CN104531573B - A kind of bacillus amyloliquefaciens and its application - Google Patents
A kind of bacillus amyloliquefaciens and its application Download PDFInfo
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Abstract
The present invention relates to a kind of bacillus amyloliquefaciens and its application, belong to technical field of microbial fermentation.The invention provides a kind of new bacterial strain；A kind of bacillus amyloliquefaciens, its preserving number are CGMCCNo.9928, and preservation date is on November 4th, 2014, and depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center（CGMCC）.The preserving number of the present invention can produce naringinase for the bacillus amyloliquefaciens of CGMCCNo.9928.Separation and Culture first of the invention has gone out to produce the solution amylase bacillus cereuss of naringinase；In its liquid fermentation liquid, naringin enzyme activity can be up to 201.12 U/mL.
Description
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens and its application, belong to technical field of microbial fermentation.
Technical background
Naringinase is a kind of important enzyme for bitter substance hydrolysis in fruit juice, is by alpha-L-Rhamnosidase（α-L-
Rhamnosidase, EC 3.2.1.40）With β-D-Glucose glycosides enzyme（β-D-glucosidase, EC3.2.1.21）The one of composition
The compound enzyme system of glycoside hydrolysis is planted, while the activity with alpha-L-Rhamnosidase and β-D-Glucose glycosides two kinds of enzymes of enzyme, naringinase
The naringin of bitterness can be acted on and generation Flavonoid substances are hydrolyzed, process is naringin first in alpha-L-rhamnoside
Rhamnose and Pu Luning are generated under enzyme effect（Prunin, and translations prunin）, three points of the bitterness about naringin of Pu Luning
One of, therefore bitterness mitigated, then general Shandong would rather be broken down into the Fructus Citri grandiss without bitterness in the presence of β-D-Glucose glycosides enzyme
Skin flavonoid substance（Predominantly naringin）.The microbe-derived aspect of naringinase is concentrated mainly on mycete（Aspergillus niger and penicillium sp
Deng）, antibacterial produce naringinase relevant report it is less.
China is Orange Producing big country, but the depth that the current processing industry subject matter in orange juice is exactly peel adds
Work Utilizing question.Contain abundant flavonoid of the peel of pomelo (predominantly Pericarpium Citri grandiss in the peel of Citrus reticulata Blanco for being taken as rubbish to process in a large number to drop to
Glycosides), pectin, dietary fiber etc., if being made full use of, ambient pressure can not only be mitigated, can also be brought benefit to the mankind.
Modern pharmacology research finds that flavonoid of the peel of pomelo has various physiologically actives：The fragility and permeability of scalable blood capillary, protection
Heart, has the effects such as antiulcer, removing toxic substances, diuresis, antibacterial, antiinflammatory heat clearing away, is also a kind of efficient Natural antioxidant, is one
Plant natural health promoting product.Therefore the deep processing of Pericarpium Citri grandiss will greatly improve the income of orchard worker, improve added value of Fructus Citri grandiss etc..
Although the research to naringinase is more and more deep, the production of only minority naringinase at present realizes industrialization,
The heavy industrialization application aspect for limiting naringinase at present has two：First subject matter is that China's shortage is adapted to
The high bacterium producing multi enzyme preparation of industrialized production, this just strongly limit application, the cultivation where the shoe pinches of high naringinase-producing strain be as
Where select outstanding mutant efficient in substantial amounts of mutagenic progeny, up to the present there is no preferable method, bacterial strain
Breeding Progress is slow, develops that new efficient screening technique is necessary, therefore is current working as to the cultivation of superior strain
Business is anxious.Second Problem is that naringin fixation techniques for enzyme also has many defects at home, used in immobilization
Reagent or carrier has high expensive, immobilization efficiency low, less stable, continuous operation are used equipment it is more complicated
Deng this is accomplished by our countries and further develops easier while more applicable process for fixation, and naringinase is applied to mandarin orange
And the de- bitter and existing relevant report of processing foreign of other products, but aborning should not on a large scale in China's naringinase
With main cause is that the country there is no commodity naringinase prepared by scale to sell, and we should strengthen with regard to breeding high-yield naringin from now on
The method of enzyme bacterial strain, and the pure enzyme of naringinase of low cost is prepared, with important practical significance.
