CN105647985B - A kind of method of biocatalysis production phosphocreatine - Google Patents
A kind of method of biocatalysis production phosphocreatine Download PDFInfo
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Abstract
A kind of method of biocatalysis production phosphocreatine, is related to the technical field of the preparation method of phosphocreatine.Method provided by the invention mainly solves the existing biocatalysis production method low yield of phosphocreatine, technical problem at high cost, this method is specially using creatine and atriphos as substrate, phosphocreatine is produced under the catalytic action of creatine kinase mutant, it is that the Leu in the 121st site in the amino acid sequence of creatine kinase parent is sported Asp that the creatine kinase mutant, which has the characteristics of amino acid sequence as shown in SEQ ID NO:3, the amino acid sequence,.The specific activity of the creatine kinase mutant is higher by 52% compared with creatine kinase parent, and it is more than 85% that this method, which makes creatine be converted into the conversion ratio of phosphocreatine, is suitable for large-scale industrial production.
Description
Technical field
The present invention relates to the technical fields of the preparation method of phosphocreatine, in particular to a kind of to utilize biological enzyme technology
Produce the Biocatalysis method of phosphocreatine.
Background technique
Phosphocreatine (phosphocreatine) is in one of muscle or other excitable tissues's (such as brain and nerve)
High energy phosphate compound is the temporary storage form of energy-rich phosphate base.The effect of phosphocreatine is very more, main function have with
Lower 7 points: (1) myocardial protective agent: phosphocreatine is widely distributed in body and respectively organizes, and 90% in musculature, phosphocreatine
It is for maintaining ATP horizontal, phosphocreatine is by open synthesis access and reduces decomposition, protects sarcolemma from lacking
Blood injury and the nucleic acid deposit for maintaining cell.It is clinically used for the Cardioprotective of cardioplegia disease and other shapes of myocardial metabolism distress
Condition.Suitable for the treatment (adjuvant treatment) of myocardial ischemia, plumpness, heart infarction and heart failure, it also can be used as various openheart surgeries；(2) delay
Muscle middle acid substance is rushed to increase suddenly；(3) phosphocreatine (CP) also participates in energy transportation, i.e., energy is carried to from mitochondria
Other positions of muscle；(4) the fortune tonic of sportsman's first choice: for increasing the physical efficiency of sportsman, raising moves into phosphocreatine
Achievement has obvious effects on, safely and effectively, without side-effects.In play, the increase of phosphocreatine (CP) level can improve training and
Games results；(5) storage form of ATP: phosphocreatine can be transferred to energy-rich phosphate ADP and generate ATP, therefore phosphocreatine
It is the storage form of ATP；(6) act on ADP and generate ATP: before internal cretinephosphokinase is reduced to zero, brain tissue is lacked
When oxygen, phosphocreatine can act on ADP and generate ATP；(7) effect of buffer: phosphocreatine is in addition to providing energy, big
It can also play buffer to the acidic materials in control muscle in strenuous practice, this is because phosphocreatine is synthesizing
Need to consume the hydrogen ion for being deposited in that lactic acid releases in muscle in big strenuous practice during ATP, and hydrogen ion in muscle
Contraction of muscle can be excessively hindered, so phosphocreatine can work as a buffer and postpone the appearance of fatigue.
In the prior art, the production method of phosphocreatine includes chemical synthesis and biological catalysis.Chemical synthesis tool
There are severe reaction conditions, fierceness, be difficult to control, generate product complexity, pollutes environment, need to use in synthesis process toxic and easy
The disadvantages of raw material of combustion；And biocatalysis rule be using creatine and ATP as substrate, under the catalysis of creatine kinase, single-minded generation
Phosphocreatine has many advantages, such as that reaction condition is mild, reaction speed is fast, specificity is strong and low-carbon environment-friendly.But since creatine swashs
The vigor of enzyme is lower, and source is limited, causes the production cost of phosphocreatine excessively high, seriously constrains phosphocreatine biocatalysis
The scale and industrialization production of method.
