CN102796751A - Mutant Pul 324 of pullulanibacillus naganoensis pullulanase and use thereof - Google Patents

Mutant Pul 324 of pullulanibacillus naganoensis pullulanase and use thereof Download PDF

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Publication number
CN102796751A
CN102796751A CN2012102984932A CN201210298493A CN102796751A CN 102796751 A CN102796751 A CN 102796751A CN 2012102984932 A CN2012102984932 A CN 2012102984932A CN 201210298493 A CN201210298493 A CN 201210298493A CN 102796751 A CN102796751 A CN 102796751A
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pul
pullulanase
mutant
enzyme
pullulanibacillus
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CN102796751B (en
Inventor
谢能中
黄日波
王青艳
秦艳
米慧芝
朱绮霞
申乃坤
朱婧
陆艳
曹薇
黎贞崇
陈东
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Guangxi Academy of Sciences
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Abstract

The invention discloses a mutant Pul 324 of a pullulanibacillus naganoensis pullulanase and a use thereof. Based on a modern enzyme engineering technology, an amino acid sequence of a pullulanibacillus naganoensis pullulanase is subjected to molecular modification. Through a PCR method, front 108 amino acid residues of a raw enzyme are deleted so that the mutant Pul 324 which can realize effective secretory expression in escherichia coli hosts is obtained. The mutant Pul 324 is inserted into pET-22b(+) so that a recombinant vector is constructed. The recombinant vector is introduced into an escherichia coli host for secretory expression of the mutant Pul 324. The mutant Pul 324 gene is smaller than a wild type gene and is conducive to plasmid stabilization. Compared with a raw enzyme, the mutant Pul 324 can realize effective secretory expression in escherichia coli hosts, realize a fermentation supernatant enzyme activity improved by 24 times and realize an endoenzyme activity improved by 40%. The mutant Pul 324 can improve a pullulanase secretory expression level of a host. An expression product can improve starch hydrolysis efficiency.

