CN110241102A - A kind of method of enzymic degradation 2,6- dihydroxy-benzoic acid - Google Patents
A kind of method of enzymic degradation 2,6- dihydroxy-benzoic acid Download PDFInfo
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Abstract
The invention discloses a kind of methods of enzymic degradation 2,6-DHBA, comprising the following steps: step 1: Sdc protein expression: step 2: the purifying of Sdc: step 3: Sdc concentration mensuration: step 4: Sdc vitality test.The present invention is using the Sdc E. coli expression strains with His label constructed that succeeded, bacterial strain is expressed using this, inducing expression is carried out to Sdc, collect thallus, through ultrasonication, after centrifugation, obtain the crude enzyme liquid of mutant enzyme, the Sdc with His6 label is purified using His-TRap affinity column, using Sdc after purification respectively under the conditions of different temperature and pH with 2, 6- dihydroxy-benzoic acid is reacted, product or reactant are detected using HPLC detection, calculate corresponding enzyme activity, determine the optimum condition of reaction, certain theories integration is provided for the development and utilization of Sdc.
Description
Technical field
The present invention relates to a kind of method more particularly to a kind of methods of enzymic degradation 2,6-DHBA.
Background technique
Salicylic acid and its derivative are widely used in fields such as medicine, chemical industry, but since its is toxic and natural degradation time
It is long, it will affect balance of nature and human health, therefore, study its biodegrading process for realizing that green production has important meaning
Justice and necessity.Moreover, decarboxylate-phenol and its derivatives of salicylic acid and its derivative, are also important organic chemical industry
Raw material has a wide range of applications in chemical industry, medical industry, can not only be used for chemically synthesized intermediate, being also important
Learn pharmaceutical raw material.Using the decarboxylic reaction of aromatic acid decarboxylase catalysis salicyclic acid derivatives, the mild, specificity with reaction condition
The denominator of high enzyme reaction can substantially reduce the energy barrier of reaction, be easier to carry out reaction.Bigcatkin willow acid decarboxylase
(Salicylic acid decarboxylase, Sdc) is also one of them, which is 2010 by Kirimura research group
A kind of irreversible enzyme found in trichosporon cutaneum Trichosporon moniliiforme WU-0401.
Other than it can be catalyzed and generate salicylic acid and its back reaction as the Kolbe-Schmitt of substrate reaction using phenol,
Bigcatkin willow acid decarboxylase can also be catalyzed 2,4- dihydroxy-benzoic acid and γ-dihydroxy-benzoic acid decarboxylation generates resorcinol, catalysis
2,3- dihydroxy-benzoic acid decarboxylations generate 1,2- dihydroxy phenol, and catalysis 4-ASA decarboxylation generates 3- amino-phenol etc.,
And it can be catalyzed their back reaction, the carboxylation that using bicarbonate as the source CO2, can be catalyzed electron rich amphyl is anti-
It answers, adds carboxyl on hydroxyl ortho position to the reaction property of can choose.By Sdc degrade gingko in noxious material-ginkgoic acid,
The result shows that Sdc can all have decarboxylation ability to the ginkgoic acid containing different substituents under the conditions of comparatively gentle, this for
It is very suitable to containing the ginkgo acid degradation there are many different substituents side chain.The above research explanation, Sdc can identify phenol hydroxyl in specific manner
Carboxyl on the ortho position of base can have more using the adjacent hydroxybenzoic acid of hydroxyl and two groups of carboxyl as decarboxylation substrate
Extensive substrate adaptability.But the enzyme for all having catalytic decarboxylation ability to salicylic acid and its multiple derivatives as one, is urged
The mechanism of action for changing decarboxylation also requires further study, such as substrate 2,6-DHBA, bigcatkin willow acid decarboxylase
The optimum temperature and optimal pH for playing catalytic action are how many, also need further to determine.
Summary of the invention
In order to solve shortcoming present in above-mentioned technology, the present invention provides a kind of enzymic degradation 2,6- dihydroxy benzenes
The method of formic acid.
