 92 to asparagine and mutated serine at position 171 to glycine.

In one embodiment of the invention, alanine at position 165 of the parent keratinase is mutated into glutamic acid, asparagine at position 292 is mutated into phenylalanine, glutamine at position 170 is mutated into threonine, leucine at position 190 is mutated into glutamine, histidine at position 92 is mutated into asparagine, and serine at position 171 is mutated into glycine.

The second object of the present invention is to provide a gene encoding the keratinase mutant.

The third purpose of the invention is to provide a recombinant plasmid carrying the gene.

The fourth purpose of the invention is to provide a recombinant bacterium for expressing the keratinase mutant.

In one embodiment of the present invention, the recombinant bacterium is a bacillus, escherichia coli, yeast or filamentous fungus as a host bacterium.

The fifth purpose of the invention is to provide a prediction, analysis and screening method for obtaining the keratinase mutant.

The sixth purpose of the invention is to provide the application of the keratinase mutant in the industries of medicine, chemical industry, feed, textile and leather making.

The keratinase mutant is applied to the industries of medicine, chemical industry, feed, textile or leather making.

The recombinant bacterium is applied to the industries of medicine, chemical industry, feed, textile or leather making.

In one embodiment of the invention, the application is specifically to ferment the recombinant bacteria to obtain the keratinase mutant, and the keratinase mutant is applied to the industries of medicine, chemical industry, feed, textile or leather making.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the invention provides a method for performing site-specific mutagenesis and multi-site combined mutagenesis on a high-flexibility Loop region, a calcium ion binding region and low-frequency amino acids in a sequence of a keratinase respectively based on computer-aided calculation and analysis. Obtaining a plurality of mutants with improved thermal stability, wherein the half-life period of the multi-site combination mutant at 60 ℃ is 66.1min, which is 3.8 times of that of wild type keratinase; the optimal reaction temperature is 60 ℃, which is 20 ℃ higher than that of the wild type. Is suitable for industrial large-scale production and has better application value and prospect. And the method for modifying the protease by using computer-aided calculation and analysis can become a general way for modifying the thermal stability of the industrial enzyme.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a three-dimensional simulation of the highly flexible Loop region of keratinase of the present invention shown in FIG. 1;

FIG. 2 is a schematic diagram showing the predicted distribution of calcium ion-binding amino acids in a three-dimensional structure;

FIG. 3 is a keratinase site amino acid frequency analysis and mutation selection;

FIGS. 4-1, 4-2 and 4-3 show the results of site-directed mutagenesis of the highly flexible Loop region of keratinase;

FIG. 5 shows the results of site-directed mutagenesis of the calcium ion-binding domain of keratinase;

FIGS. 6-1 and 6-2 show the results of low frequency amino acid mutations in keratinase;

FIG. 7 shows the mechanism of improved thermostability molecules of mutants Q170T and A165E;

FIG. 8 is a structural analysis of mutant Q190L;

FIG. 9 is a structural analysis of sequence-convergent mutants;

FIG. 10 is the result of a multi-site combinatorial mutation;

FIG. 11 is the use of keratinase in the extraction of keratin;

FIG. 12 is the use of keratinase in the degradation of feather waste;

FIG. 13 shows the use of keratinase in tanning dehairing.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1: construction of a homology model for keratinase

Homology comparison is carried out in NCBI database according to the amino acid sequence of keratinase gene kerBp, 3 crystal structures with high homology (more than 60%) are selected as templates, the 3D stereo structure of the keratinase is simulated through EasyModeller software, and the quality of the established model is evaluated by utilizing the Verify 3D function of an SAVES online analysis server (http:// service. And carrying out related analysis operation of the three-dimensional structure through PyMOL and discovery studio software.

Example 2: calculation and analysis of highly flexible Loop region of keratinase

The sequence (SEQ ID NO. 2) and structure of the keratinase are analyzed by using a FoldUnfold and IUPred protein disordered region structure prediction tool. Three sections of high-flexibility Loop regions are determined by combining homologous sequence alignment analysis and structural visualization analysis, namely Loop158-171, loop265-272 and Loop290-305 (figure 1). And further predicting and calculating the B-Factor and RMSF of the amino acids formed by the three sections of Loop regions through prediction software PredyFlexy, and screening out 22 amino acid residues with higher B-Factor and RMSF as the target of site-directed mutagenesis. Based on the 22 mutation sites on the 3 screened high-flexibility Loop, whether the mutation type is stable or not is judged by calculating the difference value (delta G) of Gibbs free energy change of the wild type and the mutation type, and the mutation amino acid type is further determined. And (3) performing 19 amino acid substitution predictions on each mutation site, constructing a virtual saturated mutant library, performing mutant screening according to the delta G value, and determining 41 mutation types of the 19 mutation sites.

