CN108396019A - A method of Pullulanase is reduced to cyclodextrin sensibility - Google Patents
A method of Pullulanase is reduced to cyclodextrin sensibility Download PDFInfo
- Publication number
- CN108396019A CN108396019A CN201810181924.4A CN201810181924A CN108396019A CN 108396019 A CN108396019 A CN 108396019A CN 201810181924 A CN201810181924 A CN 201810181924A CN 108396019 A CN108396019 A CN 108396019A
- Authority
- CN
- China
- Prior art keywords
- cyclodextrin
- pullulanase
- amino acid
- plasmid
- mutant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
- C12N9/2457—Pullulanase (3.2.1.41)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01041—Pullulanase (3.2.1.41)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of reduction Pullulanases to the method for cyclodextrin sensibility, the method carries out rationality mutation by the key amino acid to Pullulanase and cyclodextrin interaction, Pullulan enzymatic mutant is made, change the interaction between Pullulanase and cyclodextrin, to reduce sensibility of the Pullulanase to cyclodextrin, the key amino acid is that there are hydrophobic or hydrogen bond action amino acid between cyclodextrin.The method of the present invention obtains Pullulan enzymatic mutant using rite-directed mutagenesis, the interaction of Pullulanase and cyclodextrin is hindered, to improve starch utilization ratio of raw materials and beta-cyclodextrin yield.
Description
Technical field
The present invention relates to genetic engineering, enzyme engineering or technical field of food science, especially a kind of fixed by genetic engineering
Pullulanase is transformed in point mutation method, to changing the method to interact between cyclodextrin and Pullulanase.
Background technology
The hydrolysis Propiram, soluble starch, branch of Pullulanase (Pullulanase, EC 3.2.1.41) specificity form sediment
α -1 in powder and corresponding oligosaccharide, 6 glycosidic bonds generate short linear dextrin.With being used in compounding for other amylase, can carry significantly
High-starch material utilization rate, therefore be widely used in starch industry.
In glucose industry, Pullulanase has the effect of hydrolyzing alpha -1,6 glycosidic bond, therefore with before wide application
Scape can be used for preparing amylose, maltose, branched cyclodextrin, and production cyclodextrin is compounded with CGTase.In the production of cyclodextrin
In the process, by taking the production of α-CD as an example, α -1 in starch substrates cannot be hydrolyzed due to α-CGTase, α-is used alone in 6 keys
When CGTase produces α-CD, conversion ratio only has 40%-60%.Addition Pullulanase can significantly improve the conversion ratio of cyclodextrin,
But Pullulanase is easy to be inhibited by cyclodextrin so that de- by-reaction must separate progress with cyclization, cause to produce
The problems such as period length, wasting of resources.In order to shorten the production cycle, starch utilization ratio of raw materials is improved, cyclodextrin yield is increased, is used
Pullulanase produces cyclodextrin with cyclodextrin glycosyltransferase compounding " one kettle way ", therefore reduces cyclodextrin to Pullulanase
Inhibition become urgent problem to be solved.
Marshall J J, Iwamoto H, Iwamoto H et al. study cyclodextrin from enzyme activity, kinetics etc.
To the inhibiting effect of Pullulanase, it was demonstrated that cyclodextrin is the competitive inhibitor of Pullulanase.In it is rich et al. to cyclodextrin to general
The inhibiting effect of Shandong orchid enzyme has made further research, on the basis of conventional zymologic property characterizes, has studied cyclodextrin to general
The influence of Shandong orchid enzyme endogenous fluorescence and secondary structure, it was demonstrated that between cyclodextrin hydrophobic cavity and Pullulanase aromatic amino acid
Clathration be cyclodextrin inhibit the active inner principles of Pullulanase.Correlative study both domestic and external is only conceived to ring paste
The repercussion study of essence and Pullulanase, does not propose specific effective solution means.
Inventor's early period is compared by the Pullulanase sequence analysis of separate sources, derives from bacillus subtilis
Pullulanase (the NCBI accession number of (Bacillus subtilis str.168)：AF008220.1) the crystalline substance combined with cyclodextrin
Body structure (PDB：It 2E8Z) has delivered, therefore selection carries out experiment for template from the Pullulanase of bacillus subtilis and sets
Meter.After the crystal structure combined with Pullulanase to cyclodextrin parses, find out what cyclodextrin was combined with Pullulanase
Amino acid sites carry out rationality mutation design to these sites, utilize the phase of molecular simulation software mimic mutant and cyclodextrin
Interaction, from calculate chemical terms confirm these key amino acids cyclodextrin combined with Pullulanase play it is most important
Effect.To provide foundation and reference to the inhibiting effect of Pullulanase to reduce cyclodextrin by sudden change means.
Invention content
In view of the above-mentioned problems existing in the prior art, the applicant provides a kind of reduction Pullulanases to cyclodextrin
The method of sensibility.The method of the present invention obtains Pullulan enzymatic mutant using rite-directed mutagenesis, hinders Pullulanase and cyclodextrin
Interaction, to improve starch utilization ratio of raw materials and beta-cyclodextrin yield.
Technical scheme is as follows：
A method of Pullulanase is reduced to cyclodextrin sensibility, and the method passes through to Pullulanase and cyclodextrin phase
The key amino acid of interaction carries out rationality mutation, and Pullulan enzymatic mutant is made, and changes between Pullulanase and cyclodextrin
Interaction, to reduce sensibility of the Pullulanase to cyclodextrin；
The key amino acid is that there are hydrophobic or hydrogen bond action amino acid between cyclodextrin.
