CN108396019B - Method for reducing sensitivity of pullulanase to cyclodextrin - Google Patents
Method for reducing sensitivity of pullulanase to cyclodextrin Download PDFInfo
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- CN108396019B CN108396019B CN201810181924.4A CN201810181924A CN108396019B CN 108396019 B CN108396019 B CN 108396019B CN 201810181924 A CN201810181924 A CN 201810181924A CN 108396019 B CN108396019 B CN 108396019B
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
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- C12Y302/01041—Pullulanase (3.2.1.41)
Abstract
The invention discloses a method for reducing the sensitivity of pullulanase to cyclodextrin, which prepares a pullulanase mutant by carrying out rational mutation on key amino acid interacted between the pullulanase and the cyclodextrin, and changes the interaction between the pullulanase and the cyclodextrin so as to reduce the sensitivity of the pullulanase to the cyclodextrin, wherein the key amino acid is the amino acid which has hydrophobic or hydrogen bond interaction with the cyclodextrin. The method of the invention obtains the pullulanase mutant by site-directed mutagenesis, and prevents the interaction of pullulanase and cyclodextrin, thereby improving the utilization rate of raw material starch and the yield of cyclodextrin.
Description
Technical Field
The invention relates to the technical field of genetic engineering, enzyme engineering or food science, in particular to a method for modifying pullulanase by a genetic engineering site-specific mutagenesis method so as to change the interaction between cyclodextrin and pullulanase.
Background
Pullulanase (EC 3.2.1.41) specifically hydrolyzes alpha-1, 6 glycosidic bonds in pullulan, soluble starch, amylopectin and corresponding oligosaccharide to generate short straight-chain dextrin. The compound is used with other amylase, can greatly improve the utilization rate of starch raw materials, and is widely applied to the starch industry.
In the starch sugar industry, pullulanase has the function of hydrolyzing alpha-1, 6 glycosidic bonds, so the pullulanase has wide application prospect, can be used for preparing amylose, maltose and branched cyclodextrin, and can be compounded with CGTase to produce cyclodextrin. In the production process of cyclodextrin, taking the production of alpha-CD as an example, because alpha-CGTase can not hydrolyze alpha-1, 6 bonds in a starch substrate, the conversion rate is only 40% -60% when the alpha-CGTase is used alone to produce the alpha-CD. The conversion rate of cyclodextrin can be obviously improved by adding pullulanase, but pullulanase is easily inhibited by cyclodextrin, so that debranching reaction and cyclization reaction are required to be carried out separately, and the problems of long production period, resource waste and the like are caused. In order to shorten the production period, improve the utilization rate of starch as a raw material and increase the yield of cyclodextrin, the pullulanase and cyclodextrin glucosyltransferase are compounded to produce the cyclodextrin in a one-pot method, so that the reduction of the inhibition of the cyclodextrin on the pullulanase becomes a problem to be solved urgently.
Marshall J, Iwamoto H and the like research the inhibition effect of cyclodextrin on pullulanase from the aspects of enzyme activity, reaction kinetics and the like, and prove that the cyclodextrin is a competitive inhibitor of the pullulanase. The further study on the inhibition effect of cyclodextrin on pullulanase by Bo et al is carried out, and on the basis of the characterization of conventional enzymological properties, the influence of cyclodextrin on the endogenous fluorescence and secondary structure of pullulanase is studied, so that the inclusion effect between hydrophobic cavity of cyclodextrin and pullulanase aromatic amino acid is proved to be the intrinsic driving force of cyclodextrin for inhibiting the pullulanase activity. At home and abroad, only the interaction research of cyclodextrin and pullulanase is focused, and a specific effective solving means is not provided.
The inventors previously discovered that the crystal structure of pullulanase (NCBI accession number: AF008220.1) derived from Bacillus subtilis str.168 and cyclodextrin combined crystal (PDB: 2E8Z) is published through alignment of pullulanase sequences from different sources, so that the pullulanase derived from Bacillus subtilis is selected as a template for experimental design. After the crystal structure of the combination of cyclodextrin and pullulanase is analyzed, amino acid sites of the combination of cyclodextrin and pullulanase are found out, the sites are subjected to rational mutation design, molecular simulation software is utilized to simulate the interaction of mutants and cyclodextrin, and the key amino acids are proved to play a vital role in the combination of cyclodextrin and pullulanase from the aspect of computational chemistry. Thereby providing basis and reference for reducing the inhibition effect of cyclodextrin on pullulanase by mutation means.
