CN108913677B - Site-directed mutagenesis modified alkaline pullulanase and application thereof - Google Patents

Site-directed mutagenesis modified alkaline pullulanase and application thereof Download PDF

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CN108913677B
CN108913677B CN201810813706.8A CN201810813706A CN108913677B CN 108913677 B CN108913677 B CN 108913677B CN 201810813706 A CN201810813706 A CN 201810813706A CN 108913677 B CN108913677 B CN 108913677B
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CN108913677A (en
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林娟
林云
王国增
许鑫琦
黄晗
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Fuzhou University
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    • 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
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    • C12N9/2405—Glucanases
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    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)

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Abstract

The invention provides a fixed point processThe invention discloses a modified alkaline pullulanase and application thereof, and the modified alkaline pullulanase with site-specific mutation is obtained by mutating 744 th phenylalanine to alanine on the basis of amino acid shown in SEQ ID NO. 2. The mutant F744A is transformed into escherichia coli for heterologous expression, and the enzyme activity of the mutant is improved by 32.18 percent; the thermal stability is also improved, T50 30The temperature is 55.84 ℃, which is increased by 2.94 ℃ compared with the original enzyme PulSL3 Delta C; the pH stability range of the mutant F744A is wider, and more than 90% of enzyme activity can still be kept after heat preservation for 1 hour within the pH range of 5.0-10.0. The additive is applied to the detergent industry, and can obviously improve the washing effect.

Description

Site-directed mutagenesis modified alkaline pullulanase and application thereof
Technical Field
The invention relates to an alkaline pullulanase modified by site-directed mutagenesis and application thereof, belonging to the field of genetic engineering and enzyme engineering.
Background
Pullulanase can specifically cut alpha-1, 6 glycosidic bonds of starch, thereby obtaining amylose. The starch processing method is applied to the starch processing industry, and can obviously improve the utilization rate and the production efficiency of the starch. In the feed industry, at present, corn-soybean meal type daily ration is mainly used as a raw material for producing feed, but corn amylopectin is easy to form an anti-nutritional factor which is difficult to be digested by animal intestinal tracts, so that young animals cannot well absorb and utilize the feed and can also form intestinal diseases, and therefore, the pullulanase is added into the feed, the digestion, absorption and utilization of nutrient substances in the feed by the animals can be promoted, and the economic benefit is improved. The pullulanase is used as an enzyme preparation with wide application, not only can be applied to starch deep processing and feed industry, but also can be used as an effective additive to be applied to detergent industry. However, the natural strains separated from the nature have low pullulanase secretion capacity and are difficult to meet the conditions of industrial production, so that the industrial application of the natural strains is limited. Site-directed mutagenesis is an effective way for molecular modification, and is widely applied to the aspects of improving the catalytic efficiency, enzyme properties and the like of industrial enzyme preparations.
The invention utilizes a semi-rational design based on the combination of structural analysis and sequence analysis, and mutates the amino acid of the specific site of alkaline pullulanase PulSL3 delta C by a site-specific mutagenesis method to obtain a mutant with obviously improved enzyme activity and stability, and has important significance for improving the industrial application of pullulanase.
Disclosure of Invention
The invention aims to provide an alkaline pullulanase modified by site-directed mutagenesis and application thereof.
In order to realize the purpose, the following technical scheme is adopted:
the invention adopts a PCR method to obtain the source of the saline-alkali lakeAlkalibacteriumCloning from sp, SL3 strain to obtain alkaline pullulanase genepulSL3△CThe nucleotide sequence is shown as SEQ ID NO.1, the full length is 3555bp, polypeptide consisting of 1184 amino acids is coded, the sequence is shown as SEQ ID NO.2, and no signal peptide sequence exists.
Performing homologous modeling on an alkaline pullulanase PulSL3 delta C3D structure by using online software SWISS MODEL, performing online optimization on the MODEL structure by using a Chiron server, and detecting and evaluating a final structure subjected to online optimization by using methods such as PROCHECK, ERRAT, Verify-3D and the like. Mutating 744-th phenylalanine to alanine on the basis of the amino acid shown in SEQ ID NO.2 by utilizing a semi-rational design based on the combination of structural analysis and sequence analysis to obtain a mutant F744A; constructing the recombinant plasmid pET-22b (+) -F744ATransformation of host cellsE. coliBL21(DE3) to obtain genetically engineered bacteria.
The invention has the advantages that:
the invention obtains a genetically engineered bacterium with obviously improved enzyme activity and stability by modifying the molecular structure of alkaline pullulanase PulSL3 delta C by a site-directed mutagenesis method. The enzyme activity of the mutant F744A is improved by 32.18 percent, the heat stability is also improved, and T50 30The temperature is 55.84 ℃, which is increased by 2.94 ℃ compared with the original enzyme PulSL3 Delta C; the pH stability range is widened to 5.0-10.0, and more than 90% of enzyme activity can still be kept after 1 h of heat preservation (the original enzyme PulSL3 delta C can be kept after 1 h of heat preservation within the range of pH 7.0-9.0More than 80% of enzyme activity is remained).
The alkaline pullulanase is added into the detergent as an additive, so that the washing effect can be obviously improved, and the decontamination rate of composite dirt is improved by 14.21%.
Drawings
FIG. 1 shows the partial 3D structure of alkaline pullulanase PulSL3 Δ C.
FIG. 2 shows the recombinant plasmid pET-22b (+) -pulSL3△CAnd (4) constructing.
FIG. 3 shows the optimum reaction pH of mutant F744A with the original enzyme PulSL3 Δ C.
FIG. 4 shows the pH stability of mutant F744A with the original enzyme PulSL3 Δ C.
FIG. 5 shows the optimum reaction temperature of mutant F744A with the original enzyme PulSL3 Δ C.
FIG. 6 shows the thermostability of mutant F744A with the original enzyme PulSL3 Δ C.