With mycete（Aspergillus niger and penicillium sp etc.）Liquid fermentation produces naringinase and compares, and bacterial liquid fermenting and producing naringinase has
Growth cycle is short, low production cost, easily controllable growth conditionss many advantages, such as, obtained researcher in recent years and more paid close attention to.
But, the naringin enzymatic activity of antibacterial production is compared with mycete, also with a certain distance；Current institute reporter bacterium enzymatic production water
It is flat to be less than 1000 U units per ml fermentation liquids；Bacterial fermentation produces naringinase optimum temperature partial neutral environment and may limit
Its certain applications in sour environment.
Prior art（Before the present invention）The bacillus amyloliquefaciens reported are in the Biological control side of plant pathogenic fungi
Face applies more, but has no that bacillus amyloliquefaciens produce the report of naringinase.
The content of the invention
The present invention has turned out a kind of bacillus amyloliquefaciens that can produce naringinase by creative work.The present invention
Bacillus amyloliquefaciens liquid fermentation liquid in naringin enzyme activity reach 201.12 U/mL, naringinase total enzyme activity power after purification
10556U can be reached, specific enzyme activity power reaches 15609U/mg；The naringinase produced before far above the present invention, by antibacterial and mycete
Enzyme activity.In addition, the liquid fermentation liquid of the bacillus amyloliquefaciens of the present invention also contains naringin and naringenin；Wherein naringenin
It is Flavonoid substances, with health-care effecies such as blood pressure lowering, removing toxic substances, diuresis, antibacterials.The Fructus Citri grandiss that the bacillus amyloliquefaciens of the present invention are produced
Glycosides enzyme, molecular weight be 32.5 kDa, optimum operative temperature, pH be respectively 45 DEG C, 7.5.The bacillus amyloliquefaciens of the present invention
The naringinase that by antibacterial with mycete produced of the naringinase of product before the present invention is compared, and there is following special character：From Xie Dian
The part zymologic property of naringinase is produced in afnyloliquefaciens 11568（Optimum pH and pH stability, optimum temperature and temperature stabilization
Property）Deng research, show which has very big application prospect in the field such as fruit juice debitterizing of commercial application prospect, improving wine
Local flavor, the fragrance component for improving beverage and production food additive in also function to important function, to naringinase isolate and purify and
Property Quality Research provide important evidence to prepare the commercial enzyme preferably industrially applied.
Technical scheme
The invention provides a kind of new Bacillus amyloliquefaciens strain.
A kind of bacillus amyloliquefaciens（Bacillus amyloliquefacines ）11568, its preserving number is
CGMCCNo.9928, preservation date are on November 4th, 2014, and depositary institution is that China Committee for Culture Collection of Microorganisms is general
Logical microorganism center（CGMCC）.
The bacillus amyloliquefaciens 11568 of the present invention, which can produce naringinase.
Present invention also offers a kind of new application of bacillus amyloliquefaciens 11568.
Application of the bacillus amyloliquefaciens 11568 of the present invention in production naringinase.
Specifically a kind of application process is：
（1）The bacillus amyloliquefaciens 11568 of the present invention are carried out into slant culture, slant strains are obtained；
（2）Take slant strains to be inoculated in sterilized seed culture medium, the fermentation training under conditions of 40.9 DEG C, 180rpm
Foster 48h, obtains liquid fermentation liquid；
（3）Centrifugation liquid fermentation liquid, takes supernatant；
（4）Supernatant through ammonium sulfate precipitation, ion-exchange chromatography, Sephacryl S-200 gel permeation chromatographies,
Obtain naringinase.
Gained naringinase is the pure naringinase of electrophoresis that purification is 38.27；Total enzyme activity power can reach 10556U, compare enzyme
Vigor reaches 15609U/mg.The molecular weight of the naringinase for being obtained be 32.5 kDa, optimum temperature, pH be respectively 45 DEG C,
7.5。
Above-mentioned steps（1）In, the slant medium contains following component in every 1L：MgSO4 0.5g、KH2PO4
1.5g、CaCl20.1g、（NH4）2SO4 1.5g、KCl 0.5g、KNO31.5g, yeast extract 2g, orange meal 15g, agar 20g,
PH is 6.0,1 × 105 Pa 20 min of sterilizing.