Disclosure of Invention
In view of the above problems of the prior art, the present applicant provides a method for reducing the sensitivity of pullulanase to cyclodextrin. The method of the invention obtains the pullulanase mutant by site-directed mutagenesis, and prevents the interaction of pullulanase and cyclodextrin, thereby improving the utilization rate of raw material starch and the yield of cyclodextrin.
The technical scheme of the invention is as follows:
a method for reducing the sensitivity of pullulanase to cyclodextrin is characterized in that a pullulanase mutant is prepared by performing rational mutation on key amino acids of interaction of pullulanase and cyclodextrin, and the interaction between pullulanase and cyclodextrin is changed, so that the sensitivity of pullulanase to cyclodextrin is reduced;
the key amino acid is an amino acid which has hydrophobic or hydrogen bond interaction with the cyclodextrin.
The key amino acid is tryptophan, aspartic acid, arginine, asparagine and histidine.
The mutated amino acid is glycine, alanine, leucine, isoleucine, valine, proline, methionine, serine, glutamine, threonine, cysteine, glutamic acid, lysine or tyrosine.
The specific process of the rational mutation is as follows:
(1) taking a pullulanase gene as a template, selecting a plasmid, constructing an expression vector, and preparing the plasmid carrying the pullulanase gene;
(2) determining key amino acid sites for combination of pullulanase and cyclodextrin;
(3) designing mutation primers of key amino acids, and carrying out mutation by taking plasmids carrying pullulanase genes as templates to prepare mutated plasmids;
(4) and transferring the mutated plasmid into host bacteria, selecting positive monoclonal fermentation culture to obtain a pullulanase mutant enzyme solution, and separating and purifying to obtain the pullulanase mutant.
In the step (1), the plasmid is one of PMC series, pET series or pGEX series.
The host bacterium in the step (4) is gram-negative bacterium, gram-positive bacterium or fungus.
The host bacteria in the step (4) are escherichia coli, bacillus subtilis or yeast.
The separation and purification method in the step (4) is affinity chromatography, hydrophobic chromatography, ultrafiltration chromatography or gel filtration chromatography.
The cyclodextrin is one or more of alpha-CD, beta-CD and gamma-CD.
The beneficial technical effects of the invention are as follows:
through sequence comparison of pullulanases from different sources, pullulanase with a known crystal structure is used as a template, and the crystal structure is analyzed to obtain related amino acids interacting with cyclodextrin in the pullulanase. Rational mutation of the amino acids is designed, the cyclodextrin is connected to a wild type and a mutant in an abutting mode through molecular abutting software, and comparison of change of affinity of the cyclodextrin and enzyme and comparison of enzyme activity shows that mutation design results can effectively prevent the combination of the cyclodextrin and pullulanase and reduce the sensitivity of the pullulanase to the cyclodextrin, so that basis and reference are provided for reducing the inhibition effect of the cyclodextrin on the pullulanase through mutation.
According to the method, based on the analysis of the crystal structures of the enzymes and the inhibitors and the sequence comparison of enzymes from different sources, the interaction sites of the pullulanase and the cyclodextrin are found out, and the pullulanase mutant with reduced sensitivity to the cyclodextrin is obtained by utilizing site-directed mutagenesis and molecular docking analysis, so that the utilization rate of starch raw materials and the yield of the cyclodextrin are improved.
Drawings
FIG. 1 is a schematic partial view of the crystal structures of pullulanase and cyclodextrin of the present invention;
FIG. 2 shows an alignment of pullulanase sequences from different sources;
FIG. 3 is a schematic diagram of the interaction of the W437G mutant with cyclodextrin;
FIG. 4 is a schematic representation of the interaction of H477A mutant with cyclodextrin;
FIG. 5 is a schematic diagram showing the interaction of D465G mutant with cyclodextrin;
FIG. 6 shows the enzyme activities of cyclodextrin on the inhibition of wild type and W437G mutant;
FIG. 7 is a graph showing the activity of cyclodextrin on the inhibition of wild-type and H477A mutants;
FIG. 8 shows the enzyme activities of cyclodextrin on the inhibition of wild-type and D465G mutants.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
FIG. 1 is a partial schematic view of the crystal structure of the pullulanase/cyclodextrin complex of the present invention. As can be seen from the figure, the distances between tryptophan at 437 position, aspartic acid at 465 position, leucine at 468 position, asparagine at 474 position, histidine at 477 position, asparagine at 526 position and cyclodextrin hydroxyl oxygen are respectively 4.2A, 3.0A, 2.7A, 3.0A, 2.9A and 3.1A, and strong hydrogen bond interaction exists, so that the pullulanase and the cyclodextrin are closely combined to form the structural basis for inhibiting the pullulanase by the cyclodextrin.