Detailed Description
Example 1 construction of pullulanase mutant F744A
According to the 3D structural analysis (figure 1) of alkaline pullulanase PulSL3 Δ C, the 744-th phenylalanine causes a wider crack and a sharp bulge, and the structure can inhibit the hydrolysis of the second alpha-1, 6 glycosidic bond of pullulanase by the enzyme, and the hydrolysis inhibition effect on dense branched saccharides such as amylopectin, glycogen and the like is more serious. The side chain of the phenylalanine is a benzene ring with larger steric hindrance, so that the crack and the bulge are generated, and if the Phe744 is mutated into the alanine with the side chain being methyl, namely Ala744, the steric hindrance generated by the benzene ring can be greatly reduced. This mutant was designated F744A.
Design the primer containing the mutation site to recombine the plasmid pET-22b (+) -pulSL3△C(FIG. 2) as a template, the modified oligonucleotide primer-mediated site-directed mutagenesis method was used to construct an expression plasmid pET-22b (+) -containing a mutated geneF744ATransformation ofE. coliBL21(DE3) competent cells to obtain mutant recombinant engineering bacteria.
A forward primer: 5' -AATCCGGAGCCGGCAGTGAGGGAGAACC-3’；
Reverse primer: 5' -TCACTGCCGGCTCCGGATTTCAGTTCATTTCTG-3’；
Underlined bases are the sites of site-directed mutagenesis.
PCR amplification System: pET-22b (+) -pulSL3△C 1 μL，F744A-F (20 µmol/L) 1 μL，F744A-R (20 µmol/L) 1 μL，PCR Stimulant (5×) 5 μL，High Pure dNTPs(2.5 mmol/L) 4 μL，TransStart FastPfu Fly DNA Polymerase 1 μL，5×TransStart FastPfu Fly Buffer 10 μL，MgSO4 1 μL，ddH2And O is supplemented to 50 mu L.
PCR amplification conditions: pre-denaturation at 98 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 30 s, extension at 72 ℃ for 5 min, and 30 cycles; keeping the temperature at 72 ℃ for 5 min; storing at 10 deg.C. The PCR amplification product was detected by electrophoresis on a 1% agarose gel and the correct band was detected.
After completion of PCR, 1. mu.L of DMT was added to 50. mu.L of PCR amplification product, mixed well, and digested at 37 ℃ for 1 hour. Taking 1 μ L of the digestion product for transformationE.coliTop10, spread on LB solid plates containing 100. mu.g/mL Amp, cultured overnight. Selecting single colony, inoculating to LB liquid culture medium containing Amp, sucking partial bacterial liquid, detecting, extracting mutation plasmid with correct sequencing by plasmid extraction kit, and convertingE.coliBL21(DE3) competent cells to obtain mutant F744A.
The sequencing result of the mutant nucleotide sequence is shown as SEQ ID NO.3 in the sequence table, and the corresponding encoded protein amino acid sequence is shown as SEQ ID NO.4 in the sequence table.
Example 2 inducible expression and purification of pullulanase mutant F744A
Single colonies were picked from the transformation plates and inoculated into LB liquid medium containing 100. mu.g/mL Amp for overnight culture. Inoculating an overnight culture solution into 50 mL LB medium containing 100 mug/mL Amp according to 1% inoculation amount, and culturing at 37 ℃ and 180 rpm to OD600When the concentration reached 0.5, IPTG and 0.5% NaCl were added to the mixture to a final concentration of 0.1 mmol/L, and the mixture was cultured at 25 ℃ and 180 rpm for 32 hours. The fermentation broth was centrifuged at 13000 rpm for 10 min at 4 ℃ and the supernatant was taken as the crude enzyme solution.
The crude enzyme solution was first concentrated using a 10 kDa hollow fiber column and then subjected to (NH)4)2SO4Fractional precipitation, after dialysisAnd filtering the enzyme solution by a 0.22 mu m membrane to prepare a sample. First, DEAE FF (Hi Trap) as a pre-packed column was usedTM5 mL), the equilibrium buffer solution is a standard McIlvaine buffer solution with the pH value of 8.0, the elution buffer solution is a standard McIlvaine buffer solution (pH value of 8.0) dissolved with 1 mol/L NaCl, the linear gradient elution is carried out, the 280 nm ultraviolet on-line monitoring is carried out, and the protein eluent is collected in parts. The eluates containing the target protein and less impurities were mixed and dialyzed overnight against a standard McIlvaine buffer at pH 8.0 to obtain samples for further purification. (NH) was added to the sample4)2SO4The final concentration was adjusted to 1 mol/L, and the filtrate was filtered through a 0.22 μm membrane using a pre-packed column Phenyl FF (HS) (Hi Trap)TM5 mL) in an equilibrium buffer of 1 mol/L (NH)4)2SO4The standard McIlvaine buffer solution (pH 8.0) of (4), and the eluent is the standard McIlvaine buffer solution with pH 8.0, and the protein eluent is collected by sections. And (3) performing SDS-PAGE detection analysis on the enzyme solution to confirm a single band, thereby obtaining pure enzyme.
Example 3 determination of enzyme Activity and Properties of pullulanase mutant F744A
1. Enzyme activity assay of pullulanase mutant F744A
The pullulanase activity is determined by a DNS method. Incubating a mixture containing 0.9 mL of 0.5% pullulan (TCI) and 0.1 mL of enzyme solution under appropriate conditions for 10 min, adding 1.5 mL of DNS to terminate the reaction, boiling for 5 min to develop color, cooling, diluting to 10 mL with distilled water, and measuring OD540. Definition of enzyme activity unit: under certain reaction conditions, the amount of enzyme required to hydrolyze pullulan to produce 1. mu. mol of reducing sugar per minute is defined as one unit of enzyme activity U.
Compared with the original enzyme PulSL3 delta C, the enzyme activity of the pullulanase mutant F744A is improved by 32.18 percent.
