A linamarase mutant and its application

Abstract

The invention discloses a linamarase mutant and its application, belonging to the technical field of enzyme engineering. The amino acid sequence of the mutant is any one or any two or any three or four of the following (a) to (d): (a) the lysine at position 263 of the amino acid sequence shown in SEQ ID NO.1 is mutated to proline; (b) the threonine at position 53 of the amino acid sequence shown in SEQ ID NO.1 is mutated to phenylalanine; (c) the serine at position 366 of the amino acid sequence shown in SEQ ID NO.1 is mutated to arginine; (d) the valine at position 335 of the amino acid sequence shown in SEQ ID NO.1 is mutated to cysteine, and the phenylalanine at position 339 is mutated to cysteine. The invention combines the molecular design of the enzyme with the site-directed mutagenesis to obtain a mutant with significantly improved thermal stability and activity. Compared with the wild type, the mutant has significantly improved degradation efficiency of cyanogenic glycosides. The invention lays a foundation for improving the edible safety of cassava food and cyanogenic glycoside-containing food.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, and in particular to a linamarase mutant and application thereof. Background Art

[0002] Cassava (scientific name: Manihot esculenta) is an edible root tuber of the Euphorbiaceae family and the genus Manihot, and is a global food crop. Cassava contains a class of glycoside plant secondary metabolites, called cyanogenic glycosides, also known as cyanogenic glycosides. Studies have shown that cyanogenic glycosides have reproductive toxicity, developmental toxicity, and neurotoxicity, and may cause acute death at high doses. Spastic paraplegia and tropical ataxia neuropathy have been observed in people who eat cassava. The World Health Organization reported that the dietary exposure risk of cyanide mainly comes from cassava.

[0003] The cyanogenic glycosides in cassava include linamarin and linamarin, of which linamarin accounts for 90% of the total cyanogenic glycosides in cassava. The specific hydrolysis of linamarin by linamarase is a key step in removing cyanide from cassava. Previous research results show that cyanogenic glycoside compounds are relatively stable and difficult to thermally degrade during processing. The efficient degradation of cyanogenic glycosides mainly depends on the hydrolysis of linamarinase. However, the processing process is often accompanied by the inactivation of endogenous linamarinase in cassava, resulting in excessive residual cyanogenic glycosides (Zhong Y, et al. Effect of ultrasonic pretreatment oneliminating cyanogenic glycosides and hydrogen cyanide in cassava. Ultrasonics Sonochemistry, 2021, 78(1): 105742., 2021).

[0004] Linamarase belongs to glycoside hydrolysis family 1, and is a typical (α / β)8 barrel-shaped structure. Its natural molecular weight is about 70KDa, and its theoretical amino acid number is 531. It can hydrolyze the non-reducing β-D-glucose bond bound to the terminal to generate β-D-glucose and the corresponding ligand. Studies on the recombinant expression of the linamarase gene have found that the defects of the recombinant enzyme are, first, the optimal enzyme activity is 35°C, and the poor thermal stability is not conducive to the effective hydrolysis of cyanogenic glycosides. Secondly, the expression vector is not perfect, which brings difficulties to the preparation of linamarase: the eukaryotic expression system is more complex than the prokaryotic expression system, with a long expression cycle and low expression level.

[0005] Therefore, the key issue in the current enzymatic degradation of cassava cyanogenic glycosides is to improve the thermal stability and catalytic activity of linamarin based on a suitable expression vector, thereby improving the degradation efficiency of cyanogenic glycosides. Summary of the invention

[0006] The purpose of the present invention is to provide a linamarase with good thermal stability and activity and capable of efficiently degrading cyanogenic glycosides, which is applied to enzymatic degradation of plant cyanogenic glycosides to meet the requirements of processed food for biosafety.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The invention uses cassava (Manihot esculenta) linamarinase (GenBank: AAB22162.1) as a template, performs site-directed mutagenesis on it, and obtains a linamarinase mutant.

[0009] The amino acid sequence of the mutant is any one or any two or any three or four of the following (a) to (d):

[0010] (a) the lysine at position 263 of the amino acid sequence shown in SEQ ID NO.1 is mutated to proline;

[0011] (b) the threonine at position 53 of the amino acid sequence shown in SEQ ID NO.1 is mutated to phenylalanine;

[0012] (c) the serine at position 366 of the amino acid sequence shown in SEQ ID NO.1 is mutated to arginine;

[0013] (d) The valine at position 335 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to cysteine, and the phenylalanine at position 339 is mutated to cysteine.