Description

A kind of Nagano genus bacillus Pullulanase two mutants Pul324 and application thereof
Technical field
The present invention relates to biological technical field, specifically is a kind of Nagano genus bacillus Pullulanase two mutants Pul324 and application thereof.
Background technology
Starch material generally contains 60 ~ 90% pulullan, and contained 4 ~ 6% glucosyl residues of pulullan are with α-1, and the 6-glycosidic link connects.Pullulanase (Pullulanase; EC 3.2.1.41) is a kind of α-1 that can specificity cuts the pulullan tapping point; The 6-glycosidic link, thus cut whole side shoot, form an enzyme of separating of amylose starch; Improving the action effect of glycase, improving starch utilization ratio, having quite huge value aspect reducing the grain consumption, improve the quality of products and developing new product starch; In industries such as food, medicine, weaving, bioenergy, very important purposes and good market outlook are arranged, for example can utilize its production high purity glucose and fructose, high maltose syrup, alcohol fuel, and treated starch.
Along with the development of starch material deep processing industries, require zymin industry to bring in constant renewal in and improve the kind and the performance of enzyme, in recent years to satisfy industrial requirement.China begins just Pullulanase to be researched and developed since the seventies, but owing to the enzyme lower laboratory study that only limits to of living, and Pullulanase is the product that unique a kind of China still can not autonomous production in the starch refine dsugar enzyme system at present.Use at present the widest, the maximum Pullulanase of output and be derived from Denmark Novo company, the Pullulanase that the said firm produced be by acidophilia Propiram bud pole bacterium ( Bacillus acidopullulyticus) produce through submerged fermentation.In addition, abroad derive from the aerogenesis klebsiella ( Klebsiella aerogenes), subtilis ( Bacillus subtilis) and Bacillus deramificansPullulanase also obtained commercially producing.Therefore; There is a big difference apart from developed country in the research of Pullulanase in China; The complete dependence on import of industry Pullulanase; Pricing right is grasped in minority Overseas Company hand, and the market supply of monopolization has caused the high selling price of domestic Pullulanase, has limited the development of domestic industries greatly.
In order to change the dependence of China to imported product; Seek the road of a production domesticization; This research uses the enzyme engineering technology exploitation that the high-performance Pullulanase of independent intellectual property right is arranged; And utilize genetic engineering technique to make up engineering strain and carry out secreting, expressing, produce Pullulanase for large-scale industrialization and take a firm foundation.
Summary of the invention
The purpose of this invention is to provide a kind of Nagano genus bacillus Pullulanase two mutants Pul324 and application thereof.
New Pullulanase two mutants Pul324 of the present invention (SEQ ID NO. 2); It obtains through molecular modification Nagano genus bacillus Pullulanase gene (GenBank sequence number JN872757.1); Specifically be preceding 324 bases of utilizing on the modern enzyme engineering technology disappearance wild type gene, transform and obtain a new Pullulanase two mutants Pul324.Compare with protoenzyme, this two mutants effective secreting, expressing in escherichia coli host, fermented liquid supernatant is 24 times of protoenzyme, and intracellular enzyme is lived and has then been improved 40%, and its character helps reducing the production cost of Pullulanase, is suitable for suitability for industrialized production more.
A kind of Nagano genus bacillus Pullulanase mutant gene Pul324, its nucleotide sequence is shown in SEQ ID NO. 1.
Described Pullulanase mutant gene Pul324 encoded protein matter are made up of 818 amino acid, and its aminoacid sequence is shown in SEQ ID NO. 2.
Described Pullulanase mutant gene Pul324, this two mutants be through extract the Nagano genus bacillus ( Pullulanibacillus naganoensis) ATCC 53909 genomic dnas, be foundation according to the gene order of having reported among the Genbank, design the mutant gene that following primer obtains to lack preceding 324 bp bases Pul324:
Upstream primer F1 is: GTA GAATTC ACCTGCTGTAAGTAACGC
Downstream primer R1 is: GTA CTCGAG TTTACCATCAGATGGGCT
Described Pullulanase mutant gene Pul324 application of encoded protein matter in the starch hydrolytic process.
The invention still further relates to the host of containing Pullulanase two mutants Pul324 of the present invention.
New Pullulanase two mutants Pul324 of the present invention, this mutant enzyme have a purposes widely in the starch hydrolysis.
Its special being is compared with protoenzyme, two mutants Pul324 effective secreting, expressing in escherichia coli host, and the enzyme work of fermented liquid supernatant rises to original 24 times, and intracellular enzyme is lived and has then been improved 40%.Mutant gene Pul324 is littler than wild type gene, helps the stable of plasmid.
