CN106119230B - A method of circulation double-aqueous phase system isolates and purifies bromelain - Google Patents
A method of circulation double-aqueous phase system isolates and purifies bromelain Download PDFInfo
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Abstract
The present invention relates to a kind of methods that circulation double-aqueous phase system isolates and purifies bromelain, belong to the separating and purifying technology field of active ingredient of natural product；The pineapple juice of coarse extraction is obtained by pretreatment first；Then double-aqueous phase system single extraction bromelain, and the activity and protein content of upper phase bromelain are measured, judge effect of extracting；Then polymer phase is taken out, suitable distilled water is added and is diluted, adjust the pH value of solution, cloud point method reextraction bromelain simultaneously recycles polymer；Polymer after separation is used to carry out cycling extraction experiment in double-aqueous phase system next time；This method process flow is simple, at low cost, operating condition is mild, can protect the original structure and activity of target enzyme, and the recyclable recycling of polymer, environmental pollution are small.
Description
Technical field
The present invention relates to a kind of methods that circulation double-aqueous phase system isolates and purifies bromelain, and in particular to a kind of polymerization
Object-salt Bi-aqueous extraction bromelain method, belongs to the separating and purifying technology field of active ingredient of natural product.
Background technique
Bromelain derives from a kind of tropical fruit (tree) pineapple, and wherein bromelain is divided into stem spinach according to the position of extraction
Trailing plants protease and seed-timber stand.In the food industry, bromelain is optimal carnivorous rejuvenator.In Britain, about
95% meat products is processed using bromelain.The optimal reaction temperature of bromelain is lower, has very much
Conducive to its food service industry application.
Currently, bromelain isolation and purification method mainly has the precipitation method, ion-exchange chromatography, immobilized metal
Affinity chromatography etc., common issue present in these methods is: it needs continuous operation, be difficult to scale up scale, high production cost,
It easily causes environmental pollution and gained enzymatic activity is also insufficient for application more demanding to enzymatic activity in medical rank and food.
Compared with traditional separating and extracting process, aqueous two-phase extraction has operating condition mild, is easily enlarged production, and low in cost wait shows
The advantages of work.The reported aqueous two-phase for extraction and separation bromelain mainly has polyethylene glycol and poly- the third two at phase substance
Alcohol, it is this to be both greater than 100 DEG C at the minimum critical-temperature of phase-polymerization object, if extracted using this substance biologically active
Bromelain can not only destroy the bioactivity of protease, destroy its molecular structure, and being difficult to can be from polymer by object
It separates in phase, also acquires a certain degree of difficulty after extraction to polymer recovery.
Triblock polymer is that a kind of cloud point is lower, polymer of temperature sensitive type, and molecule micella is with hydrophobic polyphenylene oxide
(PPO) group is outer core and hydrophilic polyethylene oxide (PEO) group is core center, with the rising of temperature, in micella shell
PEO chain hydrauture weakens rapidly, and micellar aggregates is made to form more compact spherical structure, so as to isolate from original solution
Come.It is used for aqueous two-phase extraction, not only operating condition is mild, the original structure and characteristic of extracted substance can be protected, and
Polymer can also be with recycling and reusing, smaller to the harm of environment, while also saving cost, improves the warp of aqueous two-phase extraction
Ji benefit.
Summary of the invention
It is an object of the invention to overcome current bromelain isolation technics complex process, be difficult to scale up scale, destruction
The defects of Enzyme activity and structure, provides a kind of side of bromelain in the aqueous two-phase extraction pineapple peel based on block polymer
Method, this method process flow is simple, at low cost, operating condition is mild, can protect the original structure and activity of target enzyme, and
Polymer is recyclable to be recycled, and environmental pollution is small.
The technical solution adopted by the present invention is summarized as follows:
A method of circulation double-aqueous phase system isolates and purifies bromelain, method includes the following steps:
(1) sample pretreatment: after pineapple peel is squeezed the juice and filtered, gained filtrate is in 4 DEG C of centrifuge with 8000 revs/min
Zhongli's heart 20 minutes, more clear pineapple peel juice, the as pineapple juice of coarse extraction are obtained, measures wherein pineapple egg with folin's methods
White enzymatic activity measures protein content therein with Coomassie Brilliant Blue.