In order to solve the above technical problems, the technical solution adopted by the present invention is that: a kind of enzymic degradation 2,6- dihydroxy benzenes
The method of formic acid, comprising the following steps:
Step 1: Sdc protein expression: expression plasmid is transformed into host strain, building mutation Sdc gene expression bacterial strain,
By strain inoculated in the LB liquid medium that Amp concentration is 90~110 μ gmL-1, shaking table culture is No.1 seed liquor；It will
No.1 seed liquor is inoculated in the LB liquid medium that new Amp concentration is 90~110 μ gmL-1 in 0.8~1.5% ratio
In, shaking table culture is second seed liquid, and its OD600 is made to reach 0.4~0.8, and IPTG is then added and becomes ultimate density
For the culture solution of 0.08~0.2mM, after culture solution is induced, is centrifuged, it is spare to collect thallus；
Step 2: the purifying of Sdc: by the thallus collected in step 1 with ultrasonication buffer be resuspended, in ice bath into
Broken clasmatosis liquid is centrifuged for row ultrasonication, and supernatant is crude enzyme liquid；By crude enzyme liquid with 10~
14% SDS-PAGE detects protein expression situation, is purified using His-Trap affinity column to Sdc；
Step 3: Sdc concentration mensuration: the concentration of Bradford determination of protein concentration kit measurement purifying enzyme Sdc is used；
Step 4: it Sdc vitality test: is added and contains in the 2,6- dihydroxy-benzoic acid solution that concentration is 10~60mM
The buffer solution of 0.1mg/mLSdc, total reaction volume are 1~2mL, 25~60 DEG C of temperature, strongly acidic solution are added and terminates reaction,
Reaction solution is after 9500~10500r/min centrifugation, after 0.22 μm of membrane filtration, measures production concentration with HPLC.
Further, the step of His-Trap affinity column purifies Sdc are as follows:
I, activation system: being first turned on protein purification system power supply, after the system stabilizes, start work station, open A pump and
B pump, with MilliQ water with the speed rinse-system pipeline of 5mLmin-1 to baseline level；
II, it connects pillar: the piping connection of nickel column His-Trap FF crude and purification system is careful not to bring gas into
Then bubble is rinsed pillar with the speed of 1mLmin-1 with the MilliQ water for being more than or equal to 5 column volumes；
III, column equilibration: A pump head is put into combination buffer, and B pump head is put into elution buffer, first with being more than or equal to
The elution buffer of 5 column volumes rinses pillar with 1mLmin-1 speed, then again with the combination for being more than or equal to 5 column volumes
Buffer is balanced nickel column under same flow velocity；
IV, loading: with A pump by 5mL crude enzyme liquid on column；
V, it balances: being rinsed with the combination buffer of 15 column volumes with the speed of 0.5mLmin-1, until absorption curve
Stablize, destination protein is incorporated in nickel column at this time, and foreign protein then is rinsed；
VI, elution: being eluted with the elution buffer of 5 column volumes with the speed of 1mLmin-1, bent according to absorbing
Line collects destination protein；
VII, column regeneration: pillar is adequately rinsed with elution buffer with the speed of 1mLmin-1, is then existed
It is flushed to baseline stability with MilliQ water under same flow velocity, 20% ethyl alcohol is finally full of wherein, seals pillar after disassembly
It is placed in 4 DEG C of preservations；
VIII, close system: whole pipelines of protein purification system are full of 20% second after being rinsed well with MilliQ water
Alcohol first closes workstation program, is then shut off the power supply of all devices.
Further, in step 4 HPLC detect product resorcinol condition are as follows: chromatographic column be C18 column, with volume ratio
0.1% formic acid solution of methanol-volumetric concentration of 2:8 be mobile phase, 35 DEG C of column temperature, flow rate of mobile phase 1mL/min, Detection wavelength
278nm。
Further, in step 1 No.1 seed liquor condition of culture are as follows: temperature be 35~40 DEG C, revolving speed be 150~
11~13h of shaking table culture under conditions of 170rpm；The condition of culture of second seed liquid are as follows: temperature is at 35~40 DEG C, and revolving speed is
3~5h of shaking table culture under conditions of 150~170rpm；The inductive condition of culture solution in step 1 are as follows: temperature is 22~28 DEG C, turns
Speed induces 17~19h under conditions of being 110~130rpm；The centrifugal condition of culture solution are as follows: temperature is 3~5 DEG C, revolving speed 4800
8~12min is centrifuged under conditions of~5200rpm.
