CN108467847A

CN108467847A - Can exocytosis endo-inulinase recombined bacillus subtilis and its preparation method and application

Info

Publication number: CN108467847A
Application number: CN201810052242.3A
Authority: CN
Inventors: 高健; 姜瑞凡; 徐虹; 黄巍巍; 张丽; 薛锋; 倪浩; 梅笛芬; 张红
Original assignee: Yangcheng Institute of Technology
Current assignee: Yangcheng Institute of Technology; Yancheng Institute of Technology
Priority date: 2018-01-18
Filing date: 2018-01-18
Publication date: 2018-08-31
Anticipated expiration: 2038-01-18
Also published as: CN108467847B

Abstract

It is that the CDS sequences inuQ of endo-inulinase is overlaped with signal peptide using over-lap PCR the invention discloses a kind of recombinant plasmid, then the obtained segment containing signal peptide and large intestine withered grass shuttle plasmid pPsacB NMK links together.The invention also discloses one kind by above-mentioned recombinant plasmid transformed bacillus subtilisWB800‑RObtain can exocytosis endo-inulinase recombined bacillus subtilis.The invention also discloses the applications of the preparation method and recombined bacillus subtilis of above-mentioned recombinant plasmid and recombined bacillus subtilis.The recombined bacillus subtilis of the present invention has higher advantage than Other Engineering bacterium in terms of producing endo-inulinase, can obtain DP there is no the security risk of Escherichia coli production oligofructose₃, DP₄, DP₅The oligofructose of the degree of polymerization can prepare oligofructose by one-step fermentation, save the time, reduce the stringent condition of enzymatic.

Description

Can exocytosis endo-inulinase recombined bacillus subtilis and preparation method thereof and Using

Technical field

The present invention relates to genetic engineerings and fermentation engineering field, and in particular to one kind can exocytosis endo-inulinase Recombined bacillus subtilis and its preparation method and application

Background technology

Oligofructose is divided into sugarcane fruit and fruit fruit type oligofructose, and sugarcane fruit type oligofructose is to pass through β by 1~3 fructosyl (2-1) glycosidic bond is combined the mixture of ketose, Nystose and sugarcane sugar generated etc. with the fructosyl in sucrose；Fruit Fruit type oligofructose is under the action of endo-inulinase, by hydrolysis of inulin at the oligofructose of different polymerization degree.100 grams of dry weights There are about 60-70 grams of inulin in jerusalem artichoke, inulin is by linear β -2, the levulan of 1- glucosides chain links, and end is a sucrose Base.Therefore using jerusalem artichoke powder as raw material synanthrin restriction endonuclease hydrolysis, oligofructose slurry can be finally obtained through refined.Oligofructose is one Kind natural active matter, sugariness are 0.3-0.6 times of sucrose, have not only maintained the pure sweetening characteristics of sucrose, but also than sucrose sweet taste Clearly, it is with adjusting intestinal flora, proliferation Bifidobacterium, the absorption for promoting calcium, adjusting blood fat, immunological regulation and anti-caries tooth etc. The novel sweetener of healthcare function is known as most potential additive of new generation after the antibiotic epoch --- growth-promoting substance； It is referred to as plasmosin (PPE) in France, in dairy products, sour milk beverage, solid beverage, candy, biscuit, bread, jelly, cold It is applied in the numerous foods such as drink.

In addition to the physicochemical properties with general utility functions oligosaccharide, most noticeable physiological property is oligofructose It can be obviously improved microbial population ratio in enteron aisle, it is the activation and proliferation factor of enteral Bifidobacterium, can reduce and inhibit The generation of enteral corrupt substance inhibits the growth of harmful bacteria, adjusts enteron aisle inner equilibrium；It can promote the suction of trace elements iron, calcium It receives and utilizes, to prevent osteoporosis；Hepatotoxin can be reduced, the organic acid of anticancer can be generated in intestines, is had significant anti- Cancer function；And pure taste is sweet and dilitious, has the adipose fragrance of class and tasty and refreshing soapy feeling.The production of oligofructose in recent years The health-product markets such as product fashionable day, Europe, the United States.

Currently, synanthrin excision enzyme is focused primarily upon about the molecular biology research of dextrase in the world, it is circumscribed to synanthrin Structure and the catalyst mechanism understanding of enzyme are more deep, and relatively fewer for the research of synanthrin restriction endonuclease, are mainly limited to enzyme base The clone of cause and heterogenous expression.All gene containing alantin excision enzyme in most microorganism, but only a small number of microorganisms Gene containing restriction endonuclease in vivo, hitherto it is found that certain micro-organisms can produce synanthrin restriction endonuclease, such as Aspergillus Ficuum, Microbulbifer sp.Penicillium sp., Kluyveromy-ces marxianus, Bacillus Smithii, pseudomonas sp., Xanthomonas oryzae and Arthrobacter sp etc..It is wherein most of to be Ectoenzyme is generally used conventional treatment method and is isolated and purified, and such as centrifugation and/or ultrafiltration, inorganic salts or organic solvent are heavy Shallow lake, various column chromatographies, electrophoresis etc..Endo-inulinase is generally glycoprotein, the synanthrin restriction endonuclease of separate sources, zymologic property It differs.But when studying the heterogenous expression of endo-inulinase, it is limited only to the research of endocellular enzyme, to extracellular endo-inulinase Research it is less.

Invention content

Goal of the invention：The object of the present invention is to provide a kind of recombinant plasmid pPsacB-NMK-SP-inuQ.

Can exocytosis endo-inulinase and utilization sucrose be efficiently induction it is a further object of the present invention to provide one plant Agent and carbon source, inulin are bacillus subtilis (Bacillussubtilis) recombinant bacterium that substrate hydrolysis inulin produces oligofructose Strain WB800-R-SPnprb.

Another object of the present invention be to provide above-mentioned recombinant plasmid and recombinant bacterial strain WB800-R-SPnprb preparation method and Using.

Technical solution：The present invention provides a kind of recombinant plasmid pPsacB-NMK-SP-inuQ, and the recombinant plasmid is by big Intestines-withered grass shuttle plasmid pPsacB-NMK is mutually got continuously with the segment containing signal peptide SP and the CDS sequences inuQ of endo-inulinase It arrives；Above-mentioned signal peptide SP is SPsacB, SPsacC, SPbpr, SPamye or SPnprb, and five kinds of signal peptide sequences are as follows：

SPbpr:atgaggaaaaaaacgaaaaacagactcatcagctctgttttaagtacagttgtcatcagttca ctgctgtttccgggagcagccggggca

SPamye:atgtttgcaaaacgattcaaaacctctttactgccgttattcgctggatt tttattgctgtttcatttggttctggca

SPnprb:atgcgcaacttgaccaagacatctctattactggccggcttatgcacagcggcccaaatggt ttttgtaacacatgcctcag

SPsacB:ATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGC AGGAGGCGCAACTCAAGCGTTTGCG

SPsacC:ATGAAAAAGAGACTGATTCAAGTCATGATCATGTTCACCCTGCTGTTGACTATGGCATTTTC GGCAGATGCAGCC

Wherein, above-mentioned sequence is referring to SEQ ID NO in sequence table:14 to SEQ ID NO:18 such as show.