2. Optimum reaction pH and pH stability of pullulanase mutant F744A
Determination of optimum reaction pH: and (2) placing the pullulanase in pullulanase solutions with the temperature of 45 ℃ and different pH values (4.0-12.0) for enzymatic reaction, setting the highest enzyme activity to be 100%, calculating the relative enzyme activity under different pH values, and determining the optimum reaction pH value. Wherein the buffer solution is: a standard McIlvaine buffer solution with the pH value of 4.0-8.0, a Tris-HCl buffer solution with the pH value of 8.0-10.0, and a Gly-NaOH buffer solution with the pH value of 10.0-12.0.
Determination of pH stability: the pullulanase is placed in buffer solutions with different pH values (4.0-12.0), the temperature is kept for 1 h at 37 ℃, the enzyme activity of the pullulanase is measured under the condition that the temperature is 45 ℃ and the optimum reaction pH value is 8.0, the measured highest enzyme activity is set as 100%, and the residual enzyme activity under different pH values is calculated.
The optimum reaction pH of the mutant F744A is 8.0, which is the same as that of the original enzyme PulSL3 Δ C, but F744A has a wider pH action range under acidic conditions (FIG. 3); pH stability experiments show that the enzyme activity of over 90 percent can still be kept after the F744A is kept for 1 hour within the pH range of 5.0-10.0, and the pH stability range of the F744A is wider than that of the original PulSL3 Δ C (the enzyme activity of over 80 percent can be kept after the F744A is kept for 1 hour within the pH range of 7.0-9.0) (figure 4).
3. Optimum reaction temperature and temperature stability of pullulanase mutant F744A
Measurement of optimum reaction temperature: the pullulanase is put into pullulanase solutions with the optimal reaction pH of 8.0 and different temperatures for enzymatic reaction, the measured highest enzyme activity is set as 100%, and the relative enzyme activities at different temperatures are calculated.
Thermal stability and T50 30The determination of (1): and (3) putting the pullulanase in water baths with different temperatures for heat preservation for 30 min, measuring the enzyme activity of the pullulanase under the optimal reaction conditions (pH 8.0 and 50 ℃), setting the activity of the untreated pullulanase as 100%, and calculating the residual enzyme activity at different temperatures. The residual enzyme activity of the enzyme is 50 percent when the enzyme is kept for 30 min at a certain temperature, namely the temperature is defined as T50 30(ii) a Fitting the data of the heat stability of the pullulanase by using Sigm oil Boltzmann fit of Origin 8.0 software to obtain the T of the pullulanase50 30。
The optimum reaction temperature of the mutant F744A is 50 ℃, which is the same as that of the original enzyme PulSL3 Δ C, and the relative enzyme activity of the mutant F744A is higher within the range of 30-40 ℃ (figure 5). The results of thermal stability experiments show that the mutant F744A and the original enzyme PulSL3 delta C are stable below 47 ℃, and compared with the original enzyme PulSL3 delta C, the mutant has the advantages of high stability and low costThe residual enzyme activities of the F744A at 50 deg.C, 52 deg.C and 55 deg.C were respectively improved by 6.36%, 15.13% and 41.16% (FIG. 6). T of mutant F744A can be obtained by calculation50 3055.84 ℃ which is improved by 2.94 ℃ compared with the original enzyme PulSL3 Δ C, and shows that the mutant F744A has better thermal stability.
Example 4 use of pullulanase in detergents
1. Manufacture of artificial dirty cloth
(1) 2% potato starch solution: preparing 2% potato starch water solution, and boiling for 10 min.
(2) Protein stain solution: dissolving 1.2 g of Arabic gum powder in a small amount of water, adding 1 mL of carbon ink, grinding for 2 min, transferring the dirty solution into a 60 mL glass cup containing 6.9 g of whole milk powder aqueous solution, adding 60 mL of distilled water, homogenizing for 15 min by an emulsifying machine, adding 60 mL of prepared aqueous solution containing 12.5 g of egg liquid (egg white: egg yolk =3: 2), and continuing to homogenize for 15 min.
(3) Grease dirt liquid: preparing the soybean oil and the Sudan black into oil stain liquid according to the mass ratio of 1000:0.5, and uniformly stirring.
(4) Artificially soiling cloth: mixing the prepared 2% potato starch solution, protein dirty solution and oil dirty solution at a volume ratio of 4:4:1, homogenizing for 15 min, uniformly coating the mixed dirty solution on cotton white cloth by a scraping method, and aging in an oven at 60 deg.C for 2 h.
2. Decontamination experiment
The method is carried out on a constant-temperature water bath shaker, and the general washing conditions are as follows: the washing temperature is 40 ℃, the pH value of water is the initial pH value, the rotating speed of a shaking table is 120 rpm, the washing time is 30 min, and the hardness of water is 250 mg/kg.
The prepared dirty cloth is cut into 5 cm multiplied by 5 cm square pieces, added into a decontamination solution of a laundry detergent with the mass concentration of 2 g/L and 300 mL prepared by hard water of 250 mg/kg, preheated for 30 min on a constant temperature water bath shaking bed at 40 ℃, and each experiment group means that each beaker contains 4 cloth pieces as parallels. And comparing the whiteness difference of the stained cloth before and after washing, and calculating the decontamination rate of the detergent.
Different enzyme preparations are added into a detergent containing 2 g/L laundry detergent, the addition amounts of alpha-amylase, alkaline pullulanase, alkaline protease and lipase are respectively 0.17U/mL, 0.74U/mL, 33.33U/mL and 72U/mL, and the decontamination effect of the complex dirt caused by the compounding of the alkaline pullulanase, the amylase, the alkaline protease and the lipase is researched. The used dirty cloth is composite dirty cloth, the whiteness is measured according to a protein dirty cloth whiteness measurement method, and the decontamination rate is calculated. The experimental results are shown in table 1, the addition of the alkaline pullulanase can effectively improve the decontamination effect of the composite dirt, and the decontamination rate is improved by 14.21%.