Further, the ultrasound condition in step 2 are as follows: power 20~60%, work 2~5s, 5~8s of interval, total ultrasound
Time is 9~11min；Centrifuge separation condition are as follows: 13~17min is centrifuged under conditions of 3~5 DEG C, 13000~15000rpm.
Further, the buffer solution in step 4 are as follows: the acetic acid-sodium acetate buffer solution or pH6.0 of pH4.0~5.5~
7.0 disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution.
Further, expression plasmid is coli expression carrier pET21a-Sdc；Host strain is E.coliBL21 (DE3)
Coli strain.
Further, strongly acidic solution is any one of hydrochloric acid solution, sulfuric acid solution, nitric acid solution.
The present invention expresses bacterium using this using the Sdc E. coli expression strains with His label for the building that succeeded
Strain carries out inducing expression to Sdc, collects thallus and obtains the crude enzyme liquid of mutant enzyme after ultrasonication, centrifugation, use His-
TRap affinity column purifies the Sdc with His6 label, using Sdc after purification respectively in different temperature and pH
Under the conditions of reacted with 2,6-DHBA, product or reactant are detected using HPLC detection, calculated corresponding
Enzyme activity determines the optimum condition of reaction.
The present invention is determined by experiment the optimum condition of Sdc catalytic degradation 2,6- dihydroxy-benzoic acid.Present invention research at
Fruit deepens people to the understanding of the structure-function relationship of Sdc for further, and provides certain theory for the development and utilization of Sdc
It supports.
Detailed description of the invention
Fig. 1 be the present invention at different temperatures with the concentration relationship figure of product.
Fig. 2 be the present invention under condition of different pH with the concentration relationship figure of product.
Fig. 3 is the high-efficient liquid phase chromatogram of product resorcinol of the present invention.
Specific embodiment
The present invention will be further described in detail with reference to the specific embodiments.
A kind of method of enzymic degradation 2,6-DHBA, comprising the following steps:
Step 1: Sdc protein expression: expression plasmid is transformed into host strain, building mutation Sdc gene expression bacterial strain,
By strain inoculated in the LB liquid medium that Amp concentration is 90~110 μ gmL-1, shaking table culture is No.1 seed liquor；It will
No.1 seed liquor is inoculated in the LB liquid medium that new Amp concentration is 90~110 μ gmL-1 in 0.8~1.5% ratio
In, shaking table culture is second seed liquid, and its OD600 is made to reach 0.4~0.8, and IPTG is then added and becomes ultimate density
For the culture solution of 0.08~0.2mM, after culture solution is induced, is centrifuged, it is spare to collect thallus；
Step 2: the purifying of Sdc: by the thallus collected in step 1 with ultrasonication buffer be resuspended, in ice bath into
Broken clasmatosis liquid is centrifuged for row ultrasonication, and supernatant is crude enzyme liquid；By crude enzyme liquid with 10~
14% SDS-PAGE detects protein expression situation, is purified using His-Trap affinity column to Sdc；
Step 3: Sdc concentration mensuration: the concentration of Bradford determination of protein concentration kit measurement purifying enzyme Sdc is used；
Step 4: it Sdc vitality test: is added and contains in the 2,6- dihydroxy-benzoic acid solution that concentration is 10~60mM
The buffer solution of 0.1mg/mLSdc, total reaction volume are 1~2mL, 25~60 DEG C of temperature, strongly acidic solution are added and terminates reaction,
Reaction solution is after 9500~10500r/min centrifugation, after 0.22 μm of membrane filtration, measures production concentration with HPLC.