The key amino acid is tryptophan, aspartic acid, arginine, asparagine, histidine.
Amino acid after the mutation is glycine, alanine, leucine, isoleucine, valine, proline, egg ammonia
Acid, serine, glutamine, threonine, cysteine, glutamic acid, lysine or tyrosine.
The detailed process of rationality mutation is：
(1) using Pullulanase gene as template, plasmid, construction of expression vector is selected to be made and carry Pullulanase gene
Plasmid；
(2) the key amino acid site that Pullulanase is combined with cyclodextrin is determined；
(3) mutant primer of design key amino acid, the plasmid to carry Pullulanase gene are mutated as template, system
Plasmid after must being mutated；
(4) plasmid after mutation is transferred to host strain, chooses positive monoclonal fermented and cultured, obtains Pullulan enzymatic mutant
The Pullulan enzymatic mutant is made by isolating and purifying in enzyme solution.
Plasmid described in step (1) is one kind in PMC series, pET series or pGEX series.
Host strain described in step (4) is gramnegative bacterium, gram-positive bacterium or fungi.
Host strain described in step (4) is Escherichia coli, bacillus subtilis or saccharomycete.
The method isolated and purified described in step (4) is affinity chromatography, hydrophobic chromatography, ultrafiltration chromatography or gel mistake
Filter chromatography.
The cyclodextrin is one or more in α-CD, β-CD, γ-CD.
The present invention is beneficial to be had technical effect that：
It is brilliant using the Pullulanase of known crystal structure as template by the sequence alignment of the Pullulanase to separate sources
Body structure elucidation obtains the related amino acid to interact with cyclodextrin in Pullulanase.The rationality for designing these amino acid is prominent
Become, cyclodextrin is docked in wild type and mutant by molecular docking software, by comparing cyclodextrin and enzyme affinity
Variation and enzyme activity compare, and show that the result of mutation design can effectively hinder the combination of cyclodextrin and Pullulanase, reduce general Shandong
Blue enzyme is to cyclodextrin sensibility, to provide foundation and ginseng to the inhibiting effect of Pullulanase to reduce cyclodextrin by mutation
It examines.
The present invention is based on the sequence alignments to enzyme and inhibitor crystallographic structural analysis and separate sources enzyme, find out Pullulanase
With the site of cyclodextrin interaction, analyze to obtain the Pu Lu for reducing cyclodextrin sensibility using rite-directed mutagenesis and molecular docking
Blue enzyme mutant, to improve starch raw material utilization rate and beta-cyclodextrin yield.
Description of the drawings
Fig. 1 is Pullulanase of the present invention and cyclodextrin crystal structure partial schematic diagram；
Fig. 2 is separate sources Pullulanase sequence alignment；
Fig. 3 is W437G mutant and cyclodextrin interaction schematic diagram；
Fig. 4 is H477A mutant and cyclodextrin interaction schematic diagram；
Fig. 5 is D465G mutant and cyclodextrin interaction schematic diagram；
Fig. 6 is that cyclodextrin characterizes the enzyme activity of wild type and W437G mutant inhibiting effect；
Fig. 7 is that cyclodextrin characterizes the enzyme activity of wild type and H477A mutant inhibiting effect；
Fig. 8 is that cyclodextrin characterizes the enzyme activity of wild type and D465G mutant inhibiting effect.
Specific implementation mode
With reference to the accompanying drawings and examples, the present invention is specifically described.
Fig. 1 is Pullulanase of the present invention and cyclodextrin complexes crystal structure partial schematic diagram.It can be seen from the figure that general
437 tryptophans, 465 aspartic acids, 468 leucines, 474 asparagines, 477 groups in the orchid enzyme of Shandong
The distance between propylhomoserin, 526 asparagines and cyclodextrin hydroxy groups oxygen be respectively 4.2A, 3.0A, 2.7A, 3.0A, 2.9A,
3.1A, there are strong hydrogen bonding interactions so that Pullulanase is combined closely with cyclodextrin, is become cyclodextrin and is inhibited Pullulanase
Architecture basics.
Fig. 2 is the partial sequence comparison chart of the Pullulanase of 60 kinds of separate sources, by comparing as can be seen that in the present invention
Tryptophan, leucine, aspartic acid absolute conservation in the Pullulanase of separate sources, histidine and asparagine are in difference
It is highly conserved in the Pullulanase in source.
Embodiment 1
A method of being mutated by rationality reduces Pullulanase to cyclodextrin sensibility, and the method includes walking as follows
Suddenly：
(1) with known cyclodextrin and Pullulanase interaction crystal structure derive from bacillus subtilis
Pullulanase (the PDB of (Bacillus subtilis str.168)：It is 2E8Z) template, the accession number on NCBI is
AF008220.1, using the fully synthetic method synthesis Pullulanase gene order AmyX of chemistry.For building Bacillus coli expression
The plasmid of carrier is pET20b (+), carries T7 promoters.
(2) pET20b (+) plasmid and plasmid containing AmyX genes are subjected to NcoI and BamI double digestions, digestion production respectively
It after object is tapped and recovered, then is connected with T4 ligases, connection product is converted to E.coli DH5 α competent cells, 37 DEG C of culture 8-
12h, shake culture in picking transformant to the LB liquid medium containing 100 μ g/mL ampicillins, extracts plasmid, digestion tests
Card obtains AmyX/Pet20b (+) expression vector.