TABLE 1 Effect of pullulanase, amylase, lipase, and alkaline protease in combination for removing complex dirt
Figure DEST_PATH_IMAGE001
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
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Met Ala Asp Gly Gly Thr Ala Leu Leu Thr Val Asp Asn Gly Ser Leu
1 5 10 15
Ile Ala Glu Glu Ile Glu Thr Pro Asp Asp Ser Asp Glu Gln Leu Glu
20 25 30
Glu Thr Glu Val Glu Glu Gly Phe Phe Arg Ile His Phe Ala Ser Leu
35 40 45
Pro Ser Asp Asp Arg Glu Thr Leu Gly Leu Trp Ile Trp Asn Asp Val
50 55 60
Lys Glu Pro Ser Glu Asn Arg Gly Ala Trp Pro Asn Gly Ala Thr Ser
65 70 75 80
Phe Thr Glu Ala Val Gln Thr Asp Thr Gly Trp Thr Met Asp Ile Glu
85 90 95
Leu Glu Glu Asn Pro Gln Ser Ile Gly Phe Leu Ile Asn Ser Val Ser
100 105 110
Gly Asn Asn Leu Ser Gly Asp Ile Val Leu Arg Leu Leu Thr Ser Glu
115 120 125
Met Asn Gln Val Trp Leu Asp His Glu Thr Ala Met Thr Pro Thr Glu
130 135 140
Pro Leu Leu Glu Lys Asp Met Ile Arg Ile Asn Thr Lys Arg Asp Asn
145 150 155 160
Asn Asp Thr Asp Asp Trp Gly Leu Trp Thr Trp Asp Asp Val Ala Glu
165 170 175
Pro Thr Glu Asn Trp Pro Ala Gly Ala Gln Asp Ser Asp Gly Val Gly
180 185 190
Pro Asn Gly Thr Thr Phe Asn Leu Thr Leu Ala Glu Asp Ser Asp Gln
195 200 205
Ile Gly Phe Leu Phe Leu Asn Lys Ala Asp Gly Ser Gln Thr Arg Asp
210 215 220
Thr Thr Phe Ser Asn Leu Ser Ala His Ser Gln Leu Phe Met Arg Glu
225 230 235 240
Gly Asp Asp Thr Ile Thr Thr Asn Pro Thr Thr Val Ser Glu Ala Gly
245 250 255
Met Ile Arg Ala Glu Leu Ile Ser Glu Thr Glu Ile Glu Val Phe Phe
260 265 270
His Ser Thr Glu Gly Leu Glu Glu Ala Asp Leu Leu Glu Leu Ile Gln
275 280 285
Leu Thr Asp Ala Glu Gly Arg Asp Val Leu Phe Asp Ala Ala Val Asp
290 295 300
His Asp Arg Arg Val Val Ser Leu Thr Gly Asp Phe Ser Val Glu His
305 310 315 320
Ala Pro Thr Thr Val Thr Phe Asp Glu Thr Glu Ala Glu Ala Arg Met
325 330 335
Gly Trp Arg Leu Lys Asp Ala Leu Thr Ala Thr Asp Gly Glu Leu Gly
340 345 350
Leu Thr Phe Asn Glu Asp Gly Thr Ala Asp Leu Lys Val Trp Ser Pro
355 360 365
Ser Ala Asp Ala Val Thr Val Val Leu Thr Asp Lys Asp Asp Gln Thr
370 375 380
Val Val Val Arg Asp Asp Ile Glu Met Thr Ala Glu Glu Ser Gly Val
385 390 395 400
Trp Arg Val Val Leu Asp Glu Asp Thr Thr Gly Leu Asp Asp Val Thr
405 410 415
Gly Thr Phe Thr His Phe Ala Ile Glu Arg Asn Gly Glu Thr Val Leu
420 425 430
Ala Leu Asp Pro Thr Ala Arg Ser Met Ala Ala Trp Asn Ser Ser Asp
435 440 445
Pro Asp Asn Thr Ile Gly Lys Ala Ala Ile Val Asn Pro Ser Glu Ile
450 455 460
Gly Arg Glu Leu Asp Thr Ala Gln Ile Glu Gly Thr Asp Lys Arg Glu
465 470 475 480
Asp Ala Ile Ile Thr Glu Ile His Val Arg Asp Phe Thr Ser Asp Pro
485 490 495
Ser Ile Glu Asp Glu Leu Thr Ser Gln Phe Gly Thr Phe Ser Ala Phe
500 505 510
Ile Glu Lys Leu Asp Thr Ile Glu Ser Leu Gly Val Thr His Val Gln
515 520 525
Leu Leu Pro Val Met Ser Thr Phe Phe Ala Asn Glu Phe Glu Asn Ala
530 535 540
Glu Arg Met Leu Asp Thr Gly Ser Thr Gln Thr Asn Thr Asn Trp Gly
545 550 555 560
Thr Asp Pro Gln Ser Thr Phe Ser Leu Thr Gly Met Thr Ser Glu Asn
565 570 575
Pro Lys Asp Pro Ala Arg Arg Ile Glu Glu Phe Lys Asn Leu Ile Asp
580 585 590
Ala Ile His Ser His Gly Met Gly Val Ile Leu Asp Val Val Thr Asn
595 600 605
His Thr Ala Arg Glu His Ile Phe Glu Asp Leu Glu Pro Asn Thr Thr
610 615 620
His Phe Met Asp Ala Asp Gly Thr Ser Arg Thr Ser Phe Gly Gly Gly
625 630 635 640
Arg Leu Gly Thr Thr His Glu Met Ala Arg Arg Ile Leu Val Asp Ser
645 650 655
Ile Thr Thr Trp Val Glu Glu Thr Lys Val Asp Gly Phe Arg Phe Asp
660 665 670
Met Met Gly Asp His Asp Ala Glu Ser Ile Gln Met Ala Phe Asp Glu
675 680 685
Ala Gln Lys Leu Asn Pro Asn Ile Leu Met Ile Gly Glu Gly Trp Arg
690 695 700
Thr Phe Val Gly Asp Glu Gly Thr Glu Asp Val Met Pro Ala Asp Gln