Wherein, the segment of the CDS sequences inuQ containing signal peptide SP and endo-inulinase is obtained by over-lap PCR； The recombinant plasmid pP_sacB- NMK-SP-inuQ is by one-step cloning method by the CDS sequences containing signal peptide SP and endo-inulinase Arrange the segment and large intestine-withered grass shuttle plasmid pP of inuQ_sacB- NMK is connected.

Recombinant plasmid pP_sacB- NMK-SP-inuQ is large intestine-withered grass shuttle plasmid, in Escherichia coli and bacillus subtilis In have different replicon and replication protein, have different selection markers in two kinds of bacterium, ammonia benzyl (A) shown in Escherichia coli Resistance shows Ka Na (K) resistance in bacillus subtilis, there is the ID NO P from bacillus subtilis itself_sacBIt opens Mover and hydrolytic inulin generate the endo-inulinase gene of oligofructose.

Wherein, the preparation method of above-mentioned recombinant plasmid includes the following steps：

1) the CDS sequences of the endo-inulinase in Pseudomonas mucidolens (Pseudomonas mucidolens) are selected, if Primer is counted, endo-inulinase gene inuQ is transferred；The primer sequence such as SEQ ID NO:2 and SEQ ID NO:Shown in 3；

2) the CDS sequences inuQ of endo-inulinase and signal peptide are subjected to over-lap PCR, obtain the piece containing signal peptide SP Section；The signal peptide SP is SPsacB, SPsacC, SPbpr, SPamye or SPnprb, is verified in order to facilitate follow-up plasmid construction, Restriction enzyme site PstI and HindIII are designed into primer sequence and are synthesized when designing by overlapping PCR primers.

3) expression vector pP_sacBThe structure of-NMK-SP-inuQ：With large intestine-withered grass shuttle plasmid pP_sacB- NMK is plasmid bone Frame utilizes restriction enzyme PstI and HindIII double digestion plasmid pP_sacB- NMK obtains double digestion plasmid；

4) the double enzymes for using the segment containing signal peptide that the method for one-step cloning obtains step 2) to be obtained with step 3) Plasmid connection is cut, recombinant plasmid pP is formed_sacB-NMK-SP-inuQ。

In step 1), the source of endo-inulinase gene inuQ is Pseudomonas mucidolens (Pseudomonas Mucidolens), its sequence can be found in NCBI, and Gene Bank is AF141320.1.Sequence is as follows：

ATGCATAATACCGAAGATACCGGGCTTATTGCCTACTGGTCTTTTGACGAAGAATCGGGTAAAACAGCTGTAGATGT AATCGGGAAGATGAACGATTCTATCGATTACGTATTCAATCATGCACGATTCAAAGCATCCAGCGATCCGCAAAGAA GGAAAGGGATCAGCGGTAATGCCTTGTTGTTCGACGGATACTCGACTTGGATCAAGCGTTCAGCCGATCAGATCGGA AAACCGGAGAACGCTTTGACGCTGGAAGCATGGGTCGCGCCGAGGTCCTACGAATGGGGGGACGAACAGCGGCTTTC CGCCATCGTGAACCAGCATGATCGCGAGAAGAAAGAGGGATTTATCCTTGGTATGTACAGACACGGCACTTGGTCTT TGCAACTCGGACTCGATCACGAATGGATAGAAGTTTGGTCGGAAGACCATCCGCTTCCTAAGAACGAATGGTCTTAT GTTGTCGCTACCTATGATAAAAAAACATCTATGTTGAAGCTTGCATGCCCATCTATGTTGAAGCTTTATCTAAACGG AGTAGAGGTTGCGTCCAAACAGACCACTGCCCATTCAACGATTACTCCGTCCAGACAGGATCTGTTAATTGGTAAAA ACAACCAAGCAGTGGTACTTGCCGGCGTATTTTCGCTTAACATGTTCAATGGCTTGATCGACGAAATAAAGATCTAT AACCGAGCATTGTCATCAGATGAAATTGCGTCCTCTTTTCACCGATATTTAGTACCCTATGGTGGAAAAATACCGTC AATTCCATATGACCATTTGAAGCTTGACCGTAGTTTGCTGGCAGATGACAGACATCGGCCGCAGTACCATGTCAGTC CTCCTGCGCATTGGATGAATGAACCGCACGCTCCAATTTATTTTAACGGACAGTATCACCTGTTTTATCAGCATAAT CCGCAAGGCCCGTATTGGCATCAAATTCATTGGGGACATTGGGTTAGCGATGATCTGGTGCATTGGAGAGATCTTCC TGTCGCGTTGTCTCCGGAGAAGAACGCAGTCGATCCAGACGGGGATTGGTCCGGAAGCGCAACTTATGATGAACACG GGCTGCCGGTTCTGTTTTTTACAGCCGGAGACGATAGCGCAAAACCGAATCAAAGAGTCGGTCTTGCCAGGAGCACA TTCGCCCAAGATGGAGACAACGATTTGGTCCATTGGGTGAAGCACCCTACTCCGGTTGTCGTACAGCAGCAAGGCGT TGGAAAGTTTGGAGATTTCCGCGATCCATTCGTATGGAAAGATGGCGATACCTGGTATATGCTCGTAGGTTCGGGAA CGGATGGAGAAGGCGGAACAGCTTTAGCCTATACCTCCAAAAACTTGACGGAATGGGAGTATAGAGGACCATTTTAT ATTAGTGATCATAAGAATTATCCATACTTAGGGAAAGTATGGGAGCTTCCGGTTCTGCTTCCTTTGGGTAAAGACAA GAAAGGCCATGACAAGCATGTCTTTCTTATCAGTCCGGTAGGAGCCGGTGCGGATGTGGAGGTCTTTTATTGGATCG GAACGTTCGATAAGGAGCAGTTCCGCTTTATTCCGGATCAGAATGAGCCGCAATTAATCGATGTCGGAGATTCCCAT TTCACGGGTCCGAGTGGTATGGTCGATCCGAATACGGGACGCAAAATCCTGTTTACAATCGCGCAGGGGGAGCGGAC GCCGGCTTTGGACTATAGCGCGGGCTGGGCTCATAATGGTGGATTGCCGGTCAGCCTTTCCCTGCGTGAAGACGGAC GTCTCGGTGTGGAGCCCATTGAAGAATTGAAGTCGCTCCGCGGCAAAAAGCTTGTTTCGTTCACAAAGAAAAGTGCC GAAGAAGCTAATGATCTCCTTACCAATGTGAAGGGTGATATGCTGGAAATCATACTGGAGCTTGAGCCTGGCACAGC AAAGCAGTTCGGCATCAAAGTTCGTCGCTCCCCGGGCGGTGAAGAAGAGACGCTGCTCTACTATAATACTGAAGCCT CTACGCTGAATGTGAACCGGATGAAGACTACGCTTGATAATTTCGAACGCAGTAAAGGTATTCAAGGCGGCAAGCTG GAGTTGAACGGAGAGAACTTGAAGCTACATATTTATTTGGACCGTTCGATGATCGAAGCTTATGCCAACGGGTTAAA AAGCTTGACAACGAGAGCCTACCCGAGCAGACCAGACTCTCTGGGACTACAGATTTGGGGGGACGGAAGCGTTAGCG TTAAGTCAATGGAAGTATGGGAAATGAATTCGGCGTTCGGCCCGACTGTCTCTGCCTATATTCCTGAGCAGCACGCA GACGGAGTCCAAACCAAATAA；Referring to the SEQ ID NO in sequence table：Shown in 1；