Summary of the invention
For defect existing for the existing production method of phosphocreatine mentioned in above-mentioned background technique, the purpose of the present invention exists
In providing a kind of biocatalysis production method of high, at low cost, suitable for large-scale industrial production the phosphocreatine of yield.
To achieve the above object, inventor is to the Oryctolagus with the nucleotide sequence as shown in SEQ ID NO:1
Cuniculus creatine kinase parental gene carries out rite-directed mutagenesis, carrier appropriate is inserted into after PCR amplification, then in LB culture medium
Upper screening, to obtain the creatine kinase mutant with high catalytic activity, therefore, the present invention provides a kind of biocatalysis
The method for producing phosphocreatine, it is characterised in that: using creatine and atriphos as substrate, in the catalysis of creatine kinase mutant
Effect is lower to produce phosphocreatine, and the creatine kinase mutant has the amino acid sequence as shown in SEQ ID NO:3.
Above-mentioned creatine kinase mutant can be not purified thick enzyme form；Be also possible to through partial purification or completely it is pure
Conventional Histag method can be used in the enzyme of change, purification process；Immobilized enzyme made of curing technology is utilized if desired, can also be
Or the solidification enzyme of solid phase cell form.
Preferably, the molar ratio of the creatine and the atriphos is 20:1.
Preferably, the catalytic process also needs that MgCl is added2And sodium tripolyphosphate.
Preferably, the catalytic process is carried out in the buffer solution that pH value is 7~8.
The buffer solution is preferably Tris-HCl buffer solution.
Preferably, the creatine kinase mutant is the immobilization creatine kinase mutation being fixed on enzyme immobilization carrier
Body.
The enzyme immobilization carrier is preferably epoxy type LX-3000, silica, active carbon, bead or macroporous type
Poly- N- aminoethyl acrylamide-polyethylene.
Preferably, the additional amount of the immobilization creatine kinase mutant is 0.01-0.5g/ml buffer solution.
Preferably, the immobilization creatine kinase mutant preparation method the following steps are included:
(1) enzyme buffer liquid is washed in preparation: the mixed solution of 0.02M Tris-HCl and 0.001M EDTA, pH value 7.0；
(2) PB solution: 2.0mol/L potassium dihydrogen phosphate, pH value 7.5 is prepared；
(3) the creatine kinase mutant is diluted to 5-10mg/ml with enzyme buffer liquid of washing prepared by step (1), and will
Obtained enzyme dilution mixes in equal volume with PB solution prepared by step (2), the enzyme immobilization carrier is added, in shaking table
Reaction, the additional amount of the enzyme immobilization carrier are 10 milligrams of enzymes/gram carrier；
(4) gained reaction solution is filtered after step (3) fully reacting, and with step (1) prepare to wash enzyme buffer liquid clear
It washes to get immobilization creatine kinase mutant.
Above-mentioned creatine kinase mutant is prepared as template using the gene of Oryctolagus cuniculus creatine kinase parent
Method preferably include following steps:
(1) PCR amplification has the creatine kinase parent of the nucleotide sequence as shown in SEQ ID NO:1, amplimer pair
It is as follows
Ckm-F:5 ' GACATATGAGAGGGTTTATAATTGGT 3 ',
Ckm-R:5 ' GAGGATCCTTATTTGTCTGTCTGAGCTAAT 3 '；
(2) product of step (1) amplification is connect after digestion with restriction enzyme with carrier, obtains plasmid A；
(3) the plasmid A obtained using step (2) is template, and PCR amplification obtains F-DR segment, and amplimer is to as follows
Ckm-F:5 ' GACATATGAGAGGGTTTATAATTGGT 3 ',
121DR:5 ' TTAATAACTTCAACAACTGCTTTAGGAACCA 3 '；
(4) the plasmid A obtained using step (2) is template, and PCR amplification obtains DF-R segment, and amplimer is to as follows
121DF:5 ' GCAGTTGTTGAAGTTATTAAAGAAATAAAAGGTGT 3 ',
Ckm-R:5 ' GAGGATCCTTATTTGTCTGTCTGAGCTAAT 3 '；
(5) as template, PCR amplification obtains full the DF-R segment that the F-DR segment and step (4) obtained using step (3) obtains
Long mutant gene L121D, amplimer is to as follows
Ckm-F:5 ' GACATATGAGAGGGTTTATAATTGGT 3 ',
Ckm-R:5 ' GAGGATCCTTATTTGTCTGTCTGAGCTAAT 3 '；
(6) L121D that step (5) obtains is connect after digestion with restriction enzyme with carrier, obtains plasmid B；
(7) in the plasmid B transformed competence colibacillus bacterial cell for obtaining step (6), Multiplying culture is carried out simultaneously to conversion bacterium
Then inducing expression carries out clasmatosis processing, extract up to creatine kinase mutant.