The step of His-Trap affinity column purifies Sdc are as follows:
I, activation system: being first turned on protein purification system power supply, after the system stabilizes, start work station, open A pump and
B pump, with MilliQ water with the speed rinse-system pipeline of 5mLmin-1 to baseline level；
II, it connects pillar: the piping connection of nickel column His-Trap FF crude and purification system is careful not to bring gas into
Then bubble is rinsed pillar with the speed of 1mLmin-1 with the MilliQ water for being more than or equal to 5 column volumes；
III, column equilibration: A pump head is put into combination buffer, and B pump head is put into elution buffer, first with being more than or equal to
The elution buffer of 5 column volumes rinses pillar with 1mLmin-1 speed, then again with the combination for being more than or equal to 5 column volumes
Buffer is balanced nickel column under same flow velocity；
IV, loading: with A pump by 5mL crude enzyme liquid on column；
V, it balances: being rinsed with the combination buffer of 15 column volumes with the speed of 0.5mLmin-1, until absorption curve
Stablize, destination protein is incorporated in nickel column at this time, and foreign protein then is rinsed；
VI, elution: being eluted with the elution buffer of 5 column volumes with the speed of 1mLmin-1, bent according to absorbing
Line collects destination protein；
VII, column regeneration: pillar is adequately rinsed with elution buffer with the speed of 1mLmin-1, is then existed
It is flushed to baseline stability with MilliQ water under same flow velocity, 20% ethyl alcohol is finally full of wherein, seals pillar after disassembly
It is placed in 4 DEG C of preservations；
VIII, close system: whole pipelines of protein purification system are full of 20% second after being rinsed well with MilliQ water
Alcohol first closes workstation program, is then shut off the power supply of all devices.
HPLC detects the condition of product resorcinol in step 4 are as follows: chromatographic column is C18 column, with the methanol-of volume ratio 2:8
0.1% formic acid solution of volumetric concentration be mobile phase, 35 DEG C of column temperature, flow rate of mobile phase 1mL/min, Detection wavelength 278nm.
The condition of culture of No.1 seed liquor in step 1 are as follows: temperature is 35~40 DEG C, and revolving speed is the condition of 150~170rpm
11~13h of lower shaking table culture；The condition of culture of second seed liquid are as follows: temperature is at 35~40 DEG C, and revolving speed is 150~170rpm's
Under the conditions of 3~5h of shaking table culture；The inductive condition of culture solution in step 1 are as follows: temperature is 22~28 DEG C, revolving speed be 110~
17~19h is induced under conditions of 130rpm；The centrifugal condition of culture solution are as follows: temperature is 3~5 DEG C, revolving speed is 4800~5200rpm
Under conditions of be centrifuged 8~12min.
Ultrasound condition in step 2 are as follows: power 20~60%, work 2~5s, interval 5~8s, total ultrasonic time be 9~
11min；Centrifuge separation condition are as follows: 13~17min is centrifuged under conditions of 3~5 DEG C, 13000~15000rpm.
Buffer solution in step 4 are as follows: the acetic acid-sodium acetate buffer solution of pH4.0~5.5 or the phosphoric acid of pH6.0~7.0
Disodium hydrogen-phosphate sodium dihydrogen buffer solution.
Expression plasmid is coli expression carrier pET21a-Sdc；Host strain is E.coli BL21 (DE3) Escherichia coli
Bacterial strain.
Strongly acidic solution is any one of hydrochloric acid solution, sulfuric acid solution, nitric acid solution.
Preferably, the step of bigcatkin willow acid decarboxylase degradation 2,6-DHBA are as follows:
Step 1: Sdc protein expression: expression plasmid is transformed into E.coli BL21 (DE3) coli strain, structure
Mutation Sdc gene expression bacterial strain is built, strain inoculated is trained in the LB liquid that ampicillin (Amp) concentration is 100 μ gmL-1
It supports in base, 37 DEG C, shaking table culture 12h is as No.1 seed liquor under the speed conditions of 160rpm；No.1 seed liquor is pressed to 1% ratio
Example be inoculated in new Amp concentration be 100 μ gmL-1 LB liquid medium in, 37 DEG C, 160rpm culture about 4h as two
Number seed liquor, and make its OD600 to of about 0.6, IPTG (isopropylthiogalactoside, inducible protein expression), which is added, makes its end
Concentration is the culture solution of 0.1mM, induces 18h under conditions of 25 DEG C, 120rpm；Culture solution is through 5000rpm, 4 DEG C, centrifugation
It is spare to collect thallus by l0min.
