CN104946561B - Pullulanase active bacterial strain and its cultural method in puncture vine endophyte - Google Patents
Pullulanase active bacterial strain and its cultural method in puncture vine endophyte Download PDFInfo
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- CN104946561B CN104946561B CN201510277577.1A CN201510277577A CN104946561B CN 104946561 B CN104946561 B CN 104946561B CN 201510277577 A CN201510277577 A CN 201510277577A CN 104946561 B CN104946561 B CN 104946561B
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Abstract
The invention provides Pullulanase active bacterial strain in a kind of puncture vine endophyte, the bacterial strain is enterobacteria (Enterobacter sp.), is preserved in China typical culture collection center, and preserving number is CCTCC M 2015071.Meanwhile the invention discloses the cultural method of the bacterial strain, by enrichment culture, it is separately cultured and the step such as secondary screening obtains the bacterial strain.The present invention, by Reasonable adjustment each component content in enriched medium, is advantageous to the survival and culture of weak tendency bacterial strain using puncture vine as material；Pass through DNS methods, Pullulanase determination of activity to the bacterial strain shows, the bacterial strain has higher Pullulanase activity, the fermented and cultured 56h under the conditions of 30 DEG C, 180r/min, pH6.0, yield of enzyme, can be as good mutagenesis starting strain and the alternative bacterial strain of Pullulanase gene of structure engineering bacteria up to 4.19U/mL.
Description
Technical field
The invention belongs to microculture field, and in particular to have the bacterium of Pullulanase activity in a kind of puncture vine endophyte
Strain and its cultural method.
Background technology
Puncture vine Tribulus terrestris L. are annual herb plant, and all parts of the country are distributed, its fresh and tender battalion
Foster organ can do animal feeding-stuff, and fruit can then be used as medicine, have treatment dizziness of having a headache, distending pain in the chest and hypochondrium, breast close acute mastitis, cataract,
The effect of rubella itch.Endophyte of plant is important microorganism hereditary resource part, because of special parasitic or saprophytic ring
Border, often there is the physiological property different from edaphon.On traditional medicinal plant puncture vine endophyte research there is not yet
Report.
Pullulanase producing strains are always one of study hotspot in microbial function enzyme.Pullulanase is that one kind can be special
Property hydrolyzing alpha -1,6 glycosidic bond debranching enzyme, be both that amylopectin is degraded indispensable key enzyme, and one kind of detergent industry
Common additives.So far, most market shares of Pullulanase commodity are still by Denmark NOVO in world wide
Company is occupied.Also there are much reports on Pullulanase producing strains both at home and abroad, but most wild strain activity are relatively low,
The engineered strain built even by technique for gene engineering, also tend to be difficult to reach preferable ferment effect, enzymatic activity is stable
Property is poor.The microbial resources of nature are extremely abundant, and that is cultivated, identifies and develop by people only accounts for few part, institute
So that by the improvement of the research meanses such as test material, test method, culture medium innovation, functional enzyme is produced in constantly screening and accumulation
Bacterial strain is still a long-term and arduous research work.
At present, the screening of the Pullulanase producing strains of all kinds of document reports and authentication method are similar, major technique ring
Section is as follows：
The conventional sample type of screening Pullulanase producing strains mainly has：Conventional field soil, river, seawater, river bed and
Bottom silt, starch plant area soil etc..
Screening the most common based formulas that is separately cultured of Pullulanase producing strains is：Glutinous rice starch 25g, peptone 5g, yeast
Cream 5g, KH2PO40.5g, K2HPO40.5g, MgSO4·7H2O 0.1g, agar powder 20g, 1000mL, pH are settled to after being dissolved in water
It is worth for 6.0.
Pullulanase producing strains differential medium formula is：Peptone 10g, yeast extract 2g, red pulullan 3g, NaCl
2g, agar 20g, 1000mL, pH value 7.0 are settled to after being dissolved in water.
The method that the measure of Pullulanase activity is the most frequently used is 3,5- dinitrosalicylics acid system (DNS method).
The method of the Pullulanase producing strains system dientification of bacteria mainly includes the survey of thalline (bacterium colony) form, physiological and biochemical index
Fixed and 16S rDNA sequence analyses.
The general technology flow of test operation is：Gather test specimen --- enrichment culture --- primary dcreening operation is separately cultured ---
Discriminating, which is separately cultured (secondary screening), --- to be studied in Pullulanase determination of activity --- bacterial strain identification --- downstream.