The preparation method of new Pullulanase two mutants Pul324 of the present invention comprises the Pullulanase mutant gene PulThe steps such as mensuration that the expression of 324 acquisition, Pullulanase mutant gene, Pullulanase mutant enzyme are lived, specific as follows:
(1) Pullulanase mutant gene Pul324 acquisition
According to Pullulanase gene order information, begin to design primer transformation from 325 bit bases of protogene (GenBank sequence number JN872757.1).With the Nagano subtilis genomic dna is masterplate, uses upstream primer F1:5 ’ – GTA GAATTC(SEQ ID NO. 1 comprises one to ACCTGCTGTAAGTAACGC – 3 ' EcoRThe I restriction enzyme site) and downstream primer R1:5 ’ – GTA CTCGAG(SEQ ID NO. 2 comprises one to TTTACCATCAGATGGGCT – 3 ' XhoThe I restriction enzyme site), through polymerase chain reaction (PCR) amplification Pullulanase mutant gene.
(2) construction of recombinant plasmid and checking
Use restriction enzyme EcoRI with XhoI carries out double digestion to the Pullulanase mutant gene, is connected with same pET-22b (+) through double digestion to obtain pET22b- Pul324, transform the host E. coliDH5 α, the extraction recombinant plasmid carries out enzyme and cuts checking, and the step of going forward side by side carries out the dna sequencing analysis and confirms correct transformant.
(3) structure of engineering strain:
The recombinant plasmid transformed that checking is correct E. coliBL21 (DE3) competent cell (CaCl 2The chemical method conversion) in, utilizes the method validation positive transformant of bacterium colony PCR E. coliBL21 (DE3)/pET22b- Pul324.Through measuring, the crude enzyme liquid enzyme activity of fermented liquid supernatant is 24 times of wild-type, and intracellular enzyme is lived and then improved 40%.
[0016] outstanding substantive distinguishing features of the present invention and technique effect are:
The present invention utilizes preceding 324 bases on the modern enzyme engineering technology disappearance Nagano genus bacillus Pullulanase gene, transforms and obtains a new Pullulanase two mutants Pul324.This mutant gene is littler than wild type gene, helps the stable of plasmid.Compare with protoenzyme; Pullulanase two mutants Pul324 effective secreting, expressing in escherichia coli host, the enzyme work of fermented liquid supernatant rises to original 24 times, and intracellular enzyme is lived and has then been improved 40%; Its character helps reducing the production cost of Pullulanase, is suitable for suitability for industrialized production more.
Embodiment
The Nagano genus bacillus bacterial classification that the present invention uses can be bought from DSMZ, also can obtain through field acquisition or other approach.Microorganism culturing, gene clone, expression technology and the relevant technical scheme that the present invention adopts is mature technology commonly used in this area; The technical term that relates to also is a technical term common in this area, and therefore text of the present invention is conspicuous to those skilled in the art.Below in conjunction with embodiment technology contents of the present invention is described further, this embodiment is illustrative, rather than determinate, can not be interpreted as qualification protection scope of the present invention.
To describe in detail the present invention through embodiment below:
(1) Pullulanase mutant gene Pul234 draws Pul324 acquisition
The Nagano genus bacillus ( Pullulanibacillus naganoensis) ATCC 53909 purchase from German microbial strains preservation center, according to the Pullulanase gene order information of this bacterium, 235,325 bit bases from protogene begin to design primer respectively:
Upstream primer F2:5 ’ – GTA GAATTCCATCGATTTAAGCAAAGG – 3 '
Upstream primer F1:5 ’ – GTA GAATTCACCTGCTGTAAGTAACGC – 3 '
Downstream primer R1:5 ’ – GTA CTCGAGTTTACCATCAGATGGGCT – 3 '
Wherein upstream primer comprises one EcoRI restriction enzyme site (underscore), downstream primer comprise one XhoI restriction enzyme site (underscore).With Pullulanibacillus naganoensisATCC 53909 genomes are that masterplate carries out pcr amplification.Reaction system is: ddH 2O 41.5 μ L, 10 * buffer, 5 μ L, dNTP (2.5 mmol/L) 1 μ L, each 0.5 μ L of upstream and downstream primer (20 μ mol/L), dna profiling 1 μ L, high-fidelity DNA polymerase 0.5 μ L.The pcr amplification condition is: 94 ° of big sex change 5 min of C, 94 ° of C sex change 20 s, 61 ° of C 40 s that anneal, and 72 ° of C extend 3 min, 30 circulations, last 72 ° of C extend 10 min.Utilizing upstream primer F2 and downstream primer R1 to increase obtains Pul234 genes obtain and utilize upstream primer F1 and downstream primer R1 to increase Pul324 genes.