In step 4), recombinant plasmid pP_sacB- NMK-SP-inuQ can be respectively designated as pPsacB- according to the difference of signal peptide NMK-SPsacB-inuQ、pPsacB-NMK-SPsacC-inuQ、pPsacB-NMK-SPbpr-inuQ、pPsacB-NMK- SPamye-inuQ or pPsacB-NMK-SPnprb-inuQ.

Another aspect of the present invention additionally provide it is a kind of can exocytosis endo-inulinase recombined bacillus subtilis WB800-R-SPnprb, the recombined bacillus subtilis are obtained by above-mentioned recombinant plasmid by converting bacillus subtilis WB800-R ；Bacillus subtilis WB800-R is the deficiency for knocking out levanase SacC on the basis of genetic engineering bacterium WB800 and obtaining Bacterial strain.

It is above-mentioned can exocytosis endo-inulinase recombined bacillus subtilis WB800-R-SPnprb preparation method packet It includes：By above-mentioned recombinant plasmid pP_sacB- NMK-SP-inuQ is transformed into bacillus subtilis WB800-R, is existed by recombinant plasmid Card in bacillus subtilis WB800-R receives resistance marker screening positive transformant to get to can exocytosis endo-inulinase Recombined bacillus subtilis WB800-R-pP_sacB-NMK-SP-inuQ。

The recombinant bacterial strain bacillus subtilis WB800-R-pP of above-mentioned expression endo-inulinase_sacB-NMK-SP-inuQ The mycology property of (Bacillus subtilis) is as follows：B. subtilis cell is rod-shaped, 0.6 μm~1.0 μm of diameter, and long 1.5 μm~2.0 μm, raw in gemma, oval, sporangiocyst does not expand, and Gram-reaction is positive.37 DEG C of cultures on manganese nutrition agar plate The bacterium colony that 36h is formed is circle, and diameter 3.0mm~10.0mm, faint yellow, intermediate color depth, surfacing is rough, matt, Edge is irregular.

Preferably, by chemical transformation by above-mentioned recombinant plasmid pP_sacB- NMK-SP-inuQ is transformed into bacillus subtilis In bacterium WB800-R, to obtain preferable transformation efficiency.

Preferably, above by recombinant plasmid pP_sacBKa Nas of-the NMK-SP-inuQ in bacillus subtilis WB800-R Resistance marker screening positive transformant uses the card of a concentration of 25 μ μ of g/mL~50 g/mL to receive antibiotic.

Another aspect of the present invention provide it is above-mentioned can exocytosis endo-inulinase recombined bacillus subtilis WB800-R- pP_sacBApplications of-the NMK-SP-inuQ in preparing endo-inulinase.

Another aspect of the present invention provides a kind of preparation method of endo-inulinase, includes the following steps：

1) by the recombined bacillus subtilis WB800-R-pP_sacB- NMK-SP-inuQ is crossed to containing 25~50 μ G/mL cards receive antibiotic solid LB tablets on, 37 DEG C of incubator 12h choose single bacterium colony and connect LB culture mediums, and 37 DEG C, 200rpm shakes bacterium 12h is activated, and the recombined bacillus subtilis after activation is inoculated into the shaking flask containing culture medium with 2% inoculum concentration In, sucrose (as derivant and fermenting carbon source) is added, 32 DEG C, 200rpm, shakes bacterium, fermentation 48-72h obtains zymotic fluid；

2) it uses ammonium sulfate precipitation method slightly to carry enzyme solution to zymotic fluid progress made from step 1), then uses 4 DEG C of bag filter It is dialyzed overnight, the enzyme solution after being concentrated, 4 DEG C of super filter tube, 9000rpm is finally utilized to centrifuge 10min, obtain endo-inulinase enzyme Liquid.

Preferably, in the preparation method step 1) of above-mentioned endo-inulinase, a concentration of 5.0~20.0g/L of sucrose, producing enzyme Culture medium includes 5.0~20.0g/L sucrose, 20.0~22.0g/L yeast powders, 15.0~16.5g/L NaCl, 0.5~0.8g/L MgSO₄·7H₂O and 2.0~2.8g/L KH₂PO₄。

The present invention also provides a kind of preparation methods of oligofructose, include the following steps：

By the recombined bacillus subtilis WB800-R-pP_sacB- NMK-SP-inuQ is crossed to containing 25~50 μ g/ ML cards receive antibiotic solid LB tablets on, 37 DEG C of incubator 12h choose single bacterium colony and connect LB culture mediums, and 37 DEG C, 200rpm shakes bacterium 12h is activated, by the recombined bacillus subtilis WB800-R-pP after activation_sacB- NMK-SP-inuQ is connect with 2% inoculum concentration Kind in the shaking flask containing fermentation medium, using inulin as substrate, 5.0-20.0g/L sucrose is added, at 32 DEG C, 200rpm conditions Lower shaking flask culture 48h-60h obtains zymotic fluid, and zymotic fluid is centrifuged 10min at 12000rpm, thalline is removed, supernatant is taken to obtain Oligofructose；The fermentation medium includes 20.0~22.0g/L yeast powders, 15.0~16.5g/L NaCl, 0.5~0.8g/L MgSO₄·7H₂O, 2.0~2.8g/L KH₂PO₄。

Advantageous effect：Compared with prior art, the present invention has following advantages：

(1) bacillus subtilis is generally acknowledged food security microorganism, and recombined bacillus subtilis of the invention is not present Escherichia coli produce the security risk of oligofructose, and can obtain DP₃, DP₄, DP₅The oligofructose of the degree of polymerization；

(2) present invention obtains the efficient exocytosis endo-inulinase of one plant of energy and is derivant and carbon using sucrose for the first time Source, inulin are the bacillus subtilis that substrate hydrolysis inulin produces oligofructose.