Although have at present some production Pullulanase wild strains document report, really be able to be for industrialized production
Bacterium is seldom, so far, the most successful and extensive still only Bacillus acidopullulliticus of application.So
The scarcity of the higher wild strain of Pullulanase activity is still one of bottleneck problem for limiting the application of Pullulanase extensive exploitation, directly
Connect or indirectly have impact on bacterium mutation breeding and the structure of engineered strain of Pullulanase producing strains.With regard to China on Pullulanase
For research conditions, breakthrough achievement in research is seldom, and Pullulanase patented strain is less, is still formed without the core of competitiveness
Microorganism resource standby storehouse, the external present situation that monopoly position is in Pullulanase production field is not changed.
The content of the invention
In view of the shortcomings of the prior art, an object of the present invention is that have Propiram to provide in a kind of puncture vine endophyte
The bacterial strain and its artificial culture method of enzymatic activity.
The purpose of the present invention is achieved through the following technical solutions：
Pullulanase active bacterial strain in a kind of puncture vine endophyte provided by the invention, its bacterial strain category enterobacteria
(Enterobacter sp.), is preserved in China typical culture collection center, preservation address：China, Wuhan, Wuhan University,
Preserving number is CCTCC M 2015071, and the bacterial strain has Pullulanase active.
The cultural method of Pullulanase active bacterial strain in a kind of puncture vine endophyte provided by the invention, include following culture step
Suddenly：
Enrichment culture：Take the healthy stem of puncture vine to be cut into segment, the paraffin sealing that both ends are melted after rinsing, then use sterilized water
Rinse, it is 70% ethanol solution surface sterilization to be put into percent by volume, then is sterilized with 0.1mol/L mercuric chloride, under aseptic technique
The sealing paraffin at both ends is cut off, remaining stem is put into mortar is fully ground, and is collected in the test tube equipped with enriched medium and shakes
Bed culture.
It is separately cultured：Take appropriate enrichment culture liquid to be coated in solid separation culture medium under aseptic technique to cultivate.With
Oese chooses single bacterium colony, in the flat lining out purifying of solid separation culture medium.Then at the beginning of the method by the way that Lushi's iodine solution is added dropwise
Sieve production Pullulanase bacterial strain, selects bacterium colony small and hydrolyzes circle the greater as aimed strain.
Secondary screening：The aimed strain is inoculated in differential medium and cultivated, Lushi's iodine solution is then added dropwise, there will be hydrolysis
Circle person is defined as the bacterial strain with Pullulanase activity, while transfers in 4 DEG C of slant preservations of LB culture mediums.
In the enrichment culture step, the shaking table culture condition is 30 DEG C, 180r/min；It is described to be separately cultured in step
Condition of culture is 30 DEG C of incubated 48h；Condition of culture in the secondary screening step is 30 DEG C of incubated 48h.
The enriched medium each component concentration is：Glutinous rice starch 25g/L, trehalose 0.5g/L, peptone 10g/L, ferment
Female extract 5g/L, NaCl 9g/L, natural pH.
The solid separation culture medium each component concentration is：Amylopectin 3g/L, peptone 10g/L, yeast extract 5g/
L, NaCl 9g/L, agar powder 18g/L, pH 7.0.
The differential medium each component concentration is：Pulullan 2g/L, peptone 8g/L, yeast extract 2g/L,
NaCl 9g/L, agar powder 18g/L, pH 7.0.
The LB culture mediums each component concentration is：Tryptone 10g/L, yeast extract 5g/L, NaCl 9g/L, pH are
7.0。
The beneficial effects of the present invention are：The present invention has Pullulanase active bacterial strain using puncture vine as material, to life in it
Screened and identified, filter out the endophytic bacterial controlled effect for being capable of efficient degradation pulullan；Enriched medium passes through Reasonable adjustment
Each component content, be advantageous to the survival and culture of weak tendency bacterial strain；It is same by routine morphological, physiological and biochemical property, 16S rDNA
Source sequence and Phylogenetic analysis, Bacillus subtilis is carried out to the bacterial strain with Pullulanase activity filtered out, obtained
The general features and 16S rDAN gene orders of bacterial strain were obtained, it is determined that being under the jurisdiction of Enterobacter Enterobacter sp., was named
For enterobacteria Enterobacter sp.ZXY25；By DNS methods, the Pullulanase determination of activity to the bacterial strain shows, the bacterial strain
With higher Pullulanase activity, fermented and cultured 56h, yield of enzyme are reachable under the conditions of 30 DEG C, 180r/min, pH6.0
4.19U/mL, can be as good mutagenesis starting strain and the alternative bacterial strain of Pullulanase gene of structure engineering bacteria.