Above-mentioned steps（2）In, the seed culture medium contains following component in every 1L：
Maltose 15.1g, tryptone 10g,（NH4）2HPO420g, NaCl 10g, yeast extract 5g, pH is
7.51；
Or, naringin 10g, tryptone 10g,（NH4）2HPO420g, NaCl 10g, yeast extract 5g, pH is
7.51；
Or, MgSO4 0.5g、 KH2PO4 1.5g、 CaCl20.1g、（NH4）2SO4 1.5g、 KCl 0.5g、KNO3
1.5g, yeast extract 2, orange meal 7.5g, pH is 6.0.
Above-mentioned steps（3）In, liquid fermentation liquid centrifugation Jing after supersonic wave wall breaking；Ultrasonic frequency is 25-30Hz, is surpassed
The sound time is 10min, ultrasonic 2.5s, stops 2.5s；The rotating speed of centrifugation is 5000-6000rpm, and centrifugation time is 10-15min.
Above-mentioned steps（4）In, after ion-exchange chromatography, before gel permeation chromatography, use 0.01-0.5mol/L
NaCl carries out eluting.
The step（4）Middle ammonium sulfate precipitation is concretely comprised the following steps：Ammonium sulfate is added in supernatant so as to quality
Volumetric concentration is 0.01%-40%, is filtered to remove the precipitation of generation, then proceedes to add ammonium sulfate, to its mass-volume concentration
Till for 40%-80%, it is filtrated to get and precipitates containing naringinase.Described mass-volume concentration is referred to：Contain sulfur in per mL supernatant
The quality of sour ammonium（g）.
The step（4）Intermediate ion displacement chromatography, refers to Q Sepharose Fast Flow ion-exchange chromatographies；Its tool
Body step is：First with buffer system be 20mmol/L, the Tris-HCl buffer balance columns material of pH 8.5；After baseline balance
500 microlitres of destination protein amount of liquid is added, adsorption time is 30min；Entered with the NaCl solution of 0.01-0.5mol/L after absorption
Row gradient elution, flow velocity are set to 1mL/min, detect naringin enzyme activity and protein content in often pipe eluent.Wherein, gradient
The concrete operations of eluting are：Arranging elution program carries out gradient elution with the NaCl solution of 0.01-0.5mol/L, and flow velocity is set to
1mL/min, collects liquid.
The step（4）The concrete operations of middle gel permeation chromatography are：Fill in SephacrylS-200 post material；With
50mmol/LNaCl solution（7.2 phosphate buffer of 50mmol/L, pH is solvent）, flow velocity 0.1mL/min, balance columns material；Balance
Afterwards with same buffer eluting, flow velocity is maintained at 0.1mL/min, arranges often pipe and collects 1mL；Detection often in pipe naringin enzyme activity and
Protein content.Wherein, SephacrylS-200 is the model of chromatographic column, and the chromatographic column of unavailable other models replaces.
Can be using one or more adjustable inclined surface apparatus culture medium and seed culture medium in hydrochloric acid, sulphuric acid or phosphoric acid
pH。
The inoculum concentration of slant culture and fermentation culture can be obtained by prior art, be not required to pay performing creative labour.
The inoculum concentration of slant culture can reach the seed liquor of inoculum concentration needed for fermentation culture, and the inoculum concentration of fermentation culture is generally
0.2%（v/V）.
In the present invention, for the percentage ratio of the content of certain composition, if not otherwise specified, percentage by weight is referred both to
（w/w）.
Wherein, to the step in above-mentioned concrete application method（2）The liquid fermentation liquid for being obtained detected, testing result
For：Naringin enzyme activity reaches 201.12 U/mL, and naringinase total enzyme activity power can reach 10556U after purification, and specific enzyme activity power reaches
15609U/mg.Also containing naringin, naringin hydrolysis substrate naringenin in the liquid fermentation liquid.Naringenin is Flavonoid substances,
With health-care effecies such as blood pressure lowering, removing toxic substances, diuresis, antibacterials.
So, present invention also offers a kind of liquid fermentation liquid of bacillus amyloliquefaciens 11568.
Technical term explanation used by of the invention：
1U refers to that the naringin enzyme amount decomposed required for 1 μ g naringin substrates per minute is referred to as a naringinase enzyme activity.
Beneficial effect
Separation and Culture has gone out to produce the solution amylase bacillus cereuss 11568 of naringinase first.