[0014] In one embodiment of the present invention, the amino acid sequence of the mutant is as shown in SEQ ID NO.3, wherein the lysine at position 263 of the amino acid sequence SEQ ID NO.1 is mutated to proline.

[0015] In one embodiment of the present invention, the amino acid sequence of the mutant is as shown in SEQ ID NO.4, wherein the threonine at position 53 of the amino acid sequence SEQ ID NO.1 is mutated to phenylalanine.

[0016] In one embodiment of the present invention, the amino acid sequence of the mutant is as shown in SEQ ID NO.5, wherein the serine at position 366 of the amino acid sequence SEQ ID NO.1 is mutated to arginine.

[0017] In one embodiment of the present invention, the amino acid sequence of the mutant is as shown in SEQ ID NO.6, wherein the valine at position 335 of the amino acid sequence SEQ ID NO.1 is mutated to cysteine, and the phenylalanine at position 339 is mutated to cysteine.

[0018] In one embodiment of the present invention, the amino acid sequence of the mutant is as shown in SEQ ID NO.7, wherein the lysine at position 263 of the amino acid sequence SEQ ID NO.1 is mutated to proline, the threonine at position 53 is mutated to phenylalanine, and the serine at position 366 is mutated to arginine.

[0019] In one embodiment of the present invention, the amino acid sequence of the mutant is as shown in SEQ ID NO.8, wherein the lysine at position 263 of the amino acid sequence SEQ ID NO.1 is mutated to proline, the threonine at position 53 is mutated to phenylalanine, the serine at position 366 is mutated to arginine, the valine at position 335 is mutated to cysteine, and the phenylalanine at position 339 is mutated to cysteine.

[0020] Compared with the wild-type linamarase, the above linamarase mutants have significantly improved thermal stability and specific activity, and the mutants have significantly improved degradation efficiency of cassava cyanogenic glycosides. Specifically, compared with the wild-type linamarase, the ΔT m The specific activities of S366R, K263P-T53F-S366R and K263P-T53F-S366R-V335C-F339C were 82.4U / mg, 83.9U / mg and 85.1U / mg, which were 1.95 times, 1.99 times and 2.02 times higher than the wild type (42.1U / mg).

[0021] Another object of the present invention is to provide a method for preparing the linamarase mutant, including but not limited to a microbial synthesis method.

[0022] The present invention provides a gene encoding the linamarase mutant, and the nucleotide sequence of the gene encoding is shown in SEQ ID NOs. 9 to 14. The nucleotide sequence of the gene encoding can be modified according to the codon preference of the host cell.

[0023] The present invention provides a recombinant expression vector, which comprises an original expression plasmid and a nucleotide sequence encoding the linamarase mutant inserted into a multiple cloning site of the original expression plasmid.

[0024] Preferably, the original expression plasmid is pMAL-C2X. pMAL-C2X has a maltose binding protein solubility-promoting tag. The present invention shows that the use of the pMAL-C2X vector helps to increase the expression of the target protein, and the expression is soluble.

[0025] The present invention provides a recombinant genetically engineered bacterium, wherein the recombinant genetically engineered bacterium carries the above-mentioned recombinant expression vector. The recombinant vector transforms a host cell to obtain a recombinant genetically engineered bacterium, and the host cell can be any conventional host cell in the art. Preferably, the host bacterium is Escherichia coli.

[0026] Specifically, the above coding gene is inserted into the multiple cloning site of the pMAL-C2X vector to construct a recombinant expression vector, which is then transformed into a host cell Escherichia coli to obtain a recombinant genetically engineered bacterium, and the linamarase mutant is obtained by inducing the expression of the recombinant protein and is separated and purified.

[0027] Another object of the present invention is to provide the use of the linamarase mutant in degrading cyanogenic glycosides.

[0028] The application includes: using the enzyme extracted after the bacterial body obtained by centrifugation after fermentation and culture of the engineered bacteria containing the linamarase mutant coding gene as a catalyst to degrade plant cyanogenic glycosides under the condition of 40-50°C.

[0029] In the reaction system, a buffer solution with a pH of 5 to 7 is used as the reaction medium. Preferably, a phosphate buffer solution is used as the reaction medium. Preferably, the pH of the reaction medium is 6, and the reaction temperature is 45°C.

[0030] Specifically, the plant may be, but is not limited to, cassava, almonds, flaxseed, bamboo shoots, and sorghum. Before preparing the processed food, the raw material is first crushed and homogenized, and then an enzyme solution is added to hydrolyze the cyanogenic glycoside. After the reaction is completed, the product after enzymatic hydrolysis is obtained by drying.