(3) recombined bacillus subtilis of the invention has in terms of producing endo-inulinase than Other Engineering bacterium higher excellent Gesture；Traditional oligofructose production method is mostly enzymatic, and recombined bacillus subtilis WB800-R-SPnprb of the invention can Oligofructose is prepared by one-step fermentation biological hydrolysis conversion method, the time is saved, reduces the stringent condition of enzymatic；

(4) lactose and IPTG are compared and uses sucrose advantageously as the derivant present invention, IPTG has certain toxicity, therefore It does not select, and the addition of lactose can lead to later separation purification difficult, and while the present invention selects sucrose as derivant As carbon source so that be practically free of the by-products such as the reduced sugars such as glucose, sucrose during one-step fermentation prepares oligofructose Object is conducive to isolating and purifying for later stage fructooligosaccharide.

(5) selection signal peptide SPsacB, SPsacC, SPbpr, SPamye or SPnprb and CDS overlapping sequences are to incite somebody to action Purpose endo-inulinase takes extracellular, selected signal peptide to efficient effect in terms of heterologous protein secretion.

Description of the drawings

Fig. 1 is different transformant plasmid PCR verification electrophoretograms；

Fig. 2 is the standard curve of enzyme activity；

Fig. 3 is the fermentation enzymatic activities curve of recombinant bacterium WB800-R-SPn；

Fig. 4 is that recombinant bacterium surveys oligofructose liquid phase figure by carbon source through fermentation of inulin；

Fig. 5 is recombinant bacterium WB800-R-SPnprb fermentation diagrams；

Specific implementation mode

Reagent used in the embodiment of the present invention：

The structure of 1 recombinant plasmid of embodiment

1, the acquisition of target gene inuQ：By genome digging technology, Genbank, blastn etc. compare tool, determine The source of endo-inulinase is Pseudomonas mucidolens (Pseudomonas mucidolens) (NCBI), designs one-step cloning primer And it synthesizes, sense primer (SEQ ID NO：2)： AaaaaacgaaaaacagacctcgagATGCATAATACCGAAGATACCGGG, downstream primer： tcctgcaggtcgtcgactctAAGCTTTTATTTGGTTTGGACT(SEQ ID NO：3), PCR amplification target gene inuQ；

PCR amplification system：+ 2 μ L downstream primers+25 of 50+2 μ L sense primers of μ L=2 μ L Pseudomonas mucidolens genomic DNAs μLpfuMasterMix+19μLddH2O.Wherein, pfuMasterMix (Shanghai is after bright and beautiful chemical Science and Technology Ltd., E006-01A) Including the ingredients such as dNTP, buffering, MgSO4, pfu enzyme, Pseudomonas mucidolens genomic DNA used herein is from university of South Korea biology The Pseudomonas mucidolens (Pseudomonas mucidolens) that technical research life institute Yong Jin professors Chio give, and profit It is carried with the genomic kit (AXYGEN, AP-MN-BT-GDNA-50) that carries bought from TIANGEN Biotech (Beijing) Co., Ltd. The genomic DNA obtained.

PCR amplification condition：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 3min, cycle Number is 30.

2, SP signal peptides (signal peptide, SP) i.e. SPsacB, SPsacC, SPbpr, SPamye and SPnprb segment Acquisition；

(1) acquisition of genomic DNA：Gene group reagent is carried using what is bought from TIANGEN Biotech (Beijing) Co., Ltd. Box (AXYGEN, AP-MN-BT-GDNA-50) extracts the genome of bacillus subtilis WB800；

(2) acquisition of target fragment：Using the genomic DNA of bacillus subtilis WB800 as template, the signal of PCR acquisitions Peptide SP products；

PCR system：50μl

Template：The 2 μ l of genomic DNA of bacillus subtilis WB800

pfuMasterMix：25μl

Sense primer：2μl

Downstream primer：2μl

H2O：19μl

PCR conditions：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, and cycle-index is 30；

Sense primer is respectively：SPsacB-F(SEQ ID NO:6), SPsacC-F (SEQ ID NO:8), SPbpr-F (SEQ ID NO:4), SPamye-F (SEQ ID NO:12), SPnprb-F (SEQ ID NO:10)；

Downstream primer is respectively：SPsacB-R(SEQ ID NO:7), SPsacC-R (SEQ ID NO:9), SPbpr-R (SEQ ID NO:5), SPamye-R (SEQ ID NO:13), SPnprb-R (SEQ ID NO:11).Upstream used in PCR amplification Shown in primer sequence is specific as follows：

SPbpr-F aaaaggagcgatttactgcagATGAGGAAAAAAACGAAAAACAGAC

SPbpr-R atcttcggtattatgcatctcgagTGCCCCGGCTGCTC

SPsacB-F aaaaggagcgatttactgcagATGAACATCAAAAAGTTTGCAAAAC

SPsacB-R atcttcggtattatgcatctcgagCGCAAACGCTTGAGTTG

SPsacC-F aaaaggagcgatttactgcagATGAAAAAGAGAATGATTCAGATGGG

SPsacC-R atcttcggtattatgcatctcgagCGCAGCTGCGGAAAAGGCTT

SPnprb-F aaaaggagcgatttactgcagATGCGCAACTTGACCAAG

SPnprb-R atcttcggtattatgcatctcgagCTGCAGCTGAGGCATGTGTTAC

SPamye-F aaaaggagcgatttactgcagATGTTTGCAAAACGATTCAAAACC

SPamye-R atcttcggtattatgcatctcgagTGCCAGAACCAAATGAAACAGC

3, the acquisition of SP-inuQ segments：

By over-lap PCR by signal peptide (signal peptide, SP) SPsacB, SPsacC, SPbpr, SPamye and SPnprb overlaps to form five different segments with restriction enzyme site PstI and HindIII, SP from target gene inuQ respectively It is SPsacB, SPsacC, SPbpr, SPamye or SPnprb from bacillus subtilis.(restriction enzyme site mentioned herein is It is designed into primer, referring to the SEQ ID NO in sequence table:4~SEQ ID NO:13)

Wherein, the amplification system of over-lap PCR：+ 1 μ L SP sense primers+1 of 25+1 μ L inuQ segments of μ L=1 μ L SP segments + 12.5 μ L pfuMasterMix+8.5 μ L ddH of μ L inuQ downstream primers₂O, herein used in PCR used in five overlapping fragments Sense primer is respectively：SPsacB-F(SEQ ID NO:6), SPsacC-F (SEQ ID NO:8), SPbpr-F (SEQ ID NO: 4), SPamye-F (SEQ ID NO:12), SPnprb-F (SEQ ID NO:10)；Downstream primer used is all segment inuQ's Downstream primer, that is, SEQ ID NO:3.