(2) bromelain stability to β lactamase: by the pineapple peel juice of coarse extraction be separately stored in different pH salting liquid or
In polymer solution, after storing different time at different temperatures, enzymatic activity is measured, enzyme activity before calculating gained enzyme activity and storing
Ratio investigates the stability of bromelain with this.
Wherein, the salting liquid is K2HPO4, (NH4)3C6H5O7Or K3C6H5O7Aqueous solution, and salinity is in solution
8-15%；The polymer is L61 or L62, and polymer concentration is 5-15%, preferred polymers L61 in polymer solution；It is described
The solution of different pH is adjusted by the NaOH of the HCl and 0.1M of 0.1M, and pH range is 4-10；The storage temperature is 8-70
DEG C, preferable temperature is 8 DEG C；The time is 0.5-12 h, and the preferably time is 4 h.
(3) salt of certain mass, the pineapple of polymer and coarse extraction double-aqueous phase system single extraction bromelain: are weighed
Juice is put into the centrifuge tube of 10 mL, and a certain amount of distilled water, and regulation system pH is added, and makes 10 g of whole system quality.It is acute
After violent shock is swung, it is centrifuged 20 minutes under 2000 revs/min, until the complete split-phase of solution, polymer and object are gathered in phase,
Salt is gathered in lower phase.It is placed in 4 h in 8 DEG C of refrigerator afterwards, folin's methods measure the activity of upper phase bromelain, to obtain
The bromelain enzyme activity rate of recovery；Upper phase protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains purification of factor,
Effect of extracting is judged with this.
Wherein salt is K in the extraction system2HPO4, (NH4)3C6H5O7Or K3C6H5O7, final concentration of 8-15%；It is described poly-
Conjunction object is L61 or L62, and polymer concentration is 5-15%, preferred polymers L61 in polymer solution；The solution of the difference pH
It is adjusted by the NaOH of the HCl and 0.1M of 0.1M, pH range is 4-10, and preferably pH is 7.The protease activity rate of recoveryR a
It calculates as follows:
In formulaA t -- the upper phase enzyme activity of two phase aqueous extraction system.
A i -- total enzyme activity before extraction.
The purification of factorPFIt calculates as follows:
In formulaA t -- the upper phase enzyme activity of aqueous two-phase extraction；
A i -- total enzyme activity before extraction；
C t -- the upper phase protein content of two phase aqueous extraction system；
C i -- total protein content before extraction.
(4) it cloud point method reextraction bromelain and recycles polymer: polymer phase being taken out, suitable distillation is added
Water is diluted, and makes 4 mL of overall solution volume, and the pH value of solution is tuned into 7, is put into 35 DEG C of water-baths and is kept the temperature, and
It is centrifuged 20 minutes at 35 DEG C with 2000 revs/min, system is divided into new two-phase, and aqueous solution and object are gathered in phase, polymerization
Object is gathered in lower phase.
(5) in optimal two phase aqueous extraction system, the polymer after separation is used in double-aqueous phase system next time
Cycling extraction experiment is carried out, polymer circulation service efficiency is investigated.
Compared with prior art, the present invention have the following advantages that and the utility model has the advantages that
(1) present invention constructs temperature sensitive polymer-salt circulation double-aqueous phase system for the first time and isolates and purifies the pineapple egg in pineapple peel
White enzyme；Triblock polymer L61- salt system is used to recycle the bromelain in aqueous two-phase extraction pineapple peel for the first time.With biography
The system precipitation method, ion-exchange chromatography separation bromelain etc. are compared, which does not need to reduce using a large amount of organic solvents
Environmental pollution；Cost is relatively low for polymer used, and extraction process is simple；Enzyme is extracted from pineapple stem and meat different from traditional,
But extracted from waste pineapple peel, realize the effective use of resource.It is separated with existing polymer-salt double-aqueous phase system
Purifying bromelain is compared, and the present invention realizes recycling for temperature sensitive polymer, while having saved experimental cost, passes through
Circulation aqueous two-phase, which isolates and purifies, realizes effectively improving for the enzyme activity rate of recovery and purification of factor；It realizes resource-effective and maximizes
It utilizes, improves economic benefit.