Brief description of the drawings
Fig. 1 show hydrolysis loop graphs of the bacterial strain ZXY25 of the present invention on pulullan flat board.
Fig. 2 show bacterial strain ZXY25 of the present invention Pullulanase activity figure.
Fig. 3 show bacterial strain ZXY25 of the present invention general features figure.
Fig. 4 show phylogenetic trees of the bacterial strain ZXY25 based on 16S rDNA gene orders of the present invention.
Embodiment
Present disclosure is described in detail below in conjunction with specific embodiment.It should be noted that retouched in following embodiments
The combination of the technical characteristic or technical characteristic stated is not construed as isolated, and they can be mutually combined so as to reach
Superior technique effect.
Embodiment 1
Fig. 1 show hydrolysis loop graphs of the bacterial strain ZXY25 of the present invention on pulullan flat board；Fig. 2 show bacterium of the present invention
Strain ZXY25 Pullulanase activity figure；Fig. 3 show bacterial strain ZXY25 of the present invention general features figure；Fig. 4 show the present invention
Phylogenetic trees of the bacterial strain ZXY25 based on 16S rDNA gene orders.
1st, test material
The source of puncture vine：Pick up from Luoyang city Lip river river levee bank.
Enriched medium：Glutinous rice starch 25g, trehalose 0.5g, tryptone 10g, yeast extract 5g, NaCl 9g, steam
Distilled water is settled to 1000mL, natural pH.
Solid separation culture medium：Amylopectin 3g, tryptone 10g, yeast extract 5g, NaCl 9g, agar powder 18g,
Distilled water is settled to 1000mL, pH7.0.
Differential medium：Pulullan 2g, peptone 8g, yeast extract 2g, NaCl 9g, agar powder 18g, distilled water
It is settled to 1000mL, pH7.0.
LB culture mediums：Tryptone 10g, yeast extract 5g, NaCl 9g, distilled water are settled to 1000mL, pH7.0.
Fermentation medium：Cornstarch 25g, dusty yeast 10g, K2HPO40.5g, KH2PO40.5g, MgSO47H2O 0.5g,
FeSO40.01g, distilled water are settled to 1000mL, natural pH6.0.
2nd, cultural method
Enrichment culture：The healthy stem of puncture vine is taken, is first rinsed with running water, is cut into 5cm segments, the paraffin envelope that both ends are melted
Mouthful, with aseptic water washing three times afterwards, 70% ethanol solution surface sterilization 5min is put into, then sterilized with 0.1mol/L mercuric chloride
3min, the sealing paraffin at both ends is cut off under aseptic technique, remaining stem is put into mortar is fully ground, and is collected in dress
In the test tube for having 15mL enriched mediums, the shaking table culture 12h under the conditions of 30 DEG C, 180r/min.
It is separately cultured：Appropriate enrichment culture liquid is taken to be coated on solid separation culture medium under aseptic technique, in 30 DEG C of perseverances
Temperature culture 48h.With oese from flat board the typical single bacterium colony of picking well-grown, feature, still in solid separation culture medium
Flat lining out purifying, is carried out continuously 3 times.After numbering backup, Pullulanase bacterium is produced by the method primary dcreening operation that Lushi's iodine solution is added dropwise
Strain, bacterium colony is small and hydrolyze circle the greater as aimed strain.
Secondary screening：The target being separately cultured is purified into inoculation after differential medium, 30 DEG C of incubated 48h, is added dropwise
Lushi's iodine solution, still there is hydrolysis circle person, primarily determine that as the bacterial strain with Pullulanase activity.Transfer simultaneously in LB culture mediums
4 DEG C of slant preservations.
3rd, the activity determination method of Pullulanase bacterial strain is produced
The preparation of crude enzyme liquid：By purified inoculation in the triangular flask equipped with 25mL LB culture mediums, 30 DEG C,
180r/min shaking table culture 16h, take out 4mL bacterium solutions and transfer in the 500mL triangular flasks equipped with 50mL fermentation mediums, it is identical
Under the conditions of concussion and cultivate, every 8h sampling once.Zymotic fluid 4000r/min room temperatures are centrifuged into 20min, supernatant is thick enzyme
Liquid.