In the liquid fermentation liquid of the solution amylase bacillus cereuss 11568 of the present invention, naringin enzyme activity can be up to 201.12
U/mL。
The naringinase of the present invention, compared with the naringinase before the present invention, possesses following advantage：Optimum pH and pH stable
Property, the performance such as optimum temperature and temperature stability it is good, have greatly using front in the field such as fruit juice debitterizing of commercial application prospect
Scape, also functions to important function, in the local flavor, the fragrance component for improving beverage and production food additive for improving wine to Fructus Citri grandiss
Glycosides enzyme isolate and purify and property Quality Research provides important evidence to prepare the commercial enzyme preferably industrially applied.
The method that the present invention prepares naringinase, compared with before the present invention, antibacterial, funguses prepare the method for naringinase, has
Producing enzyme level height, short fermentation time, easily controllable growth conditionss, be easily isolated, purity is high, simple to operate, mild condition etc. is all
Many advantages, the application of entirely appropriate industrialized production.It is to crush the orange peel of 60 mesh as naringinase fermentation substrate, fully sharp
It is with house refuse garbage, cost-effective.
Preservation explanation
China Committee for Culture Collection of Microorganisms's common micro-organisms center of depositary institution
Address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica
Preservation date：On November 4th, 2014
Deposit number：CGMCCNo.9928
Classification And Nomenclature bacillus amyloliquefaciens（Bacillus amyloliquefaciens）.
Description of the drawings
The testing result of the liquid fermentation liquid that Fig. 1 is obtained by embodiment 2；
In Fig. 1,2.609 peaks represent naringin；6.582 peak is naringenin；
Fig. 2 produces naringin enzyme purification electrophoretogram by bacterial strain 11568；
In Fig. 2：M- standard protein samples；The protein band of 1- crude enzyme liquids；The protein band of 2-40 ~ 80% ammonium sulfate precipitation concentration gained；
The protein band that 3- Jing Q Sepharose Fast Flow ion-exchange chromatographies are obtained；4- Jing Sephacryl S-200 gel filtrations
The protein band that chromatography is obtained.
Specific embodiment
Below in described all specific embodiments, if no special instructions, method commonly used in the art is.In embodiment
Unreceipted particular technique or condition person, according to technology or condition described by document in the art（With reference to the phase of Puri et al.
Close document）Or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, are and can pass through commercial
The conventional products of acquisition.
1 bacillus amyloliquefaciens of embodiment, 11568 screening and separating and identification
First, bacillus amyloliquefaciens JNU002（Bacillus amyloliquefacines ）11568 screening and separating
2g collections are weighed from soil sample in all parts of the country or mouldy Pericarpium Citri Reticulatae or/and Pericarpium Citri grandiss, in the 18mL equipped with sterilization treatment
30min is vibrated in the 100mL conical flasks of normal saline, a little bead is preferably put in conical flask.Then distinguish dilute with sterilized water
Volumetric concentration is interpreted into for 1 × 10-3、1×10-4The diluent of two gradients, draws 100 μ L diluents with the pipette tips of sterilizing respectively
Coat in the selectivity plating medium prepared in advance and sterilize, 48-96h is cultivated in 30 DEG C.From selectivity flat board culture
Line on base separates single bacterium colony, and the part film-making microscopy in the single bacterium colony of picking is judged bacterial strain purity（Micro- sem observation thalline
For same morphological characteristic）.If not pure bacterium colony, repeated multiple times line is answered to separate single bacterium colony, till pure bacterium colony is obtained.Will
The pure bacterium colony for obtaining is preserved with slant medium, obtains bacterial strain pure culture body；Meanwhile, it is mild-natured in the selection of the pure bacterium colony isolated
Deca diglycol in plate culture medium（DES）Reagent, has seen whether transparent circle phenomenon, and the bacterial strain for having transparent circle is selected
Out, as the basis of follow-up secondary screening.The nutritional labeling of the selectivity plating medium is（g/L）：MgSO40.5,
KH2PO4 1.5, CaCl2 0.1,（NH4）2SO4 1.5, KCl 0.5, KNO31.5, yeast extract 2, orange meal 7.5, agar 20.