In the preparation method of above-mentioned creatine kinase mutant provided by the invention, carrier is preferably pRSET-A.Certainly,
Any other applicable carriers also can be used, such as: prokaryotic expression carrier including pUC18/ including pRSET and pES21
Cloning vector etc. including 19 and pBluscript-SK.
In the preparation method of above-mentioned creatine kinase mutant provided by the invention, creatine kinase mutant gene obtained
Any other proper method known in the art can also be used and realize in original in prokaryotic cell or eukaryocyte intracellular expression
Nucleus or eukaryocyte extracellular expression.
In the preparation method of above-mentioned creatine kinase mutant provided by the invention, the host cell of carrier be can be including big
Prokaryotic cell including enterobacteria is also possible to the eukaryocyte including saccharomyces cerevisiae and including finishing red saccharomyces pastorianus.
The utility model has the advantages that
The biocatalysis production method of phosphocreatine provided by the invention, which is removed, has traditional Biocatalysis method institute jointly
Except the advantages that reaction condition having is mild, reaction speed is fast, specificity is strong and low-carbon environment-friendly, current existing phosphorus is also had both
The advantages of yield not available for the Biocatalysis method of creatine acid is high, at low cost, suitable for large-scale industrial production.After aforementioned
In terms of the acquisition of a part of advantage is mainly derived from following two in the present invention: 1, through enzyme assay, the present invention is manually lured
The specific activity of the creatine kinase mutant with the amino acid sequence as shown in SEQ ID NO:3 of acquisition is led compared with creatine kinase parent
Originally it is higher by 52%；2, the creatine kinase mutant by immobilization has higher vigor and better stability, and enzyme can be improved
Reuse rate.The conversion ratio that Biocatalysis method provided by the invention makes creatine be converted into phosphocreatine it has been confirmed by experiments that is super
Cross 85%.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below, and following embodiment is to solution of the invention
It releases, the person that is not specified actual conditions the invention is not limited to following embodiment, in embodiment, routinely condition or manufacturer suggest
Condition carry out.
Embodiment 1
The amplification of creatine kinase parental gene and clone
Creatine kinase parent means the creatine kinase (CKM) from Oryctolagus cuniculus, nucleotide sequence
(GenBank NM_001082239 is referred to) as shown in SEQ ID NO:1, amino acid sequence (reference as shown in SEQ ID NO:2
GenBank NP_001075708).According to the gene order design primer of creatine kinase parent to ckm-F:5 '
GACATATGAGAGGGTTTATAATTGGT 3 ' and ckm-R:5 ' GAGGATCCTTATTTGTCTGTCTGAGCTAAT 3 '.With drawing
Object expands creatine kinase encoding gene to ckm-F and ckm-R from Oryctolagus cuniculus cDNA library.
Amplification condition are as follows: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH4)2SO4, 2mM MgSO4,
0.1%Triton X-100,50mM dATP, 50mM dTTP, 50mM dCTP, 50mM dGTP, 400nM primer ckm-F,
400nM primer ckm-R, 100ng cDNA, 1.0U Pfu archaeal dna polymerase (Promega, USA), then with sterile water tune reactant
It accumulates to 50ml.