[0031] Preferably, in the reaction system, the amount of the catalyst used is 4 to 5 U / mL.

[0032] Preferably, the hydrolysis time is 40 to 90 minutes.

[0033] The culture medium used for the recombinant engineered bacteria can be a culture medium in the art that can grow the transformant and produce the linamarase of the present invention. When the host cell of the engineered bacteria is Escherichia coli Rosetta-gami2 (DE3), the expression vector is pMAL-C2X, and the fermentation medium is LB medium, the fermentation culture conditions are: 25-37°C culture until OD 600 When the pH is 0.6-0.8, add 0.1-1.0 mM IPTG and induce at 25-37°C and 200-250 rpm / min for 2-18 h.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention combines the molecular design of the enzyme with site-directed mutagenesis to obtain mutants with significantly improved thermal stability and activity, especially the mutant K263P-T53F-S366R-V335C-F339C, whose optimum temperature is 45°C, which is 10°C higher than the optimum temperature of 35°C of the wild type, and whose specific activity is 85.1U / mg, which is 2.02 times that of the wild type (42.1U / mg).

[0036] (2) The present invention can be used to effectively degrade cyanogenic glycosides of cassava. Under the same enzyme addition concentration conditions, the degradation rate of cyanogenic glycosides of the mutant is 85%, which is 1.54 times higher than that of the wild type. The mutant of the present invention lays a foundation for improving the edible safety of cassava food and food containing cyanogenic glycosides.

[0037] (3) The present invention realizes soluble and efficient expression of linamarase by using a prokaryotic expression system through vector design and expression condition screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 are the melting temperatures and specific activities of the disulfide bond mutants.

[0039] Figure 2 It is the RMSF diagram of iterative molecular dynamics simulation, where A is the first round of molecular dynamics simulation, B is the second round of molecular dynamics simulation, and C is the third round of molecular dynamics simulation.

[0040] Figure 3 are the melting temperatures and specific activities of the single-site saturation mutants.

[0041] Figure 4 The optimal temperature (A) and optimal pH (B) of mutants K263P-T53F-S366R and K263P-T53F-S366R-V335C-F339C.

[0042] Figure 5This is the protein conformation of the mutant K263P-T53F-S366R-V335C-F339C.

[0043] Figure 6 It is the electrophoresis diagram of the double enzyme digestion product of the recombinant vector, where M is the DNA marker, channel 1 is pMAL-c2X-mebgl, channel 2 is pET 28a-mebgl, channel 3 is pET 28a-sumo-mebgl, channel 4 is pCold-mebgl, channel 5 is pET 32a-mebgl, and channel 6 is pET 50b-mebgl.

[0044] Figure 7 This is the electrophoresis diagram of colony PCR products, where M is DNA marker, A is pET 28a-mebgl, B is pCold-mebgl, C is pET 28a-sumo-mebgl, D is pMAL-c2X-mebgl, E is pET 32a-mebgl, and F is pET 50b-mebgl.

[0045] Figure 8 The expression of recombinant total protein (A) and soluble protein (B) of different vectors, where channels 1 and 2 are pET28a-mebgl, channels 3 and 4 are pET 28a-sumo-mebgl, channels 5 and 6 are pCold-mebgl, channels 7 and 8 are pMal-C2X-mebgl, channels 9 and 10 are pET 32a-mebgl, and channels 11 and 12 are pET 50b-mebgl. The left side of each group is the control without inducer.

[0046] Fig. 9 This is a diagram for optimizing the inducing expression conditions, where A is the effect of temperature on the induced expression, channels 1-4 are 37°C, 1 and 2 are total protein, 3 and 4 are soluble protein, channels 5-8 are 25°C, 5 and 6 are total protein, 7 and 8 are soluble protein, and the left side of each group is a control without inducer; B is the effect of the concentration of inducer IPTG added on the induced expression, channels 1-6 are 0, 0.1, 0.2, 0.4, 0.8, and 1.0 mM IPTG, respectively; C is the effect of time on the induced expression, channels 1-7 are induced for 0, 1, 2, 3, 4, 5, and 6 h, respectively.

[0047] Fig.10 This is an investigation of the amount of enzyme added, where CK is a control without adding recombinant enzyme; the investigation of the amount of enzyme added was carried out at 45°C and pH 6.0 for 90 minutes.