Resulting amplified production is carried out agarose gel electrophoresis detect, detect the amplified production size and fit like a glove with the target gene fragment size.The PCR product carries out purifying with DNA purification kit (day root company).Concrete steps reference dna product purification test kit specification sheets.With restriction enzyme EcoR I and Xho I the Pullulanase mutant gene of purifying is carried out double digestion; Be connected with same pET-22b (+) through double digestion and obtain pET22b-pul234 and pET22b-pul324; Transform host E.coli DH5 α; The extraction recombinant plasmid carries out enzyme and cuts checking, and the step of going forward side by side carries out the dna sequencing analysis and confirms correct transformant.
The recombinant plasmid transformed that checking is correct E. coliBL21 (DE3) competent cell (CaCl 2The chemical method conversion) in, utilizes the method validation positive transformant of bacterium colony PCR E. coliBL21 (DE3)/pET22b- Pul234 draws E. coliBL21 (DE3)/pET22b- Pul324.Be inoculated in 5 mL liquid LB substratum (containing 100 μ g/mL penbritins) with making up successful engineering strain, 37 ° of C cultivate 10 h.Get the liquid seeds of 0.5 mL and inoculate 50 mL liquid LB substratum (containing 100 μ g/mL penbritins), 37 ° of C cultivate 2-3 h, work as OD 620When being 0.4 left and right sides, add 1 mM IPTG, place 37 ° of C to induce then, times 12 h.
The present invention adopts the DNS reagent color developing method to measure the Pullulanase vigor.Its principle is that Pullulanase hydrolysis pulullan substrate obtains reducing sugar, reducing sugar and DNS reagent contained 3, the 5-dinitrosalicylic acid altogether produces henna complex compound in the heat back, the depth of its color is directly proportional with the amount of reducing sugar within the specific limits.Use the spectrophotometry light absorption value can calculate the vigor of Pullulanase.
The step of enzyme activity determination is: get 1mL fermented liquid 12,000r/min is centrifugal, and 3min obtains clear enzyme solution.(100mM pH4.75), adds clear enzyme solution on the 100 μ L then, is incubated 15min in 60 ℃ of water-baths in centrifuge tube, to add 100 μ L concentration and be the sodium acetate buffer of 1% (w/v) Propiram.The ice bath termination reaction adds excessive DNS (300 μ L) reagent then, boiling water bath colour developing 5min, and OD is measured in the cooling back 540, can calculate extracellular enzyme and live.The thalline of bacterial sediment suspends with 200 μ L sterilized waters, adds an amount of Triton X-100 and N,O-Diacetylmuramidase, 37 ℃ of water-bath 30min, and 12, the centrifugal 3min of 000r/min gets supernatant and analyzes intracellular enzyme work as stated above.Compare with protoenzyme, Pullulanase two mutants Pul324 effective secreting, expressing in escherichia coli host, the enzyme work of fermented liquid supernatant rises to original 24 times, and intracellular enzyme is lived and has then been improved 40%.
In the starch-liquefying process, saccharifying enzyme is the α-1 of main hydrolysis amylose starch then, the 4-glycosidic link.And Pullulanase can specificity cuts the α-1 of pulullan tapping point; The 6-glycosidic link; Thereby cut whole side shoot; Form amylose starch and be convenient to the action effect of other glycase, improve the starch hydrolysis efficiency, cut down the consumption of energy, improve the quality of products and the aspect of developing new product has higher value starch.In the mashing process, suitably the saccharifying enzyme Dextrozyme DX catalyzed reaction 0.5,1.0,1.5 and the 2.0h of dilution produce 0.758,1.502,2.219 and the 2.628g/L reducing sugar respectively.As the auxiliary Pullulanase two mutants Pul324 that adds, under the constant situation of other condition, the concentration of reduced sugar of generation is increased to 1.071,2.307,3.145 and 3.902g/L, and the hydrolysis efficiency increase rate is all greater than 40%.This shows that the auxiliary Pullulanase two mutants Pul324 that adds can effectively improve the starch hydrolysis efficiency really.
This invention has important economic benefit, and the special property of two mutants helps reducing the production cost of Pullulanase, is suitable for suitability for industrialized production more.It should be understood that; For a person skilled in the art; Can improve or conversion according to above-mentioned explanation; For example, described wild-type Pullulanase except deriving from the Nagano genus bacillus, can also derive from aerogenesis klebsiella, subtilis, bacillus megaterium, bacillus pumilus, Bacillus licheniformis, bacillus cereus, Bacillus deramificans, Bacillus acidopullulyticusDeng bacterial strain.Described carrier is suitable in hosts such as subtilis, Bacillus licheniformis, pichia spp, expressing.Also can Pullulanase two mutants of the present invention be changed in protokaryon or the eucaryon host, to realize the expression of Pullulanase two mutants through electrotransformation, protoplast transformation method etc.Perhaps the aminoacid sequence of two mutants is transformed through enzyme engineering technology.And all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention.
  