(3) plasmid AmyX/Pet20b (+) is transformed into E.coli BL21 (DE3) host strain, is applied to the mould of benzyl containing ammonia
On the LB tablets of plain (100mg/mL), 37 DEG C of culture 8h are named as AmyX/Pet20b (+)/BL21 (DE3).Picking single bacterium drops down onto
In LB liquid medium, 37 DEG C are incubated overnight, and strain is preserved with glycerine.
(4) parsing obtains depositing with cyclodextrin on Pullulanase from the crystal structure of bacillus subtilis Pullulanase
In amino acid sites W437, D465, R468, N474, H477, N526 of interaction, pass through the Pullulanase to separate sources
Sequence alignment, W437, D465, R468, N474, H477, N526 key amino acid absolute conservation's N474, H477 key amino acid
It is highly conserved.
Mutant primer is designed, the absolute conservation in 437 sites and the tryptophan (W) with benzene ring structure are sported to minimum nothing
The glycine (G) of side chain, mutant primer is as shown in table 1 below；
Table 1
Note：Underscore is mutating alkali yl
Rite-directed mutagenesis is carried out using PCR, PCR reaction systems are：5 × Primer STAR GXL Buffer, 10 μ L；
DNTPMixture (2.5mM), 4 μ L；Forward primer (10 μM), 1.5 μ L；Reverse primer (10 μM), 1.5 μ L；Template DNA
(10ng/ μ L), 1 μ L；Distilled water is added to 50 μ L in Prime STAR GXL DNA Polymerase (1.25 μ/μ L), 1 μ L.
PCR amplification condition is：98 DEG C of pre-degeneration 3min then carry out 30 and recycle (98 DEG C of 20s, 60 DEG C of 30s, 68 DEG C of 6min), 68 DEG C
Continue to extend 10min.
PCR product is digested through Dpn I (Thermo Fisher companies), is transformed into bacillus coli DH 5 alpha competent cell, is felt
It is incubated overnight in LB solid mediums (containing 100 μ g/mL ampicillins) by state cell, picking monoclonal is in LB liquid medium
In (contain 100 μ g/mL ampicillins) culture, extract plasmid, correct plasmid be sequenced and converts to expressive host Escherichia coli
BL21(DE3)。
(5) picking is transferred to the positive monoclonal of expressive host e. coli bl21 (DE3) and (contains 100 μ in LB liquid medium
G/mL), 37 DEG C, 200rpm cultivates 8-12h, and inoculum concentration 5% is seeded to TB culture mediums (ampicillin for containing 100 μ g/mL)；
37 DEG C, 200rpm is cultivated to OD=0.6, the IPTG of final concentration 0.2mM is added, 25 DEG C, 160rpm induced expression 96h will ferment
4 DEG C of liquid, 10000g centrifuge 10Min, collect supernatant.
(6) by the ultra-filtration centrifuge tube of fermented supernatant fluid 10kDa, 4000g centrifuges 10Min, initial concentration separation.It will concentration
Zymotic fluid cross 0.22 μm of filter membrane, purified with nickel affinity chromatography, whole process of purification carries out under cryogenic.
Nickel affinity chromatography step：
1. balancing：With 20mM Tris-HCl, the buffer solution balance nickel column of 500mM NaCl, pH7.5；
2. loading：Pretreated sample is with the flow velocity loading of 1mL/min；
3. cleaning：With 20mM Tris-HCl, the buffer solution for cleaning foreign protein of 500mM NaCl, pH7.5；
4. eluting：Purpose egg is eluted with the buffer solution of 20mM Tris-HCl, 500mM NaCl, 300mM imidazoles, pH7.5
In vain, Detection wavelength 280nm is collected containing the active eluent of Pullulanase to get to the purifying enzyme of mutant W437G.
(7) Pullulanase enzyme activity determination uses 3,5- dinitrosalicylics acid system (DNS).Pullulanase is under certain conditions
Catalyzing hydrolysis pulullan polysaccharide generates reduced sugar, and 3,5- dinitrosalicylic acids restore under heat condition with reduced sugar and generate brownish red
Amido complex compound, shade is directly proportional to reduction sugar amount in a certain range, can be measured under 540nm wavelength, calculates enzyme activity.
Enzyme-activity unit defines：The enzyme amount that catalysis per minute generates the reduced sugar for being equivalent to 1 μm of ol glucose reducing power is defined as a work
Unit of force.
Enzyme activity determination step：
A. it preheats：It takes the pulullan polysaccharide solution (pH 6.0) of 1mL 1% in centrifuge tube, is placed in 45 DEG C of water-bath and protects
Warm 10min；
B. it reacts：The enzyme solution of 0.1mL is added, shakes mixing, accurate timing 30min is added 1.5mL DNS and terminates reaction,
Boiling water bath 10min, cools down immediately.
C. it measures：Light absorption value is surveyed under the conditions of 540nm and calculates vigor.
(8) molecular docking
Using Autodock vina 1.1.2 (http://vina.scripps.edu) software study wild type and prominent
The binding force of variant enzyme and cyclodextrin.Docking input file is generated with AutoDockTools 1.5.6 softwares, molecular docking
Parameter is：Center_x=126.601, center_y=72.661, center_z=127.473；Size_x=15, size_y
=15, size_z=15.When being docked with vina, default parameters, obtained best model pymol1.7.6 softwares are selected
It is analyzed.