[0048] Fig.11This study explored the degradation of cyanogenic glycosides by mutants and wild types, where WT is the wild type. The degradation study was carried out at an enzyme addition concentration of 47 mg / L and pH 6.0. DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0051] The materials and reagents involved in the following examples are:

[0052] 1. Strains and vectors

[0053] Escherichia coli strain E. coli DH5α, E. coli strain E. coli Rosetta-gami2 (DE3), and plasmid pMAL-C2X were all purchased from commercial sources.

[0054] The expression vector pMAL-C2X-mebgl-Twin Strep II was constructed in this study. It was obtained by homologous recombination with the target gene linamarase (GenBank: AAB22162.1) using plasmid pMAL-C2X as a template. The target gene sequence is SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.1, from which the signal peptide sequence is removed. There is a maltose binding protein solubility tag before the 5' end of the target gene to help the soluble expression of the target protein. To help the efficient purification of the target protein, the twin-strep II tag purification tag is connected to the 3' end of the target gene.

[0055] 2. Reagents

[0056] 2×Phanta Flash Master Mix (Dye Plus) high-fidelity enzyme, ClonExpress II One Step Cloning Kit, Mut Express MultiS Fast Mutagenesis Kit V2, and FastPure Plasmid Mini Kit were purchased from Nanjing Novozymes Biotechnology Co., Ltd.; Gravity flow strep-tactin XT4flow and 10×Buffer BXT were purchased from IBA Life Sciences; SYPRO orange dye was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; GBW(E)080115 water cyanide component analysis standard substance (cyanide) was purchased from China Institute of Metrology; linamarin (98%) and radix lotioside (97%) standards were purchased from TRC Company, Canada.

[0057] 3. Culture medium

[0058] LB medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride (pH 7.0). Solid medium was supplemented with 1.5% agar powder.

[0059] 4. Solution

[0060] Twin-strep II affinity chromatography binding buffer (Washing buffer): 10mM Tris-HCl, 150mMNaCl, 1mM EDTA, adjusted to pH 8.0.

[0061] Isonicotinic acid-barbituric acid color developer (20g / L): After 6g of sodium hydroxide is heated and dissolved in 400mL of water, 10g of isonicotinic acid is added. After heating and dissolving, 5g of barbituric acid is added. Heat until completely dissolved, cool, and dilute to 500mL.

[0062] Example 1 Disulfide bond mutant design

[0063] The disulfide bond of linamarase (GenBank: AAB22162.1) was designed online by DSDBASE-MODIP (http: / / caps.ncbs.res.in / iws / modip.html) and Disulfideby Design2 (http: / / cptweb.cpt.wayne.edu / DbD2 / ), and four pairs of disulfide bond mutants were screened: S260-D265, V335-F339, A364-F369, S201-S213. Primers were designed to construct four pairs of disulfide bond mutants, and the primer sequences are shown in Table 1.

[0064] Table 1 Disulfide bond mutant primer sequences

[0065]

[0066] Taking S260C-D265C as an example, the construction process of the mutant is as follows:

[0067] (1) Using the constructed expression vector pMAL-C2X-mebgl-Twin Strep II as a template, PCR amplification was first performed using upstream and downstream primers S260C-D265C. The PCR amplification reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3. The PCR amplification results were verified by agarose gel electrophoresis.

[0068] Table 2 PCR reaction system

[0069]

[0070] Table 3 PCR reaction program

[0071]

[0072]

[0073] (2) Perform Dpn I digestion. The digestion reaction system is shown in Table 4. After gently tapping to mix, centrifuge briefly to collect the contents at the bottom of the tube, and place in a PCR amplification instrument at 37° C. for 2 h.

[0074] Table 4 Digestion reaction system

[0075]

[0076] (3) Perform a recombination reaction. The recombination reaction system is shown in Table 5. After gently tapping to mix, centrifuge briefly to collect the tube bottom, and place it on a PCR amplification instrument at 37°C for 30 minutes.

[0077] Table 5 Recombination reaction system

[0078]

[0079] (4) For transformation of the recombinant product, thaw the competent cells DH5α in iceberg, take 10 μL of the recombinant product and add it to 100 μL of the competent cells, flick the tube wall to mix, and place on ice for 30 minutes. After heat shock at 42°C for 45 seconds, immediately place on ice to cool for 2 minutes. Add 900 μL of antibiotic-free LB medium and culture at 37°C with shaking for 1 hour (200 rpm). Spread the bacteria on LB solid medium containing ampicillin (100 μg / mL) and culture overnight at 37°C in an incubator.