Over-lap PCR amplification condition is：94 DEG C of denaturation 30s, 55 DEG C of annealing 1min, 72 DEG C extend 3min, cycle-index 30.

4, it connects：After restriction enzyme PstI and HindIII double digestion plasmid pPsacB-NMK recovery purifyings, utilize The ClonExpress bought from Nanjing Vazyme Biotechnology Co., Ltd.^TM II One Step Cloning Kit(C112- 01), SP-inuQ segments and the plasmid pPsacB-NMK after digestion are attached.

Linked system：20 μ L=4 μ Lbuffer+2 μ L one-step cloning ligase ExnaseTM II+4 μ L SP-inuQ+6 μ L pPsacB-NMK+4μL ddH₂O), 37 DEG C of water-bath 30min.

PPsacB-NMK in the step is pP43-NMK plasmids (Nanjing University of Technology Xu Hong professors seminar give) On the basis of be transformed and obtain, specific transformation process is：

1) extraction Bacillus subtilis genes group DNA；The gene bought using TIANGEN Biotech (Beijing) Co., Ltd. Group extracts kit (AXYGEN, AP-MN-BT-GDNA-50) extracts the genomic DNA of bacillus subtilis WB800；

2) acquisition of target fragment PsacB：It is same using step 1 using Bacillus subtilis genes group DNA as template PCR conditions carry out PCR amplification and obtain PsacB segments；

PCR system is：50μl

Template：2 μ l of Bacillus subtilis genes group DNA

pfuMasterMix 25μl

Sense primer PsacB-F：2μl

Downstream primer PsacB-R：2μl

H2O：19μl

PCR reaction conditions：94 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, cycle Number is 30.

3) BamHI and PstI digestions pP43-NMK plasmid fragments；

4) it connects：Target fragment PsacB is connect with the pP43-NMK plasmid fragments after digestion obtain pPsacB-NMK and be worn Shuttle plasmid；The connector of condition of contact and linked system with the SP-inuQ segments and the plasmid pPsacB-NMK after digestion of step 4 System.

The ClonExpress that the kit is bought from Nanjing Vazyme Biotechnology Co., Ltd.^TM II One Step During Cloning Kit (C112-01), this PCR, used sense primer PsacB-F and downstream primer PsacB-R difference For

PsacB-F：taacacatgcctcagGGATTCGACCTTTTTAACCCATCA；

PsacB-R：gaccatgattacgccCTGCAGTAAATCGCTCCTTTTTTA；

5, it converts：SP-inuQ segments and the linked systems connected of the plasmid pPsacB-NMK after digestion are put on ice 30min is set, is then transformed into the reaction solution connected in E. coli competent DH5a by 42 DEG C of heat-shock transformed methods, 37 DEG C Recovery 1h, is applied to the LB solid mediums containing 100 μ g/mL ammonia benzyl antibiotic, and 37 DEG C of culture 12h screen transformant (target Recombinant plasmid is E. coli-B shuttle plasmid, and the resistance with ammonia benzyl in Escherichia coli shows that Ka Na is anti-in withered grass Property), choosing 4-6 transformant is transferred in the LB liquid medium containing 100 μ g/mL ammonia benzyl antibiotic, 37 DEG C of shaking table cultures Remaining bacterium solution is taken 4mL to centrifuge, outwells supernatant, plasmid kit is carried using purchase by 12h after glycerol tube preserves strain (AXYGEN, AxyPrepTM Plasmid Miniprep Kit 250-prep) extracts the plasmid in transformant.When with inuQ When the SP of overlapping is respectively SPsacB, SPsacC, SPbpr, SPamye or SPnprb, the plasmid name of corresponding structure is respectively pPsacB-NMK-SPsacB-inuQ、pPsacB-NMK-SPsacC-inuQ、pPsacB-NMK-SPbpr-inuQ、pPsacB- NMK-SPamye-inuQ or pPsacB-NMK-SPnprb-inuQ.It is respectively using plasmid as template, with the primer of segment inuQ (referring to the SEQ ID NO in sequence table：2 and SEQ ID NO：3) it is primer, use and the identical PCR conditions of step 1 and journey Sequence, the nucleic acid electrophoresis proof diagram that PCR product obtains are as shown in Figure 1.

2 recombinant plasmid transformed bacillus subtilis WB800-R of embodiment obtains recombined bacillus subtilis WB800-R- pP_sacB-NMK-SP-inuQ

Since bacillus subtilis is gram-positive bacteria, selected method is chemical transformation, and transformation efficiency is higher. Bacillus subtilis WB800-R competence is made using chemical reagent, is distributed into 500 μ L/ pipes, by the verification obtained of embodiment 1 Correct plasmid pPsacB-NMK-SPsacB-inuQ, pPsacB-NMK-SPsacC-inuQ, pPsacB-NMK-SPbpr- InuQ, pPsacB-NMK-SPamye-inuQ or pPsacB-NMK-SPnprb-inuQ are added separately to be placed in different pipes In 500 μ L bacillus subtilis WB800-R competence, 37 DEG C, 200rpm shakes bacterium 90min, is finally respectively coated card and receives antibiosis On the final concentration of 20 μ g/mL LB tablets of element, it is individually positioned at 37 DEG C and cultivates, choose transformant, and be inoculated into respectively containing Ka Na In antibiotic liquid culture medium, 37 DEG C, 200rpm shakes bacterium 12h, respectively in glycerol tube -80 DEG C preserve and bacterial strains and be named as WB800-R-pP_sacB-NMK-SPsacB-inuQ、WB800-R-pPsacB-NMK-SPsacC-inuQ、WB800-R-pPsacB- NMK-SPbpr-inuQ, WB800-R-pPsacB-NMK-SPamye-inuQ and WB800-R-pPsacB-NMK-SPnprb- inuQ。

Wherein, bacillus subtilis WB800-R is that (Nanjing University of Technology Xu Hong teaches project in genetic engineering bacterium WB800 Group give) on the basis of, using the method for homologous single-crossover, the volume that plasmid PKSV7 knocks out levanase is knocked out using Thermo-sensitive Code gene SacC, obtains deficient strain, because of the presence of levanase, the oligofructose that can obtain hydrolysis hydrolyzes achievement Sugar, therefore oligofructose in order to obtain need the levanase for knocking out bacillus subtilis itself.