Step 2: the purifying of Sdc: by the thallus collected in step 1 with ultrasonication buffer be resuspended, in ice bath into
Row ultrasonication, ultrasound condition are as follows: power 40%, work 3s, interval 7s, and total ultrasonic time is 10min.(according to the actual situation,
Optimize ultrasonication condition, keep minute most short, reduce calorific value, guarantee the activity of enzyme, general 10S circulation primary) it is broken
Clasmatosis liquid afterwards is centrifuged 15min under conditions of 4 DEG C, 14000rpm, and supernatant is crude enzyme liquid.Crude enzyme liquid is with 12%
SDS-PAGE detects protein expression situation, is purified using His-Trap affinity column to Sdc.
His-Trap histidine mark affinity column is a kind of nickel ion protein purification column, to histidine tag
Protein has the affinity of specificity, containing histidine tag (His6Tag), facilitates the purifying for carrying out enzyme, at present extensively
Applied to protein purification.The all liq that this step uses could use after being required to use 0.22 μM of membrane filtration, in order to avoid
Block pillar.
The step of His-Trap affinity column purifies Sdc are as follows:
I, activation system: being first turned on protein purification system power supply, after the system stabilizes, start work station, open A pump and
B pump, with MilliQ water (ultrapure water) with the speed rinse-system pipeline of 5mLmin-1 to baseline level；
II, it connects pillar: the piping connection of nickel column His-Trap FF crude and purification system is careful not to bring gas into
Then bubble is rinsed pillar with the speed of 1mLmin-1 with the MilliQ water for being more than or equal to 5 column volumes；
III, column equilibration: A pump head is put into combination buffer, and B pump head is put into elution buffer, first with being more than or equal to
The elution buffer of 5 column volumes rinses pillar with 1mLmin-1 speed, then again with the combination for being more than or equal to 5 column volumes
Buffer is balanced nickel column under same flow velocity；
Wherein, combination buffer: 50mM sodium phosphate, 500mM NaCl, 20mM imidazoles, pH8.0 use 0.22 μm of filter membrane mistake
It is spare after filter.
Elution buffer: 50mM sodium phosphate, 500mM NaCl, 400mM imidazoles, pH8.0, after 0.22 μm of membrane filtration
It is spare.
IV, loading: will be on about 5mL crude enzyme liquid on column with A pump；
V, it balances: being rinsed with the combination buffer of about 15 column volumes with the speed of 0.5mLmin-1, until absorbing
Curve is stablized, and destination protein is incorporated in nickel column at this time, and foreign protein then is rinsed；
VI, elution: it is eluted with the elution buffer of about 5 column volumes with the speed of 1mLmin-1, according to suction
Curve is received to collect destination protein；
VII, column regeneration: pillar is adequately rinsed with elution buffer with the speed of 1mLmin-1, is then existed
It is flushed to baseline stability with MilliQ water under same flow velocity, 20% ethyl alcohol is finally full of wherein, seals pillar after disassembly
It is placed in 4 DEG C of preservations；
VIII, close system: whole pipelines of protein purification system are full of 20% second after being rinsed well with MilliQ water
Alcohol first closes workstation program, is then shut off the power supply of all devices.
Step 3: Sdc concentration mensuration: the concentration of Bradford determination of protein concentration kit measurement purifying enzyme is used.
Step 4: Sdc vitality test: containing 0.1mg/mL's in the acetic acid-sodium acetate buffer solution (concentration 0.2M) of pH5.5
Sdc, substrate 2,6-DHBA solution concentration are 30mM, total reaction volume 1mL, and 40 DEG C of temperature, reaction carries out 1h, adds
Enter 100 μ L 12M HCl and terminate reaction, reaction solution after 0.22 μm of membrane filtration, is surveyed after 10000r/min is centrifuged with HPLC
Determine production concentration.Sdc is as catalyst degradation 2,6- dihydroxy-benzoic acid.The concentration for measuring product is determined according to production concentration
The optimal pH and optimum temperature of bigcatkin willow acid decarboxylase catalytic degradation 2,6- dihydroxy-benzoic acid.