705 710 715 720
Asp Trp Met Gln His Thr Gln Ala Val Gly Ser Phe Ser Asp Asp Phe
725 730 735
Arg Asn Glu Leu Lys Ser Gly Phe Gly Ser Glu Gly Glu Pro Arg Phe
740 745 750
Ile Thr Gly Gly Thr Arg Ser Ile Gln Arg Ile Phe Asp Asn Leu Thr
755 760 765
Ala Asn Pro His Asn Phe Met Ala Thr Asp Pro Gly Asp Val Val Pro
770 775 780
Thr Ile Ala Ala His Asp Asn Leu Thr Leu His Asp Val Ile Ala Gln
785 790 795 800
Ser Ile Gln Lys Asp Pro Glu Thr His Gln Glu Glu Ile His Gln Arg
805 810 815
Ile Arg Leu Gly Asn Leu Met Val Leu Thr Ser Gln Gly Thr Pro Phe
820 825 830
Val His Ala Gly Gln Glu Thr Gly Arg Thr Lys Gln Phe Arg Asp Pro
835 840 845
Asp Phe Ile Glu Pro Val Ala Asn Asp Gln Val Pro Thr Lys Ser Thr
850 855 860
Phe Met Thr Asp Glu Asp Gly Asn Pro Phe Leu Thr Pro Thr Phe Ile
865 870 875 880
His Asp Ser Thr Asp Ser Thr Asp Ala Val Asn Arg Phe Glu Trp Asp
885 890 895
Lys Val Thr Asp Ala Glu Ala Thr Pro Ile Asn Thr Gln Thr Gln Ser
900 905 910
Thr Thr Ser Gly Leu Ile Ala Leu Arg Arg Ser Thr Asp Ala Phe Ser
915 920 925
Lys Gly Thr Met Glu Glu Ile Ala Asp Met Val Ser Leu Val Asp Ala
930 935 940
Pro Glu Ile Glu Asp Glu Asp Leu Val Ile Val Thr Arg Ala Glu Asp
945 950 955 960
Ser Asn Gly Asp Arg Thr Thr Val Phe Val Asn Ala Asp Asp Ser Glu
965 970 975
Arg Thr Leu Thr Leu Asp Ser Asp Leu Thr Glu Gly His Val Leu Val
980 985 990
Asp Ser Gln Gln Ala Gly Thr Arg Ala Ile Ala Arg Pro Glu Gly Ile
995 1000 1005
Thr Val Glu Gln Ala Gly Val Thr Leu Ala Pro Leu Thr Ala Ser
1010 1015 1020
Val Val Leu Leu Thr Asp Arg Glu Ile Glu Pro Val Glu Glu Ser
1025 1030 1035
Asp Glu Asp Gly Asp Glu Gly Thr Asp Pro Gly Asn Gly Glu Gln
1040 1045 1050
Pro Gly Gly Glu Ser Gly Pro Gly Thr Asp Gln Gly Ser Asp Gly
1055 1060 1065
Asp Asp Pro Val Ser Gly Gly Glu Glu Thr Ala Asp Pro Glu Arg
1070 1075 1080
Asp Ala Glu Gly Asp Asp Thr Pro Glu Asp Asp Thr Asp Leu Ser
1085 1090 1095
Glu Asp Pro Gly Ala Gly Gln Asp Ser Gly Asp Ser Ile Ala Asp
1100 1105 1110
Gly Asp Gln Gly His Ser Asp Gly Pro Leu Asp Gly Pro Asp Gly
1115 1120 1125
Asp Glu Thr Gly Lys Ala Glu Gly Asp Ser Ser Glu Ser Gln Thr
1130 1135 1140
Gly Glu Gln Asn Gly Glu Arg Leu Pro Ser Thr Ala Thr Leu Leu
1145 1150 1155
Trp Thr Val Gly Ala Val Gly Leu Met Ser Leu Leu Thr Gly Val
1160 1165 1170
Val Val Arg Gln Ile Lys Lys Lys Asn Lys Thr
1175 1180
<210> 3
<211> 3555
<212> DNA
<213> Artificial sequence
<400> 3
atggcagacg gcggaacagc actcctgaca gtggataacg ggtcactgat tgcggaagaa 60
attgagacac cggatgattc agatgaacag ctagaagaaa ccgaggtgga agaaggattt 120
tttcgcatcc attttgcgtc gcttccgtca gatgatcgcg agactcttgg gctctggatc 180
tggaacgatg taaaagagcc ttctgaaaac agaggggcat ggccgaatgg ggcgacctca 240
tttacagagg ctgttcagac cgactacggc tggtatatgg atattgaatt ggaagagaat 300
ccacagtcga ttggcttcct tatcaattcc gtctcgggga acaatctgtc aggagatatc 360
gtcctgcggc ttctgacttc tgagatgaat caggtatggc ttgatcatga gtatgccatg 420
acaccttatg agcctttact tgaaaaggac atgatccgga tcaactacaa acgagacaac 480
aatgactacg atgactgggg cctctggacc tgggacgatg tggcagaacc aacagaaaac 540
tggcctgcag gcgctcagga cagcgatggc gtcgggccta acggaaccta ttttaatctt 600
acgctggcag aagattccga tcagatcggt tttctgttcc tgaataaagc tgatggttcc 660
cagacccggg attacacatt ctcgaacttg tccgctcaca gccagctatt tatgcgtgaa 720
ggagacgata ctatttatac caatccgtac tatgtcagtg aagccggcat gatccgagcc 780
gagctgattt ctgaaactga aatcgaagtg tttttccatt cgacagaagg actggaggag 840
gctgatctgc ttgaattaat tcagctgacc gatgcagaag gacgagacgt gctatttgat 900
gcggctgtcg atcatgaccg acgagtcgtt agtctgaccg gtgacttctc agtggagcat 960
gctccttata ccgtgacttt tgacgaaacg gaagcagaag cgcgaatggg ctggcgcctg 1020
aaggatgcgc tctatgccta tgacggagaa cttggtctga cttttaacga agacgggacc 1080
gctgatctga aagtctggtc tccaagtgcc gacgcagtga cggtcgtttt atatgataaa 1140