Follow-up secondary screening：The bacterial strain for having transparent circle is carried out into shake-flask culture, the big bacterial strain of transparent circle is chosen as aimed strain,
It is named as：Bacillus amyloliquefaciens 11568；The nutritional labeling of Shake flask medium used is（g/L）：MgSO4 0.5, KH2PO4
1.5, CaCl2 0.1,（NH4）2SO41.5, KCl 0.5, KNO31.5, yeast extract 2, orange meal 7.5, pH are 6.0.
2nd, bacillus amyloliquefaciens（Bacillus amyloliquefacines ）11568 identifications
1. morphological characteristic
In LB culture medium flat plates culture 48 hours at 40 DEG C, bacterium colony surface is smooth, moistening, and edge is irregular, gram dye
Color is positive, 2 ~ 15mm of colony diameter, can spread growth, and bacterium colony is transparent, and without parasporal crystal, bacterium colony is without projection.It is flat in LB culture medium
It is in rod-short that thalline is cultivated on plate.The nutritional labeling of the LB culture medium flat plates culture is（g/L）：Yeast extract 5, NaCl
10, tryptone 10.
2.16S rDNA sequence analysis：
Few sequence nucleotide primer is that funguses PCR expands universal primer：
Forward primer ITS1：5'-TCCGTAGGTGAACCTGCGG-3', as shown in SEQ ID NO.2；
Downstream primer ITS4：5'-TCCTCCGCTTATTGATATGC -3'；As shown in SEQ ID NO.3；
The 16S rDNA of the bacterium are expanded using PCR technologies；
The concrete operations of pcr amplification reaction are as follows（The operation not illustrated is routine operation）：
Reaction system is sequentially added into PE tubules；Open PCR amplification instrument amplification；Instrument is closed after completing reaction；Take out anti-
Liquid is answered to enter row agarose gel electrophoresis inspection DNA amplification
Various reagents and consumption involved by pcr amplification reaction system：Pcr amplification reaction system：0.5 μ L template DNAs, 1.0
μ L dNTP, 0.5 μ L Taq enzymes, each 1.0 μ L of primer, moisturizing to 50 μ L.
Pcr amplification reaction condition:94 DEG C of 4 min of preheating, each circulation include：94 DEG C of degeneration 30s, 56 DEG C of 30 s of annealing,
72 DEG C of extension 6min, 30 circulations, finally again with 72 DEG C, 10 min.
Pcr amplification product entrusts AudioCodes prosperous company in Beijing to complete examining order.
PCR primer is sequenced Jing after agarose gel electrophoresiies inspection, and DNA sequence table is：SEQ ID NO.1.
3. physiological and biochemical property
According to《Primary Jie Shi handbooks》（R.E. Buchanan, basic this grade of N.E. are compiled, Science Press, and 1984）Bacterial strain is entered
Row Physiology and biochemistry is identified（It is shown in Table 1）, its physiological and biochemical property matched with bacillus amyloliquefaciens.
Table 1：Biochemical reactions table
Preparation of the embodiment 2 containing naringinase, bacillus amyloliquefaciens 11568 liquid fermentation liquid
（1）Slant culture：Bacillus amyloliquefaciens 11568 are inoculated on slant medium, are cultivated 48 hours at 40 DEG C,
Obtain inclined-plane bacterial strain.Slant medium used（g/L）For：MgSO4 0.5、KH2PO4 1.5、CaCl20.1、（NH4）2SO4 1.5、
KCl 0.5, KNO3 1.5, yeast extract 2, orange meal 15, agar 20, pH 6.0,1 × 105Pa 20 min of sterilizing.
（2）Liquid shake-flask fermentation：Will inclined-plane bacterial strain with 0.2%（v/V）Inoculum concentration be inoculated into liquid submerged culture base
In, 40.9 DEG C of cultivation temperature, shaking speed 180r/min, incubation time 48h；Obtain liquid fermentation liquid.Liquid submerged culture base（g/
L）For：MgSO4 0.5、KH2PO4 1.5、CaCl20.1、（NH4）2SO4 1.5、 KCl 0.5、KNO31.5th, yeast extract 2, Fructus Citri tangerinae
Corium farinosum 7.5, pH 6.0,1 × 105Pa 20 min of sterilizing.
（3）By high performance liquid chromatography（Water：Methanol=1:1）Determine the composition of liquid fermentation liquid, testing result such as Fig. 1：
Contain naringin, naringenin in liquid fermentation liquid.