Pcr amplification reaction program are as follows: 95 DEG C 3 minutes, 35 circle circulation: 95 DEG C 50 seconds, 50 DEG C 30 seconds and 72 DEG C 1 minute, most
72 DEG C 10 minutes afterwards.The product of amplification is after restriction enzyme NdeI and BamHI digestion and through same restriction enzyme
Carrier pRSET-A (being originated from Invitrogen, USA) connection of NdeI and BamHI digestion, obtains plasmid pRSET-ckm.It is surveyed through DNA
Sequence, determines the nucleotide sequence of the creatine kinase being cloned, and is specifically shown in SEQ ID NO:1 in sequence table, corresponding amino
Acid sequence is the SEQ ID NO:2 in sequence table.
Embodiment 2
The preparation of creatine kinase mutant
With plasmid pRSET-ckm (see embodiment 1) for template, design primer is to 121DF:5 ' GCAGTTGTTGAAGTTAT
TAAAGAAATAAAAGGTGT 3 ' and 121DR:5 ' TTAATAACTTCAACAACTGCTTTAGGAACCA 3 '.
F-DR segment is expanded with primer pair ckm-F and 121DR, expands DF-R segment with primer pair 121DF and ckm-R.Expand
Increase reaction condition are as follows: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH4)2SO4, 2mM MgSO4, 0.1%
Triton X-100,50mM dATP, 50mM dTTP, 50mM dCTP, 50mM dGTP, 1.5U Pfu archaeal dna polymerase
(Promega, USA), 20ng pRSET-ckm and 400nM primer ckm-F and 400nM primer 121DR is (alternatively, 400nM draws
Object 121DF and 400nM primer ckm-R), with sterile water tune reaction volume to 50 microlitres.
Pcr amplification reaction program are as follows: 95 DEG C 3 minutes, 35 circle circulation: 95 DEG C 50 seconds, 52 DEG C 30 seconds and 72 DEG C 3 minutes, most
72 DEG C 5 minutes afterwards.It is separated through 1% agarose gel electrophoresis and commercial reagents box is used to recycle, respectively obtain F-DR segment and DF-R piece
Section.
Then full-length gene, amplification reaction condition are expanded are as follows: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM
(NH4)2SO4, 2mM MgSO4, 0.1%Triton X-100,50mM dATP, 50mM dTTP, 50mM dCTP, 50mM dGTP,
400nM primer ckm-F and 400nM ckm-R, 1.5U Pfu archaeal dna polymerase, 20ng F-DR segment and 20ng DF-R segment,
With sterile water tune reaction volume to 50 microlitres.
Pcr amplification reaction program are as follows: 95 DEG C 3 minutes, 35 circle circulation: 95 DEG C 50 seconds, 52 DEG C 30 seconds and 72 DEG C 3 minutes, most
72 DEG C 5 minutes afterwards.It is separated through 1% agarose gel electrophoresis and commercial reagents box is used to recycle, obtain overall length mutated gene L121D.
L121D segment is recycled and after restriction enzyme NdeI and BamHI digestion and through same restriction enzyme
The carrier pRSET-A of NdeI with BamHI digestion is connected, and obtains plasmid pRSET-L121D.Plasmid pRSET-L121D is transferred to impression
State bacterial cell E.coli BL21 determines SEQ ID NO:3 institute of the amino acid sequence of L121D such as in sequence table through DNA sequencing
Show.