Buffer solution is the acetic acid-sodium acetate buffer solution of pH4.0~5.5 or the Na2HPO4-NaH2PO4 of pH6.0~7.0
Buffer.Strongly acidic solution is any one of hydrochloric acid solution, sulfuric acid solution, nitric acid solution.The purpose of strongly acidic solution is added
It is to terminate reaction, pH value becomes larger suddenly inactivates Sdc.
The condition of HPLC detection product resorcinol are as follows: chromatographic column is C18 column, with methanol-volumetric concentration of volume ratio 2:8
0.1% formic acid solution be mobile phase, 35 DEG C of column temperature, flow rate of mobile phase 1mL/min, Detection wavelength 278nm.
It is measured under different temperatures, different pH value respectively, the product isophthalic two that Sdc degradation 2,6-DHBA obtains
The concentration of phenol.
The optimal pH of enzyme measures: measurement Sdc is in the different bufferings that pH is respectively 4.0,4.5,5.0,5.5,6.0,6.5,7.0
Degradation capability in liquid.
Enzyme optimal reactive temperature: measurement Sdc is in 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C of not equalities of temperature
Degradation capability under degree.
Table 1 is the production concentration under different temperatures
| Temperature/DEG C | 25 | 30 | 35 | 40 | 45 | 50 | 55 | 60 |
| Resorcinol/mM | 2.5 | 9.0 | 14 | 30 | 17 | 16 | 13 | 5 |
Table 2 is the production concentration under condition of different pH
| PH value | 4.0 | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 |
| Resorcinol/mM | 2.5 | 13 | 15 | 30 | 18 | 12 | 3.0 |
The optimum temperature of Sdc degradation 2,6-DHBA is 40 DEG C it can be seen from table 1, table 2, and optimal pH is
5.5, the concentration of product resorcinol is maximum at this time, as shown in Figure 1 and Figure 2.
The present invention respectively 25~60 DEG C temperature range and pH4.0~7.0 under conditions of, measurement Sdc degrade 2,6- bis-
The enzyme activity of hydroxybenzoic acid, determine Sdc optimum temperature and most suitable action pH, compare Sdc in different temperatures, difference pH
Under the conditions of the different substrates of catalysis optimal condition, provide data reference for its industrial applications.
Above embodiment is not limitation of the present invention, and the present invention is also not limited to the example above, this technology neck
The variations, modifications, additions or substitutions that the technical staff in domain is made within the scope of technical solution of the present invention, also belong to this hair
Bright protection scope.
Claims (8)
1. a kind of method of enzymic degradation 2,6-DHBA, it is characterised in that: the described method comprises the following steps:
Step 1: Sdc protein expression: expression plasmid is transformed into host strain, building mutation Sdc gene expression bacterial strain, by bacterium
Strain is inoculated in the LB liquid medium that Amp concentration is 90~110 μ gmL-1, and shaking table culture is No.1 seed liquor；By No.1
Seed liquor is inoculated in the LB liquid medium that new Amp concentration is 90~110 μ gmL-1 in 0.8~1.5% ratio, is shaken
Bed culture is second seed liquid, and its OD600 is made to reach 0.4~0.8, and it is 0.08 that IPTG, which is then added, and becomes ultimate density
After culture solution is induced, is centrifuged, it is spare to collect thallus for the culture solution of~0.2mM；
Step 2: the purifying of Sdc: the thallus collected in step 1 ultrasonication buffer is resuspended, is surpassed in ice bath
Sound is broken, broken clasmatosis liquid is centrifuged, supernatant is crude enzyme liquid；By crude enzyme liquid with 10~14%
SDS-PAGE detects protein expression situation, is purified using His-Trap affinity column to Sdc；
Step 3: Sdc concentration mensuration: the concentration of Bradford determination of protein concentration kit measurement purifying enzyme Sdc is used；
Step 4: it Sdc vitality test: is added in the 2,6- dihydroxy-benzoic acid solution that concentration is 10~60mM and contains 0.1mg/
The buffer solution of mLSdc, total reaction volume are 1~2mL, 25~60 DEG C of temperature, strongly acidic solution are added and terminates reaction, reaction solution
After 9500~10500r/min centrifugation, after 0.22 μm of membrane filtration, production concentration is measured with HPLC.