Example 3: prediction and analysis of calcium ion binding region of keratinase

The amino acid sequence of the keratinase is analyzed by an on-line prediction tool IonCom, 37 potential amino acid residues related to calcium ion binding are predicted, the amino acids are further positioned in a homologous model structure of the keratinase and are divided into 4 groups from the space position (figure 2), and a conserved calcium ion binding site CaI consisting of amino acids D149, L183, D184, N185, T186, I187, G188, V189 and L190 is determined by observing the characteristics of the 4 groups on a three-dimensional structure. Analysis of the CaI region of the keratinase conserved calcium ion binding site shows that D149, L183, I187, V189 and N185 are conserved amino acids of the keratinase and calcium ion binding site, wherein calcium ions are directly coordinated with D149, L183, I187 and V189 residues, and other amino acid residues comprising D184, T186, G188 and L190 which form a cavernous Loop structure play a role of a supporting structure. Therefore, the experiment carries out mutation on the above non-conservative amino acid residues so as to enhance the binding capacity of the site to calcium ions or better maintain the stability of the structure.

Example 4: calculation and analysis of "Low frequency" amino acids of keratinase

The keratinase KerBp amino acid sequence (FSATA format) is submitted to an online homologous sequence alignment analysis software Consenssus Finder, the low-frequency amino acid selection range is less than 10%, and the amino acid residue with low frequency of appearance in the target protein sequence is replaced by the high-frequency amino acid at the position in the homologous sequence. Based on the analysis and prediction results, 20 mutation sites and mutation amino acids were determined (see FIG. 3).

Example 5: construction of keratinase mutants

Site-directed mutagenesis primers (as shown in Table 1) were designed based on the sequence of keratinase KerBp (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO. 2), and whole-plasmid one-step reverse PCR was performed on plasmid KerBp/pMA5 carrying the keratinase KerBp gene. The purified recovered PCR product was digested with Dpn I enzyme to remove the template plasmid. After the digestion reaction is finished, the reaction is carried out for 10min at 70 ℃ for enzyme deactivation treatment, then the mixture is immediately placed on ice for cooling, and 10 mu L of digestion products are taken and transformed into a clone host E.coli JM 109. Carrying out colony PCR verification on the transformant, selecting a transformant which is verified to be correct for sequencing verification, finally converting the correctly constructed site-directed mutant plasmid into a host cell B.subtilis WB600 to obtain a recombinant bacterium with a target mutation, and selecting the transformant for sequencing verification.

TABLE 1 primer design for site-directed mutagenesis of keratinase

Note: italicized as the site of mutation

Example 6: keratinase activity assay

The activity of the keratinase is determined by taking 1% (w/v) soluble keratin as a substrate, and the specific method is as follows:

adding 0.5mL of substrate to 0.5mL of enzyme solution diluted with Tris-HCl pH 9.0, incubating in a water bath at 50 ℃ for 20min, adding 1mL of 4-th-content TCA solution to terminate the reaction, centrifuging the sample at 12,000rpm for 5min, collecting 1mL of centrifuged supernatant, and adding 5mL of 0.4M Na ₂ CO ₃ And 1mL of a forskolin phenol reagent, heating in a water bath at 40 ℃ for 20min for color development after uniformly mixing, cooling to room temperature, and measuring the absorbance at 660 nm. In the control group, the enzyme solution and the TCA solution are mixed, incubated in a water bath at 50 ℃ for 20min, and then the substrate is added, and the rest operations are consistent with those of the experimental group. All experiments were performed on 3 replicates and the final results were expressed as mean ± standard deviation, calculated using the STDEV formula in Excel.

Definition of enzyme activity: in the reaction system, enzyme liquid hydrolyzes the substrate to ensure that each increase of 0.01 of absorbance value at 660nm is one enzyme activity unit, U.mL ^-1 。

The result of enzyme activity measurement shows that the mutants N164V, N164C, N164I, A165E, A165G, Q170T, Q170A, G262K, N263C, S264M, S267C, S267H, S267E, S270P, S270W, N291C, N291T, N292F, V293C, L190Q, H92N, H144D, S171G and T282V better retain enzyme activity, wherein the keratinase activity of the mutant N164C is improved by 13% (the experimental results are shown in figure 4-1, figure 4-2 and figure 4-3); the enzyme activity of L190Q is improved by 69% (the experimental result is shown in figure 5); the enzymatic activity of S171G was increased by 30% (see FIGS. 6-1 and 6-2 for experimental results).