Embodiment 3

Recombined bacillus subtilis WB800-R-pP_sacB- NMK-SP-inuQ fermentation production endo-inulinases：

First, by the correct recombined bacillus subtilis WB800-R-pP of verification obtained of embodiment 2_sacB-NMK- SPsacB-inuQ、WB800-R-pPsacB-NMK-SPsacC-inuQ、WB800-R-pPsacB-NMK-SPbpr-inuQ、 WB800-R-pPsacB-NMK-SPamye-inuQ and WB800-R-pPsacB-NMK-SPnprb-inuQ is drawn by glycerol tube respectively Line receives the solid LB media of antibiotic to 25 μ g/mL cards, and 37 DEG C of stationary culture 12h choose single bacterium colony access and contain 25 μ g/mL cards It receives in the liquid LB of antibiotic, 37 DEG C, 200rpm shakes bacterium 12h activation, the culture medium obtained according to optimization (comprising 5.0~ 20.0g/L sucrose, 20.0~22.0g/L yeast powders, 15.0~16.5g/L NaCl, 0.5~0.8g/L MgSO₄·7H₂O, 2.0 ~2.8g/L KH₂PO₄), culture medium is prepared, by the recombined bacillus subtilis WB800-R- containing unlike signal peptide pP_sacB-NMK-SPsacB-inuQ、WB800-R-pPsacB-NMK-SPsacC-inuQ、WB800-R-pPsacB-NMK- SPbpr-inuQ, WB800-R-pPsacB-NMK-SPamye-inuQ and WB800-R-pPsacB-NMK-SPnprb-inuQ distinguish It is inoculated in 50mL LB seed culture mediums, 32 DEG C, is inoculated in culture medium respectively after 200rpm cultures 12h, 5.0- 20.0g/L sucrose inductions and as carbon source, 32 DEG C, 200rpm cultures sample 1mL every 6h, and 12000rpm centrifuges 1min, takes Clearly, after diluting 20 times, 200 μ L enzyme solutions and 800 μ L2% inulin, 55 DEG C of reaction 30min is taken to inactivate 10min, take 25 μ L reaction solutions Then plus 550 μ L RO water with 50 μ L DNS (dinitrosalicylic acid) mixings, and 5min is boiled in boiling water, take 200 μ L points to enzyme On target, light absorption value is surveyed at 540nm.All samplings are handled with same method, standard working curve meter according to Fig.2, Calculating enzymatic activities, (standard working curve is worked it out according to the light absorption value of the glucose of various concentration, and abscissa represents grape The concentration of sugar, unit g/L, ordinate indicate it in the light absorption value of 540nm, unit Abs), make the production of each recombinant bacterium Enzyme curve is as shown in figure 3, and selected optimal expression recombinant bacterium is WB800-R-pPsacB-NMK-SPnprb-inuQ, highest enzyme Living is 22.26U/mL.Enzyme activity is defined as：1U is equal to the enzyme amount needed for 1min 1 μm of ol reduced sugar of generation.

4 optimal recombinant bacterium WB800-R-pPsacB-NMK-SPnprb-inuQ one-step fermentations of embodiment prepare oligofructose：

Embodiment 3 is optimized to the highest recombinant bacterium WB800-R-pPsacB-NMK-SPnprb-inuQ scribing line of obtained enzyme activity Onto the solid LB tablets for receiving antibiotic containing 25~50 μ g/mL cards, 37 DEG C of incubator 12h choose single bacterium colony and connect LB culture mediums, 200rpm cultivates 12h under the conditions of 32 DEG C, and 2% inoculum concentration is transferred to the 500mL containing 100mL fermentation mediums and substrate inulin In shaking flask, the fermentation medium is 20.0g/L yeast powders, 15.0g/L NaCl, 0.5g/L MgSO₄·7H₂O, 2.0g/L KH₂PO₄, 5.0-20.0g/L sucrose inductions are added and as carbon source, a concentration of 90.0g/L of substrate inulin is sampled, sample every 6h 0.22 μm of water system film is crossed, HPLC is walked, utilizes nh 2 column NH₂Oligofructose yield is detected, mobile phase is 70% acetonitrile, column temperature 30 DEG C, flow velocity 0.8mL/min, detector is Composition distribution RI.By Composition distribution, liquid phase figure such as attached drawing 4 has been marked in figure Go out, different degree of polymerization DP₃, DP₄, DP₅, NH₂The appearance time of column is related with molecular weight, and molecular weight is smaller, it is more early go out Peak, corresponding peak are DP respectively₃, DP₄, DP₅, according to liquid phase as a result, drawing out the Yield mapping of oligofructose, as shown in Figure 5.Fig. 5 The change of production trend of different polymerization degree and the curve of total oligofructose are shown respectively, inulin is measured in above-mentioned zymotic fluid Inscribe enzyme activity 42.04 ± 0.93U/mL, 90.0g/L inulin substrate obtains 40.08 ± 0.68g/L of oligofructose, the degree of polymerization point It Wei not DP₃、DP₄And DP₅, IOSs represents oligofructose DP in Fig. 5₃, DP₄, DP₅Total output.It is contained only in by-product on a small quantity about 4.05 ± 0.59g/L fructose is practically free of the reduced sugars such as glucose, sucrose, is conducive to isolating and purifying for later stage fructooligosaccharide.