FIG. 2 is a partial sequence alignment chart of 60 pullulanases from different sources, and it can be seen from the alignment that tryptophan, leucine and aspartic acid in the present invention are absolutely conserved among pullulanases from different sources, and histidine and asparagine are highly conserved among pullulanases from different sources.
Example 1
A method of reducing the sensitivity of pullulanase to cyclodextrins by rational mutation, said method comprising the steps of:
(1) a pullulanase gene sequence AmyX is synthesized by a chemical total synthesis method by taking pullulanase (PDB: 2E8Z) which is derived from Bacillus subtilis str.168 and has a known crystal structure of interaction between cyclodextrin and pullulanase as a template and the accession number of the pullulanase on NCBI is AF 008220.1. The plasmid used to construct the E.coli expression vector was pET20b (+) carrying the T7 promoter.
(2) Carrying out NcoI and BamHI double enzyme digestion on pET20b (+) plasmid and plasmid containing AmyX gene respectively, tapping and recovering the enzyme digestion product, then connecting with T4 ligase, transforming the connecting product into E.coli DH5 alpha competent cells, culturing for 8-12h at 37 ℃, picking transformants into a liquid LB culture medium containing 100 mu g/mL ampicillin, carrying out shake culture, extracting the plasmid, and carrying out enzyme digestion verification to obtain the AmyX/Pet20b (+) expression vector.
(3) Plasmid AmyX/Pet20b (+) was transformed into E.coli BL21(DE3) host bacteria, spread on LB plate containing ampicillin (100mg/mL), and cultured at 37 ℃ for 8h, named AmyX/Pet20b (+)/BL21(DE 3). Picking single colony to liquid LB culture medium, culturing overnight at 37 deg.C, and preserving strain with glycerol.
(4) Analyzing a crystal structure derived from the bacillus subtilis pullulanase to obtain amino acid sites W437, D465, R468, N474, H477 and N526 of the pullulanase interacting with cyclodextrin, and comparing sequences of the pullulanase from different sources to obtain the key amino acids absolutely conserved by the key amino acids of W437, D465, R468, N474, H477 and N526 and highly conserved by the key amino acids of N474 and H477.
Designing a mutation primer, mutating tryptophan (W) which is absolutely conserved at position 437 and has a benzene ring structure into minimum glycine (G) without a side chain, wherein the mutation primer is shown in the following table 1;
TABLE 1
Note: underlined are the mutated bases
Performing site-directed mutagenesis by using PCR, wherein the PCR reaction system is as follows: 5 × Primer STAR GXL Buffer, 10 μ L; dNTPMixture (2.5mM), 4. mu.L; forward primer (10. mu.M), 1.5. mu.L; reverse primer (10. mu.M), 1.5. mu.L; template DNA (10 ng/. mu.L), 1. mu.L; prime STAR GXL DNA Polymerase (1.25. mu.l/. mu.l), 1. mu.l, add double distilled water to 50. mu.l. The PCR amplification conditions were: pre-denaturation at 98 ℃ for 3min followed by 30 cycles (98 ℃ for 20s, 60 ℃ for 30s, 68 ℃ for 6min) and extension at 68 ℃ for 10 min.
The PCR product was digested with Dpn I (Thermo Fisher Co.), transformed into E.coli DH 5. alpha. competent cells, which were cultured overnight in LB solid medium (containing 100. mu.g/mL ampicillin), and a single clone was picked up and cultured in LB liquid medium (containing 100. mu.g/mL ampicillin), and plasmids were extracted and the correctly sequenced plasmids were transformed into the expression host E.coli BL21(DE 3).
(5) Selecting a positive monoclonal transferred into an expression host escherichia coli BL21(DE3), culturing the positive monoclonal in an LB liquid culture medium (containing 100 mu g/mL) at 37 ℃ and 200rpm for 8-12h, wherein the inoculation amount is 5%, and inoculating the positive monoclonal into a TB culture medium (containing 100 mu g/mL ampicillin); culturing at 37 deg.C and 200rpm until OD is 0.6, adding IPTG with final concentration of 0.2mM, inducing expression at 25 deg.C and 160rpm for 96h, centrifuging the fermentation broth at 4 deg.C and 10000g for 10Min, and collecting supernatant.
(6) And (4) performing primary concentration and separation on the fermentation supernatant by using a 10kDa ultrafiltration centrifugal tube and 4000g for centrifugation for 10 Min. The concentrated fermentation broth was passed through a 0.22 μm filter and purified by nickel affinity chromatography, the whole purification process being carried out at low temperature.