2. the method for enzymic degradation 2,6-DHBA according to claim 1, it is characterised in that: the His-
The step of Trap affinity column purifies Sdc are as follows:
I, activation system: being first turned on protein purification system power supply, after the system stabilizes, starts work station, opens A pump and B pump,
With MilliQ water with the speed rinse-system pipeline of 5mLmin-1 to baseline level；
II, it connects pillar: the piping connection of nickel column His-Trap FF crude and purification system is careful not to bring bubble into, so
Pillar is rinsed with the speed of 1mLmin-1 with the MilliQ water for being more than or equal to 5 column volumes afterwards；
III, column equilibration: A pump head is put into combination buffer, and B pump head is put into elution buffer, first with more than or equal to 5
The elution buffer of column volume rinses pillar with 1mLmin-1 speed, then slow with the combination for being more than or equal to 5 column volumes again
Fliud flushing is balanced nickel column under same flow velocity；
IV, loading: with A pump by 5mL crude enzyme liquid on column；
V, it balances: being rinsed with the combination buffer of 15 column volumes with the speed of 0.5mLmin-1, until absorption curve is stablized,
Destination protein is incorporated in nickel column at this time, and foreign protein then is rinsed；
VI, elution: being eluted with the elution buffer of 5 column volumes with the speed of 1mLmin-1, according to absorption curve come
Collect destination protein；
VII, column regeneration: pillar is adequately rinsed with elution buffer with the speed of 1mLmin-1, then same
Flow velocity under with MilliQ water be flushed to baseline stability, be finally full of 20% ethyl alcohol wherein, pillar sealed after disassembly and is placed in 4
DEG C save；
VIII, close system: whole pipelines of protein purification system are full of 20% ethyl alcohol after being rinsed well with MilliQ water, first
Workstation program is closed, the power supply of all devices is then shut off.
3. the method for enzymic degradation 2,6-DHBA according to claim 2, it is characterised in that: the step 4
The condition of middle HPLC detection product resorcinol are as follows: chromatographic column is C18 column, with methanol-volumetric concentration 0.1% of volume ratio 2:8
Formic acid solution is mobile phase, 35 DEG C of column temperature, flow rate of mobile phase 1mL/min, Detection wavelength 278nm.
4. the method for enzymic degradation 2,6-DHBA according to claim 3, it is characterised in that: the step 1
The condition of culture of middle No.1 seed liquor are as follows: temperature be 35~40 DEG C, revolving speed be 150~170rpm under conditions of shaking table culture 11~
13h；The condition of culture of second seed liquid are as follows: temperature is at 35~40 DEG C, and revolving speed is shaking table culture under conditions of 150~170rpm
3~5h；The inductive condition of culture solution in step 1 are as follows: temperature is 22~28 DEG C, revolving speed induces under conditions of being 110~130rpm
17~19h；The centrifugal condition of culture solution are as follows: under conditions of temperature is 3~5 DEG C, revolving speed is 4800~5200rpm be centrifuged 8~
12min。
5. the method for enzymic degradation 2,6-DHBA according to claim 4, it is characterised in that: the step 2
In ultrasound condition are as follows: power 20~60%, work 2~5s, interval 5~8s, total ultrasonic time be 9~11min；Centrifuge separation
Condition are as follows: 13~17min is centrifuged under conditions of 3~5 DEG C, 13000~15000rpm.
6. the method for enzymic degradation 2,6-DHBA according to claim 5, it is characterised in that: the step 4
In buffer solution are as follows: the acetic acid-sodium acetate buffer solution of pH4.0~5.5 or disodium hydrogen phosphate-biphosphate of pH6.0~7.0
Sodium buffer.
7. the method for enzymic degradation 2,6-DHBA according to claim 6, it is characterised in that: the expression matter
Grain is coli expression carrier pET21a-Sdc；The host strain is E.coli BL21 (DE3) coli strain.
8. the method for described in any item enzymic degradation 2,6-DHBAs according to claim 1~7, it is characterised in that:
The strongly acidic solution is any one of hydrochloric acid solution, sulfuric acid solution, nitric acid solution.
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