gatgatcaga ctgtcgttgt cagagatgat atcgaaatga ctgccgagga gtcaggcgtg 1200
tggcgtgttg ttcttgatga agacacgacc ggactggacg atgtgacggg gtacttctac 1260
cattttgcga ttgaaagaaa tggcgaaacc gtcctggcac tggatcctta tgcccgctca 1320
atggccgcat ggaacagcag tgatccggat aattacatcg gcaaagctgc catcgtgaat 1380
ccgagtgaga tcggtcggga actggattat gctcagatcg aaggctatga caagcgcgaa 1440
gatgcgatca tttatgaaat tcatgtcaga gacttcacat ccgatccgtc aattgaagat 1500
gaactgacca gtcagttcgg gacgttcagt gctttcattg aaaagctcga ttatatcgaa 1560
agtctgggtg tgacccatgt tcagctgctt ccggtcatga gctatttctt tgcaaatgaa 1620
ttcgaaaacg ccgaacggat gttggactac ggatcgactc agaccaacta taactggggt 1680
tacgatccgc agagctactt ctctctgacg ggaatgtatt cagaaaatcc aaaagacccg 1740
gcgagacgca ttgaagaatt caaaaatctg atcgatgcga ttcattccca cggcatgggt 1800
gtcattcttg atgtggtcta taatcacacg gcacgggagc acatttttga agatctggaa 1860
ccgaactatt accatttcat ggatgcggac ggcacgtcac gaacaagttt cggaggcggc 1920
cgactaggca ccactcatga gatggcccgc cgtattctgg tcgattcgat cacatactgg 1980
gtggaagaat ataaagttga cggcttccgc ttcgatatga tgggcgatca cgatgccgaa 2040
agcattcaga tggcatttga cgaagcacag aaactgaatc cgaatatcct gatgatcggt 2100
gagggctggc ggacctttgt cggtgacgaa ggctatgaag acgtcatgcc ggctgaccag 2160
gactggatgc agcacacaca agccgttgga tcgttctcgg atgacttcag aaatgaactg 2220
aaatccggag gcggcagtga gggagaaccg cgctttatta ccggaggcac acggtccatc 2280
caacgaattt ttgacaatct gacagccaat ccacataact ttatggcgac agatccgggc 2340
gatgtggtcc cttatatcgc ggcgcacgac aacctgacgc ttcatgatgt catcgctcaa 2400
agcattcaga aagatccgga ataccaccag gaagagatcc atcagcgtat ccgtctgggc 2460
aatctgatgg tgctgacttc tcagggcacg ccattcgtcc atgccggtca ggaatacgga 2520
cggaccaagc aattccgtga tcctgacttt atcgaacctg tagcaaacga tcaggttcct 2580
tacaagtcga cattcatgac agatgaagac gggaatccgt tcctttaccc gtatttcatt 2640
catgactcat acgattcaac ggatgcggtc aaccgttttg aatgggataa agtgacggat 2700
gctgaagctt atccgattaa tacgcagacc cagtcttaca catctggtct gattgcattg 2760
cggcgcagta cggatgcctt cagtaaagga acgatggaag agatcgcgga catggtgtcg 2820
ctagtggatg cgccggaaat cgaggacgaa gacctggtta tcgtctatcg tgcagaagat 2880
tccaatggcg atcgttacta cgtctttgtg aatgcagatg attccgaacg aacgctgaca 2940
cttgattctg atttgactga agggcacgtc ctggtcgata gtcagcaggc cggcacacga 3000
gcgatcgcca gaccagaagg catcacggtc gaacaggctg gtgtcacgct ggctcctttg 3060
acggcatccg tggttttact tacggataga gaaattgagc cggttgaaga gagtgacgaa 3120
gatggcgatg aaggaactga cccaggcaac ggggaacagc ctggtggaga atctggacca 3180
ggaacagatc agggatccga tggcgacgat cctgtctcag gtggcgaaga aacggctgat 3240
ccagaaagag atgccgaggg tgatgattat ccggaagacg acactgattt gtctgaggat 3300
cctggtgctg gtcaggatag tggagattcg atagctgatg gcgaccaagg tcattcagat 3360
ggtccactag atgggccgga tggtgatgaa acaggcaaag cggaaggcga ttcttctgaa 3420
tcccagactg gtgagcagaa cggagaaagg ctcccctcta cagcaaccct actctggact 3480
gtaggagcag tcggactgat gagcctcttg acaggggtag tggtcagaca gatcaaaaag 3540
aaaaataaga cataa 3555
<210> 4
<211> 1184
<212> PRT
<213> Artificial sequence
<400> 4
Met Ala Asp Gly Gly Thr Ala Leu Leu Thr Val Asp Asn Gly Ser Leu
1 5 10 15
Ile Ala Glu Glu Ile Glu Thr Pro Asp Asp Ser Asp Glu Gln Leu Glu
20 25 30
Glu Thr Glu Val Glu Glu Gly Phe Phe Arg Ile His Phe Ala Ser Leu
35 40 45
Pro Ser Asp Asp Arg Glu Thr Leu Gly Leu Trp Ile Trp Asn Asp Val
50 55 60
Lys Glu Pro Ser Glu Asn Arg Gly Ala Trp Pro Asn Gly Ala Thr Ser
65 70 75 80
Phe Thr Glu Ala Val Gln Thr Asp Thr Gly Trp Thr Met Asp Ile Glu
85 90 95
Leu Glu Glu Asn Pro Gln Ser Ile Gly Phe Leu Ile Asn Ser Val Ser
100 105 110
Gly Asn Asn Leu Ser Gly Asp Ile Val Leu Arg Leu Leu Thr Ser Glu
115 120 125
Met Asn Gln Val Trp Leu Asp His Glu Thr Ala Met Thr Pro Thr Glu
130 135 140
Pro Leu Leu Glu Lys Asp Met Ile Arg Ile Asn Thr Lys Arg Asp Asn