Nickel affinity chromatography step:
balancing: equilibrating the nickel column with 20mM Tris-HCl, 500mM NaCl, pH7.5 buffer;
sample loading: the pretreated sample is loaded at a flow rate of 1 mL/min;
thirdly, cleaning: washing the hybrid protein with 20mM Tris-HCl, 500mM NaCl, pH7.5 buffer solution;
and fourthly, elution: eluting the target protein by using a buffer solution of 20mM Tris-HCl, 500mM NaCl, 300mM imidazole and pH7.5, wherein the detection wavelength is 280nm, and collecting the eluent containing the pullulanase activity to obtain the purified enzyme of the mutant W437G.
(7) The pullulanase activity is measured by a 3, 5-dinitrosalicylic acid method (DNS). The pullulanase catalyzes and hydrolyzes pullulan polysaccharide to generate reducing sugar under certain conditions, the 3, 5-dinitrosalicylic acid and the reducing sugar are reduced to generate a brownish red amino complex under thermal conditions, the color depth is in direct proportion to the amount of the reducing sugar within a certain range, and the enzyme activity can be measured and calculated under the wavelength of 540 nm. Definition of enzyme activity unit: the amount of enzyme that catalyzes the production of reducing sugars equivalent to 1. mu. mol of glucose reducing power per minute is defined as one activity unit.
Enzyme activity determination:
A. preheating: putting 1mL of 1% pullulan solution (pH 6.0) into a centrifuge tube, and placing on a water bath at 45 ℃ for heat preservation for 10 min;
B. reaction: adding 0.1mL of enzyme solution, shaking and mixing uniformly, timing accurately for 30min, adding 1.5mL of DNS to terminate the reaction, boiling in a water bath for 10min, and immediately cooling.
C. Measurement: absorbance was measured at 540nm and viability was calculated.
(8) Molecular docking
The binding force of the wild-type and mutant enzymes to cyclodextrin was studied using Autodock vina 1.1.2(http:// vina. script. edu) software. Generating a docking input file by using AutoDockTools 1.5.6 software, wherein the parameters of molecular docking are as follows: center _ x-126.601, center _ y-72.661, and center _ z-127.473; size _ x 15, size _ y 15, and size _ z 15. When the vina is used for docking, default parameters are selected, and the obtained optimal model is analyzed by pymol1.7.6 software.
Pullulanase (PDB: 2E8Z) derived from Bacillus subtilis str.168, which has a crystal structure known to interact with pullulanase, was used as a template, and its accession number at NCBI was AF 008220.1. Through analysis of the crystal structure, amino acid positions W437, D465, R468, N474, H477 and N526 of pullulanase interacting with cyclodextrin are obtained, and through sequence alignment of pullulanase from different sources, key amino acids of W437, D465, R468, N474, H477 and N526 are absolutely conserved and key amino acids of N474 and H477 are highly conserved. Selecting 437-position tryptophan which is absolutely conservative and has a benzene ring structure and is 4.2A away from hydroxyl oxygen of cyclodextrin to perform mutation, and selecting minimum amino acid glycine without a side chain structure as the mutated amino acid. Schematic representation of the docking of the mutants with cyclodextrins is shown in FIG. 3. The affinity between the wild type pullulanase and cyclodextrin was-6.8 kcal/mol, while the affinity between the mutant and cyclodextrin was-6.7 kcal/mol, and it can be seen that the affinity between the W437G mutant and cyclodextrin was decreased. In addition, the closest distance between the mutated glycine and the cyclodextrin is 6.0A, and the distance is increased. By combining the two points, the sensitivity of the pullulanase to the cyclodextrin can be reduced, a theoretical basis is provided for reducing the inhibition effect of the cyclodextrin to the pullulanase by mutation, and the pullulanase inhibitor can be further used for reducing the inhibition effect of the cyclodextrin to the pullulanase, so that the utilization rate of starch raw materials and the yield of the cyclodextrin can be improved.
The sensitivity of the pullulanase to cyclodextrin is further characterized by enzyme activity, the experimental result is shown in fig. 6, and compared with the wild pure enzyme, the mutant can find that: the inhibition effect of the three cyclodextrins on the mutant pullulanase is obviously reduced compared with that of the wild pullulanase, and the effect of reducing the sensitivity of the pullulanase on the cyclodextrins is achieved.
Example 2
A method of reducing the sensitivity of pullulanase to cyclodextrins by rational mutation, said method comprising the steps of:
(1) a pullulanase gene sequence AmyX is synthesized by a chemical total synthesis method by taking pullulanase (PDB: 2E8Z) which is derived from Bacillus subtilis str.168 and has a known crystal structure of interaction between cyclodextrin and pullulanase as a template and the accession number of the pullulanase on NCBI is AF 008220.1. The plasmid used to construct the E.coli expression vector was pET20b (+) carrying the T7 promoter.