[0080] (5) The recombinant vector was identified, transformants were selected for bacterial liquid PCR identification, plasmids were extracted, and the extracted plasmids were double-digested with SacI / XhoI and sent to Qingke Biotechnology for sequencing identification. The construction process of other mutants was the same as that of S260C-D265C, except that the PCR amplification primers were replaced with the corresponding mutant primers. Finally, 4 pairs of disulfide bond mutant recombinant vectors were obtained.

[0081] (6) Recombinant engineered bacteria were constructed. The recombinant vectors of the four pairs of disulfide bond mutants were transformed into competent Escherichia coli Rosetta-gami2 (DE3), and spread on LB solid culture medium containing ampicillin (100 μg / mL) to obtain positive recombinant engineered bacteria and obtain induced expression strains of the mutants.

[0082] (7) Expression and purification of the target protein. The recombinant engineered bacteria were inoculated into LB medium (100 μg / mL ampicillin, 20 μg / mL chloramphenicol) and cultured at 37°C until OD 600 When the pH was 0.6-0.8, 0.3 mM IPTG was added and induced at 37°C and 200-250 rpm / min for 3 h. Centrifugation was performed at 8000 rpm for 10 min to collect the cells.

[0083] The bacterial cells were resuspended in washing buffer (1 / 10, g / V) and disrupted using an ultrasonic cell disruptor. After centrifugation at 12000 rpm for 1 h, the supernatant protein solution was collected, the supernatant was adsorbed by strep tactin XT 4Flow pre-packed column, washed with washing buffer, eluted with eluting buffer, concentrated by 10 kDa ultrafiltration tube, and the protein concentration was determined by Bradford solution and identified by SDS-PAGE electrophoresis.

[0084] (8) To measure the enzyme activity of the mutant, 50 μL of enzyme solution was placed in 900 μL of 100 mM phosphate buffer (pH 6.0), and 10 mM linamarin solution was added and immediately reacted in a water bath at 30°C for 15 min. 1 mL of 0.1 mol / L NaOH was immediately added to terminate the reaction, and 3 mL of 1 mol / L KH2PO4 was added. 200 μL of 10 g / L chloramine T was added and reacted at room temperature for 3 min. 5 mL of 20 g / L isonicotinic acid-barbituric acid colorimetric reagent was added and reacted at room temperature for 15 min. The absorbance was measured colorimetrically at a wavelength of 600 nm. The enzyme activity unit is defined as: under the above reaction conditions, the amount of enzyme used to generate 1 μmol of cyanide ion per minute is one activity unit (U). The specific activity is defined as the activity per unit mass of enzyme (U / mg).

[0085] (9) The melting temperature (T m) determination: The melting temperature (T m ), take 20 μL of enzyme solution in buffer (10mM Tris-HCl, 150mM NaCl, pH 8.0), add 5 μL of 100-fold diluted SYPRO orange fuel, and place in PCR instrument for reaction. The reaction procedure is: the reaction temperature is gradually increased from 25°C to 90°C within 40 minutes. The excitation wavelength and emission wavelength are 490nm and 575nm respectively. The melting temperature (T) is obtained according to the Boltzmann model formula m ).

[0086] The thermal stability and activity results of the 4 disulfide bond mutants are shown in Figure 1 As shown in the figure, the thermal stability of V335C-F339C was significantly improved, and the Tm was increased by 2.9°C compared with the wild type. At the same time, the specific activity of the mutant V335C-F339C did not change significantly, while the specific activity of the mutant A364C-F369C decreased.

[0087] Example 2 Identification of key residues

[0088] (1) Three-dimensional structure simulation of linamarase

[0089] Homology modeling of linamarase (GenBank: AAB22162.1) and mutants was performed using the AlphaFold2 server (https: / / github.com / deepmind / alphafold accessed on 2022). Enter the target protein sequence and select the code to execute the program. In addition to the default parameters, the number of model cycles (number_recycles) and the number of models generated per run (num_moldels) were set to 5, and AMBER energy optimization was performed during modeling. The visual structure of the model was observed using PyMOL. In the subsequent calculation process, the pdb file constructed by AlphaFold was used as the protein structure data.

[0090] (2) Sequence alignment

[0091] In order to avoid the negative impact caused by destroying the highly conserved sequence of the protein, CD-search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) was used to analyze the conserved region of the protein. NCBI BLAST was used for sequence alignment analysis. CLUSTAL O (1.2.4) multiple sequence alignment was used to perform multiple sequence alignment of proteins with high similarity.