145 150 155 160
Asn Asp Thr Asp Asp Trp Gly Leu Trp Thr Trp Asp Asp Val Ala Glu
165 170 175
Pro Thr Glu Asn Trp Pro Ala Gly Ala Gln Asp Ser Asp Gly Val Gly
180 185 190
Pro Asn Gly Thr Thr Phe Asn Leu Thr Leu Ala Glu Asp Ser Asp Gln
195 200 205
Ile Gly Phe Leu Phe Leu Asn Lys Ala Asp Gly Ser Gln Thr Arg Asp
210 215 220
Thr Thr Phe Ser Asn Leu Ser Ala His Ser Gln Leu Phe Met Arg Glu
225 230 235 240
Gly Asp Asp Thr Ile Thr Thr Asn Pro Thr Thr Val Ser Glu Ala Gly
245 250 255
Met Ile Arg Ala Glu Leu Ile Ser Glu Thr Glu Ile Glu Val Phe Phe
260 265 270
His Ser Thr Glu Gly Leu Glu Glu Ala Asp Leu Leu Glu Leu Ile Gln
275 280 285
Leu Thr Asp Ala Glu Gly Arg Asp Val Leu Phe Asp Ala Ala Val Asp
290 295 300
His Asp Arg Arg Val Val Ser Leu Thr Gly Asp Phe Ser Val Glu His
305 310 315 320
Ala Pro Thr Thr Val Thr Phe Asp Glu Thr Glu Ala Glu Ala Arg Met
325 330 335
Gly Trp Arg Leu Lys Asp Ala Leu Thr Ala Thr Asp Gly Glu Leu Gly
340 345 350
Leu Thr Phe Asn Glu Asp Gly Thr Ala Asp Leu Lys Val Trp Ser Pro
355 360 365
Ser Ala Asp Ala Val Thr Val Val Leu Thr Asp Lys Asp Asp Gln Thr
370 375 380
Val Val Val Arg Asp Asp Ile Glu Met Thr Ala Glu Glu Ser Gly Val
385 390 395 400
Trp Arg Val Val Leu Asp Glu Asp Thr Thr Gly Leu Asp Asp Val Thr
405 410 415
Gly Thr Phe Thr His Phe Ala Ile Glu Arg Asn Gly Glu Thr Val Leu
420 425 430
Ala Leu Asp Pro Thr Ala Arg Ser Met Ala Ala Trp Asn Ser Ser Asp
435 440 445
Pro Asp Asn Thr Ile Gly Lys Ala Ala Ile Val Asn Pro Ser Glu Ile
450 455 460
Gly Arg Glu Leu Asp Thr Ala Gln Ile Glu Gly Thr Asp Lys Arg Glu
465 470 475 480
Asp Ala Ile Ile Thr Glu Ile His Val Arg Asp Phe Thr Ser Asp Pro
485 490 495
Ser Ile Glu Asp Glu Leu Thr Ser Gln Phe Gly Thr Phe Ser Ala Phe
500 505 510
Ile Glu Lys Leu Asp Thr Ile Glu Ser Leu Gly Val Thr His Val Gln
515 520 525
Leu Leu Pro Val Met Ser Thr Phe Phe Ala Asn Glu Phe Glu Asn Ala
530 535 540
Glu Arg Met Leu Asp Thr Gly Ser Thr Gln Thr Asn Thr Asn Trp Gly
545 550 555 560
Thr Asp Pro Gln Ser Thr Phe Ser Leu Thr Gly Met Thr Ser Glu Asn
565 570 575
Pro Lys Asp Pro Ala Arg Arg Ile Glu Glu Phe Lys Asn Leu Ile Asp
580 585 590
Ala Ile His Ser His Gly Met Gly Val Ile Leu Asp Val Val Thr Asn
595 600 605
His Thr Ala Arg Glu His Ile Phe Glu Asp Leu Glu Pro Asn Thr Thr
610 615 620
His Phe Met Asp Ala Asp Gly Thr Ser Arg Thr Ser Phe Gly Gly Gly
625 630 635 640
Arg Leu Gly Thr Thr His Glu Met Ala Arg Arg Ile Leu Val Asp Ser
645 650 655
Ile Thr Thr Trp Val Glu Glu Thr Lys Val Asp Gly Phe Arg Phe Asp
660 665 670
Met Met Gly Asp His Asp Ala Glu Ser Ile Gln Met Ala Phe Asp Glu
675 680 685
Ala Gln Lys Leu Asn Pro Asn Ile Leu Met Ile Gly Glu Gly Trp Arg
690 695 700
Thr Phe Val Gly Asp Glu Gly Thr Glu Asp Val Met Pro Ala Asp Gln
705 710 715 720
Asp Trp Met Gln His Thr Gln Ala Val Gly Ser Phe Ser Asp Asp Phe
725 730 735
Arg Asn Glu Leu Lys Ser Gly Phe Gly Ser Glu Gly Glu Pro Arg Phe
740 745 750
Ile Thr Gly Gly Thr Arg Ser Ile Gln Arg Ile Phe Asp Asn Leu Thr
755 760 765
Ala Asn Pro His Asn Ala Met Ala Thr Asp Pro Gly Asp Val Val Pro
770 775 780
Thr Ile Ala Ala His Asp Asn Leu Thr Leu His Asp Val Ile Ala Gln
785 790 795 800
Ser Ile Gln Lys Asp Pro Glu Thr His Gln Glu Glu Ile His Gln Arg
805 810 815
Ile Arg Leu Gly Asn Leu Met Val Leu Thr Ser Gln Gly Thr Pro Phe
820 825 830
Val His Ala Gly Gln Glu Thr Gly Arg Thr Lys Gln Phe Arg Asp Pro
835 840 845
Asp Phe Ile Glu Pro Val Ala Asn Asp Gln Val Pro Thr Lys Ser Thr
850 855 860
Phe Met Thr Asp Glu Asp Gly Asn Pro Phe Leu Thr Pro Thr Phe Ile
865 870 875 880
His Asp Ser Thr Asp Ser Thr Asp Ala Val Asn Arg Phe Glu Trp Asp
885 890 895
Lys Val Thr Asp Ala Glu Ala Thr Pro Ile Asn Thr Gln Thr Gln Ser
900 905 910