The crystal structure to be interacted with known cyclodextrin and Pullulanase derives from bacillus subtilis (Bacillus
Subtilis str.168) Pullulanase (PDB：2E8Z) it is template, the accession number on NCBI is AF008220.1.It is logical
Cross the parsing to its crystal structure, obtain on Pullulanase with cyclodextrin exist interaction amino acid sites W437, D465,
R468, N474, H477, N526, by the sequence alignment of the Pullulanase to separate sources, W437, D465, R468, N474,
H477, N526 key amino acid absolute conservation's N474, H477 key amino acid are highly conserved.It selects absolute conservation and carries phenyl ring
Distance is that 437 tryptophans of 4.2A are mutated between structure, with cyclodextrin hydroxy groups oxygen, and mutating acid selects unprotected side chain
The minimum amino acids Glycine of structure.The schematic diagram that mutant is docked with cyclodextrin is as shown in Figure 3.Wild type Pullulanase and ring
Affinity between dextrin is -6.8kcal/mol, and the affinity between mutant and cyclodextrin is -6.7kcal/mol, can
To find out the reduction of the affinity between W437G mutant and cyclodextrin.In addition to this, between the glycine and cyclodextrin after mutation
Nearest distance is 6.0A, and distance becomes remote.It is this 2 points comprehensive, it may be said that bright Pullulanase reduces the sensibility of cyclodextrin, has
Theoretical foundation is provided to the inhibiting effect of Pullulanase conducive to cyclodextrin is reduced for mutation, and then can be used for reducing cyclodextrin pair
The inhibiting effect of Pullulanase, so as to improve starch raw material utilization rate and beta-cyclodextrin yield.
Sensibility of the Pullulanase to cyclodextrin further is characterized with enzyme activity, experimental result is listed in Fig. 6, by mutant and open country
The raw pure enzyme of type is compared, it can be found that：Three kinds of cyclodextrin are more aobvious than wild type Pullulanase to the inhibiting effect of mutant Pullulanase
Writing reduces, and has achieved the effect that reduce Pullulanase to cyclodextrin sensibility.
Embodiment 2
A method of being mutated by rationality reduces Pullulanase to cyclodextrin sensibility, and the method includes walking as follows
Suddenly：
(1) with known cyclodextrin and Pullulanase interaction crystal structure derive from bacillus subtilis
Pullulanase (the PDB of (Bacillus subtilis str.168)：It is 2E8Z) template, the accession number on NCBI is
AF008220.1, using the fully synthetic method synthesis Pullulanase gene order AmyX of chemistry.For building Bacillus coli expression
The plasmid of carrier is pET20b (+), carries T7 promoters.
(2) pET20b (+) plasmid and plasmid containing AmyX genes are subjected to NcoI and BamI double digestions, digestion production respectively
It after object is tapped and recovered, then is connected with T4 ligases, connection product is converted to E.coli DH5 α competent cells, 37 DEG C of culture 8-
12h, shake culture in picking transformant to the LB liquid medium containing 100 μ g/mL ampicillins, extracts plasmid, digestion tests
Card obtains AmyX/Pet20b (+) expression vector.
(3) plasmid AmyX/Pet20b (+) is transformed into E.coli BL21 (DE3) host strain, is applied to the mould of benzyl containing ammonia
On the LB tablets of plain (100mg/mL), 37 DEG C of culture 8h are named as AmyX/Pet20b (+)/BL21 (DE3).Picking single bacterium drops down onto
In LB liquid medium, 37 DEG C are incubated overnight, and strain is preserved with glycerine.
(4) parsing obtains depositing with cyclodextrin on Pullulanase from the crystal structure of bacillus subtilis Pullulanase
In amino acid sites W437, D465, R468, N474, H477, N526 of interaction, pass through the Pullulanase to separate sources
Sequence alignment, W437, D465, R468, N474, H477, N526 key amino acid absolute conservation's N474, H477 key amino acid
It is highly conserved.
Mutant primer is designed, it is minimum that the highly conserved of 477 sites and the histidine (H) with imidazole ring structure are sported band
The alanine (A) of side chain, mutant primer is as shown in table 2 below；
Table 2
Note：Underscore is mutating alkali yl
Rite-directed mutagenesis is carried out using PCR, PCR reaction systems are：5 × Primer STAR GXL Buffer, 10 μ L；
DNTPMixture (2.5mM), 4 μ L；Forward primer (10 μM), 1.5 μ L；Reverse primer (10 μM), 1.5 μ L；Template DNA
(10ng/ μ L), 1 μ L；Distilled water is added to 50 μ L in Prime STAR GXL DNA Polymerase (1.25 μ/μ L), 1 μ L.
PCR amplification condition is：98 DEG C of pre-degeneration 3min then carry out 30 and recycle (98 DEG C of 20s, 60 DEG C of 30s, 68 DEG C of 6min), 68 DEG C
Continue to extend 10min.