(2) in the double-aqueous phase system of existing extraction bromelain, polyethylene glycol or polypropylene glycol are usual component,
In contrast, temperature sensitive polymer used in the present invention is three block temperature sensitive polymer L61, there is lower minimum Critical Solution
Temperature makes phase separation temperature that can form double-aqueous phase system at 8 DEG C, is conducive to isolating and purifying for bromelain after salt is added,
Its original structure and activity can be effectively kept not to be destroyed in low temperature.Therefore the higher enzyme activity rate of recovery 62.5% and purifying are obtained
The factor 9.21.
(3) temperature-sensing property for utilizing triblock polymer L61 can be heated above the separation of its cloud point Shi Huiyu object, because
This can enter in double-aqueous phase system next time bromelain is recycled, continuous extraction, realize it is resource-effective and
It maximally utilizes, improves economic benefit.
Detailed description of the invention
Fig. 1 is the schematic diagram of the method for the present invention concrete operations.
Specific embodiment
Following embodiment be in order to make it will be understood by those skilled in the art that the present invention, but in any way limit this
Invention.Without departing from the essence of the present invention, what those skilled in the art can make is any obvious
Replacement or modification is improved to all belong to the scope of protection of the present invention.
Below with reference to specific example, the present invention is further illustrated.
Embodiment 1:
(1) sample pretreatment: after pineapple peel is squeezed the juice and filtered, gained filtrate is in 4 DEG C of centrifuge with 8000 revs/min
Zhongli's heart 20 minutes, more clear pineapple peel juice is obtained, wherein bromelain enzymatic activity is 90 with folin's methods measurement, with examining horse
This brilliant blue method measures protein content therein.
(2) bromelain stability to β lactamase: the pineapple peel juice of coarse extraction is separately stored in pH=4, concentration is 8%
K2HPO4, (NH4)3C6H5O7And K3C6H5O7In solution, after 0.5 h is stored at 15 DEG C, enzymatic activity, gained enzyme activity and storage are measured
Enzyme activity ratio is respectively 65.3%, 61.4% and 60.2% before depositing, it follows that enzyme activity decreases in the present context for storage, pineapple
Protease has certain loss, but in K2HPO4It is lost in system minimum.
(3) double-aqueous phase system single extraction bromelain: certain mass is separately added into the centrifuge tube of three 10 mL
K2HPO4, (NH4)3C6H5O7And K3C6H5O7, and be separately added into polymer L61 and 4mL pineapple peel juice, distilled water is added and adjusts
Section system pH=4 make 10 g of extraction system quality.In three kinds of L61- K2HPO4/(NH4)3C6H5O7/K3C6H5O7Extraction system
In, final concentration of 8%, the L61 final concentration of 8% of salt.After mixed solution is acutely shaken, 20 points are centrifuged under 2000 revs/min
Clock, until the complete split-phase of solution, polymer and object are gathered in phase, and salt is gathered in lower phase.It is placed in 8 DEG C of refrigerator afterwards
In 4 h, folin's methods measure the activity of upper phase bromelain, to obtain bromelain activity recovery point in three systems
It Wei 44.9%, 38.1% and 43.4%；Upper phase protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains three
Purification of factor is respectively 5.43,4.79 and 4.62 in system.
(4) it cloud point method reextraction bromelain and recycles polymer: polymer phase being taken out, suitable distillation is added
Water is diluted, and makes 4 mL of overall solution volume, and the pH value of solution is tuned into 7, is put into 35 DEG C of water-baths and is kept the temperature, and
It is centrifuged 20 minutes at 35 DEG C with 2000 revs/min, system is divided into new two-phase, and aqueous solution and object are gathered in phase, gathers
It closes object and is gathered in lower phase.Folin's methods measure the activity of bromelain, so that it is secondary to obtain bromelain enzyme activity in three systems
The rate of recovery is respectively 41.2%, 32.5% and 39.1%；Protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains
Reextraction purification of factor is respectively 5.97,6.45 and 5.78 in three systems.
Embodiment 2:
(1) sample pretreatment: after pineapple peel is squeezed the juice and is filtered, gained filtrate in 4 DEG C of centrifuge with 8000 turns/
Minutes centrifugation 20 minutes, more clear pineapple peel juice is obtained, wherein bromelain enzymatic activity is 90 with folin's methods measurement, with examining
Mas bright blue method measures protein content therein.