Sequence table

<110>Yancheng Institute Of Technology

<120>Can exocytosis endo-inulinase recombined bacillus subtilis and its preparation method and application

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2331

<212> DNA

<213>The enzyme gene inuQ (inuQ) of endo-inulinase

<400> 1

atgcataata ccgaagatac cgggcttatt gcctactggt cttttgacga agaatcgggt 60

aaaacagctg tagatgtaat cgggaagatg aacgattcta tcgattacgt attcaatcat 120

gcacgattca aagcatccag cgatccgcaa agaaggaaag ggatcagcgg taatgccttg 180

ttgttcgacg gatactcgac ttggatcaag cgttcagccg atcagatcgg aaaaccggag 240

aacgctttga cgctggaagc atgggtcgcg ccgaggtcct acgaatgggg ggacgaacag 300

cggctttccg ccatcgtgaa ccagcatgat cgcgagaaga aagagggatt tatccttggt 360

atgtacagac acggcacttg gtctttgcaa ctcggactcg atcacgaatg gatagaagtt 420

tggtcggaag accatccgct tcctaagaac gaatggtctt atgttgtcgc tacctatgat 480

aaaaaaacat ctatgttgaa gcttgcatgc ccatctatgt tgaagcttta tctaaacgga 540

gtagaggttg cgtccaaaca gaccactgcc cattcaacga ttactccgtc cagacaggat 600

ctgttaattg gtaaaaacaa ccaagcagtg gtacttgccg gcgtattttc gcttaacatg 660

ttcaatggct tgatcgacga aataaagatc tataaccgag cattgtcatc agatgaaatt 720

gcgtcctctt ttcaccgata tttagtaccc tatggtggaa aaataccgtc aattccatat 780

gaccatttga agcttgaccg tagtttgctg gcagatgaca gacatcggcc gcagtaccat 840

gtcagtcctc ctgcgcattg gatgaatgaa ccgcacgctc caatttattt taacggacag 900

tatcacctgt tttatcagca taatccgcaa ggcccgtatt ggcatcaaat tcattgggga 960

cattgggtta gcgatgatct ggtgcattgg agagatcttc ctgtcgcgtt gtctccggag 1020

aagaacgcag tcgatccaga cggggattgg tccggaagcg caacttatga tgaacacggg 1080

ctgccggttc tgttttttac agccggagac gatagcgcaa aaccgaatca aagagtcggt 1140

cttgccagga gcacattcgc ccaagatgga gacaacgatt tggtccattg ggtgaagcac 1200

cctactccgg ttgtcgtaca gcagcaaggc gttggaaagt ttggagattt ccgcgatcca 1260

ttcgtatgga aagatggcga tacctggtat atgctcgtag gttcgggaac ggatggagaa 1320

ggcggaacag ctttagccta tacctccaaa aacttgacgg aatgggagta tagaggacca 1380

ttttatatta gtgatcataa gaattatcca tacttaggga aagtatggga gcttccggtt 1440

ctgcttcctt tgggtaaaga caagaaaggc catgacaagc atgtctttct tatcagtccg 1500

gtaggagccg gtgcggatgt ggaggtcttt tattggatcg gaacgttcga taaggagcag 1560

ttccgcttta ttccggatca gaatgagccg caattaatcg atgtcggaga ttcccatttc 1620

acgggtccga gtggtatggt cgatccgaat acgggacgca aaatcctgtt tacaatcgcg 1680

cagggggagc ggacgccggc tttggactat agcgcgggct gggctcataa tggtggattg 1740

ccggtcagcc tttccctgcg tgaagacgga cgtctcggtg tggagcccat tgaagaattg 1800

aagtcgctcc gcggcaaaaa gcttgtttcg ttcacaaaga aaagtgccga agaagctaat 1860

gatctcctta ccaatgtgaa gggtgatatg ctggaaatca tactggagct tgagcctggc 1920

acagcaaagc agttcggcat caaagttcgt cgctccccgg gcggtgaaga agagacgctg 1980

ctctactata atactgaagc ctctacgctg aatgtgaacc ggatgaagac tacgcttgat 2040

aatttcgaac gcagtaaagg tattcaaggc ggcaagctgg agttgaacgg agagaacttg 2100

aagctacata tttatttgga ccgttcgatg atcgaagctt atgccaacgg gttaaaaagc 2160

ttgacaacga gagcctaccc gagcagacca gactctctgg gactacagat ttggggggac 2220

ggaagcgtta gcgttaagtc aatggaagta tgggaaatga attcggcgtt cggcccgact 2280

gtctctgcct atattcctga gcagcacgca gacggagtcc aaaccaaata a 2331

<210> 2

<211> 48

<212> DNA

<213>Upstream primer sequence (Artificial Sequence)

<400> 2

aaaaaacgaa aaacagacct cgagatgcat aataccgaag ataccggg 48

<210> 3

<211> 42

<212> DNA

<213>Downstream primer sequence (Artificial Sequence)

<400> 3

tcctgcaggt cgtcgactct aagcttttat ttggtttgga ct 42

<210> 4

<211> 46

<212> DNA

<213>Sense primer SPbpr-F (Artificial Sequence)

<400> 4

aaaaggagcg atttactgca gatgaggaaa aaaacgaaaa acagac 46

<210> 5

<211> 38

<212> DNA

<213>Downstream primer SPbpr-R (Artificial Sequence)

<400> 5

atcttcggta ttatgcatct cgagtgcccc ggctgctc 38

<210> 6

<211> 46

<212> DNA

<213>Sense primer SPsacB-F (Artificial Sequence)

<400> 6

aaaaggagcg atttactgca gatgaacatc aaaaagtttg caaaac 46

<210> 7

<211> 41

<212> DNA

<213>Downstream primer SPsacB-R (Artificial Sequence)

<400> 7

atcttcggta ttatgcatct cgagcgcaaa cgcttgagtt g 41

<210> 8

<211> 47

<212> DNA

<213>Sense primer SPsacC-F (Artificial Sequence)

<400> 8

aaaaggagcg atttactgca gatgaaaaag agaatgattc agatggg 47

<210> 9

<211> 44

<212> DNA

<213>Downstream primer SPsacC-R (Artificial Sequence)

<400> 9

atcttcggta ttatgcatct cgagcgcagc tgcggaaaag gctt 44

<210> 10

<211> 39

<212> DNA

<213>Sense primer SPnprb-F (Artificial Sequence)

<400> 10

aaaaggagcg atttactgca gatgcgcaac ttgaccaag 39

<210> 11

<211> 46

<212> DNA

<213>Downstream primer SPnprb-R (Artificial Sequence)

<400> 11

atcttcggta ttatgcatct cgagctgcag ctgaggcatg tgttac 46

<210> 12

<211> 45

<212> DNA

<213>Sense primer SPamye-F (Artificial Sequence)

<400> 12

aaaaggagcg atttactgca gatgtttgca aaacgattca aaacc 45

<210> 13

<211> 46

<212> DNA

<213>Downstream primer SPamye-R (Artificial Sequence)

<400> 13

atcttcggta ttatgcatct cgagtgccag aaccaaatga aacagc 46

<210> 14

<211> 90

<212> DNA

<213> SPbpr(Artificial Sequence)