（4）The confirmation of naringinase and the measure of enzyme activity：Using improvement Davis methods, 1 g/ L Fructus Citri grandiss are added in test tube
2 mL of skin glycosides titer, 0. 2 mol/ L pH5.5 acetate buffer solution 1.9mL, preheats 5 min in 40 DEG C of thermostat water baths, then
Add 0.1 mL liquid shake-flask fermentation liquid, 40 DEG C of 30 min of isothermal reaction；Obtain enzymolysis solution.Take 0.1 mL enzymolysis solutions and add 90%
The NaOH solution 0.5mL of diglycol 5mL, 1mol/L, distilled water 0.4mL, and take 0.1 mL distilled water and do blank
（Distilled water is enzyme activity Basis with the difference of enzymolysis solution absorbance）.After 40 DEG C of 15 min of water bath with thermostatic control, at 420 nm
Survey absorbance.So that, under the conditions of 40 DEG C, pH5.5, the enzyme amount needed for 1 μ g naringins of hydrolysis per minute is 1 enzyme activity unit
（U）.Measure step（2）The naringinase enzyme activity of the liquid fermentation liquid of preparation is 197.83U/mL.
Preparation of the embodiment 3 containing naringinase, bacillus amyloliquefaciens 11568 liquid fermentation liquid
By 2 step of embodiment（1）Cultured inclined-plane bacterial strain is with 0.2%（v/V）Inoculum concentration be inoculated into equipped with liquid culture
In the 250ml conical flasks of base, liquid shake-flask fermentation, 40.9 DEG C of fermentation temperature, 180 r/min of shaking speed, fermentation time are carried out
48 h；Obtain liquid fermentation liquid.Liquid culture based component（g/L）：Naringin 10, tryptone 10,（NH4）2HPO4 20、NaCl
10th, yeast extract 5；PH 7.51,1 × 105Pa 20 min of sterilizing.
（3）By high performance liquid chromatography（Water：Methanol=1:1）Determine the composition of liquid fermentation liquid, testing result such as Fig. 1：
Contain naringin, naringenin in liquid fermentation liquid.
（4）Assay method according to the naringinase enzyme activity of embodiment 2 determines the liquid fermentation liquid prepared by the present embodiment
Naringin enzyme activity；Testing result is：Naringin enzyme activity reaches 140 U/mL.
Preparation of the embodiment 4 containing naringinase, bacillus amyloliquefaciens 11568 liquid fermentation liquid
By 2 step of embodiment（1）Cultured inclined-plane bacterial strain is with 0.2%（v/V）Inoculum concentration be inoculated into equipped with liquid culture
In the 250ml conical flasks of base, liquid shake-flask fermentation, 40.9 DEG C of fermentation temperature, 180 r/min of shaking speed, fermentation time are carried out
48 h；Obtain liquid fermentation liquid.Liquid culture based component（g/L）：Maltose 15.1, tryptone 10,（NH4）2HPO4 20、
NaCl 10, yeast extract 5；PH 7.51,1 × 105Pa 20 min of sterilizing.
（3）By high performance liquid chromatography（Water：Methanol=1:1）Determine the composition of liquid fermentation liquid, testing result such as Fig. 1：
Contain naringin, naringenin in liquid fermentation liquid.
（4）Assay method according to the naringinase enzyme activity of embodiment 2 determines the liquid fermentation liquid prepared by the present embodiment
Naringin enzyme activity；Testing result is：Naringin enzyme activity reaches 201.12 U/mL, at home and abroad locates in naringinase correlational study
In higher producing enzyme level.
5 bacillus amyloliquefaciens of embodiment, 11568 liquid fermentation produces naringinase
By centrifugation after liquid fermentation liquid supersonic wave wall breaking, supernatant is taken.Wherein, ultrasonic frequency is 25Hz, ultrasound
Time is 10min, ultrasonic 2.5s, stops 2.5s；The rotating speed of centrifugation is 5000rpm, and centrifugation time is 10min.
By supernatant ammonium sulfate precipitation.Concrete operations are：Ammonium sulfate is added in supernatant so as to which quality volume is dense
Spend for 40%, be filtered to remove the precipitation of generation, then ammonium sulfate continuously added in filtrate, to its mass-volume concentration be
Till 40%-80%, it is filtrated to get and precipitates containing naringinase.Described mass-volume concentration means：Contain in per mL supernatant
The quality of ammonium sulfate（g）.