(2) Carrying out NcoI and BamHI double enzyme digestion on pET20b (+) plasmid and plasmid containing AmyX gene respectively, tapping and recovering the enzyme digestion product, then connecting with T4 ligase, transforming the connecting product into E.coli DH5 alpha competent cells, culturing for 8-12h at 37 ℃, picking transformants into a liquid LB culture medium containing 100 mu g/mL ampicillin, carrying out shake culture, extracting the plasmid, and carrying out enzyme digestion verification to obtain the AmyX/Pet20b (+) expression vector.
(3) Plasmid AmyX/Pet20b (+) was transformed into E.coli BL21(DE3) host bacteria, spread on LB plate containing ampicillin (100mg/mL), and cultured at 37 ℃ for 8h, named AmyX/Pet20b (+)/BL21(DE 3). Picking single colony to liquid LB culture medium, culturing overnight at 37 deg.C, and preserving strain with glycerol.
(4) Analyzing a crystal structure derived from the bacillus subtilis pullulanase to obtain amino acid sites W437, D465, R468, N474, H477 and N526 of the pullulanase interacting with cyclodextrin, and comparing sequences of the pullulanase from different sources to obtain the key amino acids absolutely conserved by the key amino acids of W437, D465, R468, N474, H477 and N526 and highly conserved by the key amino acids of N474 and H477.
Designing a mutation primer, mutating histidine (H) with imidazole ring structure at position 477 into alanine (A) with minimum side chain, wherein the mutation primer is shown in the following table 2;
TABLE 2
Note: underlined are the mutated bases
Performing site-directed mutagenesis by using PCR, wherein the PCR reaction system is as follows: 5 × Primer STAR GXL Buffer, 10 μ L; dNTPMixture (2.5mM), 4. mu.L; forward primer (10. mu.M), 1.5. mu.L; reverse primer (10. mu.M), 1.5. mu.L; template DNA (10 ng/. mu.L), 1. mu.L; prime STAR GXL DNA Polymerase (1.25. mu.l/. mu.l), 1. mu.l, add double distilled water to 50. mu.l. The PCR amplification conditions were: pre-denaturation at 98 ℃ for 3min followed by 30 cycles (98 ℃ for 20s, 60 ℃ for 30s, 68 ℃ for 6min) and extension at 68 ℃ for 10 min.
The PCR product was digested with Dpn I (Thermo Fisher Co.), transformed into E.coli DH 5. alpha. competent cells, which were cultured overnight in LB solid medium (containing 100. mu.g/mL ampicillin), and a single clone was picked up and cultured in LB liquid medium (containing 100. mu.g/mL ampicillin), and plasmids were extracted and the correctly sequenced plasmids were transformed into the expression host E.coli BL21(DE 3).
(5) Selecting a positive monoclonal transferred into an expression host escherichia coli BL21(DE3), culturing the positive monoclonal in an LB liquid culture medium (containing 100 mu g/mL) at 37 ℃ and 200rpm for 8-12h, wherein the inoculation amount is 5%, and inoculating the positive monoclonal into a TB culture medium (containing 100 mu g/mL ampicillin); culturing at 37 deg.C and 200rpm until OD is 0.6, adding IPTG with final concentration of 0.2mM, inducing expression at 25 deg.C and 160rpm for 96h, centrifuging the fermentation broth at 4 deg.C and 10000g for 10Min, and collecting supernatant.
(6) And (4) performing primary concentration and separation on the fermentation supernatant by using a 10kDa ultrafiltration centrifugal tube and 4000g for centrifugation for 10 Min. The concentrated fermentation broth was passed through a 0.22 μm filter and purified by nickel affinity chromatography, the whole purification process being carried out at low temperature.
Nickel affinity chromatography step:
balancing: equilibrating the nickel column with 20mM Tris-HCl, 500mM NaCl, pH7.5 buffer;
sample loading: the pretreated sample is loaded at a flow rate of 1 mL/min;
thirdly, cleaning: washing the hybrid protein with 20mM Tris-HCl, 500mM NaCl, pH7.5 buffer solution;
and fourthly, elution: eluting the target protein by using a buffer solution of 20mM Tris-HCl, 500mM NaCl, 300mM imidazole and pH7.5, detecting the wavelength at 280nm, and collecting the eluent containing the pullulanase activity to obtain the purified enzyme of the mutant H477A.
