CN102808006A - Method for producing phosphocreatine by microbial enzyme method - Google Patents
Method for producing phosphocreatine by microbial enzyme method Download PDFInfo
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- CN102808006A CN102808006A CN2012103018258A CN201210301825A CN102808006A CN 102808006 A CN102808006 A CN 102808006A CN 2012103018258 A CN2012103018258 A CN 2012103018258A CN 201210301825 A CN201210301825 A CN 201210301825A CN 102808006 A CN102808006 A CN 102808006A
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Abstract
The invention discloses a method for producing phosphocreatine by converting creatine and triphosadenine by a microbial enzyme method. According to the method, a genetic engineering method is adopted; recombinant escherichia coli BL21(DE3)-pET21a(+)-CPK which expresses creatine kinase efficiently is constructed; and the reaction of the creatine and the triphosadenine is catalyzed to produce the phosphocreatine by taking fermentation bacteria or lysate as an enzyme source. The invention provides a new green production process method for the phosphocreatine, which has the advantages of mild conditions, short period, few impurities in an enzymatic system, convenience in separation and extraction of products, simple in process steps, safety in operation and the like; and the method has a very good industrial application prospect.
Description
Technical field
The invention belongs to biotechnology and produce the technical field of phosphocreatine, relate to the method that transforms creatine and Triphosaden production phosphocreatine through microbial enzyme method.
Background technology
Phosphocreatine (Creatine phosphate, CP or Phosphocreatine, PCr or PC) also claim creatine phosphate, is the important derivatives of creatine, in Mammals muscle, separates obtaining in nineteen twenty-seven.In the cell of high-energy conversion, phosphocreatine has following two kinds of effects at least: the one, and as intracellular energy carrier, the 2nd, as a kind of energy snubber agent.Phosphocreatine has been widely used in all kinds of cardiopathic control medicines as the intravital a kind of high energy stored substance of people, also can be used as nutritious prod and directly takes, and is widely used in the clinical diagnosis as biochemical preparation simultaneously.
The preparation method of phosphocreatine mainly contains at present:
(1) classical approach is to be starting raw material with the creatine, in NaOH solution, drips POCl3 and carries out condensation reaction, obtains disodium creatine phosphate through barium or calcium salt exchange purifying.The soluble barium salt that this method is introduced has intensive toxicity, therefore produces the disodium creatine phosphate of injection medicine for needs, and possibly there is certain risk in this technology.
(2) organic synthesis method is through preparation Phosphorylcreatinine midbody, recrystallization purifying, and the hydrogenation debenzylation obtains the creatinine sodium phosphate then, and further the heating open loop gets disodium creatine phosphate in aqueous sodium hydroxide solution.This method steps is many, cost is high, introduces hydrogen simultaneously, has certain danger in the operation.
(3) biotransformation method be with microorganism cells or the enzyme that from organism, extracts as biological catalyst, utilize the specificity of enzyme to prepare phosphocreatine, have the reaction conditions gentleness, an advantage such as speed of response is fast and specificity is strong.The Chinese patent CN02129203.5 of existing report adopts the Tn that from rabbit muscle, extracts, and is raw material with phosphoric acid and creatine, and biological enzyme synthesizes disodium creatine phosphate.But because the Tn enzyme activity that he adopts is lower, and receive the source restriction, can not macro preparation, therefore do not have industrialization value.
Tn (EC2.7.3.2); Usually be present in cells of tissues such as heart, muscle and the brain slurry and plastosome of animal; It is the important kinases that and intracellular energy running, Muscle contraction, TP regeneration have direct relation; Commentaries on classics phosphoryl reaction between its reversible ground catalysis creatine and the ATP, reaction formula is as shown in Figure 1.
