CN104480098A - Creatine kinase immobilization method suitable for phosphocreatine enzyme-method production process - Google Patents
Creatine kinase immobilization method suitable for phosphocreatine enzyme-method production process Download PDFInfo
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- CN104480098A CN104480098A CN201410716674.1A CN201410716674A CN104480098A CN 104480098 A CN104480098 A CN 104480098A CN 201410716674 A CN201410716674 A CN 201410716674A CN 104480098 A CN104480098 A CN 104480098A
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- polyvinyl alcohol
- creatine kinase
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- sodium alginate
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Abstract
The invention relates to a transformation for creatine kinase of rabbit muscle by virtue of an enzyme immobilization technology, so as to improve the performance of creatine kinase used as a reaction catalyzing enzyme in phosphokinase production. The invention discloses an enzyme immobilization method, wherein p-polyvinyl alcohol and sodium alginate are used as raw materials and are mixed according to a certain ratio to produce gel, thus immobilizing creatine kinase. The method disclosed by the invention has the following advantages: the repeated usage rate of creatine kinase can be greatly increased through the transformation, the production efficiency is increased, the production cost is reduced, and the method is of an important significance for promoting the enzyme-method phosphocreatine industry.
Description
Technical field
The invention belongs to the transformable technical field of bioengineering of creatine kinase, be specifically related to the Production by Enzymes enzyme demand transforming to be applicable to phosphocreatine by enzyme immobilization technology to rabbit flesh creatine kinase.
Background technology
In the cytoplasm that creatine kinase (EC 2.7.3.2) is present in the tissues such as the heart of animal, muscle and brain usually and plastosome; be that one operates with intracellular energy, Muscle contraction, ATP regenerate the important kinases having direct relation, its phosphoryl that turns reversibly between catalysis creatine and ATP reacts.In phosphocreatine route of synthesis, play the effect that reversible catalysis creatine forms phosphocreatine, need ATP to provide phosphoryl in positive reaction process, phosphocreatine contains energy-rich bond, has energy storage effect, therefore can compensate the concentration of body ATP.
Phosphocreatine; a kind of high-energy phosphate compound in muscle or other excitability tissues (as brain and nerve); it is the temporary storage form of energy-rich phosphate base; it can as myocardial cell protection agent and Power supply thing; for the auxiliary therapy such as clinical operation and myocardial ischemia; effective, security is high, and social demand is large.The advantages such as at present in the production technology of phosphocreatine, biological enzyme synthetic technology has non-environmental-pollution due to it, and reaction conditions is gentle, and the fast and specificity of speed of response is strong, have become the research emphasis of phosphocreatine manufacture.
Biological enzyme is produced in phosphocreatine technique, and the key of technology is the use of creatine kinase.At present, the production for this enzyme is extracted and is difficult to mass-producing, and the reasons such as enzyme utilising efficiency is low cause phosphocreatine production cost too high, and thus this technology never obtains industrialization development.Therefore find a kind of can improve this enzyme stability and the processing method of Reusability rate particularly important.Way current in current industry is by creatine kinase immobilization, improves stability and the repeat usage of enzyme, reduces the cost of production enzyme.
Summary of the invention
The present invention proposes a kind of method of immobilization creatine kinase, is more suitable for the industrial demand of phosphocreatine to make this enzyme.It can improve the repeat usage of creatine kinase in Production by Enzymes phosphocreatine technique further, and then improves phosphocreatine production efficiency, reduces production cost, thus lays the foundation for the industrialization of Production by Enzymes phosphocreatine.
Technical scheme of the present invention is mainly and utilizes enzyme immobilization technology, with to polyvinyl alcohol and sodium alginate for starting material, utilize best creatine kinase immobilization technology, to reaching the object improving creatine kinase performance in phosphocreatine production practice, enhance productivity further, reduce production cost.
The detailed process of creatine kinase immobilization technology is:
(1) take and be added in hac buffer (pH 4.0 ~ 5.0) polyvinyl alcohol, be heated to 80 DEG C, mixing, until dissolve completely polyvinyl alcohol.
(2) get the sodium alginate soln being dissolved in acetate buffer solution (pH 4.0 ~ 5.0) of certain volume, join in polyvinyl alcohol solution, slowly stir 45 min.
(3) room temperature is cooled to polyvinyl alcohol and mixed solution of sodium alginate, adds a certain amount of creatine kinase storage liquid, mix.
(4) will to polyvinyl alcohol, sodium alginate and creatine kinase mixed solution, dropwise join low temperature precooling 2.0% ~ 7.0% boric acid and 0.5% ~ 2.0% CaCl
2in mixed solution, be cured reaction.In the curing process, the concentration to polyvinyl alcohol added is 5% ~ 20%, and the concentration range of sodium alginate is 0.05% ~ 2.0%, the amount of creatine kinase is 2000 ~ 5000U, immobilization temperature is 4 ~ 10 DEG C, and the time is 4 ~ 10h, and immobilization process pH value is 4.0 ~ 5.0.