(2) pineapple peel juice of coarse extraction bromelain stability to β lactamase: is separately stored in pH=10, concentration 15%
K2HPO4, (NH4)3C6H5O7And K3C6H5O7In solution, after 12 h are stored at 70 DEG C, measure enzymatic activity, gained enzyme activity with
Enzyme activity ratio is 2.5%, 2.1% and 1.8% before storing.Therefore, long-time storage can make enzymatic activity bright under high temperature, high pH environment
Aobvious decline, bromelain loss are more.
(3) double-aqueous phase system single extraction bromelain: certain mass is separately added into the centrifuge tube of three 10 mL
K2HPO4, (NH4)3C6H5O7And K3C6H5O7, and be separately added into polymer L61 and 4mL pineapple peel juice, distilled water is added and adjusts
Section system pH=10 make 10 g of extraction system quality.In three kinds of L61- K2HPO4/(NH4)3C6H5O7/K3C6H5O7Extractor body
In system, final concentration of 15%, the L61 final concentration of 12% of salt.After mixed solution is acutely shaken, it is centrifuged under 2000 revs/min
20 minutes, until the complete split-phase of solution, polymer and object were gathered in phase, and salt is gathered in lower phase.It is placed in 8 DEG C afterwards
4 h in refrigerator, folin's methods measure the activity of upper phase bromelain, recycle to obtain bromelain enzymatic activity in three systems
Rate is respectively 58.1%, 50.1% and 42%；Upper phase protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains three
Purification of factor is respectively 6.19,5.07 and 4.86 in a system.
(4) it cloud point method reextraction bromelain and recycles polymer: polymer phase being taken out, suitable distillation is added
Water is diluted, and makes 4 mL of overall solution volume, and the pH value of solution is tuned into 7, is put into 35 DEG C of water-baths and is kept the temperature, and
It is centrifuged 20 minutes at 35 DEG C with 2000 revs/min, system is divided into new two-phase, and aqueous solution and object are gathered in phase, gathers
It closes object and is gathered in lower phase.Folin's methods measure the activity of bromelain, so that it is secondary to obtain bromelain enzyme activity in three systems
The rate of recovery is respectively 52.9%, 48.1% and 38.7%；Protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains
Reextraction purification of factor is respectively 7.8,6.51 and 5.24 in three systems.
Embodiment 3:
(1) sample pretreatment: after pineapple peel is squeezed the juice and is filtered, gained filtrate in 4 DEG C of centrifuge with 8000 turns/
Minutes centrifugation 20 minutes, more clear pineapple peel juice is obtained, wherein bromelain enzymatic activity is 90 with folin's methods measurement, with examining
Mas bright blue method measures protein content therein.
(2) pineapple peel juice of coarse extraction bromelain stability to β lactamase: is separately stored in the 12% of pH=7.5
K2HPO4, (NH4)3C6H5O7And K3C6H5O7In solution, after 4 h are stored at 8 DEG C, enzymatic activity, gained enzyme activity and storage are measured
Preceding enzyme activity ratio is 82.6%, 65.6% and 62.5%.Therefore, storing enzyme activity in three kinds of salting liquids in the present context slightly reduces, spinach
The loss of trailing plants protease is less, and the pH, temperature and time in the environment are suitable storage environment；And in K2HPO4It is lost in solution
At least, i.e. K2HPO4It is most weak to the inhibition of enzymatic activity.
(3) double-aqueous phase system single extraction bromelain: certain mass is separately added into the centrifuge tube of three 10 mL
K2HPO4, (NH4)3C6H5O7And K3C6H5O7, and be separately added into polymer L61 and 4mL pineapple peel juice, distilled water is added and adjusts
Section system pH=7 make 10 g of extraction system quality.In three kinds of L61- K2HPO4/(NH4)3C6H5O7/K3C6H5O7Extraction system
In, final concentration of 12%, the L61 final concentration of 10% of salt.After mixed solution is acutely shaken, 20 are centrifuged under 2000 revs/min
Minute, until the complete split-phase of solution, polymer and object are gathered in phase, and salt is gathered in lower phase.It is placed in 8 DEG C of ice afterwards
4 h in case, folin's methods measure the activity of upper phase bromelain, to obtain the bromelain enzyme activity rate of recovery point in three systems
It Wei 54.37%, 46.22% and 49.74%；Upper phase protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains three
Purification of factor is respectively 6.12,5.53 and 5.31 in a system.