But; The Tn that traditional enzyme process adopts generally extracts from animal muscle; The thick enzyme system of this employing animal muscle cytoplasm preparation certainly will be introduced more foreign protein in reaction system as the enzyme source, will bring difficulty to the separation-extraction technology after transforming; Also there is invention to adopt 3-phoshoglyceric acid as initial substrate; Employing comprises the prozyme system of Tn; But because the enzyme of participation reaction is more; The desired optimum condition of various enzymes is different, therefore can not guarantee to satisfy simultaneously the optimum condition of all enzymes, finally is the productive rate that influences phosphocreatine.
Summary of the invention
The object of the invention is exactly to solve the problems referred to above that exist in the existing phosphocreatine technology of preparing, and the green method for transformation of the microbial enzyme method production phosphocreatine that a kind of cost is low, productive rate is high, environment friendly and pollution-free is provided.
The method that microbial enzyme method provided by the invention is produced phosphocreatine comprises: zymophyte somatocyte or lysate with the gene engineering recombinant bacterium of expressing Tn are the enzyme source; With creatine and Triphosaden is substrate; Under room temperature to 45 ℃; PH9 to 11 to 5h, produces phosphocreatine through enzymatic conversion method reaction 0.5.Wherein, in the enzymatic reaction liquid, the add-on of substrate creatine is 5 to 15g/L, and the add-on of Triphosaden is 2.5 to 10g/L, and the addition in enzyme source is 300 to 900U/L, and the add-on of cofactor anhydrous magnesium sulfate is 32.5 to 95mmol/L.
The consumption of substrate creatine is preferably 10g/L, and the consumption of Triphosaden is preferably 7.5g/L, and the addition in enzyme source is preferably 450U/L; The addition of anhydrous magnesium sulfate is preferably 65mmol/L; Preferred invert point is 37 ℃, preferably transforms pH9, preferred transformation time 3h.
The gene engineering recombinant bacterium of described expression Tn is e. coli bl21 (DE3)-pET21a (+)-CPK, and expression vector is pET21a (+)-CPK recombinant expression vector, and expressing in the reorganization somatic cells has highly active Tn.
The host bacterium of the gene engineering recombinant bacterium of described expression L-arginase is an e. coli bl21, and expression vector is pET21a (+)-CPK recombinant expression vector.
The present invention has made up bacillus coli gene engineering strain BL21 (DE3)-pET21a (+)-CPK that efficiently expresses Tn voluntarily; Promptly from mouse muscle, extract total RNA; Structure contains the bacillus coli gene engineering bacteria of mouse Tn gene, and the Tn enzyme activity of this somatic cells can reach 30U/g.Collect thalline, carry out the crude enzyme liquid that ultrasonic disruption obtains Tn, enzyme work is about 6U/mL.
In embodiments of the present invention; Adopt HPLC to measure the content of phosphocreatine; Concrete grammar is following: performance liquid chromatography adopts the C18 post, and (150 * 4.6mm) measure the content of phosphocreatine; This liquid-phase condition is: with acetonitrile-phosphoric acid salt (5:95) is moving phase, and wherein the phosphoric acid salt prescription is 0.2% potassium primary phosphate and 0.08% 4-butyl ammonium hydrogen sulfate; Sample size 10 μ L; Overall flow rate is 1.0mL/min; Detect wavelength 210nm; Column temperature is 25 ℃.
Advantage of the present invention and beneficial effect:
The present invention is from substrate creatine and Triphosaden; Fermentation thalline or lysate that utilization efficiently expresses bacillus coli gene engineering bacteria BL21-pET21a (+)-CPK of Tn are the enzyme source; Enzyme process catalysis creatine and Triphosaden are produced phosphocreatine; Have few, the advantages such as the product separation and Extraction convenient, process step simple, operational safety of impurity in mild condition, cycle weak point, the enzymatic system, good commercial application prospect is arranged.