The invention provides a kind of enzyme catalyst transformation type that can be applied to phosphocreatine enzyme process production technique, add the reusable rate of enzyme, reduce production cost, non-environmental-pollution.These advantages are all that the creatine kinase of the present invention's transformation can be widely used in providing guarantee in phosphocreatine enzyme process industrial production.
Below in conjunction with specific examples, the present invention is set forth further, but these examples are not used for limiting the scope of the invention.
Embodiment
Embodiment 1
1, take 10 g to be added in 50 mL hac buffers (pH 4.0) polyvinyl alcohol, be heated to 80 DEG C, mixing, until dissolve completely polyvinyl alcohol.
2, get 2.0% sodium alginate soln that 50 mL are dissolved in acetate buffer solution, join in polyvinyl alcohol solution, slowly stir 45min.
3, room temperature is cooled to polyvinyl alcohol and mixed solution of sodium alginate, adds 110 U/L creatine kinase storage liquid 25mL, mix.
4, will to polyvinyl alcohol, sodium alginate and creatine kinase mixed solution, dropwise join low temperature precooling 5% boric acid and 1% CaCl
2in mixed solution, be cured reaction.In the curing process, temperature remains on 4 DEG C, and the time is 4h.
Be 41U/mg through improved immobilization creatine kinase enzyme activity, pH vigor scope of this transformation enzyme expands to 4 ~ 8 by original 5 ~ 7, shows all to increase significantly to the stability of the conditions such as soda acid; The optimal reactive temperature of this transformation enzyme is 42 DEG C, improves about 5 DEG C than wild resolvase; Its temperature vigor scope rises to 15 ~ 50 DEG C by 15 ~ 35 DEG C; Transformation period of reusing of this transformation enzyme is 46 times, has good to reuse stability; This transformation enzyme is compared with wild resolvase, and the transformation period at 60 DEG C is 10.5 hours, and thermostability specific ionization enzyme improves about 6 times; The preservation transformation period at 4 ~ 10 DEG C is 150 ~ 200 days.
Embodiment 2
1, take 5g to be added in 50mL hac buffer (pH 4.0) polyvinyl alcohol, be heated to 80 DEG C, mixing, until dissolve completely polyvinyl alcohol.
2, get 2.0% sodium alginate soln that 50 mL are dissolved in acetate buffer solution, join in polyvinyl alcohol solution, slowly stir 45 min.
3, room temperature is cooled to polyvinyl alcohol and mixed solution of sodium alginate, adds 121 U/L creatine kinase storage liquid 25 mL, mix.
4, will to polyvinyl alcohol, sodium alginate and creatine kinase mixed solution, dropwise join low temperature precooling 2% boric acid and 0.5% CaCl
2in mixed solution, be cured reaction.In the curing process, temperature remains on 4 DEG C, and the time is 4h.
Be 52 U/mg through improved immobilization creatine kinase enzyme activity, pH vigor scope of this transformation enzyme expands to 4 ~ 8 by 5 ~ 7 of original resolvase, shows all to increase significantly to the stability of the conditions such as soda acid; The optimal reactive temperature of this transformation enzyme is 45 DEG C, improves about 10 DEG C than wild resolvase; Its temperature vigor scope rises to 15 ~ 50 DEG C by 15 ~ 35 DEG C; Transformation period of reusing of this transformation enzyme is 50 times, has good to reuse stability; This transformation enzyme is compared with wild resolvase, and the transformation period at 60 DEG C is 12.5 hours, and thermostability specific ionization enzyme improves about 7 times; The preservation transformation period at 4 ~ 10 DEG C is 250 ~ 300 days.
Embodiment 3
1, take 5 g to be added in 50 mL hac buffers (pH 4.0) polyvinyl alcohol, be heated to 80 DEG C, mixing, until dissolve completely polyvinyl alcohol.
2, get 1.0% sodium alginate soln that 50 mL are dissolved in acetate buffer solution, join in polyvinyl alcohol solution, slowly stir 45 min.
3, room temperature is cooled to polyvinyl alcohol and mixed solution of sodium alginate, adds 80 U/L creatine kinase storage liquid 25mL, mix.
4, will to polyvinyl alcohol, sodium alginate and creatine kinase mixed solution, dropwise join low temperature precooling 6% boric acid and 1.5% CaCl
2in mixed solution, be cured reaction.In the curing process, temperature remains on 4 DEG C, and the time is 4h.
Be 50U/mg through improved immobilization creatine kinase enzyme activity, pH vigor scope of this transformation enzyme expands to 4 ~ 9 by original 5 ~ 7, shows all to increase significantly to the stability of the conditions such as soda acid; The optimal reactive temperature of this transformation enzyme is 44 DEG C, improves about 10 DEG C than wild resolvase; Its temperature vigor scope rises to 15 ~ 65 DEG C by 15 ~ 35 DEG C; Transformation period of reusing of this transformation enzyme is 75 times, has good to reuse stability; This transformation enzyme is compared with wild resolvase, and the transformation period at 60 DEG C is 18 hours, and thermostability specific ionization enzyme improves about 10 times; The preservation transformation period at 4 ~ 10 DEG C is 250 ~ 300 days.