(4) it cloud point method reextraction bromelain and recycles polymer: polymer phase being taken out, suitable distillation is added
Water is diluted, and makes 4 mL of overall solution volume, and the pH value of solution is tuned into 7, is put into 35 DEG C of water-baths and is kept the temperature, and
It is centrifuged 20 minutes at 35 DEG C with 2000 revs/min, system is divided into new two-phase, and aqueous solution and object are gathered in phase, gathers
It closes object and is gathered in lower phase.Folin's methods measure the activity of bromelain, so that it is secondary to obtain bromelain enzyme activity in three systems
The rate of recovery is respectively 49.3%, 42.8% and 43.5%；Protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains
Reextraction purification of factor is respectively 7.31,6.5 and 6.43 in three systems.
Embodiment 4:
(1) sample pretreatment: after pineapple peel is squeezed the juice and is filtered, gained filtrate in 4 DEG C of centrifuge with 8000 turns/
Minute centrifugation 20 minutes, obtains more clear pineapple peel juice, measures wherein bromelain enzymatic activity with folin's methods, uses coomassie
Brilliant blue method measures protein content therein.
(2) pineapple peel juice of coarse extraction bromelain stability to β lactamase: is separately stored in 5% L61 of pH=7.5
In 5%L62 solution, after 4 h are stored at 8 DEG C, measure enzymatic activity, gained enzyme activity and store before enzyme activity ratio be 89.9% and
80.2%.Therefore, it stores in the polymer solution of low concentration in the present context, enzyme activity slightly reduces, and bromelain loses very
Few, i.e. low concentration polymer is weaker to the inhibition of enzymatic activity.
(3) double-aqueous phase system single extraction bromelain: certain mass is separately added into the centrifuge tube of three 10 mL
K2HPO4, (NH4)3C6H5O7And K3C6H5O7, and be separately added into polymer L61 and 4mL pineapple peel juice, distilled water is added and adjusts
Section system pH=7 make 10 g of extraction system quality.In three kinds of L61- K2HPO4/(NH4)3C6H5O7/K3C6H5O7Extraction system
In, final concentration of 10%, the L61 final concentration of 5% of salt.After mixed solution is acutely shaken, 20 points are centrifuged under 2000 revs/min
Clock, until the complete split-phase of solution, polymer and object are gathered in phase, and salt is gathered in lower phase.It is placed in 8 DEG C of refrigerator afterwards
In 4 h, folin's methods measure the activity of upper phase bromelain, distinguish to obtain the bromelain enzyme activity rate of recovery in three systems
It is 43.5%, 37.8% and 42.7%；Upper phase protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains three individuals
Purification of factor is respectively 4.86,4.32 and 3.4 in system.
(4) it cloud point method reextraction bromelain and recycles polymer: polymer phase being taken out, suitable distillation is added
Water is diluted, and makes 4 mL of overall solution volume, and the pH value of solution is tuned into 7, is put into 35 DEG C of water-baths and is kept the temperature, and
It is centrifuged 20 minutes at 35 DEG C with 2000 revs/min, system is divided into new two-phase, and aqueous solution and object are gathered in phase, gathers
It closes object and is gathered in lower phase.Folin's methods measure the activity of bromelain, so that it is secondary to obtain bromelain enzyme activity in three systems
The rate of recovery is respectively 39.7%, 34.2% and 37.8%；Protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains
Reextraction purification of factor is respectively 5.85,5.7 and 5.57 in three systems.
Embodiment 5:
(1) sample pretreatment: after pineapple peel is squeezed the juice and is filtered, gained filtrate in 4 DEG C of centrifuge with 8000 turns/
Minute centrifugation 20 minutes, obtains more clear pineapple peel juice, measures wherein bromelain enzymatic activity with folin's methods, uses coomassie
Brilliant blue method measures protein content therein.