(7) The pullulanase activity is measured by a 3, 5-dinitrosalicylic acid method (DNS). The pullulanase catalyzes and hydrolyzes pullulan polysaccharide to generate reducing sugar under certain conditions, the 3, 5-dinitrosalicylic acid and the reducing sugar are reduced to generate a brownish red amino complex under thermal conditions, the color depth is in direct proportion to the amount of the reducing sugar within a certain range, and the enzyme activity can be measured and calculated under the wavelength of 540 nm. Definition of enzyme activity unit: the amount of enzyme that catalyzes the production of reducing sugars equivalent to 1. mu. mol of glucose reducing power per minute is defined as one activity unit.
Enzyme activity determination:
A. preheating: putting 1mL of 1% pullulan solution (pH 6.0) into a centrifuge tube, and placing on a water bath at 45 ℃ for heat preservation for 10 min;
B. reaction: adding 0.1mL of enzyme solution, shaking and mixing uniformly, timing accurately for 30min, adding 1.5mL of DNS to terminate the reaction, boiling in a water bath for 10min, and immediately cooling.
C. Measurement: absorbance was measured at 540nm and viability was calculated.
(8) Molecular docking
The binding force of the wild-type and mutant enzymes to cyclodextrin was studied using Autodock vina 1.1.2(http:// vina. script. edu) software. Generating a docking input file by using AutoDockTools 1.5.6 software, wherein the parameters of molecular docking are as follows: center _ x-126.601, center _ y-72.661, and center _ z-127.473; size _ x 15, size _ y 15, and size _ z 15. When the vina is used for docking, default parameters are selected, and the obtained optimal model is analyzed by pymol1.7.6 software.
Pullulanase (PDB: 2E8Z) derived from Bacillus subtilis str.168, which has a crystal structure known to interact with pullulanase, was used as a template, and its accession number at NCBI was AF 008220.1. Through analysis of the crystal structure, amino acid positions W437, D465, R468, N474, H477 and N526 of pullulanase interacting with cyclodextrin are obtained, and through sequence alignment of pullulanase from different sources, key amino acids of W437, D465, R468, N474, H477 and N526 are absolutely conserved and key amino acids of N474 and H477 are highly conserved. Selecting histidine 477 with highly conserved imidazole ring structure and 2.9A distance with cyclodextrin hydroxyl oxygen for mutation, and selecting minimum amino acid alanine with side chain structure for mutation. Schematic representation of the docking of the mutants with cyclodextrins is shown in FIG. 4. The affinity between the wild type pullulanase and cyclodextrin was-6.8 kcal/mol, while the affinity between the mutant and cyclodextrin was-6.7 kcal/mol, it can be seen that the affinity between the H477A mutant and cyclodextrin was decreased. In addition, the closest distance between the mutated alanine and the cyclodextrin is 5.3A, which is far away. By combining the two points, the sensitivity of the pullulanase to the cyclodextrin can be reduced, a theoretical basis is provided for reducing the inhibition effect of the cyclodextrin to the pullulanase by mutation, and the pullulanase inhibitor can be further used for reducing the inhibition effect of the cyclodextrin to the pullulanase, so that the utilization rate of starch raw materials and the yield of the cyclodextrin can be improved.
The sensitivity of the pullulanase to cyclodextrin is further characterized by enzyme activity, the experimental result is shown in fig. 7, and compared with the wild pure enzyme, the mutant can find that: the inhibition effect of the three cyclodextrins on the mutant pullulanase is obviously reduced compared with that of the wild pullulanase, and the effect of reducing the sensitivity of the pullulanase on the cyclodextrins is achieved.
Example 3
A method of reducing the sensitivity of pullulanase to cyclodextrins by rational mutation, said method comprising the steps of:
(1) a pullulanase gene sequence AmyX is synthesized by a chemical total synthesis method by taking pullulanase (PDB: 2E8Z) which is derived from Bacillus subtilis str.168 and has a known crystal structure of interaction between cyclodextrin and pullulanase as a template and the accession number of the pullulanase on NCBI is AF 008220.1. The plasmid used to construct the E.coli expression vector was pET20b (+) carrying the T7 promoter.
(2) Carrying out NcoI and BamHI double enzyme digestion on pET20b (+) plasmid and plasmid containing AmyX gene respectively, tapping and recovering the enzyme digestion product, then connecting with T4 ligase, transforming the connecting product into E.coli DH5 alpha competent cells, culturing for 8-12h at 37 ℃, picking transformants into a liquid LB culture medium containing 100 mu g/mL ampicillin, carrying out shake culture, extracting the plasmid, and carrying out enzyme digestion verification to obtain the AmyX/Pet20b (+) expression vector.