[0092] (3) Molecular dynamics simulation

[0093] Molecular dynamics simulations were performed using Gromacs 2022. The initial conformation of the protein was fixed at the center of a cube composed of an SPC / E water molecule model, at least 1 nm away from the edge of the box, and the force field was amber99sb-ildn. The protein charge was neutralized by adding sodium or chloride ions. Energy minimization was performed using the steepest descent algorithm with a step size of 0.01 nm and a maximum number of steps of 50,000 steps. The simulation was stopped when the maximum force was less than 1000 kJ / mol / nm. Pre-equilibrium for NVT and NPT was performed successively at 330 K and 1 atm, with a step size of 2 fs and an equilibrium time of 100 ps. After successful pre-equilibrium, molecular dynamics simulations were performed at 330 K and 1 atm, with a step size of 2 fs and a total simulation time of 100 ns. The active residues in the first round of simulation were replaced with alanine (alanine replaced with serine) for the second round of simulation, and so on until no new active residues appeared.

[0094] like Figure 2 As shown, the simulation results found that the flexible regions of the protein are mainly distributed in the regions from lysine at position 51 to isoleucine at position 57, from threonine at position 305 to glutamate at position 308, from aspartic acid at position 261 to valine at position 264, and from tyrosine at position 365 to tryptophan at position 368. Among them, three active regions are distributed on the surface of the protein, one is distributed at the entrance of the pocket, and the other region is distributed from cysteine ​​at position 183 to alanine at position 184 inside the protein.

[0095] Example 3 Virtual Saturation Mutation

[0096] FoldX5 (https: / / foldxsuite.crg.eu / command / PositionScan) was used to perform saturation mutations on all residues of the protein, and the folding free energy ΔΔG of all single-point mutants was calculated. The folding free energy of the protein was calculated and analyzed by the stability module, and the virtual saturation mutation was analyzed by the mutation module. Combined with the hot spot residues of the protein, potential beneficial single-point mutants were screened. Combined with the conservative domain analysis and sequence alignment analysis, conservative residues were excluded from mutation. The mutants screened by free energy need to be further structurally examined to eliminate some chemically unstable mutants. The main review criteria include: whether hydrogen bonds and salt bridges are destroyed, especially in the hydrophobic interior of the protein, whether the hydrophobic residues are located on the solvent contact surface, whether an internal cavity is formed, and whether there is serious damage to the spatial structure. Based on the above review, 13 mutants were finally screened. According to the mutant construction method in Example 1, the following 13 mutants were constructed respectively, and the mutant primers are shown in Table 6 below.

[0097] Table 6 Primer sequences for single point mutations

[0098]

[0099]

[0100] According to the enzyme activity and melting temperature determination method in Example 1, the thermal stability and activity of the 13 mutants were verified. Figure 3 As shown in Figure 2, the melting temperatures of S183P, S183Q, S183F, K263P, T53F, and S366R are significantly increased, ΔT m The ΔT of S366R was 3.5°C, 3.7°C, 2.8°C, 3.2°C, 3.3°C and 1.9°C respectively. However, the specific activity of S183P, S183Q and S183F decreased significantly. The specific activity of K263P and T53F did not decrease significantly while the thermal stability was improved. m The temperature increased by 1.9℃, but the specific activity was significantly improved (82.4U / mg), which was 1.95 times higher than WT (42.1U / mg).

[0101] Therefore, we focused on the three mutant residues with significantly improved stability or activity. K263P, T53F and S366R are far apart in space, and we combined them to construct the mutant K263P-T53F-S366R. The mutant K263P-T53F-S366R and the disulfide bond mutants obtained above were combined to construct the mutant K263P-T53F-S366R-V335C-F339C, and further enzymatic properties and kinetics were analyzed. The results are as follows: Figure 4 As shown in Table 7, the optimum temperature of the mutant is about 45°C, which is higher than the optimum temperature of the wild type at 35°C. The optimum pH of the mutant is not different from that of the wild type. m ,t 1 / 2 (60℃), K m , K cat , specific activity, and melting temperature of the combined mutations were further increased. The ΔT m The temperature increased by 3.9℃ and 5.5℃ respectively. 1 / 2 (60℃) increased by 2.25 and 3.40 times, respectively. The specific activities of mutants K263P-T53F-S366R and K263P-T53F-S366R-V335C-F339C were 83.9 and 85.1 U / mg, respectively, which were 1.99 and 2.02 times that of WT, respectively. The Michaelis constant of the mutants was smaller than that of WT, K cat / K mThe ratio increases.