Description of drawings
Fig. 1 is the reaction synoptic diagram that Tn catalysis creatine generates phosphocreatine;
Fig. 2 is a HPLC method liquid phase collection of illustrative plates of measuring phosphocreatine (among the figure shown in the A) and typical curve (among the figure shown in the B);
Fig. 3 is a conversion condition of investigating phosphocreatine;
A: the relation of temperature of reaction and transformation efficiency; The relation of B:pH and transformation efficiency; C: the relation of reaction times and transformation efficiency; D: the relation of Triphosaden addition and transformation efficiency; E: the relation of creatine addition and transformation efficiency; F: the relation of enzyme amount and transformation efficiency; G: the relation of anhydrous magnesium sulfate addition and transformation efficiency.
Fig. 4 is under optimum conversion condition, the content of phosphocreatine in conversion fluid when HPLC method assaying reaction finishes.
Embodiment
One, the mensuration of Tn vigor
Adopt chemical staining method to measure the Tn vigor, principle is under certain condition, and the reverse catalysis phosphocreatine of Tn generates creatine, utilizes the reaction of creatine and naphthyl alcohol and di-acetyl, generates red compound.Within the specific limits, the red depth is directly proportional with creatine, with the creatine reference liquid colorimetric of same processing, tries to achieve enzyme activity.
Specific operation process is following:
The preparation of 1 reagent: substrate is mixed in (1)
1. pH7.4 tris-HCI buffer: take by weighing Tutofusin tris 2.4g, add zero(ppm) water and be settled to 100mL, add 0.2N hydrochloric acid 88.8mL and anhydrous magnesium sulfate 0.34g again, regulate pH to 7.4.
2. 12mM phosphocreatine solution: take by weighing Disodium phosphocreatine 36mg, adding distil water is settled to 100mL, regulates pH to 7.4.
3. 1mM ADP solution: take by weighing ADP sodium salt 0.233g, add zero(ppm) water and be settled to 100mL, regulate pH to 7.4.
Face with before, get above-mentioned 1., 2., 3. each 10mL of reagent mixes, and is the mixing substrate.
(2) 0.3N barium hydroxide solution
Take by weighing hydrated barta 4.73g, adding distil water is settled to 100mL.
(3) 5% solution of zinc sulfate
Take by weighing zinc sulfate 8.82g, adding distil water is settled to 100mL.
(4) standard creatine solution (1.7 μ M/mL)
Accurately take by weighing anhydrous creatine 22.3mg, adding distil water is settled to 100mL.
(5) di-acetyl solution
Be made into 1% solution earlier, place 4 ℃ of preservations of brown bottle, face the time spent with 20 times of distilled water dilutings.
(6) store alkaline solution
Take by weighing 30g sodium hydroxide, the 64g soda ash light, adding distil water is settled to 500mL.
(7) naphthyl alcohol solution
Take by weighing naphthyl alcohol 1g, adopt above-mentioned storage alkaline solution to make it dissolving, and be settled to 100mL.
2 measurement operation steps (as shown in table 1 below)
Table 1 unit: mL
The enzyme activity definition: under above-mentioned reaction conditions, in 37 ℃, it is an enzyme activity unit that 1min catalysis forms the needed enzyme amount of 1 μ mol creatine.