(3) Plasmid AmyX/Pet20b (+) was transformed into E.coli BL21(DE3) host bacteria, spread on LB plate containing ampicillin (100mg/mL), and cultured at 37 ℃ for 8h, named AmyX/Pet20b (+)/BL21(DE 3). Picking single colony to liquid LB culture medium, culturing overnight at 37 deg.C, and preserving strain with glycerol.
(4) Analyzing a crystal structure derived from the bacillus subtilis pullulanase to obtain amino acid sites W437, D465, R468, N474, H477 and N526 of the pullulanase interacting with cyclodextrin, and comparing sequences of the pullulanase from different sources to obtain the key amino acids absolutely conserved by the key amino acids of W437, D465, R468, N474, H477 and N526 and highly conserved by the key amino acids of N474 and H477.
Designing a mutation primer, and mutating highly conserved aspartic acid (D) with a side chain structure at the 465 site into minimum amino acid glycine (G) without a side chain, wherein the mutation primer is shown in the following table 3;
TABLE 3
Note: underlined are the mutated bases
Performing site-directed mutagenesis by using PCR, wherein the PCR reaction system is as follows: 5 × Primer STAR GXL Buffer, 10 μ L; dNTPMixture (2.5mM), 4. mu.L; forward primer (10. mu.M), 1.5. mu.L; reverse primer (10. mu.M), 1.5. mu.L; template DNA (10 ng/. mu.L), 1. mu.L; prime STAR GXL DNA Polymerase (1.25. mu.l/. mu.l), 1. mu.l, add double distilled water to 50. mu.l. The PCR amplification conditions were: pre-denaturation at 98 ℃ for 3min followed by 30 cycles (98 ℃ for 20s, 60 ℃ for 30s, 68 ℃ for 6min) and extension at 68 ℃ for 10 min.
The PCR product was digested with Dpn I (Thermo Fisher Co.), transformed into E.coli DH 5. alpha. competent cells, which were cultured overnight in LB solid medium (containing 100. mu.g/mL ampicillin), and a single clone was picked up and cultured in LB liquid medium (containing 100. mu.g/mL ampicillin), and plasmids were extracted and the correctly sequenced plasmids were transformed into the expression host E.coli BL21(DE 3).
(5) Selecting a positive monoclonal transferred into an expression host escherichia coli BL21(DE3), culturing the positive monoclonal in an LB liquid culture medium (containing 100 mu g/mL) at 37 ℃ and 200rpm for 8-12h, wherein the inoculation amount is 5%, and inoculating the positive monoclonal into a TB culture medium (containing 100 mu g/mL ampicillin); culturing at 37 deg.C and 200rpm until OD is 0.6, adding IPTG with final concentration of 0.2mM, inducing expression at 25 deg.C and 160rpm for 96h, centrifuging the fermentation broth at 4 deg.C and 10000g for 10Min, and collecting supernatant.
(6) And (4) performing primary concentration and separation on the fermentation supernatant by using a 10kDa ultrafiltration centrifugal tube and 4000g for centrifugation for 10 Min. The concentrated fermentation broth was passed through a 0.22 μm filter and purified by nickel affinity chromatography, the whole purification process being carried out at low temperature.
Nickel affinity chromatography step:
balancing: equilibrating the nickel column with 20mM Tris-HCl, 500mM NaCl, pH7.5 buffer;
sample loading: the pretreated sample is loaded at a flow rate of 1 mL/min;
thirdly, cleaning: washing the hybrid protein with 20mM Tris-HCl, 500mM NaCl, pH7.5 buffer solution;
and fourthly, elution: eluting the target protein by using a buffer solution of 20mM Tris-HCl, 500mM NaCl, 300mM imidazole and pH7.5, wherein the detection wavelength is 280nm, and collecting the eluent containing the pullulanase activity to obtain the purified enzyme of the mutant D465G.
(7) The pullulanase activity is measured by a 3, 5-dinitrosalicylic acid method (DNS). The pullulanase catalyzes and hydrolyzes pullulan polysaccharide to generate reducing sugar under certain conditions, the 3, 5-dinitrosalicylic acid and the reducing sugar are reduced to generate a brownish red amino complex under thermal conditions, the color depth is in direct proportion to the amount of the reducing sugar within a certain range, and the enzyme activity can be measured and calculated under the wavelength of 540 nm. Definition of enzyme activity unit: the amount of enzyme that catalyzes the production of reducing sugars equivalent to 1. mu. mol of glucose reducing power per minute is defined as one activity unit.
Enzyme activity determination:
A. preheating: putting 1mL of 1% pullulan solution (pH 6.0) into a centrifuge tube, and placing on a water bath at 45 ℃ for heat preservation for 10 min;
B. reaction: adding 0.1mL of enzyme solution, shaking and mixing uniformly, timing accurately for 30min, adding 1.5mL of DNS to terminate the reaction, boiling in a water bath for 10min, and immediately cooling.
C. Measurement: absorbance was measured at 540nm and viability was calculated.
(8) Molecular docking
The binding force of the wild-type and mutant enzymes to cyclodextrin was studied using Autodock vina 1.1.2(http:// vina. script. edu) software. Generating a docking input file by using AutoDockTools 1.5.6 software, wherein the parameters of molecular docking are as follows: center _ x-126.601, center _ y-72.661, and center _ z-127.473; size _ x 15, size _ y 15, and size _ z 15. When the vina is used for docking, default parameters are selected, and the obtained optimal model is analyzed by pymol1.7.6 software.
Pullulanase (PDB: 2E8Z) derived from Bacillus subtilis str.168, which has a crystal structure known to interact with pullulanase, was used as a template, and its accession number at NCBI was AF 008220.1. Through analysis of the crystal structure, amino acid positions W437, D465, R468, N474, H477 and N526 of pullulanase interacting with cyclodextrin are obtained, and through sequence alignment of pullulanase from different sources, key amino acids of W437, D465, R468, N474, H477 and N526 are absolutely conserved and key amino acids of N474 and H477 are highly conserved. Selecting 465-bit aspartic acid which is absolutely conservative and has a side chain structure and is 3.0A away from cyclodextrin hydroxyl oxygen for mutation, and selecting the minimum amino acid glycine without the side chain structure as the mutated amino acid. Schematic representation of the docking of the mutants with cyclodextrins is shown in FIG. 5. The affinity between the wild type pullulanase and cyclodextrin was-6.8 kcal/mol, while the affinity between the mutant and cyclodextrin was-6.7 kcal/mol, it can be seen that the affinity between the D465G mutant and cyclodextrin was reduced. In addition, the closest distance between the mutated alanine and the cyclodextrin is 5.4A, which is far away. By combining the two points, the sensitivity of the pullulanase to the cyclodextrin can be reduced, a theoretical basis is provided for reducing the inhibition effect of the cyclodextrin to the pullulanase by mutation, and the pullulanase inhibitor can be further used for reducing the inhibition effect of the cyclodextrin to the pullulanase, so that the utilization rate of starch raw materials and the yield of the cyclodextrin can be improved.
The sensitivity of the pullulanase to cyclodextrin is further characterized by enzyme activity, the experimental result is shown in fig. 8, and compared with the wild pure enzyme, the mutant can find that: the inhibition effect of the three cyclodextrins on the mutant pullulanase is obviously reduced compared with that of the wild pullulanase, and the effect of reducing the sensitivity of the pullulanase on the cyclodextrins is achieved.
Claims (6)
1. A method for reducing the sensitivity of pullulanase to cyclodextrin is characterized in that a pullulanase mutant is prepared by performing rational mutation on key amino acids of interaction of the pullulanase and the cyclodextrin, and the interaction between the pullulanase and the cyclodextrin is changed, so that the sensitivity of the pullulanase to the cyclodextrin is reduced;
the key amino acid is an amino acid which has hydrophobic or hydrogen bond interaction with cyclodextrin;
the mutant is W437G, H477A or D465G;
the specific process of the rational mutation is as follows:
(1) taking a pullulanase gene as a template, selecting a plasmid, constructing an expression vector, and preparing the plasmid carrying the pullulanase gene;
(2) determining key amino acid sites for combination of pullulanase and cyclodextrin;
(3) designing mutation primers of key amino acids, and carrying out mutation by taking plasmids carrying pullulanase genes as templates to prepare mutated plasmids;
(4) and transferring the mutated plasmid into host bacteria, selecting positive monoclonal fermentation culture to obtain a pullulanase mutant enzyme solution, and separating and purifying to obtain the pullulanase mutant.
2. The method according to claim 1, wherein the plasmid in step (1) is one of PMC series, pET series or pGEX series.
3. The method according to claim 1, wherein the host bacterium in step (4) is a gram-negative bacterium, a gram-positive bacterium or a fungus.
4. The method according to claim 1, wherein the host bacterium in the step (4) is Escherichia coli, Bacillus subtilis or yeast.
5. The method according to claim 1, wherein the separation and purification method in step (4) is affinity chromatography, hydrophobic chromatography, ultrafiltration chromatography or gel filtration chromatography.
6. The method of claim 1, wherein the cyclodextrin is one or more of α -CD, β -CD, and γ -CD.
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