Sequence table
< 110>Guangxi Academy Of Sciences
< 120>a kind of Nagano genus bacillus Pullulanase two mutants Pul324 and application thereof
<160>?2
<170>?PatentIn?version?3.5
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<211>?2457
<212>?DNA
<213>The Nagano genus bacillus ( Pullulanibacillus naganoensis)
<400>?1
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gatgttttca?attattatag?cgggctgatt?catcttcgtc?ttgatcaccc?agccttccgc 2160
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<213>The Nagano genus bacillus ( Pullulanibacillus naganoensis)
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IAAERPMVQK?FIIDSLKFWV?NEYHVDGFRF?DLMALLGKDT?MSKAATQLHA?IDPGIALYGE 540
PWTGGTSALP?ADQLLTKGAQ?KGMGVAVFND?NLRNGLDGSV?FDSSAQGFAT?GATGLTDAIK 600
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GIPFMQGGEE?MLRTKGGNDN?SYNAGDVVNE?FDWSRKAQYP?DVFNYYSGLI?HLRLDHPAFR 720
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ATSGKVGEST?LGQAEGSVQV?PGISMMILHQ?EVSPSDGK 818
Figure IDA00002038577100011
Figure IDA00002038577100021

Claims (4)

1. Nagano genus bacillus Pullulanase mutant gene Pul324, it is characterized in that its nucleotide sequence is shown in SEQ ID NO. 1.
2. Pullulanase mutant gene according to claim 1 Pul324 encoded protein matter is characterized in that, are made up of 818 amino acid, and its aminoacid sequence is shown in SEQ ID NO. 2.
3. Pullulanase mutant gene according to claim 1 Pul324, it is characterized in that, this two mutants be through extract the Nagano genus bacillus ( Pullulanibacillus naganoensis) ATCC 53909 genomic dnas, be foundation according to the gene order of having reported among the Genbank, design the mutant gene that following primer obtains to lack preceding 324 bp bases Pul324:
Upstream primer F1 is: GTA GAATTC ACCTGCTGTAAGTAACGC
Downstream primer R1 is: GTA CTCGAG TTTACCATCAGATGGGCT.
4. Pullulanase mutant gene according to claim 1 Pul324 application of encoded protein matter in the starch hydrolytic process.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450553A (en) * 2014-09-17 2015-03-25 上海大学 Pullulanibacillus pueri and culture method thereof
CN104893998A (en) * 2014-10-10 2015-09-09 上海大学 Puer aureobacidium pullulans and cultivation method thereof
CN105132305A (en) * 2015-07-09 2015-12-09 上海大学 Pseudomonas strain and screening method thereof
CN105734034A (en) * 2016-04-25 2016-07-06 江南大学 Method for improving catalytic performance of pullulanase with truncated flexible residues
CN108913677A (en) * 2018-07-23 2018-11-30 福州大学 A kind of Fixedpoint mutation modified alkaline pullulanase and its application
CN114790451A (en) * 2020-12-01 2022-07-26 天津科技大学 Pullulanase and application thereof
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106084016B (en) * 2016-03-07 2020-03-20 南宁邦尔克生物技术有限责任公司 Signal peptide mutant capable of improving expression quantity of recombinant pullulanase and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011076123A1 (en) * 2009-12-22 2011-06-30 Novozymes A/S Compositions comprising boosting polypeptide and starch degrading enzyme and uses thereof
CN102120971A (en) * 2010-12-02 2011-07-13 天津工业生物技术研究所 Pullulanase-producing bacterium, heat-resisting pullulanase produced from same, and coding gene of pullulanase-producing bacterium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011076123A1 (en) * 2009-12-22 2011-06-30 Novozymes A/S Compositions comprising boosting polypeptide and starch degrading enzyme and uses thereof
CN102120971A (en) * 2010-12-02 2011-07-13 天津工业生物技术研究所 Pullulanase-producing bacterium, heat-resisting pullulanase produced from same, and coding gene of pullulanase-producing bacterium

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
李美蓉等: "蜡样芽孢杆菌GXBC-3三个普鲁兰酶基因的表达及其酶学特性", 《生物工程学报》, vol. 28, no. 4, 25 April 2012 (2012-04-25) *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450553A (en) * 2014-09-17 2015-03-25 上海大学 Pullulanibacillus pueri and culture method thereof
CN104450553B (en) * 2014-09-17 2017-06-06 上海大学 A kind of general Shandong indigo plant bacterium in Pu'er and its cultural method
CN104893998A (en) * 2014-10-10 2015-09-09 上海大学 Puer aureobacidium pullulans and cultivation method thereof
CN105132305A (en) * 2015-07-09 2015-12-09 上海大学 Pseudomonas strain and screening method thereof
CN105734034A (en) * 2016-04-25 2016-07-06 江南大学 Method for improving catalytic performance of pullulanase with truncated flexible residues
CN108913677A (en) * 2018-07-23 2018-11-30 福州大学 A kind of Fixedpoint mutation modified alkaline pullulanase and its application
CN114790451A (en) * 2020-12-01 2022-07-26 天津科技大学 Pullulanase and application thereof
CN114790451B (en) * 2020-12-01 2023-12-19 天津科技大学 Pullulanase and application thereof
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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