Embodiment 3
The extraction and purification of enzyme
By the plasmid pRSET-ckm of the parental gene containing creatine kinase and the plasmid of the mutant gene containing creatine kinase
PRSET-L121D distinguishes transformed competence colibacillus bacterial cell E.coli BL21, in Luria broth (LB) plate (card containing 50mg/L
That mycin) it is 37 DEG C of cultures 24 hours upper.Inoculation individually be cloned in 5 milliliters of LB liquid mediums (kanamycins containing 50mg/L) in
30 DEG C culture 20-24 hours.Thalline were collected by centrifugation, and is suspended in 1 milliliter of 100mM Tris-HCl buffer (pH 7.5).So
Ultrasonic treatment bacterial cell is used afterwards.Centrifugation (10 DEG C, 17,800g, 10 minutes) simultaneously collects supernatant, i.e., obtains creatine kinase respectively
The thick leach protein (or crude extract) of parent and creatine kinase mutant.
Embodiment 4
The measurement of enzymatic activity
Prepare substrate solution: the MgCl of ATP, 20mM of creatine (Creatine), 5mM containing 100mM2, 40mM trimerization phosphorus
Sour sodium and 100mM Tris-HCl buffer adjust pH to 7.5.400 microlitres of substrate solution are taken, is then respectively adding 100 microlitres
Creatine kinase parent and 100 microlitres of creatine kinase mutant carry out reacting for 10 minutes in 37 DEG C.Centrifugation (10 DEG C, 17,
800g, 15 minutes) and supernatant is collected, phosphocreatine contains in the supernatant as obtained by high pressure liquid chromatography (HPLC) measurement
Amount.One unit specific enzyme activity is defined as converting micromole's creatine per minute under the above conditions being enzyme needed for phosphocreatine
Amount.Measurement result are as follows: the special Sexual potency of creatine kinase parent is 3.5U/mg, and the special Sexual potency of creatine kinase mutant is
5.3U/mg.Zymoprotein concentration, measurement result are measured with sds polyacrylamide gel electrophoresis are as follows: the enzyme of creatine kinase parent is than living
Property be 100, the specific enzyme activity of creatine kinase mutant is 152.
Embodiment 5
The preparation of immobilization creatine kinase mutant
The thick enzyme of creatine kinase mutant is taken, (0.02M Tris-HCl/0.001M EDTA, pH 7.0 is molten with enzyme buffer liquid is washed
Liquid) protein content is diluted to as 5-10mg/ml.By enzyme dilution and PB solution (2.0mol/L potassium dihydrogen phosphate, pH 7.5) etc.
Volume mixture is added enzyme immobilization carrier LX-3000 (10 milligrams of enzymes/gram carrier), in shaking table (revolving speed 100rpm) 25 DEG C it is anti-
It answers 20 hours.Sock filtration is used after the reaction was completed, is cleaned 5-6 times with enzyme buffer liquid is washed, is obtained immobilization creatine kinase mutant.
Embodiment 6
Phosphocreatine is prepared with immobilization creatine kinase mutant
Prepare substrate solution: the MgCl of ATP, 20mM of creatine (Creatine), 5mM containing 100mM2, 40mM trimerization phosphorus
Sour sodium and 100mM Tris-HCl buffer adjust pH to 7.5.1 milliliter of substrate solution is taken, 0.05 gram of immobilization creatine is then added
Kinase mutants.Reaction 2-20 hours is carried out in 37 DEG C.Centrifugation (10 DEG C, 17,800g, 15 minutes) simultaneously collects supernatant.Pass through
The content of phosphocreatine in high pressure liquid chromatography (HPLC) measurement gained supernatant.The result shows that: creatine is converted into phosphocreatine
Conversion ratio be more than 85%.
Claims (8)
1. a kind of method of biocatalysis production phosphocreatine, it is characterised in that: using creatine and atriphos as substrate,
Phosphocreatine, the amino acid sequence of the creatine kinase mutant such as SEQ ID are produced under the catalytic action of creatine kinase mutant
Shown in NO:3, the catalytic process also needs that MgCl2 and sodium tripolyphosphate is added.
2. the method for biocatalysis according to claim 1 production phosphocreatine, it is characterised in that: the creatine with it is described
The molar ratio of atriphos is 20:1.
3. the method for biocatalysis according to claim 1 production phosphocreatine, it is characterised in that: the catalytic process is
It is carried out in the buffer solution that pH value is 7 ~ 8.