Example 7: mutant screening and evaluation of thermostability

The correctly constructed mutants are streaked by a milk LB flat plate to separate out single colonies, and the single colonies with obvious transparent circles are picked up to be filled with a liquid LB culture medium (50 mu g.mL) ^-1 Kanr) for 8-12h, and the mutants were screened initially by enzyme activity assay. The preliminarily screened mutant strain was further inoculated to TB fermentation medium (50. Mu.g.mL) at an inoculum size of 3% ^-1 Kanr) and sampling every 12h to determine the enzyme activity for re-screening. Heat-treating each mutant recombinase at 50 deg.C, 60 deg.C, 70 deg.C for 60min, and determining residual enzyme activity; the mutants were incubated at 60 ℃ for 10min, 30min, 60min and 90min, respectively, and the inactivation half-lives of the mutants at 60 ℃ were determined.

Through the verification of a site-directed mutagenesis experiment on a high-flexibility Loop region of keratinase, two mutants A165E and Q170T with improved thermal stability are obtained, and the half-life of the keratinase at 60 ℃ is respectively improved to 28.9min and 38.5min from 17.3min before mutagenesis on the basis of keeping the enzyme activity (the experimental result is shown in figure 4-1, figure 4-2 and figure 4-3).

By mutating the non-conservative amino acid site of the calcium ion binding region CaI of keratinase, an advantageous mutant L190Q with improved keratinase activity and stability is obtained by screening, and the half-life period at 60 ℃ is improved to 28.3min (see the experimental result in figure 5).

By replacing amino acid residues in the keratinase sequence which occur less frequently than 10% with "high frequency amino acids" at that position in the homologous sequence. Totally 20 mutants are constructed, and two mutants H92N and S171G with synchronously improved enzyme activity and thermal stability are obtained through screening (the experimental results are shown in figure 6-1 and figure 6-2).

Example 8: molecular mechanism for improving heat stability of mutant

In order to explore a molecular mechanism for improving the thermal stability of a mutation site, the intermolecular forces before and after mutation of the modified site are analyzed, and the result shows that after the mutation of the 170 site from glutamine to threonine, a hydrogen bond with shorter length and stronger acting force is newly formed between the 170 site and aspartic acid at the 205 site, and the hydrogen bond effect is presumed to be one of the main reasons for improving the thermal stability of the Q170T mutant. Similarly, after the 165 site is mutated from alanine to glutamic acid, the hydrogen bond distance formed between the 165 site and the 143 site amino acid residue and between the 165 site and the 164 site amino acid residue is shortened, the acting force is stronger, and the stability of the protein can be better maintained (the experimental result is shown in fig. 7).

The results of the amino acid mutation of the calcium ion binding site show that the stability of the mutants G188N and G188Q is improved, but the enzyme activity is greatly reduced, and the calcium ion binding is considered as a regulation mechanism, so that the calcium ion binding plays a switching role in switching between stable and unstable states when different biological requirements of the protease are regulated. Thus the mutants G188N and G188Q may improve thermostability by enhancing interaction with calcium ions, but prevent activation of the enzyme by calcium ions. The mutant L190Q is relatively far away from calcium ions in space, the influence on the calcium ions is small after mutation, but the amido bonds of the mutant L190Q are easy to form intramolecular or intermolecular hydrogen bonds, so that the stability of the enzyme protein is improved (the experimental result is shown in figure 8).

Screening and obtaining 3 mutants H92N, H144D and S171G which improve the heat stability or expression quantity from the constructed 20 sequence isotropic mutants, wherein the mutation sites are all positioned on the surface of the protein as can be observed from a 3D homology model of the keratinase (see figure 9), wherein the mutant S171G mutates serine into glycine with the simplest structure, the transmembrane resistance of the glycine is reduced, the serine is hydrophilic amino acid, and the side chain of the glycine is between polar and nonpolar, so that the transmembrane secretion of the glycine is facilitated; in addition, glycine can be ionized in water, can form stable hydrogen bonds with water molecules, and is favorable for improving the stability of protein. Similarly, H92N mutates basic histidine into hydrophilic asparagine, which is beneficial to improving the stability of the asparagine in solution.

Example 9: multiple site combinatorial mutagenesis

The above-mentioned studies confirmed that 6 mutants A165E and Q170T, N292F, L190Q, H92N and S171G, which improve the thermostability of keratinase KerBp on the basis of better enzyme activity retention, were located on the surface of highly flexible Loop158-171, loop290-305, calcium ion binding site CaI region and enzyme protein, respectively. The mutation of a single region or site has certain limitation on the heat stability improvement effect of the whole protein, so that the sites are subjected to combined mutation so as to further improve the heat stability of the keratinase through a superposition effect. The results show that the inactivation half-life of the mutant at 60 ℃ is increased from 17.3min to 38.5min, 48.9min, 50.7min and 66.1min respectively, and the optimal reaction temperature is increased from 40 ℃ to 60 ℃ (the experimental results are shown in figure 10).