(2) pineapple peel juice of coarse extraction bromelain stability to β lactamase: is separately stored in 15% L61 of pH=7.5
In 15%L62 solution, after 4 h are stored at 8 DEG C, enzymatic activity is measured, enzyme activity ratio is 69.08% before gained enzyme activity and storage
With 56.8%.Therefore, it stores in high concentration polymer solution in the present context, enzyme activity reduces, and bromelain has certain damage
It loses, i.e. high concentration polymer is stronger to the inhibition of enzymatic activity.
(3) double-aqueous phase system single extraction bromelain: certain mass is separately added into the centrifuge tube of three 10 mL
K2HPO4, (NH4)3C6H5O7And K3C6H5O7, and be separately added into polymer L61 and 4mL pineapple peel juice, distilled water is added and adjusts
Section system pH=7 make 10 g of extraction system quality.In three kinds of L61- K2HPO4/(NH4)3C6H5O7/K3C6H5O7Extraction system
In, final concentration of 10%, the L61 final concentration of 15% of salt.After mixed solution is acutely shaken, 20 are centrifuged under 2000 revs/min
Minute, until the complete split-phase of solution, polymer and object are gathered in phase, and salt is gathered in lower phase.It is placed in 8 DEG C of ice afterwards
4 h in case, folin's methods measure the activity of upper phase bromelain, to obtain the bromelain enzyme activity rate of recovery point in three systems
It Wei 60.1%, 53.2% and 57.2%；Upper phase protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains three
Purification of factor is respectively 6.5,6.08 and 4.76 in system.Therefore, bromelain loss is less under this condition, obtained enzyme
Purity is also higher.
(4) it cloud point method reextraction bromelain and recycles polymer: polymer phase being taken out, suitable distillation is added
Water is diluted, and makes 4 mL of overall solution volume, and the pH value of solution is tuned into 7, is put into 35 DEG C of water-baths and is kept the temperature, and
It is centrifuged 20 minutes at 35 DEG C with 2000 revs/min, system is divided into new two-phase, and aqueous solution and object are gathered in phase, gathers
It closes object and is gathered in lower phase.It is respectively 55.6% to obtain bromelain enzyme activity secondary recovery rate in three systems, 45.7% He
53.9%；Protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains reextraction purification of factor in three systems
Respectively 7.5,6.73 and 7.94.Therefore, some losses of bromelain under the conditions of the reextraction, but obtain the purity of enzyme compared with
It is high.
Embodiment 6:
(1) sample pretreatment: after pineapple peel is squeezed the juice and is filtered, gained filtrate in 4 DEG C of centrifuge with 8000 turns/
Minute centrifugation 20 minutes, obtains more clear pineapple peel juice, measures wherein bromelain enzymatic activity with folin's methods, uses coomassie
Brilliant blue method measures protein content therein.
(2) pineapple peel juice of coarse extraction bromelain stability to β lactamase: is separately stored in 12% L61 of pH=7.5
In 12%L62 solution, after 4 h are stored at 8 DEG C, measure enzymatic activity, gained enzyme activity and store before enzyme activity ratio be 72.6% and
65.1%.Therefore, it storing in polymer solution in the present context, enzyme activity slightly reduces, and bromelain has less loss, and
It is lost less in L61 system, i.e. polymer L61 is weaker to the inhibition of enzymatic activity.
(3) double-aqueous phase system single extraction bromelain: certain mass is separately added into the centrifuge tube of three 10 mL
K2HPO4, (NH4)3C6H5O7And K3C6H5O7, and be separately added into polymer L61 and 4mL pineapple peel juice, distilled water is added and adjusts
Section system pH=7 make 10 g of extraction system quality.In three kinds of L61- K2HPO4/(NH4)3C6H5O7/K3C6H5O7Extraction system
In, final concentration of 12%, the L61 final concentration of 12% of salt.After mixed solution is acutely shaken, 20 are centrifuged under 2000 revs/min
Minute, until the complete split-phase of solution, polymer and object are gathered in phase, and salt is gathered in lower phase.It is placed in 8 DEG C of ice afterwards
4 h in case, folin's methods measure the activity of upper phase bromelain, to obtain the bromelain enzyme activity rate of recovery point in three systems
It Wei 68.6%, 60.27% and 64.84%；Upper phase protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains three
Purification of factor is respectively 6.53,5.87 and 5.37 in a system.Therefore, bromelain loss seldom, obtains under this condition
The purity of enzyme is also higher.