4. the method for biocatalysis according to claim 3 production phosphocreatine, it is characterised in that: the buffer solution is
Tris-HCl buffer solution.
5. the method for biocatalysis production phosphocreatine according to claim 1, it is characterised in that: the creatine kinase is prominent
Variant is the immobilization creatine kinase mutant being fixed on enzyme immobilization carrier.
6. the method for biocatalysis production phosphocreatine according to claim 5, it is characterised in that: the enzyme immobilization carries
Body is epoxy type LX-3000, silica, active carbon, bead or the poly- N- aminoethyl acrylamide-polyethylene of macroporous type.
7. the method for biocatalysis production phosphocreatine according to claim 5, which is characterized in that the immobilization creatine
The additional amount of kinase mutants is 0.01-0.5g/ml buffer solution.
8. the method for biocatalysis production phosphocreatine according to claim 5, which is characterized in that the immobilization creatine
The preparation methods of kinase mutants the following steps are included:
(1) enzyme buffer liquid is washed in preparation: the mixed solution of 0.02M Tris-HCl and 0.001M EDTA, pH value 7.0；
(2) PB solution: 2.0mol/L potassium dihydrogen phosphate, pH value 7.5 is prepared；
(3) the creatine kinase mutant is diluted to 5-10mg/ml with enzyme buffer liquid of washing prepared by step (1), and will obtained
Enzyme dilution mixed in equal volume with PB solution prepared by step (2), add the enzyme immobilization carrier, it is anti-in shaking table
It answers, the additional amount of the enzyme immobilization carrier is 10 milligrams of enzymes/gram carrier；
(4) gained reaction solution is filtered after step (3) fully reacting, and is cleaned with enzyme buffer liquid of washing prepared by step (1), i.e.,
Obtain immobilization creatine kinase mutant.
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| CN102808006A (en) * | 2012-08-23 | 2012-12-05 | 天津启仁医药科技有限公司 | Method for producing phosphocreatine by microbial enzyme method |
| CN102911928A (en) * | 2012-07-06 | 2013-02-06 | 西南药业股份有限公司 | Immobilized method of creatine kinase |
| CN104357420A (en) * | 2014-12-02 | 2015-02-18 | 杭州清科生物科技有限公司 | Creatine kinase mutant applicable to phosphocreatine enzymic method production process |
| CN104480098A (en) * | 2014-12-02 | 2015-04-01 | 杭州清科生物科技有限公司 | Creatine kinase immobilization method suitable for phosphocreatine enzyme-method production process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911928A (en) * | 2012-07-06 | 2013-02-06 | 西南药业股份有限公司 | Immobilized method of creatine kinase |
| CN102808006A (en) * | 2012-08-23 | 2012-12-05 | 天津启仁医药科技有限公司 | Method for producing phosphocreatine by microbial enzyme method |
| CN104357420A (en) * | 2014-12-02 | 2015-02-18 | 杭州清科生物科技有限公司 | Creatine kinase mutant applicable to phosphocreatine enzymic method production process |
| CN104480098A (en) * | 2014-12-02 | 2015-04-01 | 杭州清科生物科技有限公司 | Creatine kinase immobilization method suitable for phosphocreatine enzyme-method production process |
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| Role of the linker between the N- and C-terminal domains in the stability and folding of rabbit muscle creatine kinase;Hua-Wei He等;《The International Journal of Biochemistry & Cell Biology》;20070522;第39卷;摘要、第1817页左栏第2段至第1818页右栏第1页 |
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| CN105647986A (en) | 2016-06-08 |
| CN105647986B (en) | 2019-05-03 |
| CN105671096B (en) | 2019-05-07 |
| CN107109379B (en) | 2021-02-23 |
| WO2016169307A1 (en) | 2016-10-27 |
| CN105647985A (en) | 2016-06-08 |
| CN105671096A (en) | 2016-06-15 |
| CN107109379A (en) | 2017-08-29 |
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