Thr Thr Ser Gly Leu Ile Ala Leu Arg Arg Ser Thr Asp Ala Phe Ser
915 920 925
Lys Gly Thr Met Glu Glu Ile Ala Asp Met Val Ser Leu Val Asp Ala
930 935 940
Pro Glu Ile Glu Asp Glu Asp Leu Val Ile Val Thr Arg Ala Glu Asp
945 950 955 960
Ser Asn Gly Asp Arg Thr Thr Val Phe Val Asn Ala Asp Asp Ser Glu
965 970 975
Arg Thr Leu Thr Leu Asp Ser Asp Leu Thr Glu Gly His Val Leu Val
980 985 990
Asp Ser Gln Gln Ala Gly Thr Arg Ala Ile Ala Arg Pro Glu Gly Ile
995 1000 1005
Thr Val Glu Gln Ala Gly Val Thr Leu Ala Pro Leu Thr Ala Ser
1010 1015 1020
Val Val Leu Leu Thr Asp Arg Glu Ile Glu Pro Val Glu Glu Ser
1025 1030 1035
Asp Glu Asp Gly Asp Glu Gly Thr Asp Pro Gly Asn Gly Glu Gln
1040 1045 1050
Pro Gly Gly Glu Ser Gly Pro Gly Thr Asp Gln Gly Ser Asp Gly
1055 1060 1065
Asp Asp Pro Val Ser Gly Gly Glu Glu Thr Ala Asp Pro Glu Arg
1070 1075 1080
Asp Ala Glu Gly Asp Asp Thr Pro Glu Asp Asp Thr Asp Leu Ser
1085 1090 1095
Glu Asp Pro Gly Ala Gly Gln Asp Ser Gly Asp Ser Ile Ala Asp
1100 1105 1110
Gly Asp Gln Gly His Ser Asp Gly Pro Leu Asp Gly Pro Asp Gly
1115 1120 1125
Asp Glu Thr Gly Lys Ala Glu Gly Asp Ser Ser Glu Ser Gln Thr
1130 1135 1140
Gly Glu Gln Asn Gly Glu Arg Leu Pro Ser Thr Ala Thr Leu Leu
1145 1150 1155
Trp Thr Val Gly Ala Val Gly Leu Met Ser Leu Leu Thr Gly Val
1160 1165 1170
Val Val Arg Gln Ile Lys Lys Lys Asn Lys Thr
1175 1180
<210> 5
<211> 28
<212> DNA
<213> Artificial sequence
<400> 5
aatccggagc cggcagtgag ggagaacc 28
<210> 6
<211> 33
<212> DNA
<213> Artificial sequence
<400> 6
tcactgccgg ctccggattt cagttcattt ctg 33

Claims (5)

1. An alkaline pullulanase modified by site-directed mutagenesis, which is characterized in that: the alkaline pullulanase is obtained by mutating 744 th phenylalanine to alanine on the basis of the amino acid shown in SEQ ID NO. 2.
2. The gene for coding the site-directed mutagenesis modified alkaline pullulanase of claim 1, the sequence of which is shown in SEQ ID NO. 3.
3. A vector comprising the gene of claim 2.
4. A genetically engineered bacterium comprising the vector of claim 3.
5. Use of the site-directed mutagenesis modified alkaline pullulanase of claim 1 in the preparation of a detergent.
CN201810813706.8A 2018-07-23 2018-07-23 Site-directed mutagenesis modified alkaline pullulanase and application thereof Active CN108913677B (en)

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JP3025625B2 (en) * 1995-05-10 2000-03-27 花王株式会社 Alkaline pullulanase gene having alkaline α-amylase activity
JP4372986B2 (en) * 2000-10-03 2009-11-25 花王株式会社 Alkaline pullulanase
AR069167A1 (en) * 2007-11-05 2010-01-06 Danisco Us Inc Genencor Div ALPHA-AMYLASE VARIANTS OF BACILLUS LICHENIFORMIS WITH INCREASED THERMOSTABILITY AND / OR DEPENDENCE ON DECREASED CALCIUM
CN102796751B (en) * 2012-08-21 2013-06-19 广西科学院 Mutant Pul 324 of pullulanibacillus naganoensis pullulanase and use thereof
CN103194434B (en) * 2013-04-15 2014-08-13 山东天力药业有限公司 Novel sulfolobus solfataricus trehalose hydrolase, gene of hydrolase, recombinant expression vector containing gene, and recombinant bacterium, and preparation of hydrolase
CN104357427A (en) * 2014-11-11 2015-02-18 福州大学 High-temperature-resistant alkaline salt-tolerant xylanase XynSL4 as well as gene and application thereof
ES2903092T3 (en) * 2015-07-21 2022-03-31 Novozymes As Polypeptides having pullulanase activity suitable for use in liquefaction
WO2017139697A1 (en) * 2016-02-10 2017-08-17 Synlogic, Inc. Bacteria engineered to treat diseases associated with hyperammonemia
CN107164346B (en) * 2017-07-18 2019-09-13 福州大学 A kind of alkalinity salt tolerant Pullulanase PulA and its gene and application
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