<400> 14

atgaggaaaa aaacgaaaaa cagactcatc agctctgttt taagtacagt tgtcatcagt 60

tcactgctgt ttccgggagc agccggggca 90

<210> 15

<211> 78

<212> DNA

<213> SPamye(Artificial Sequence)

<400> 15

atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60

tttcatttgg ttctggca 78

<210> 16

<211> 82

<212> DNA

<213> SPnprb(Artificial Sequence)

<400> 16

atgcgcaact tgaccaagac atctctatta ctggccggct tatgcacagc ggcccaaatg 60

gtttttgtaa cacatgcctc ag 82

<210> 17

<211> 87

<212> DNA

<213> SPsacB(Artificial Sequence)

<400> 17

atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 60

gcaggaggcg caactcaagc gtttgcg 87

<210> 18

<211> 75

<212> DNA

<213> SPsacC(Artificial Sequence)

<400> 18

atgaaaaaga gactgattca agtcatgatc atgttcaccc tgctgttgac tatggcattt 60

tcggcagatg cagcc 75

<210> 19

<211> 39

<212> DNA

<213> PsacB-F (Artificial Sequence)

<400> 19

taacacatgc ctcagggatt cgaccttttt aacccatca 39

<210> 20

<211> 39

<212> DNA

<213> PsacB-R(Artificial Sequence)

<400> 20

gaccatgatt acgccctgca gtaaatcgct cctttttta 39

Claims

1. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid by large intestine-withered grass shuttle plasmid pPsacB-NMK with contain Signal peptide SP is connected to obtain with the segment of the CDS sequences inuQ of endo-inulinase, the signal peptide SP be SPsacB, SPsacC, SPbpr, SPamye or SPnprb.

2. the construction method of recombinant plasmid described in claim 1, which is characterized in that this approach includes the following steps：

1）The CDS sequences of endo-inulinase in selected Pseudomonas mucidolens, design and synthesize primer, are transferred by PCR amplification The gene inuQ of endo-inulinase；The primer sequence such as SEQ ID NO:2 and SEQ ID NO:Shown in 3；

2）The CDS sequences inuQ of endo-inulinase and signal peptide are subjected to over-lap PCR, obtain the segment containing signal peptide SP；Institute It is SPsacB, SPsacC, SPbpr, SPamye or SPnprb to state signal peptide SP；

3）The structure of expression vector pPsacB-NMK-SPinuQ：Using large intestine-withered grass shuttle plasmid pPsacB-NMK as plasmid bone Frame obtains double digestion plasmid using restriction enzyme PstI and HindIII double digestion plasmid pPsacB-NMK；

4）Using the method for one-step cloning by step 2）The segment containing signal peptide SP obtained and step 3）The double digestion of acquisition Plasmid connects, and forms recombinant plasmid pP_sacB-NMK-SP-inuQ。

3. it is a kind of can exocytosis endo-inulinase recombined bacillus subtilis, which is characterized in that the recombinant bacillus gemma bar Bacterium is by recombinant plasmid described in claim 1 by converting bacillus subtilisWB800-RIt obtains；The bacillus subtilisWB800-RIt is in genetic engineering bacteriumWB800On the basis of knock out the obtained deficient strains of levanase SacC.

4. it is a kind of prepare described in claim 3 can exocytosis endo-inulinase recombined bacillus subtilis method, It is characterized in that, this method includes：By the recombinant plasmid transformed into the bacillus subtilisWB800-RIn, by described heavy Group plasmid is in the bacillus subtilisWB800-RIn card receive resistance marker screening positive transformant to get to it is described can born of the same parents The recombined bacillus subtilis of outer secretion endo-inulinase.

5. according to the method described in claim 4, it is characterized in that, by chemical transformation by the recombinant plasmid transformed into withered Careless bacillusWB800-RIn.

6. according to the method described in claim 4, it is characterized in that, it is described by recombinant plasmid in bacillus subtilisWB800- RIn card to receive resistance marker screening positive transformant be to receive antibiotic using the card of a concentration of 25 μ μ of g/mL ~ 50 g/mL to be sieved Choosing.

7. described in claim 3 can exocytosis endo-inulinase recombined bacillus subtilis prepare endo-inulinase and Prepare the application in oligofructose.

8. a kind of preparation method of endo-inulinase, which is characterized in that the preparation method includes the following steps：

1）Recombined bacillus subtilis described in claim 3 is crossed to and receives the solid LB of antibiotic containing 25 ~ 50 μ g/mL cards On tablet, 37 DEG C of 12 h of incubator choose single bacterium colony and connect LB culture mediums, and 37 DEG C, 200 rpm shake 12 h of bacterium and activated, and will activate Recombined bacillus subtilis afterwards is inoculated into 2% inoculum concentration in the shaking flask containing culture medium, addition sucrose, 32 DEG C, and 200 Rpm, shakes bacterium, and fermentation 48-72 h obtain zymotic fluid；

2）Using ammonium sulfate precipitation method to step 1）Zymotic fluid progress obtained slightly carries enzyme solution, then uses 4 DEG C of bag filter overnight Dialysis, the enzyme solution after being concentrated finally utilize 4 DEG C of super filter tube, 9000 rpm to centrifuge 10min, obtain endo-inulinase enzyme solution.

9. preparation method according to claim 8, which is characterized in that a concentration of 5.0 ~ 20.0 g/L of the sucrose, institute It includes 5.0 ~ 20.0 g/L sucrose, 20.0~22.0 g/L yeast powders, 15.0~16.5 g/L NaCl, 0.5 to state culture medium ~0.8 g/L MgSO₄·7H₂O and 2.0~2.8 g/L KH₂PO₄。

10. a kind of preparation method of oligofructose, which is characterized in that the preparation method includes the following steps：By claim 3 institute The recombined bacillus subtilis stated be crossed to received containing 25 ~ 50 μ g/mL cards antibiotic solid LB tablets on, 37 DEG C of incubators 12 H chooses single bacterium colony and connects LB culture mediums, and 37 DEG C, 200 rpm shake 12 h of bacterium and activated, by the recombined bacillus subtilis after activation It is inoculated into the shaking flask containing fermentation medium with 2% inoculum concentration, using inulin as substrate, 5.0-20.0 g/L sucrose is added, 32 DEG C, shaking flask culture 48h-60h obtains zymotic fluid under the conditions of 200rpm, and zymotic fluid is centrifuged 10min at 12000rpm, removes bacterium Body takes supernatant to obtain oligofructose；The fermentation medium includes 20.0~22.0g/L yeast powders, 15.0~16.5 g/L NaCl, 0.5~0.8 g/L MgSO₄·7H₂O, 2.0~2.8 g/L KH₂PO₄。