PCR product is digested through Dpn I (Thermo Fisher companies), is transformed into bacillus coli DH 5 alpha competent cell, is felt
It is incubated overnight in LB solid mediums (containing 100 μ g/mL ampicillins) by state cell, picking monoclonal is in LB liquid medium
In (contain 100 μ g/mL ampicillins) culture, extract plasmid, correct plasmid be sequenced and converts to expressive host Escherichia coli
BL21(DE3)。
(5) picking is transferred to the positive monoclonal of expressive host e. coli bl21 (DE3) and (contains 100 μ in LB liquid medium
G/mL), 37 DEG C, 200rpm cultivates 8-12h, and inoculum concentration 5% is seeded to TB culture mediums (ampicillin for containing 100 μ g/mL)；
37 DEG C, 200rpm is cultivated to OD=0.6, the IPTG of final concentration 0.2mM is added, 25 DEG C, 160rpm induced expression 96h will ferment
4 DEG C of liquid, 10000g centrifuge 10Min, collect supernatant.
(6) by the ultra-filtration centrifuge tube of fermented supernatant fluid 10kDa, 4000g centrifuges 10Min, initial concentration separation.It will concentration
Zymotic fluid cross 0.22 μm of filter membrane, purified with nickel affinity chromatography, whole process of purification carries out under cryogenic.
Nickel affinity chromatography step：
1. balancing：With 20mM Tris-HCl, the buffer solution balance nickel column of 500mM NaCl, pH7.5；
2. loading：Pretreated sample is with the flow velocity loading of 1mL/min；
3. cleaning：With 20mM Tris-HCl, the buffer solution for cleaning foreign protein of 500mM NaCl, pH7.5；
4. eluting：Purpose egg is eluted with the buffer solution of 20mM Tris-HCl, 500mM NaCl, 300mM imidazoles, pH7.5
In vain, Detection wavelength 280nm is collected containing the active eluent of Pullulanase to get to the purifying enzyme of mutant H477A.
(7) Pullulanase enzyme activity determination uses 3,5- dinitrosalicylics acid system (DNS).Pullulanase is under certain conditions
Catalyzing hydrolysis pulullan polysaccharide generates reduced sugar, and 3,5- dinitrosalicylic acids restore under heat condition with reduced sugar and generate brownish red
Amido complex compound, shade is directly proportional to reduction sugar amount in a certain range, can be measured under 540nm wavelength, calculates enzyme activity.
Enzyme-activity unit defines：The enzyme amount that catalysis per minute generates the reduced sugar for being equivalent to 1 μm of ol glucose reducing power is defined as a work
Unit of force.
Enzyme activity determination step：
A. it preheats：It takes the pulullan polysaccharide solution (pH 6.0) of 1mL 1% in centrifuge tube, is placed in 45 DEG C of water-bath and protects
Warm 10min；
B. it reacts：The enzyme solution of 0.1mL is added, shakes mixing, accurate timing 30min is added 1.5mL DNS and terminates reaction,
Boiling water bath 10min, cools down immediately.
C. it measures：Light absorption value is surveyed under the conditions of 540nm and calculates vigor.
(8) molecular docking
Using Autodock vina 1.1.2 (http://vina.scripps.edu) software study wild type and prominent
The binding force of variant enzyme and cyclodextrin.Docking input file is generated with AutoDockTools 1.5.6 softwares, molecular docking
Parameter is：Center_x=126.601, center_y=72.661, center_z=127.473；Size_x=15, size_y
=15, size_z=15.When being docked with vina, default parameters, obtained best model pymol1.7.6 softwares are selected
It is analyzed.
The crystal structure to be interacted with known cyclodextrin and Pullulanase derives from bacillus subtilis (Bacillus
Subtilis str.168) Pullulanase (PDB：2E8Z) it is template, the accession number on NCBI is AF008220.1.It is logical
Cross the parsing to its crystal structure, obtain on Pullulanase with cyclodextrin exist interaction amino acid sites W437, D465,
R468, N474, H477, N526, by the sequence alignment of the Pullulanase to separate sources, W437, D465, R468, N474,
H477, N526 key amino acid absolute conservation's N474, H477 key amino acid are highly conserved.It selects highly conserved and carries imidazoles
Distance is that 477 histidines of 2.9A are mutated between ring structure, with cyclodextrin hydroxy groups oxygen, and mutating acid selects side chain
The minimum amino acid alanine of structure.The schematic diagram that mutant is docked with cyclodextrin is as shown in Figure 4.Wild type Pullulanase and ring
Affinity between dextrin is -6.8kcal/mol, and the affinity between mutant and cyclodextrin is -6.7kcal/mol, can
To find out the reduction of the affinity between H477A mutant and cyclodextrin.In addition to this, between the alanine and cyclodextrin after mutation
Nearest distance is 5.3A, and distance becomes remote.It is this 2 points comprehensive, it may be said that bright Pullulanase reduces the sensibility of cyclodextrin, has
Theoretical foundation is provided to the inhibiting effect of Pullulanase conducive to cyclodextrin is reduced for mutation, and then can be used for reducing cyclodextrin pair
The inhibiting effect of Pullulanase, so as to improve starch raw material utilization rate and beta-cyclodextrin yield.
Sensibility of the Pullulanase to cyclodextrin further is characterized with enzyme activity, experimental result is listed in Fig. 7, by mutant and open country
The raw pure enzyme of type is compared, it can be found that：Three kinds of cyclodextrin are more aobvious than wild type Pullulanase to the inhibiting effect of mutant Pullulanase
Writing reduces, and has achieved the effect that reduce Pullulanase to cyclodextrin sensibility.