[0102] Table 7 Enzymatic parameters of mutants

[0103]

[0104]

[0105] In the following content, the expression conditions of mutant K263P-T53F-S366R-V335C-F339C were optimized. The protein conformation of mutant K263P-T53F-S366R-V335C-F339C is shown in Figure 5 shown.

[0106] Example 4 Expression vector screening

[0107] The construction of pET28a(+)-mebgl, pET28a-SUMO-mebgl, pCold-GST-mebgl, pMAL-C2X-mebgl, pET32a(+)-mebgl and pET50b(+)-mebgl recombinant plasmids comprises the following steps: double-digesting plasmids pET28a(+), pET28a-SUMO, pCold-GST, pMAL-C2X, pET32a(+) and pET50b(+) with SalI / HindIII, NheI / XhoI, SacI / XhoI, SmaI / XhoI, NcoI / XhoI and SmaI / SacI, respectively, and recovering the plasmids by gel to prepare a linearized vector, which is then connected to a target gene fragment containing a homologous sequence at the end of the linearized vector to obtain a circularized connection product. pMAL-C2X-mebgl-Twin StrepII was amplified using plasmid pMAL-C2X-mebgl as template. The amplification primers of the target gene are shown in Table 8.

[0108] Table 8 Target gene amplification primers

[0109]

[0110]

[0111] Each ligation product was transformed into the cloning strain E. coli DH5α. After overnight culture, the colonies on the transformation plate were picked for PCR identification. After the remaining bacterial solution was expanded, the plasmid was extracted and double enzyme digestion was performed for identification. The plasmid with the correct test result was recovered and sent to Qingke Biotech for sequencing.

[0112] The recombinant plasmid with correct sequencing was transformed into expression strains E. coli Rosetta-gami2 (DE3) and E. coli BL21 (DE3) to construct expression strains. The bacterial solution PCR was performed with the corresponding primers, and the products were identified by agarose gel electrophoresis. The recombinant engineered bacteria were inoculated in LB medium (100 μg / mL ampicillin, 20 μg / mL chloramphenicol) and cultured at 37°C until OD 600 When the pH was 0.6-0.8, 0.3 mM IPTG was added and induced at 37°C and 200-250 rpm / min for 3 h. Centrifugation was performed at 8000 rpm for 10 min to collect the cells.

[0113] The bacterial cells were resuspended in washing buffer (1 / 10, g / V) and disrupted using an ultrasonic cell disruptor. After centrifugation at 12000 rpm for 1 h, the supernatant protein solution was collected, the supernatant was adsorbed by strep tactin XT 4Flow pre-packed column, washed with washing buffer, eluted with eluting buffer, concentrated by 10 kDa ultrafiltration tube, and the protein concentration was determined by Bradford solution and identified by SDS-PAGE electrophoresis.

[0114] like Figure 6 The results of double enzyme digestion of each recombinant vector are shown in Figure 2. The recombinant plasmids were transformed into expression strains, such as Figure 7 is the result of bacterial solution PCR identification. The expression of all constructed vectors was compared, such as Figure 8 As shown in the figure, from the perspective of total protein expression, the target proteins of pCold-mebgl and pMAL-c2X-mebgl were significantly expressed, and the other vectors also expressed some target proteins. Further analysis of the soluble expression of the target proteins showed that only the soluble expression of pMAL-c2X-mebgl was significant.

[0115] Example 5 Optimization of expression conditions

[0116] Different expression conditions of the recombinant vector pMAL-C2X-mebgl-Twin Strep II were optimized, including expression temperature, induction agent concentration, induction time, etc. The temperatures were 15, 20, 25, and 37°C, the IPTG concentrations were 0, 0.1, 0.3, 0.5, and 1.0 mM, and the expression times were 0, 0.5, 1, 2, 4, 8, 12, and 24 h. Fig. 9The results showed that the protein expression at 37°C was significantly higher than that at 25°C. The IPTG concentration had no effect on the expression. The expression reached the highest level when the expression time was 3h. The bacterial growth curve showed that the fermentation density increased exponentially when the inducer IPTG was added around 3h of culture, and the bacterial growth slowed down after 9h of culture. In summary, the expression conditions determined were: 37°C, 0.1mM IPTG for 3h.