Naringinase precipitation 0.4mol/L NaCl will be contained（The concentration of NaCl can also be 00.1-0.5mol/L）Washed
It is de-；The concrete operations of eluting are：First with buffer system be 20mmol/L, the Tris-HCl buffer balance columns of pH 8.5
Material；500 microlitres of destination protein amount of liquid is added after baseline balance, adsorption time is 30min；0.01-0.5mol/ is used after absorption
The NaCl solution of L carries out gradient elution, and flow velocity is set to 1mL/min, detects naringin enzyme activity and protein in often pipe eluent
Content.
After by eluting containing naringinase precipitate, carry out successively Q Sepharose Fast Flow ion-exchange chromatographies,
SephacrylS-200 gel permeation chromatographies, that is, obtain naringinase.
Which concretely comprises the following steps：Fill in SephacrylS-200 post material；Use 50mmol/LNaCl solution（50mmol/L, pH
7.2 phosphate buffers are solvent）, flow velocity 0.1mL/min, balance columns material；With same buffer eluting after balance, flow velocity is maintained at
0.1mL/min, arranges often pipe and collects 1mL；Detect naringin enzyme activity and protein content in often pipe；Wherein, SephacrylS-
200 is the model of chromatographic column, and the chromatographic column of unavailable other models replaces.
Respectively liquid fermentation liquid prepared by embodiment 2,3 or 4 is carried out separating, purification, obtain purification for 38.27
The pure naringinase of electrophoresis；Purity and Rate activity are shown in Table 2.
Table 2
。
Jing SDS-PAGE can measure the molecular weight of the enzyme and be 32.5 kDa, and SDS-PAGE results are shown in Fig. 2.
<110>Beijing Technology and Business University
<120>A kind of bacillus amyloliquefaciens and its application
<160>3
<210>1
<211>1443
<212>DNA
<213>Bacillus amyloliquefaciens（Bacillus amyloliquefaciens）11568
<400>1
CCTCTGTCAC TTCGGCGGCT GGCTCCATAA AGGTTACCTC ACCGACTTCG GGTGTTACAA 60
ACTCTCGTGG TGTGACGGGC GGTGTGTACA AGGCCCGGGA ACGTATTCAC CGCGGCATGC 120
TGATCCGCGA TTACTAGCGA TTCCAGCTTC ACGCAGTCGA GTTGCAGACT GCGATCCGAA 180
CTGAGAACAG ATTTGTGGGA TTGGCTTAAC CTCGCGGTTT CGCTGCCCTT TGTTCTGTCC 240
ATTGTAGCAC GTGTGTAGCC CAGGTCATAA GGGGCATGAT GATTTGACGT CATCCCCACC 300
TTCCTCCGGT TTGTCACCGG CAGTCACCTT AGAGTGCCCA ACTGAATGCT GGCAACTAAG 360
ATCAAGGGTT GCGCTCGTTG CGGGACTTAA CCCAACATCT CACGACACGA GCTGACGACA 420
ACCATGCACC ACCTGTCACT CTGCCCCCGA AGGGGACGTC CTATCTCTAG GATTGTCAGA 480
GGATGTCAAG ACCTGGTAAG GTTCTTCGCG TTGCTTCGAA TTAAACCACA TGCTCCACCG 540
CTTGTGCGGG CCCCCGTCAA TTCCTTTGAG TTTCAGTCTT GCGACCGTAC TCCCCAGGCG 600
GAGTGCTTAA TGCGTTAGCT GCAGCACTAA GGGGCGGAAA CCCCCTAACA CTTAGCACTC 660
ATCGTTTACG GCGTGGACTA CCAGGGTATC TAATCCTGTT CGCTCCCCAC GCTTTCGCTC 720
CTCAGCGTCA GTTACAGACC AGAGAGTCGC CTTCGCCACT GGTGTTCCTC CACATCTCTA 780
CGCATTTCAC CGCTACACGT GGAATTCCAC TCTCCTCTTC TGCACTCAAG TTCCCCAGTT 840
TCCAATGACC CTCCCCGGTT GAGCCGGGGG CTTTCACATC AGACTTAAGA AACCGCCTGC 900
GAGCCCTTTA CGCCCAATAA TTCCGGACAA CGCTTGCCAC CTACGTATTA CCGCGGCTGC 960
TGGCACGTAG TTAGCCGTGG CTTTCTGGTT AGGTACCGTC AAGGTGCCGC CCTATTTGAA 1020