The foregoing is only the present invention's other embodiment, instead of whole embodiments, these embodiments are not in order to limit the present invention.Those skilled in the art are not making the every other possibility embodiment obtained under creative work prerequisite, and the part made embodiment is without departing from theon the basis of the spirit of the present invention revised and improved, and all belongs to the scope of protection of present invention.
Claims (5)
1. be applicable to a remodeling method for the creatine kinase of phosphocreatine production and application, it is characterized in that:
Utilize enzyme immobilization technology, with to polyvinyl alcohol and sodium alginate for starting material, being mixed polyvinyl alcohol gel particle according to a certain percentage, is carrier immobilized creatine kinase with it.
2. enzyme immobilization technology according to claim 1, is characterized in that, said method comprising the steps of:
(1) getting some grams is added in hac buffer to polyvinyl alcohol, is heated to 80 DEG C, and mixing, until dissolve completely polyvinyl alcohol;
(2) get the sodium alginate soln being dissolved in acetate buffer solution of certain volume, join in (1) solution, slowly stir 45min;
(3) room temperature is cooled to polyvinyl alcohol and mixed solution of sodium alginate, adds a certain amount of creatine kinase storage liquid, mix;
(4) mixed solution of previous step is dropwise joined low temperature precooling boric acid and CaCl by syringe
2in mixed solution, 150rpm shakes up 15min, is transferred in 10% metabisulfite solution, is cured reaction 4 ~ 10h, completes solidification; The gel particle of solidification uses the acetate buffer solution of pH 4.0 ~ 5.0 to wash, and is stored in this damping fluid.
3. enzyme immobilization step according to claim 2, is characterized in that:
In (1) and (2) solution, the pH scope of hac buffer is 4.0 ~ 5.0.
4. enzyme immobilization step according to claim 2, is characterized in that:
In order to obtain best in quality to polyvinyl alcohol gel particle, in (3) solution, be 5% ~ 20% to the concentration range of polyvinyl alcohol the best, the optimum concentration range of sodium alginate is 0.05% ~ 2.0%.
5. enzyme immobilization step according to claim 2, is characterized in that:
In (4) solution, the concentration range of boric acid is 2.0% ~ 7.0%, CaCl
2concentration range be 0.5% ~ 2.0%, immobilized temperature is 4 ~ 10 DEG C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647986A (en) * | 2015-04-23 | 2016-06-08 | 邦泰生物工程(深圳)有限公司 | Method for preparing phosphocreatine by biological catalysis |
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| CN101348782A (en) * | 2008-08-30 | 2009-01-21 | 兰州大学 | Macroreticular polyvinyl alcohol bead carrier and preparation thereof |
| CN102021163A (en) * | 2009-09-21 | 2011-04-20 | 江苏同和涂装机械有限公司 | Polyvinyl alcohol immobilization and microorganism preparation method |
| CN102911928A (en) * | 2012-07-06 | 2013-02-06 | 西南药业股份有限公司 | Immobilized method of creatine kinase |
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| CN101348782A (en) * | 2008-08-30 | 2009-01-21 | 兰州大学 | Macroreticular polyvinyl alcohol bead carrier and preparation thereof |
| CN102021163A (en) * | 2009-09-21 | 2011-04-20 | 江苏同和涂装机械有限公司 | Polyvinyl alcohol immobilization and microorganism preparation method |
| CN102911928A (en) * | 2012-07-06 | 2013-02-06 | 西南药业股份有限公司 | Immobilized method of creatine kinase |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647986A (en) * | 2015-04-23 | 2016-06-08 | 邦泰生物工程(深圳)有限公司 | Method for preparing phosphocreatine by biological catalysis |
| CN105647985A (en) * | 2015-04-23 | 2016-06-08 | 邦泰生物工程(深圳)有限公司 | Method for producing phosphocreatine by biological catalysis |
| CN105671096A (en) * | 2015-04-23 | 2016-06-15 | 邦泰生物工程(深圳)有限公司 | Method for producing creatine phosphate by biological catalysis |
| CN105647986B (en) * | 2015-04-23 | 2019-05-03 | 邦泰生物工程(深圳)有限公司 | The method of biocatalysis production phosphocreatine |
| CN105671096B (en) * | 2015-04-23 | 2019-05-07 | 邦泰生物工程(深圳)有限公司 | A method of phosphocreatine is produced by biocatalysis |
| CN105647985B (en) * | 2015-04-23 | 2019-05-07 | 邦泰生物工程(深圳)有限公司 | A kind of method of biocatalysis production phosphocreatine |
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