Enzyme assay is carried out to secondary screening bacterial strain using DNS methods.It is specific as follows：In 1mL 0.5% pulullan solution
Middle addition 1mL phosphate buffers (pH6.0), add 1mL crude enzyme liquids, after 60 DEG C of water-bath 30min, add DNS 1.5mL, boiling water
5min is bathed, taking-up is cooled down rapidly with flowing water, finally in 520nm wavelength measurement light absorption values.One enzyme-activity unit is defined as：Corresponding
Under reaction condition, the enzyme amount needed for 1 μm of ol glucose is discharged after degraded pulullan per minute, is represented with 1U/mL.
4th, the authentication method of bacterial strain
The Biochemical Indexes method of 4.1 bacterial strains
Assay method is with reference to Liu Guosheng chief editors'《Microbiology Experiment technology》With Pan Chunmei, Zhang Xiaojing chief editor's《Micro- life
Thing technology》, identification reference《The outstanding Bacteria Identification handbook (the 8th edition) of uncle》.
The 16S rDNA analysis methods of 4.2 bacterial strains
By the inoculation with Pullulanase activity in LB fluid nutrient mediums, 30 DEG C, 180r/min shaking table culture 12h,
Obtain bacterium solution.Using TaKaRa MiniBEST Bacterial Genomic DNA Extraction kit Ver.2.0 reagents
Box extracts genomic DNA.
16S rDNA pcr amplification primer things：Forward primer 27F：5′-AGAGTTTGATCCTGGCTCAG-3′
Reverse primer 1492R：5′-TAGGGTTACCTTGTTACGACTT-3′
Primer is synthesized by the prosperous bio tech ltd of Beijing AudioCodes.
PCR amplification system (20 μ L)：10 × Ex Taq PCR buffer 2 μ L, dNTP (10mmol/mL) 1.6 μ L, it is positive
With reverse primer (20 μm/mL) each 1 μ L, 1 μ L, Ex Taq archaeal dna polymerases of DNA profiling 0.2 μ L, ddH2O 13.2μL.PCR expands
Increase program：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 50 DEG C of annealing 45s, 72 DEG C of extension 1min 30s, are circulated 30 times；72℃
Final extension 7min.Expanding fragment length is 1.5kb or so, and PCR primer cuts glue after 1% agarose gel electrophoresis, is used
TaKaRa MiniBEST Agarose Gel DNA Extraction kit Ver.3.0 kits carry out glue reclaim, recovery production
Thing and TaKaRa pMDTM18-T Vecter carriers connect, by recombinant plasmid transformed in DH5 α competent escherichia coli cells, 37
DEG C it is incubated overnight, is screened through blue hickie, send Beijing AudioCodes prosperous bio tech ltd's sequencing to positive colony.
Using MEGA6.06 softwares carry out Multiple Sequence Alignment, select Neighbor-Joining method constructing systems develop into
Change tree.
5th, result of the test
By enrichment culture, it is separately cultured and secondary screening, obtains one plant of pulullan degraded hydrolysis and enclose big and significant bacterial strain,
As shown in figure 1, tentatively judging that it has higher Pullulanase activity, it is defined as aimed strain, strain number ZXY25.
5.1 Pullulanase active bacterial strain ZXY25 enzyme assay
Pullulanase determination of activity is carried out to ZXY25 bacterial strains using DNS methods, the results showed that, the Pullulanase of ZXY25 bacterial strains
Activity is up to 4.19U/mL, confirms with good Pullulanase activity, as shown in Fig. 2 the Pullulanase of ZXY25 bacterial strains
Activity is ideal, is the good wild starting strain of mutation breeding, has larger development and application potentiality.
The identification of 5.2 ZXY25 bacterial strains
(1) colony characteristicses of ZXY25 bacterial strains
ZXY25 bacterial strains are straight profiled bat bacterium, and thalline is long 1.3-2.9 μm, wide 0.5-1.2 μm, have flagellum and pod membrane, can move,
Colony edge on pulullan flat board is neat, surface wettability, low projection, bacterium colony blood red, and zymotic fluid is also in blood red, such as
Shown in Fig. 3.
(2) Biochemical Indexes of ZXY25 bacterial strains
Assay method is with reference to Liu Guosheng chief editors'《Microbiology Experiment technology》With Pan Chunmei, Zhang Xiaojing chief editor's《Micro- life
Thing technology》, identification reference《The outstanding Bacteria Identification handbook (the 8th edition) of uncle》.