(4) it cloud point method reextraction bromelain and recycles polymer: polymer phase being taken out, suitable distillation is added
Water is diluted, and makes 4 mL of overall solution volume, and the pH value of solution is tuned into 7, is put into 35 DEG C of water-baths and is kept the temperature, and
It is centrifuged 20 minutes at 35 DEG C with 2000 revs/min, system is divided into new two-phase, and aqueous solution and object are gathered in phase, gathers
It closes object and is gathered in lower phase.It is respectively 62.5% to obtain bromelain enzyme activity secondary recovery rate in three systems, 55.7% He
60.6%；Protein content is measured with Coomassie Brilliant Blue, the activity of desmoenzyme obtains reextraction purification of factor in three systems
Respectively 9.21,7.54 and 8.21.Therefore, bromelain loss is less under the conditions of the reextraction, obtains the purity of enzyme
It is very high, especially in L61- K2HPO4Effect is best in system.
(5) in this embodiment final concentration of 12% L61 and 12% K2HPO4Extraction system, to bromelain enzyme activity
Ult rec can reach 62.5%, and purification of factor 9.21 is the system of optimal aqueous two-phase extraction bromelain.Herein
On the basis of, the polymer after separation is re-used for carry out cycling extraction experiment in new double-aqueous phase system, recycles two with this
Secondary, obtaining the final enzyme activity rate of recovery is respectively 61.8% and 60.4%, it was demonstrated that polymer L61 has circulation well in the system
Effect of extracting.
In test, K is selected2HPO4It is that can use because it is with relatively high ability of saltouing as split-phase system
Measure it is less in the case where quickly reduce cloud point, temperature needed for reaching separation system.From the point of view of isolating and purifying the enzyme activity rate of recovery, with
Other split-phase salt are compared, and K is used2HPO4When, the relatively high enzyme activity rate of recovery can be obtained.This may be due to bromelain
Isoelectric point 9.55, and a certain amount of K2HPO4The pH value of solution just nearby, is conducive to point of bromelain in this way
From.
Claims (2)
1. a kind of method that circulation double-aqueous phase system isolates and purifies bromelain, which is characterized in that the circulation aqueous two-phase body
System is that polymer-salt recycles two phase aqueous extraction system,
This method carries out in accordance with the following steps:
(1) sample pretreatment: obtaining the pineapple juice of coarse extraction, measures wherein bromelain enzymatic activity with folin's methods, uses coomassie
Brilliant blue method measures protein content therein；
(2) double-aqueous phase system single extraction bromelain: weighing the salt of certain mass, and the pineapple juice of polymer and coarse extraction is put
Enter in centrifuge tube, distilled water, and regulation system pH is added；Acutely it is centrifuged after concussion, until the complete split-phase of solution, polymer and mesh
Mark object is gathered in phase, and salt is gathered in lower phase, is then placed into 8 DEG C of placement 4h in refrigerator, the work of phase bromelain in measurement
Property, to obtain the bromelain enzyme activity rate of recovery；The activity of phase protein content in measurement, desmoenzyme obtains purification of factor,
Effect of extracting is judged with this；The salt is K2HPO4、 (NH4)3C6H5O7Or K3C6H5O7；The final concentration of 8- of salt in system
15%；
The polymer is L61, and the concentration of polymer is 5-15% in system；The system pH is 4-10；
(3) cloud point method reextraction bromelain and recycle polymer: polymer phase taken out, be added suitable distilled water into
Row dilution, adjusts the pH value of solution, is put into water-bath and keeps the temperature, and be centrifuged, system is divided into new two-phase, aqueous solution and object
It is gathered in phase, polymer aggregational is in lower phase；The pH of the solution is 7；In the water-bath holding temperature be 35 DEG C, it is described from
Heart condition is at 35 DEG C, and 2000 revs/min are centrifuged 20 minutes；
(4) polymer after separation is used to carry out cycling extraction experiment in double-aqueous phase system next time.
2. a kind of method that circulation double-aqueous phase system isolates and purifies bromelain according to claim 1, feature exist
In system pH described in step (2) is 7.
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