Embodiment 3
A method of being mutated by rationality reduces Pullulanase to cyclodextrin sensibility, and the method includes walking as follows
Suddenly：
(1) with known cyclodextrin and Pullulanase interaction crystal structure derive from bacillus subtilis
Pullulanase (the PDB of (Bacillus subtilis str.168)：It is 2E8Z) template, the accession number on NCBI is
AF008220.1, using the fully synthetic method synthesis Pullulanase gene order AmyX of chemistry.For building Bacillus coli expression
The plasmid of carrier is pET20b (+), carries T7 promoters.
(2) pET20b (+) plasmid and plasmid containing AmyX genes are subjected to NcoI and BamI double digestions, digestion production respectively
It after object is tapped and recovered, then is connected with T4 ligases, connection product is converted to E.coli DH5 α competent cells, 37 DEG C of culture 8-
12h, shake culture in picking transformant to the LB liquid medium containing 100 μ g/mL ampicillins, extracts plasmid, digestion tests
Card obtains AmyX/Pet20b (+) expression vector.
(3) plasmid AmyX/Pet20b (+) is transformed into E.coli BL21 (DE3) host strain, is applied to the mould of benzyl containing ammonia
On the LB tablets of plain (100mg/mL), 37 DEG C of culture 8h are named as AmyX/Pet20b (+)/BL21 (DE3).Picking single bacterium drops down onto
In LB liquid medium, 37 DEG C are incubated overnight, and strain is preserved with glycerine.
(4) parsing obtains depositing with cyclodextrin on Pullulanase from the crystal structure of bacillus subtilis Pullulanase
In amino acid sites W437, D465, R468, N474, H477, N526 of interaction, pass through the Pullulanase to separate sources
Sequence alignment, W437, D465, R468, N474, H477, N526 key amino acid absolute conservation's N474, H477 key amino acid
It is highly conserved.
Mutant primer is designed, the highly conserved of 465 sites and the aspartic acid (D) with side-chain structure are sported into unprotected side chain
Minimum amino acids Glycine (G), mutant primer is as shown in table 3 below；
Table 3
Note：Underscore is mutating alkali yl
Rite-directed mutagenesis is carried out using PCR, PCR reaction systems are：5 × Primer STAR GXL Buffer, 10 μ L；
DNTPMixture (2.5mM), 4 μ L；Forward primer (10 μM), 1.5 μ L；Reverse primer (10 μM), 1.5 μ L；Template DNA
(10ng/ μ L), 1 μ L；Distilled water is added to 50 μ L in Prime STAR GXL DNA Polymerase (1.25 μ/μ L), 1 μ L.
PCR amplification condition is：98 DEG C of pre-degeneration 3min then carry out 30 and recycle (98 DEG C of 20s, 60 DEG C of 30s, 68 DEG C of 6min), 68 DEG C
Continue to extend 10min.
PCR product is digested through Dpn I (Thermo Fisher companies), is transformed into bacillus coli DH 5 alpha competent cell, is felt
It is incubated overnight in LB solid mediums (containing 100 μ g/mL ampicillins) by state cell, picking monoclonal is in LB liquid medium
In (contain 100 μ g/mL ampicillins) culture, extract plasmid, correct plasmid be sequenced and converts to expressive host Escherichia coli
BL21(DE3)。
(5) picking is transferred to the positive monoclonal of expressive host e. coli bl21 (DE3) and (contains 100 μ in LB liquid medium
G/mL), 37 DEG C, 200rpm cultivates 8-12h, and inoculum concentration 5% is seeded to TB culture mediums (ampicillin for containing 100 μ g/mL)；
37 DEG C, 200rpm is cultivated to OD=0.6, the IPTG of final concentration 0.2mM is added, 25 DEG C, 160rpm induced expression 96h will ferment
4 DEG C of liquid, 10000g centrifuge 10Min, collect supernatant.
(6) by the ultra-filtration centrifuge tube of fermented supernatant fluid 10kDa, 4000g centrifuges 10Min, initial concentration separation.It will concentration
Zymotic fluid cross 0.22 μm of filter membrane, purified with nickel affinity chromatography, whole process of purification carries out under cryogenic.
Nickel affinity chromatography step：
1. balancing：With 20mM Tris-HCl, the buffer solution balance nickel column of 500mM NaCl, pH7.5；
2. loading：Pretreated sample is with the flow velocity loading of 1mL/min；
3. cleaning：With 20mM Tris-HCl, the buffer solution for cleaning foreign protein of 500mM NaCl, pH7.5；
4. eluting：Purpose egg is eluted with the buffer solution of 20mM Tris-HCl, 500mM NaCl, 300mM imidazoles, pH7.5
In vain, Detection wavelength 280nm is collected containing the active eluent of Pullulanase to get to the purifying enzyme of mutant D465G.
(7) Pullulanase enzyme activity determination uses 3,5- dinitrosalicylics acid system (DNS).Pullulanase is under certain conditions
Catalyzing hydrolysis pulullan polysaccharide generates reduced sugar, and 3,5- dinitrosalicylic acids restore under heat condition with reduced sugar and generate brownish red
Amido complex compound, shade is directly proportional to reduction sugar amount in a certain range, can be measured under 540nm wavelength, calculates enzyme activity.
Enzyme-activity unit defines：The enzyme amount that catalysis per minute generates the reduced sugar for being equivalent to 1 μm of ol glucose reducing power is defined as a work
Unit of force.
Enzyme activity determination step：
A. it preheats：It takes the pulullan polysaccharide solution (pH 6.0) of 1mL 1% in centrifuge tube, is placed in 45 DEG C of water-bath and protects
Warm 10min；
B. it reacts：The enzyme solution of 0.1mL is added, shakes mixing, accurate timing 30min is added 1.5mL DNS and terminates reaction,
Boiling water bath 10min, cools down immediately.