Measurement result shows, Gram-negative, amphimicrobian, no gemma, clark and Lubsreaction, indole reaction, phenylpropyl alcohol
Propylhomoserin dehydrogenase, DNA enzymatic, urase and indole reaction are negative, lactose, V-P reactions, citrate reacting positive, gelatin liquefaction, no
Produce hydrogen sulfide.It is initially identified as Enterobacter Enterobacter.
(3) the 16S rDNA identifications of ZXY25 bacterial strains
By ZXY25 inoculations in LB fluid nutrient mediums, 32 DEG C, 200r/min shaking table culture 16h, bacterium solution is obtained.Using
TaKaRa MiniBEST Bacterial Genomic DNA Extraction kit Ver.2.0 kits extract genome
DNA。
The 16S rDNA pcr amplification primers things used for：
Forward primer, 27F：5′-AGAGTTTGATCCTGGCTCAG-3′
Reverse primer, 1492R：5′-TAGGGTTACCTTGTTACGACTT-3′
PCR amplification system (20 μ L)：10 × Ex Taq PCR buffer 2 μ L, dNTP (10mmol/mL) 1.6 μ L, it is positive
With reverse primer (20 μm/mL) each 1 μ L, 1 μ L, Ex Taq archaeal dna polymerases of DNA profiling 0.2 μ L, ddH2O 13.2μL.PCR expands
Increase program：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 50 DEG C of annealing 45s, 72 DEG C of extension 1min 30s, are circulated 30 times；72℃
Final extension 7min.Expanding fragment length is 1.5kb or so, and PCR primer cuts glue after 1% agarose gel electrophoresis, is used
TaKaRa MiniBEST Agarose Gel DNA Extraction kit Ver.3.0 kits carry out glue reclaim, recovery production
Thing and TaKaRa pMDTM18-T Vecter carriers connect, by recombinant plasmid transformed in DH5 α competent escherichia coli cells, 37
DEG C it is incubated overnight, is screened through blue hickie, positive colony sequencing.
The ZXY25 bacterial strain 16S rDNA sequence lengths of acquisition are 1503bp, and sequence is as follows：
TAGGGTTACCTTGTTACGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCT
ACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCT
GATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGA
GGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCC
ATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTG
GCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCA
GCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCT
TCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGC
GGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGAC
ATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTG
TCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCC
CTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAG
ACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGG
AGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCAATCGCCAAGGTTATTAACCTTAACGCCTTCCTCCTCGCTGAAAG
TACTTTACAACCCGAAGGCCTTCTTCATACACGCGGC
ATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAG
TTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCTA
ATCCCATCTGGGCACATCTGATGGCATGAGGCCCGAAGGTCCCCCACTTTGGTCTTGCGACGTTATGCGGTATTAGC
TACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCGA
GAGCAAGCTCTCTGTGTTACCGCTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCTGAGCCAGGATCAAA
CTC
Homology is carried out by NCBI blast on-line analyses function to the 16S rDNA gene orders of ZXY25 bacterial strains to search
Rope, and chadogram is developed with the Neighbor-Joining methods constructing system in MEGA6.06 softwares, as shown in Figure 4.As a result
Show, ZXY25 and clostridium perfringen Enterobacter aerogenes gather for one, illustrate that the two affiliation connects the most
Closely.Synthetical cultivation feature, physiological and biochemical property and 16S rDNA the sequencing results, determine that ZXY25 is under the jurisdiction of Enterobacter
Enterobacter, it is named as enterobacteria Enterobacter sp.ZXY25.
Although having been presented for some embodiments of the present invention herein, it will be appreciated by those of skill in the art that
Without departing from the spirit of the invention, the embodiments herein can be changed.Above-described embodiment be it is exemplary, no
Restriction that should be using the embodiments herein as interest field of the present invention.
Claims (1)
1. Pullulanase active bacterial strain in a kind of puncture vine endophyte, it is characterised in that the bacterial strain is under the jurisdiction of enterobacteria
(Enterobacter sp.), is preserved in China typical culture collection center, and preserving number is CCTCC M 2015071, the bacterium
Strain has Pullulanase activity.
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| CN102604861A (en) * | 2012-02-16 | 2012-07-25 | 复旦大学 | Pullulanase, pullulanase producing strain and application of the pullulanase |
| CN102676437A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Producing method of extracellular pullulanase and special microorganism thereof |
| CN103525852A (en) * | 2013-10-24 | 2014-01-22 | 江南大学 | High-throughput screening method of recombinant bacteria based on combination of self-induction culture and chromatic substrate |
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