[0117] Example 6 Study on the reaction conditions of mutants degrading cyanogenic glycosides

[0118] Peel the cleaned cassava and use a grinder to break the cassava flesh into a homogenate. Add different proportions of enzyme solution (U / mL, enzyme activity / volume) to the cassava pulp and hydrolyze it at different temperatures for a certain time. Dry it in an electric hot air drying oven at 55°C, grind it with a dry grinder until it passes through an 80-mesh steel sieve, and obtain cassava flour after cyanogenic glycoside degradation. Detect the cyanogenic glycoside content in the degraded cassava flour to obtain the cyanogenic glycoside clearance rate. The calculation formula for the cyanogenic glycoside clearance rate is: clearance rate = (original cassava cyanogenic glycoside content - residual cyanogenic glycoside content) / original cassava cyanogenic glycoside content.

[0119] The cyanogenic glycosides are detected by LC-MS / MS method.

[0120] The liquid phase conditions are:

[0121] a) Chromatographic column: UPLC C18 (50mm×2.1mm, 1.8μm) chromatographic column, or chromatographic column with equivalent performance;

[0122] b) Mobile phase and elution conditions are shown in Table 1;

[0123] c) Flow rate: 0.2 mL / min;

[0124] d) Column temperature: 30°C;

[0125] e) Injection volume: 10 μL.

[0126] Table 9 Mobile phase and gradient elution reference conditions

[0127]

[0128] The mass spectrometry conditions were:

[0129] a) Ion source: electrospray ion source;

[0130] b) Scanning mode: positive ion scanning mode;

[0131] c) Drying gas temperature: 325°C;

[0132] d) Drying gas flow rate: 5L / min;

[0133] e) Sheath gas temperature: 350°C;

[0134] f) Sheath gas flow rate: 11 L / min;

[0135] g) Atomizing gas pressure: 45MPa;

[0136] h) Capillary voltage: 3500V;

[0137] i) Detection method: multiple reaction monitoring mode;

[0138] The monitored ion pairs and collision energies are shown in Table 10.

[0139] Table 10 Retention time, monitoring ion pairs, collision energy and fragmentation voltage of cyanogenic glycosides

[0140]

[0141]

[0142] The results are as follows Fig.10 and Fig.11 As shown in the figure, when the addition amount was 4U / mL, the clearance rate of cyanogenic glycosides was 84.8%, and the corresponding enzyme addition concentration was 47mg / L. Under the same enzyme addition concentration, the clearance rate of cyanogenic glycosides of the wild type was 55.1% when it was degraded at 35℃ for 90min. The mutant had the highest clearance rate of cyanogenic glycosides at 45℃, with a clearance value of 85% at 90min. The average clearance rate of cyanogenic glycosides of the mutant within 90min was 0.203μmol / min / mg enzyme, the clearance rate was 73% at 40min, and the average clearance rate of cyanogenic glycosides within 40min was 0.393μmol / min / mg enzyme. In summary, compared with the wild type, under the same enzyme addition concentration, the highest hydrolysis rate of the mutant was 1.54 times higher than that of the wild type.

[0143] The above examples are only some specific embodiments of the present invention, and the present invention is not limited to the above embodiments. All equivalent modifications or changes that can be directly derived or associated with the contents disclosed by ordinary technicians in the field should be considered to be covered by the protection scope of the present invention.

Claims

1. A linamarase mutant, characterized in that: The amino acid sequence of the mutant is shown in any one of SEQ ID NO.3, SEQ ID NO.7 and SEQ ID NO.

8.

2. A recombinant expression vector, characterized in that: The recombinant expression vector comprises an original expression plasmid and a nucleotide sequence encoding the linamarase mutant according to claim 1 inserted into the multiple cloning site of the original expression plasmid.

3. The recombinant expression vector according to claim 2, characterized in that The original expression plasmid was pMAL-C2X.

4. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria carries the recombinant expression vector as claimed in claim 2 or 3.

5. The recombinant genetically engineered bacterium according to claim 4, characterized in that: The host bacteria is Escherichia coli.

6. Use of the linamarinase mutant according to claim 1 in degrading linamarin.

7. The use according to claim 6, characterized in that The application includes: using the enzyme extracted after the bacterial body obtained by centrifugation after fermentation and culture of the engineered bacteria containing the linamarinase mutant coding gene as a catalyst to degrade plant linamarin at 40-50°C.

8. The use according to claim 7, characterized in that The plant is cassava, almond, flax seed, bamboo shoot or sorghum; in the reaction system, the dosage of the catalyst is 4-5 U / mL.

9. The use according to claim 8, characterized in that The degradation time is 40~90 min.

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