C. it measures：Light absorption value is surveyed under the conditions of 540nm and calculates vigor.
(8) molecular docking
Using Autodock vina 1.1.2 (http://vina.scripps.edu) software study wild type and prominent
The binding force of variant enzyme and cyclodextrin.Docking input file is generated with AutoDockTools 1.5.6 softwares, molecular docking
Parameter is：Center_x=126.601, center_y=72.661, center_z=127.473；Size_x=15, size_y
=15, size_z=15.When being docked with vina, default parameters, obtained best model pymol1.7.6 softwares are selected
It is analyzed.
The crystal structure to be interacted with known cyclodextrin and Pullulanase derives from bacillus subtilis (Bacillus
Subtilis str.168) Pullulanase (PDB：2E8Z) it is template, the accession number on NCBI is AF008220.1.It is logical
Cross the parsing to its crystal structure, obtain on Pullulanase with cyclodextrin exist interaction amino acid sites W437, D465,
R468, N474, H477, N526, by the sequence alignment of the Pullulanase to separate sources, W437, D465, R468, N474,
H477, N526 key amino acid absolute conservation's N474, H477 key amino acid are highly conserved.It selects absolute conservation and carries side chain
Distance is that 465 aspartic acids of 3.0A are mutated between structure, with cyclodextrin hydroxy groups oxygen, and mutating acid is selected without side
The minimum amino acids Glycine of chain structure.The schematic diagram that mutant is docked with cyclodextrin is as shown in Figure 5.Wild type Pullulanase with
Affinity between cyclodextrin is -6.8kcal/mol, and the affinity between mutant and cyclodextrin is -6.7kcal/mol,
It can be seen that the affinity between D465G mutant and cyclodextrin reduces.In addition to this, the alanine after mutation and cyclodextrin it
Between nearest distance be 5.4A, distance becomes remote.It is this 2 points comprehensive, it may be said that bright Pullulanase reduces the sensibility of cyclodextrin,
Be conducive to reduces cyclodextrin for mutation provides theoretical foundation to the inhibiting effect of Pullulanase, and then can be used for reducing cyclodextrin
To the inhibiting effect of Pullulanase, so as to improve starch raw material utilization rate and beta-cyclodextrin yield.
Sensibility of the Pullulanase to cyclodextrin further is characterized with enzyme activity, experimental result is listed in Fig. 8, by mutant and open country
The raw pure enzyme of type is compared, it can be found that：Three kinds of cyclodextrin are more aobvious than wild type Pullulanase to the inhibiting effect of mutant Pullulanase
Writing reduces, and has achieved the effect that reduce Pullulanase to cyclodextrin sensibility.
Claims (9)
1. a kind of Pullulanase that reduces is to the method for cyclodextrin sensibility, it is characterised in that the method by Pullulanase with
The key amino acid of cyclodextrin interaction carries out rationality mutation, and Pullulan enzymatic mutant is made, and changes Pullulanase and is pasted with ring
Interaction between essence, to reduce sensibility of the Pullulanase to cyclodextrin；
The key amino acid is that there are hydrophobic or hydrogen bond action amino acid between cyclodextrin.
2. according to the method described in claim 1, it is characterized in that the key amino acid is tryptophan, aspartic acid, smart ammonia
Acid, asparagine, histidine.
3. according to the method described in claim 1, it is characterized in that amino acid after the mutation be glycine, it is alanine, bright
Propylhomoserin, valine, proline, methionine, serine, glutamine, threonine, cysteine, glutamic acid, relies isoleucine
Propylhomoserin or tyrosine.
4. according to the method described in claim 1, it is characterized in that the detailed process of rationality mutation is：
(1) using Pullulanase gene as template, select plasmid, construction of expression vector that the plasmid for carrying Pullulanase gene is made；
(2) the key amino acid site that Pullulanase is combined with cyclodextrin is determined；
(3) mutant primer of design key amino acid, the plasmid to carry Pullulanase gene are mutated as template, are made prominent
Plasmid after change；
(4) plasmid after mutation is transferred to host strain, chooses positive monoclonal fermented and cultured, obtains Pullulan enzymatic mutant enzyme
The Pullulan enzymatic mutant is made by isolating and purifying in liquid.
5. according to the method described in claim 4, it is characterized in that plasmid described in step (1) be PMC series, pET series or
One kind in pGEX series.
6. according to the method described in claim 4, it is characterized in that host strain described in step (4) be gramnegative bacterium,
Gram-positive bacterium or fungi.
7. according to the method described in claim 4, it is characterized in that host strain described in step (4) is Escherichia coli, withered grass bud
Spore bacillus or saccharomycete.
8. according to the method described in claim 4, it is characterized in that the method isolated and purified described in step (4) is affinity chromatography
Method, hydrophobic chromatography, ultrafiltration chromatography or gel-filtration chromatography.