Example 10: application of keratinase in extraction of active keratin

The wool is washed twice with detergent to remove dust and other impurities, and then the detergent is washed away to obtain clean wool. The scoured and dried wool was degreased with acetone in a soxhlet extractor for 24 hours and then washed to remove the residual solvent. The defatted wool was thoroughly dried and cut to pieces for use. 5g of chopped degreased wool is immersed in 100mL of aqueous solution, the pH value is adjusted to 10.0 by NaOH, and the mixture is stirred for 1h at 65 ℃ to swell wool fibers. Adding 1% glutathione, and continuously stirring at 65 deg.C for 2-3h to pretreat wool, and opening disulfide bond in wool. The temperature was reduced to 50 ℃ and 100kU of keratinase solution was added and stirred continuously for 48h. The enzyme was inactivated by heating at 90 ℃ for 10 min. Centrifuging the hydrolysate at 4 deg.C and 8000 Xg for 20min, collecting supernatant, dialyzing with 10kDa dialysis bag at 4 deg.C for 48 hr, and replacing distilled water every 4 hr.

The results show that soluble wool keratin with molecular weight of about 45kDa and 28kDa is obtained by extraction, the keratin extract has good biocompatibility, shows obvious cell proliferation and migration promoting effect and has strong oxidation resistance (the experimental results are shown in figure 11). The keratin has potential application value in the field of medical biomaterials.

Example 11: application of keratinase in feather waste degradation

Collecting feather waste, cleaning, drying and shearing for later use. Weighing 1g of feather, placing the feather in a 500mL Erlenmeyer flask, adding 50mL of L-cysteine solution with the concentration of 0.4% and the pH value of 10, treating for 3h at 85 ℃, adding 50mL of keratinase solution into each flask after the temperature is reduced to room temperature, and carrying out enzymolysis treatment at 40 ℃ and 220 rpm. The results show that keratinase can effectively degrade feathers and obtain a plurality of essential amino acids (the experimental results are shown in figure 12). The keratinase solves the problem of recycling feather waste, and the degradation product rich in various amino acids is also a high-quality feed additive.

Example 12: application of keratinase in tanning depilation

Placing the sheepskin cut into the size of about 6cm multiplied by 6cm into the crude enzyme solution containing 10000U activity of keratinase, incubating at 37 ℃ under 200rpm, and taking out regularly to detect the sheepskin unhairing condition. After the hair removal is finished, the sheepskin is washed clean by tap water, and the hair removal effect is further analyzed by a stereo microscope and the like. Since the depilation effect is not quantified, an observation and analysis means combining sensory evaluation and microscopic observation is adopted. The hair removal effect is good by three grades: force is not needed, and the hair can be easily torn off; the hair removal effect is general: the wool can be torn off by applying force; failure to depilate: the wool can not be torn off by force. The processing result shows that the keratinase can remove the hair of the sheepskin thoroughly, and the sheepskin which is removed by the enzyme method has light color, soft texture and smooth hand feeling. Observation through a stereomicroscope showed that the grain surface of the dehaired skins was clear and intact (results are shown in FIG. 13).

SEQ ID NO.1：

SEQ ID NO.2：

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Various other modifications and alterations will occur to those skilled in the art upon reading the foregoing description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

SEQUENCE LISTING

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Claims

1. A keratinase mutant with improved heat stability is characterized in that a keratinase with an amino acid sequence shown as SEQ ID NO.2 is used as a parent, and the keratinase mutant comprises at least one mutation of alanine at the 165 th site of the parent keratinase to glutamic acid, asparagine at the 292 th site to phenylalanine, glutamine at the 170 th site to threonine, leucine at the 190 th site to glutamine, histidine at the 92 th site to asparagine and serine at the 171 th site to glycine.

2. The keratinase mutant according to claim 1, wherein alanine at position 165, asparagine at position 292, glutamine at position 170, leucine at position 190, histidine at position 92, asparagine and serine at position 171 are mutated to glutamic acid, phenylalanine at position 292, threonine at position 292, glutamine at position 190, and glycine at position 171 of a parent keratinase.

3. A gene encoding the keratinase mutant of any of claims 1-2.

4. A recombinant plasmid carrying the gene of claim 3.

5. A recombinant bacterium expressing the keratinase mutant of any one of claims 1-2.

6. The recombinant strain of claim 5, wherein the recombinant strain is a host strain selected from the group consisting of Bacillus, escherichia coli, yeast and filamentous fungi.

7. Use of the keratinase mutant according to any of claims 1-2 for keratin extraction, feather waste degradation, tanning dehairing.

8. The recombinant bacterium of claim 6, wherein the recombinant bacterium is used for keratin extraction, feather waste degradation and leather making and unhairing; the application specifically comprises the steps of fermenting the recombinant bacteria to obtain a keratinase mutant, and applying the keratinase mutant to keratin extraction, feather waste degradation and leather making and unhairing.