CGGCACTTGT TCTTCCCTAA CAACAGAGCT TTACGATCCG AAAACCTTCA TCACTCACGC 1080
GGCGTTGCTC CGTCAGACTT TCGTCCATTG CGGAAGATTC CCTACTGCTG CCTCCCGTAG 1140
GAGTCTGGGC CGTGTCTCAG TCCCAGTGTG GCCGATCACC CTCTCAGGTC GGCTACGCAT 1200
CGTCGCCTTG GTGAGCCGTT ACCTCACCAA CTAGCTAATG CGCCGCGGGT CCATCTGTAA 1260
GTGGTAGCCG AAGCCACCTT TTATGTCTGA ACCATGCGGT TCAGACAACC ATCCGGTATT 1320
AGCCCCGGTT TCCCGGAGTT ATCCCAGTCT TACAGGCAGG TTACCCACGT GTTACTCACC 1380
CGTCCGCCGC TAACATCAGG GAGCAAGCTC CCATCTGTCC GCTCGACTTG CATGTATAGG 1440
CAC 1443
<210>2
<211>19
<212>DNA
<213>Artificial sequence
<400>2
TCCGTAGGTG AACCTGCGG 19
<210>3
<211>20
<212>DNA
<213>Artificial sequence
<400>3
TCCTCCGCTT ATTGATATGC 20
Claims (5)
1. a kind of bacillus amyloliquefaciens（Bacillus amyloliquefacines ）11568, its preserving number is
CGMCCNo.9928。
2. application of the bacillus amyloliquefaciens 11568 described in a kind of claim 1 in production naringinase.
3. a kind of liquid fermentation liquid, it is characterised in that its preparation method comprises the steps：
（1）Bacillus amyloliquefaciens 11568 are carried out into slant culture, slant strains are obtained；
（2）Take slant strains to be inoculated in sterilized seed culture medium, the fermentation culture under conditions of 40.9 DEG C, 180rpm
48h, obtains liquid fermentation liquid.
4. liquid fermentation liquid according to claim 3, it is characterised in that above-mentioned steps（1）In, what the slant culture was used
Slant medium, contains following component in every 1L：MgSO4 0.5g、KH2PO4 1.5g、CaCl20.1g、（NH4）2SO4 1.5g、
KCl 0.5g、KNO31.5g, yeast extract 2g, orange meal 15g, agar 20g, pH are 6.0,1 × 105Pa 20 min of sterilizing.
5. liquid fermentation liquid according to claim 3, it is characterised in that above-mentioned steps（2）In, the seed culture medium, often
Contain following component in 1L：
Maltose 15.1g, tryptone 10g,（NH4）2HPO420g, NaCl 10g, yeast extract 5g, pH is 7.51；
Or, naringin 10g, tryptone 10g,（NH4）2HPO420g, NaCl 10g, yeast extract 5g, pH is 7.51；
Or, MgSO4 0.5g、 KH2PO4 1.5g、 CaCl20.1g、（NH4）2SO4 1.5g、KCl 0.5g、KNO3 1.5g、
Yeast extract 2g, orange meal 7.5g, pH is 6.0.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0599159A2 (en) * | 1992-11-27 | 1994-06-01 | Hoechst Aktiengesellschaft | Alpha-L-rhamnosidase for obtaining rhamnose, process for the preparation and use |
| CN103060287A (en) * | 2013-01-10 | 2013-04-24 | 集美大学 | Naringinase fermentation medium |
| CN103789332A (en) * | 2014-03-10 | 2014-05-14 | 湖北工业大学 | Construction of yeast strain having naringinase producing and recycling functions |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0599159A2 (en) * | 1992-11-27 | 1994-06-01 | Hoechst Aktiengesellschaft | Alpha-L-rhamnosidase for obtaining rhamnose, process for the preparation and use |
| CN103060287A (en) * | 2013-01-10 | 2013-04-24 | 集美大学 | Naringinase fermentation medium |
| CN103789332A (en) * | 2014-03-10 | 2014-05-14 | 湖北工业大学 | Construction of yeast strain having naringinase producing and recycling functions |
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