9. according to the method described in claim 1, it is characterized in that the cyclodextrin be α-CD, β-CD, one kind in γ-CD or
It is a variety of.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810181924.4A CN108396019B (en) | 2018-03-01 | 2018-03-01 | Method for reducing sensitivity of pullulanase to cyclodextrin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810181924.4A CN108396019B (en) | 2018-03-01 | 2018-03-01 | Method for reducing sensitivity of pullulanase to cyclodextrin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108396019A true CN108396019A (en) | 2018-08-14 |
| CN108396019B CN108396019B (en) | 2021-08-03 |
Family
ID=63091857
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810181924.4A Active CN108396019B (en) | 2018-03-01 | 2018-03-01 | Method for reducing sensitivity of pullulanase to cyclodextrin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108396019B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112941056A (en) * | 2021-02-24 | 2021-06-11 | 长春大学 | Starch pullulanase mutant and application thereof |
| CN114934035A (en) * | 2022-04-06 | 2022-08-23 | 江西省科学院微生物研究所(江西省流域生态研究所) | Thermophilic acidic III type pullulanase mutant with improved starch degradation capability as well as preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008153805A2 (en) * | 2007-05-30 | 2008-12-18 | Danisco Us, Inc., Genencor Division | Improved variants of the bacillus licheniformis alpha-amylase |
| CN105219746A (en) * | 2015-10-14 | 2016-01-06 | 江南大学 | A kind of cyclomaltodextrin glucanotransferase mutant suppressing to weaken by beta-cyclodextrin |
-
2018
- 2018-03-01 CN CN201810181924.4A patent/CN108396019B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008153805A2 (en) * | 2007-05-30 | 2008-12-18 | Danisco Us, Inc., Genencor Division | Improved variants of the bacillus licheniformis alpha-amylase |
| CN105219746A (en) * | 2015-10-14 | 2016-01-06 | 江南大学 | A kind of cyclomaltodextrin glucanotransferase mutant suppressing to weaken by beta-cyclodextrin |
Non-Patent Citations (1)
| Title |
|---|
| 于博等: "环糊精及其衍生物对普鲁兰酶抑制机理的研究", 《食品工业科技》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112941056A (en) * | 2021-02-24 | 2021-06-11 | 长春大学 | Starch pullulanase mutant and application thereof |
| CN112941056B (en) * | 2021-02-24 | 2022-11-18 | 长春大学 | Starch pullulanase mutant and application thereof |
| CN114934035A (en) * | 2022-04-06 | 2022-08-23 | 江西省科学院微生物研究所(江西省流域生态研究所) | Thermophilic acidic III type pullulanase mutant with improved starch degradation capability as well as preparation method and application thereof |
| CN114934035B (en) * | 2022-04-06 | 2023-03-28 | 江西省科学院微生物研究所(江西省流域生态研究所) | Thermophilic acidic III type pullulanase mutant with improved starch degradation capacity as well as preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108396019B (en) | 2021-08-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108384770B (en) | A method of cyclodextrin is reduced to Pullulanase inhibiting effect | |
| CN108034645B (en) | Preparation and application of cyclodextrin glucosyltransferase mutant | |
| CN103484443B (en) | A kind of Pullulan enzymatic mutant and preparation method thereof | |
| Cheng et al. | High-level extracellular production of α-cyclodextrin glycosyltransferase with recombinant Escherichia coli BL21 (DE3) | |
| CN102120971B (en) | Pullulanase-producing bacterium, heat-resisting pullulanase produced from same, and coding gene of pullulanase-producing bacterium | |
| CN108018268B (en) | Cyclodextrin glucosyltransferase mutant for improving AA-2G yield | |
| CN104531629B (en) | A kind of yclodextrin glycosyltransferase mutant of raising AA 2G conversion ratios | |
| Duan et al. | Enhancing the cyclodextrin production by synchronous utilization of isoamylase and α-CGTase | |
| CN108486080B (en) | Cyclodextrin glucosyltransferase and preparation method thereof | |
| CN102994468B (en) | Cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and preparation method thereof | |
| CN108531466B (en) | Cyclodextrin glucosyltransferase with improved product specificity and preparation method thereof | |
| CN102676557B (en) | Encoding gene of type I pullulanase as well as recombinant expression and application thereof | |
| Wu et al. | Catalytic hydrolysis of starch for biohydrogen production by using a newly identified amylase from a marine bacterium Catenovulum sp. X3 | |
| CN108396019A (en) | A method of Pullulanase is reduced to cyclodextrin sensibility | |
| CN110157688A (en) | A kind of linear maltooligosacchaeides generation enzyme mutant for producing maltopentaose ability and improving | |
| CN109825489A (en) | A kind of beta amylase and its application with hypersecretion ability | |
| Ren et al. | Purification and characterization of a highly selective sucrose isomerase from Erwinia rhapontici NX-5 | |
| Duan et al. | Expression, biochemical and structural characterization of high-specific-activity β-amylase from Bacillus aryabhattai GEL-09 for application in starch hydrolysis | |
| CN112980906A (en) | Enzyme composition for preparing beta-nicotinamide mononucleotide and application thereof | |
| CN108753746B (en) | Maltooligosyl trehalose synthase mutant with improved thermal stability | |
| CN113025590A (en) | Optimization method for improving secretion expression of cyclodextrin glucosyltransferase and application thereof | |
| CN110184259A (en) | A kind of anaerobic spore-bearing bacilli source Pullulan enzymatic mutant and its application | |
| Ara et al. | A CGTase with high coupling activity using γ-cyclodextrin isolated from a novel strain clustering under the genus Carboxydocella | |
| CN103966180A (en) | Method for improving cyclization activity of cyclodextrin glucosyltransferase | |
| CN111944784B (en) | Maltogenic oligosaccharide based seaweed hydrolase mutant with improved heat stability and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |