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  • 专利说明
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    • 专利摘要:
    The present application relates to the field of enzyme engmeenng, especially relates to a pullulanase as well as preparation and use thereof The pullulanase and coding gene thereof were obtained by random mutation by using the Error-prone PCR technique on the gene of wild-type pullulanase to obtain a mutant PLUM. The enzyme activity of the mutant PLUM was improved by 57.03% compared with the wild-type pullulanase PLUM.
    公开号:NL2021281A
    申请号:NL2021281
    申请日:2018-07-11
    公开日:2018-08-07
    发明作者:Wang Yu；Ban Litong；Huang Liang；Sun Ning；Yang Hongpeng
    申请人:Beijing Jinhong Tianbang Information Tech Co Ltd；
    IPC主号:
    专利说明:

    Technical Field
    The present disclosure relates to the enzyme engineering technical field, especially relates to a pullulanase as well as preparation and use thereof.
    Background
    A pullulanase is a starch debranching enzyme, which can hydrolyze alpha-1,6 glucosidic bond of polysaccharides so that the amylose can be converted into amylopectin to the maximum extent. The pullulanase has very important application in the starch processing industry, and it can improve on a large scale the utilization rate and the production efficiency of the starch, and is relatively well applied to production of glucose syrup, maltose syrup and beer.
    In the production of glucose, pullulanase and glucamylase are used together for a saccharification process, and the yield of glucose is increased by reducing the content of the oligosaccharide by using the pullulanase, and the dosage of the glucamylase can be reduced.
    The pullulanase can also be used in the beer industry, it can be added in the saccharification or fermentation process to improve the fermentation capability of beer wort.
    In 1961, Aerobacter aerogenes which can produce pullulanase was first found by people, and good enzymatic properties of the pullulanase were reported. Since then, a variety of microorganisms capable of producing pullulanase were found by researchers in various countries through extensive research, such as Bacillus Cereus var. Mycoides, Bacillus Acidopullulyticus, Bacillus Subtilis, and Clostridum Themosulfurogenes. But most of the strains for producing pullulanase at present have no industrial value. The present disclosure will provide a high-activity pullulanase by means of genetic engineering.
    Summary of the Invention
    In order to achieve the aim, the present disclosure provides a pullulanase mutant and a gene thereof. According to the method disclosed by the invention, an Error-prone
    PCR (polymerase chain reaction) technology is used, so that the pullulanase coding gene pul from Bacillus Acidopullulyticus is subjected to random mutation to obtain a pullulanase mutant gene pulm. The specific enzyme activity of the mutant is increased by 57.03% compared with that of the original gene, and a high-activity pullulanase was obtained by expressing in Pichia Pastoris.
    Detailed Description
    In the present discloure, the following definitions are adopted:
    1. The amino acid and DNA nucleic acid sequence naming method:
    The amino acid residues are named by the three-letter code form specified by the IUPAC nomenclature. The DNA nucleic acid sequence adopts a universally accepted
    IUPAC nomenclature.
    2. Identification of pullulanase mutant
    The form of “original amino acid position substituted amino acid” was adopted to express the mutated amino acid in pullulanase mutant, such as Gly547Cys, represents that the amino acid which position number is 547 is Gly in the original pullulanase but is replaced to Cys in the pullulanase mutant, the number of the positions corresponds to the amino acid sequence number of the wild type pullulanase in SEQ ID NO: 2.
    In the present disclosure, the original pullulanase is represented by PUL, the amino acids sequence is shown in SEQ ID NO: 2. The mutated pullulanase is represented by PULM, the amino acids sequence is shown in SEQ ID NO:4. The gene encoding PUL is represented by pul, shown in SEQ ID NO: 1, the gene encoding PULM is represented by pulm, shown in SEQ ID NO: 3.
    PullulanaseNucleotideAmino acidsNo. 1639No. 1646No. 1867No. 547No. 549No. 623PULGACGlyAsnLeu
    PULMTCTCysThrPhe
    The host cell used for expressing the pullulanase mutant is Pichia Pastoris SMD 1168, the expression vector is pGAPZaCo
    Beneficial effects:
    1. According to the method disclosed by the invention, the wild type pullulanase 5 gene is randomly mutated by using Error-prone PCR technology, the pullulanase mutant
    PLUM is obtained, and the specific enzyme activity of the pullulanase mutant PLUM is improved by 57.03% compared with that of the wild-type pullulanase.
    2. The mutant site of the pullulanase mutant mentioned in the invention provides a new direction and revelation for research of pullulanase in future.
    Brief Description of Figures
    Figurel is a Error-prone PCR product electrophoresis image; wherein,M is marker; plum is the product of Error-prone PCR;
    Figure2 is a double-enzyme digestion verification graph of pGAPZaC-/ w//zz; wherein, M is marker; Vector is the product of double-enzyme digestion.
    Examples
    In order to enable the purpose, the technical scheme and the advantages of the invention to be clearer, the present invention is further described in detail with reference to specific examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not intended to limit the present invention.
    Examplel Obtaining of the genome DNA of Bacillus Acidopullulyticus
    The wild type pullulanase mature peptide gene pul was from Bacillus
    Acidopullulyticus stored in the applicant’s laboratory. The genome DNA was extracted by the method comprising the following steps:
    (1) an inoculating loop of Bacillus Acidopullulyticus was picked from a flat plate and inoculated into a medium, then cultured at 30°C, 200 rpm for one night;
    (2) the culture solution was centrifuged at 12000r/min for 10 minutes, and then the thallus was harvested in the centrifuge tube;
    (3) 1 mL solution I(50mmol/L Glucose; 25mmol/L Tris-HCl, pH8.0; lOmmol/L EDTA, pH8.0), 150pL lysozyme solution were added to the thallus, and digested at 37°C for 30 minutes;
    (4) 300 pL solutionII (0.2mol/L NaOH; 1% SDS) was added thereto, then the 5 centrifuge tube was inverted for 5 minutes;
    (5) equal volume of solution III (saturated phenol: chloroform= 1: 1) was added, mixed uniformly, and then the mixed solution was centrifuged at the room temperature, 12000r/min for 10 minutes, then the supernatant was transferred to another clean EP tube, and the organic phase and protein precipitate in lower layer were abandoned;
    (6) the above supernatant was repeatedly extracted twice, and extracted with equal volume of chloroform for one time, so as to remove the trace amounts of phenol;
    (7) a DNA was precipitated by adding 2-fold volume of absolute ethyl alcohol, then centrifuging at 12000 r/min for 10 minutes, removing the resulting supernatant, and washing the resulting precipitate with 70% ethanol (500pL) for two times;
    (8) the EP tube was inverted and aired on a fdter paper, then the DNA was dissolved by TE buffer solution, and preserved at -20 °C for later use.
    Example2 Obtaining of the mutator gene of pullulanase
    1. random mutation
    Random mutation was carried out on the basis of the error-prone PCR (polymerase chain reaction) technology, and a high-activity pullulanase gene was obtained (TaKaRa Taq DNA polymerase was used).
    Primers were designed as follows:
    Forward primer Pl(SEQ ID No.5):
    5’-TAAGAAGGAGATATACCATGGACAGCACCAGTACCAAGGTCATC-3’
    Reverse primer P2( SEQ ID No.6):
    ’ -GTGGTGGTGGTGGTGCTCGAGTTACTGCTTAAGGATC A AAGTGGAGA-3 ’ The amplification template was the genome DNA obtained in example 1, the reaction system of the amplification was as follows:
    10x Taq PCR buffer5 pLdNTPs(2mmol/L each)5 pLforward primer Pl(10 pmol/L)1.5 pLReverse primer P2( 10 pmol/L)1.5 pL25 mmol/L MgCLllpL5 mmol/L MnCL5pLAmplification template (genome DNA)20 pmolTaqDNA polymerase1 pLddELOcomplement to 50 pL
    The amplification conditions were as follows: the reaction system was pre-denatured at 95°C for 3 minutes; denatured at 95°C for 60 seconds; and annealed at 61 °C for 60 seconds, extended at 72 °C for 180 seconds; after 30 cycles, the reaction system was incubated at 72°C for 10 minutes, and then stored at 4 °C.
    The PCR amplification product was detected by 1.0% agarose gel electrophoresis, a band of about 2800 bp was observed (fig. 1). The PCR amplification product does not need to be treated, which can be instantly used for the constaiction of recombinant vector, and also can be stored for a long time at -20°C.
    2. Linearization of the expression vector
    A conventional restriction enzyme of Takara was used to linearize the PET-28 plasmid, the reaction system was as follows:
    Neo I5pLXhol5pL10*K bufferlOpL0.1 % BSAlOpLpET-28a5 pgddELOcomplement to lOOpL
    The linearization conditions are as follows: the reaction system was incubated at °C for 3 hours; at 65 °C for 20 minutes, and then stored at 4 °C. The linearized product can be immediately used for the construction of expression vector and can also s be stored for a long time at -20°C.
    3. Construction of expression vector library
    The pul mutant expression vector library was constructed by using the one-step ligase of ClonExpress II of Vazyme to connect the error-prone PCR product with the linearized PET-28a. In order to ensure sufficient storage capacity, five connecting reactions were carried out at the same time, the connecting system was 100pL in total.
    The connecting system comprised the following components:
    5*CE II buffer4pLError-prone PCR product112 ngLinearized PET-28a110 ngExnase II2pLddH2Ocomplement to 20pL
    Note: the connecting system was prepared in an ice bath.
    The reaction conditions were as follows: the connecting system was incubated at
    37°C for 30 minutes; and at 4 °C for 5 minutes.
    After the reaction was finished, the product can be stored at 4°C in a short term or stored at -20°Cin a long term.
    4. Construction of pul mutant expression strain library
    20pL of the pul mutant expression vector obtained in step3 was transformed into the expression strain Escherichia coli BL21 in the following manner:
    The competent cells of E.coli BL21 (100pL for each) were taken out from -80°C, and placed on ice to be dissolved, then immediately 20pL of the pul mutant expression vector was added thereto in an aseptic environment after dissolving. Then, the system was placed on ice for 30 minutes, and heat shocked by a water bath for 90s at 42°C, cooled on ice for 1.5 min. Afterwards, 900pL of LB medium was added thereto, and a pre-cultivation was performed at 37°C, 200r/min for 30 min, then a centrifugation was carried out for 2 min at the speed of 3000 rpm. The supernate of 600pL was skimmed, and the residual supernate were uniformly mixed with the resulting thallus sediment through blowing-suction by a pipettor to obtain a concentrated bacterial solution.
    Further, each lOOpL of the concentrated bacterial solution was spreaded on an LB flat plate with kanamycin resistance, each group was provided with four parallel, and all inverted on the constant-temperature incubator to subject to a cultivation at 37°C for one night.
    Finally, 20 flat plates with recombinant strain were obtained, sealed with sealing film and placed at 4 °C for short-term storing.
    5. Screening of the high-activity pullulanase gene
    At least 2000 positive transformants were selected from the 20 flat plates with recombinant strain of step 4, and each of the positive transformants was divided to two parts, one was inoculated into a new flat plate with kanamycin resistance which used for strain preservation; meanwhile, the other was inoculated in a 96-well plate with 200pL LB liquid medium for each well (which contains 30pg/mL of kanamycin).
    The flat plate used for strain preservation was cultured overnight at 37 °C, and then sealed with a sealing film, stored at 4°C.
    The 96-well plates were cultured at 37°C, 200r/min. When ODf,oo reached 0.6, each of the wells was added with IPTG (final concentration of 1 mmol/L), and then an induction was performed at 16°C for 16h followed by a centrifugation at 4°C, 4000 r/min for 15 minutes to obtain the thallus. The obtained thallus was resuspended in 15 mL of pre-cooled PBS buffer solution with pH 7.4, and a cell disruption treatment was performed to crush cells by using a low-temperature ultrahigh-pressure continuous flow cell disruption instrument. After the disruption treatment was completed, a centrifugation was carried out at 4°C, 12000 r/min for 45 minutes to collect the supernate to obtain a crude enzyme solution. Then, an enzyme activity measurement was carried out on the crude enzyme solution. The measurement result showed that the enzyme activity of the mutantpulm is the highest, it was improved by 57.03% compared with that of the wild type pul. The mutant pulm was sent to Beijing Huada Gene Science and Technology Co., Ltd. to finish the gene sequencing by using a universal primer T7/T7 ter, the sequencing result showed that the mutator g&cie-pulm has a nucleotide sequence as shown in SEQ ID NO.3 which encoded an amino acid with Gly547Cys,
    Asn549Thr and Leu623Phe compared with the PUL.
    Example3 Construction of a recombinant Pichia Pastoris freely expressing the pullulanase mutant
    The high-activity pullulanase gene (pulrn) was linked with a Pichia Pastoris 5 secretory expression vector-pGAPZaC to construct the recombinant expression vector pGAPZaC-pz/Z/w, and tansformed into the Pichia Pastoris .
    1. Construction of the recombinant expression vector pGAPZaC-/W/w
    The pGAPZaC was a fusion expression vector and provided with an alpha factor secreting signal peptide which can secrete protein out of pichia pastoris cells made the protein convenient to be purified; meanwhile, the Zeocin resistant gene on pGAPZaC was used for preserving and screening of recombinant strains;
    TheMCS (multiple cloning site) of pGAPZaC include EcoPl, PmR, Xhol, Notl and ,7vd. EcoPUXbal were chosen to construct the recombinant expression vector pGAPZaC-pz/Zm. Primers used to amplify pulm and add restriction enzyme cutting site were as follows:
    Forward primer P5(SEQ ID No.7):
    GGAATTCGACAGCACCAGTACCAAGGTCATC (Contains the EcoRl site)
    Reverse primer P6(SEQ ID No. 8):
    GCTCTAGATTACTGCTTAAGGATCAA AGTGGAGA ( Contains the Xbal site)
    The amplification template was pET28a-/ w/m, the amplification system was as follows ( The Pyrobest DNA Polymerase of Takara was used in this system )
    Amplification template DNA500 ngPyrobest DNAPolymerase(5 U/pL)0.25pL10x Pyrobest buffer II5 pLdNTPs(2.5 mmol/L each)4 pLForward primer P5(10 pmol/L)1.5 pLReverse primer P6(10 pmol/L)1.5 pLddfUOcomplement to 50 pL
    The amplification conditions were as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds; annealing at 61 °C for 60 seconds, and extension at 72°C for 180 seconds. This process repeats for 30 cycles. Then, incubation was performed at 72°C for 10 minutes. And the product was stored at 4°C. The PCR amplification product (pulm) was detected by 1.0% agarose gel electrophoresis, a band of about 2800 bp was observed. After being purified by the DNA Purification Kit the PCR amplification product can be immediately used for the constaiction of the recombinant expression vector, and also can be stored for a long time at -20°C.
    The purified pulm and the pGAPZaC were respectively subjected to enzyme digestion by using the EcoRMXbal, the enzyme digestion system was as follows:
    EcoR I5pLXbal5pL10*M buffer(from TaKaRa)lOpLDNA5 pgddH2Ocomplement to lOOpL
    1.0% agarose gel electrophoresis was used to separate the enzyme digestion product, the pGAPZaC and pulm fragments were recycled by gel extraction and linked by T4 ligase overnight. The connecting system was as follows:
    T4 ligaselpL10* T4 Buffer2.5pLpGAPZaC0.03 pmolpulm0.3 pmolddH2Ocomplement to 25pL
    After the preparation of the connecting system was completed, the system was kept at 16°C for 16 hours for connecting, and then stored at 4°C. Thus, the recombinant expression vector pGAPZaC-/w//« was completed.
    The heat shock method was used to transform the pGAPZaC -pulm into E. coli
    DH5a competent cells. And the competent cells were spreaded onto the LB flat plate with Zeocin resistance and then cultured overnight. Then, the positive transformants were selected for plasmids extraction, then the plasmids were verified by enzyme digestion (fig.2) and sequencing was done to confirm the right recombinant expression vector pG APZaC-/ ////» was obtained.
    2. Construction and screening of recombinant strain expressing high activity pullulanase mutant (1) Preparation of the linearized plasmid DNA
    Before transformed into the Pichia Pastoris, the recombinant expression vector pGAPZaC-/ ////// needed to be linearized, so that the integration efficiency of the plasmid on the Pichia Pastoris chromosome will be improved. And the linearization was completed by restriction endonuclease BspH I.
    (2) The transformation of linearized pGAPZaC-/ w//w into pichia pastoris, identification of positive transformant, and screening of pullulanase strain with high-productivity
    CD 80pL of Pichia Pastoris SMD 1168 competent cells and 10pg of the linearized pGAPZaC-jra/wz were added to a 1.5 ml pre-cooled centrifuge tube and mixed evenly, then transferred into a pre-cooled conversion cup;
    CD the conversion cup of stepCD was placed in a ice bath for 5 min, and a electroporation was carried out on Pichia Pastoris SMD 1168 according to the parameters recommended by the electroporation device;
    (3) after the pulse, 1 ml of pre-cooled 1 mol/L sorbitol solution was added into the conversion cup immediately to obtain a transformation solution, then the transformation solution was transfered into a new 1.5mL centrifuge tube;
    CD after a static culture at 30 °C for 1.5h, 200 pL of the transformation solution was sucked and spread onto the MD medium;
    © a cultivation was carried out at 30°C until the transformants appeared.
    (6) a single colony of the transformants was selected and dissolved in 10pL of deionized water to get the bacterial suspension. 2pL of the bacterial suspension was taken and added with Lyticcase to react for lOmin at 30°C. Then, the resulting reaction solution was placed into a refrigerator with the temperature of -80 °C to be frozen for 10 min, so that the cell wall of the yeast was cracked to release the genome. The released io genome was used as a template for PCR. The positive transformant was identified by taking Pichia Pastoris SMD 1168 with empty pGAPZaC as a control.
    @ on the basis that the positive transformant has been identified, screening of the high geneticin-resistant transformant was performed by using flat plate containing different concentrations of geneticin, and then the enzyme activity of the pullulanase of the high geneticin-resistant transformant was measured respectively to obtain the high-yield strain SMD 1168/ pGAPZaC-pitlm of pullulanase .
    Example4 Expression and preparation of pullulanase mutant by SMD 1168/ pGAPZaC -pulm
    The recombinant bacterium SMD 1168/ pGAPZaC -pulm was inoculated to a YPD liquid medium and cultured at 30°C, 250r/min for 24 h. Then the culture was transferred into a fresh BMGY medium at the inoculation amount of 1%, and cultured at 30°C, 250r/min for 24 hours, and a centrifugation was carried out for 5 minutes at 6000 r/min to obtain the thallus. Then, the thallus was transferred into a BMMY medium, and cultured at 30°C, 250 r/min for 120 hours to obtain the crude enzyme liquid of the pullulanase. Then a high-activity pullulanase was precipitated by salt fractionation of the crude enzyme liquid, wherein the protein precipitate was collected, dissolved, desalted by dialysis, and treated by an ion exchange chromatography and a gel chromatography, and then the freeze drying, so as that the high-activity pullulanase pure enzyme powder was obtained.
    About 183mg pure enzyme powder of pullulanase was obtained by every liter of culture medium.
    Example5 Determination of the pullulanase activity
    1. Assay method
    DNS method: 50pL appropriately diluted pullulanase solution was added to 450pL buffer(the 5% pulullan solution and the buffer solution with pH5.0 were uniformly mixed in a ratio of 1: 8), and then sufficient mixing was done to react 30 min at 50°C, and then 500pL DNS solution was added to terminate the reaction. A water bath was carried out for 10 minutes at 100°C, then OD540 values were measured.
    2. Result
    The specific activity of PUL and PULM were assayed as 370 U/mg and 581 U/mg, it was shown that the specific activity has been improved by 57.03% after the mutation.
    Definition of specific enzyme activity: under determination conditions, the enzyme 5 required by generating 1 pmol of reducing sugar (Glucose) per minute from the hydrolyzation of pulullan is defined as an enzyme activity unit (U), the specific enzyme activity means the number of enzyme activity unit in per unit weight of protein, which was generally represented by U/mg protein.
    The SMD 1168/ pGAPZaC-/w/m and Bacillus Acidopulhdyticus were fermented as 10 the method described in example 4, and then the pullulanase activity of the fermentation broth thereof was meausured. It was shown that SMD 1168/ pGAPZaC-/w//« was
    106.4U/ml and Bacillus Acidopulhdyticus was 7.4U/mL.
    The following clauses are not the claims, but include contemplated embodiments. The 15 Applicant hereby gives notice that new claims may be formulated to such clauses and/or combinations of such clauses and/or features taken from the description or claims, e.g., during prosecution of the present application or of any further application derived therefrom.
    1. A pullulanase mutant, wherein, the amino acid sequence of said mutant is shown in
    SEQ ID NO.4.
    2. The pullulanase mutant according to clause 1, wherein its expression vector is pGAPZaC.
    3. The pullulanase mutant according to clause 1, wherein its host cell is Pichia Pastoris
    GS115.
    4. A coding gene of the pullulanase mutant according to clause 1.
    5. The coding gene according to clause 4, wherein the nucleotide sequence of said gene is shown in SEQ ID NO.3.
    6. A use of the pullulanase mutant according to clause 1.
    7. A use of the coding gene of the pullulanase mutant according to clause 4.
    The above embodiments only express several embodiments of the present invention, the description is specific and detailed, but is not to be construed as limiting the scope of the patent. It should be noted that for one of ordinary skill in the art, the above embodiments can also make a plurality of deformation, combinations and improvements without departing from the concept of the present invention, thereof, all of which belong to the scope of protection of this patent. Therefore, the protection scope of the invention should be determined by the appended claims.
    权利要求:
    Claims (7)
    [1]
    CONCLUSIONS
    A pullulanase mutant, wherein the amino acid sequence of the mutant is shown in SEQ ID NO. 4.
    5
    [2]
    The pullulanase mutant according to claim 1, wherein the expression vector is pGAPZaC.
    [3]
    The pullulanase mutant according to claim 1, wherein its host cell is Pichia Pastoris GS115.
    [4]
    A coding gene for the pullulanase mutant according to claim 1.
    [5]
    Encoding gene according to claim 4, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 3.
    [6]
    Use of the pullulanase mutant according to claim 1.
    [7]
    Use of the coding gene for the pullulanase mutant according to claim 4.
    1/1
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    同族专利:
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    JP2006174841A|2006-01-16|2006-07-06|Kao Corp|Mutated pullulanase|
    CN103484443B|2012-07-23|2015-12-09|江南大学|A kind of Pullulan enzymatic mutant and preparation method thereof|
    CN106434604A|2016-12-14|2017-02-22|曹书华|Pullulanase mutant with improved catalytic performance|CN109321552B|2018-10-11|2021-01-22|山东隆科特酶制剂有限公司|Novel pullulanase, gene thereof, engineering bacteria and preparation method|
    法律状态:
    2022-03-02| MM| Lapsed because of non-payment of the annual fee|Effective date: 20210801 |
    优先权:
    申请号 | 申请日 | 专利标题
    CN201810527491.3A|CN108374004A|2018-05-29|2018-05-29|A kind of Pullulanase and its application|
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