Two, phosphocreatine Determination on content--HPLC method is measured the content of phosphocreatine
Performance liquid chromatography adopts the C18 post, and (150 * 4.6mm) measure the content of phosphocreatine; This liquid-phase condition is: performance liquid chromatography adopts the C18 post, and (150 * 4.6mm) measure the content of phosphocreatine; This liquid-phase condition is: with acetonitrile-phosphoric acid salt (5:95) is moving phase, and wherein the phosphoric acid salt prescription is 0.2% potassium primary phosphate and 0.08% 4-butyl ammonium hydrogen sulfate; Sample size 10 μ L; Overall flow rate is 1.0mL/min; Detect wavelength 210nm; Column temperature is 25 ℃.(like Fig. 2 A)
Accurately prepare the phosphocreatine reference liquid of series concentration, continuous sample introduction 5 times calculates peak area and also averages, and (X mmol/L) is X-coordinate, and peak area (Y) is an ordinate zou drawing standard curve with phosphocreatine concentration.The phosphocreatine that enzymatic reaction produced is through suitably measuring with HPLC after the dilution, and the establishing criteria curve carries out accurate quantification.(like Fig. 2 B)
The expression of colibacillary structure of embodiment 1 genetic engineering bacterium and reorganization Tn
The Tn gene order of the mouse muscle of including according to ncbi database, design upstream primer CPK1:5 '-GGGAAT TCC ATA TGC CGT TCG GCA ACA CCC ACA AC-3 ' (SEQ ID No.1) and downstream primer CPK2:5 '-CCG CTC GAG CTT CTG CGC GGG GAT CAT GTC GTC G-3 ' (SEQ ID No.2).Extract the total RNA of mice skeletal, reverse transcription prepares cDNA.With mice skeletal cDNA is template, and pcr amplification obtains the Tn gene.Adopt gene engineering method, make up recombinant expression plasmid pET-21a (+)-CPK.Recombinant expression vector pET-21a (+)-CPK is converted into E.coli BL21 (DE3), obtains engineering strain.
Positive recombinant bacterial strain is inoculated in 4mL contains incubated overnight in the LB substratum of penbritin 100 μ g/mL; Inserting 100mL with 1% switching amount subsequently contains in the fresh LB substratum of penbritin 100 μ g/mL; 37 ℃ of shaking culture 3h; Then, adding final concentration is the IPTG induced liquid of 1mM, and specificity is induced the expression of Tn.
The gained nutrient solution is in 4 ℃ following 6; The centrifugal 10min of 000rpm collects thalline, obtains to express the gene engineering recombinant bacterium (BL21 (DE3)-pET21a (+)-CPK) of Tn; Centrifugal again after phosphoric acid buffer (pH8.0) the suspension washing with 50mmol/L, collect thalline as zymogenic cell.
The fermentation of embodiment 2 reorganization Tn gene engineering recombinant bacteriums
Recombination bacillus coli among the embodiment 1 is inoculated in 4mL contains in the LB substratum of 100 μ g/mL penbritins incubated overnight as primary seed solution; Insert 100mL with 1% switching amount subsequently and contain in the LB substratum of 100 μ g/mL penbritins, in 37 ℃, 200rpm cultivates 8-12h as secondary seed solution; Secondary seed solution is transferred in the 100L fermentor tank with 1% inoculum size, and fermention medium is fresh LB substratum, 37 ℃ of culture temperature, initial speed 300rpm, initial pH7.2.Timing sampling, when bacterium liquid OD600nm reached 1.0-1.2, the adding final concentration was that the IPTG induced liquid of 1mM is induced.4 ℃ of following jar fermented liquids down 3, the centrifugal 15min of 000rpm collects somatic cells, obtains expressing the zymophyte somatocyte of the gene engineering recombinant bacterium of Tn, measures by method one, and enzyme work can reach 30U/g.
The lysate preparation of embodiment 3 reorganization Tn gene engineering recombinant bacteriums
According to embodiment 2 fermentation reorganization Tn gene engineering recombinant bacteriums, obtain the fermentation thalline, collect thalline and carry out the crude enzyme liquid that ultrasonic disruption obtains Tn, measure its enzyme activity according to method one, enzyme work is about 6U/mL.
The investigation of temperature in embodiment 4 conversion reactions
As proenzyme, in every liter transformation system, add enzyme amount 300U/L with the lysate of the zymophyte somatocyte of the foregoing description 2 preparation or embodiment 3 preparations respectively; Substrate creatine 5g/L, Triphosaden 3.5g/L, anhydrous magnesium sulfate 50mmol/L; Transform pH value 10, transformation time 0.5h.When investigating 30-45 ℃ of invert point to the influence of Triphosaden transformation efficiency (because the unit price of Triphosaden is about twenties times of creatine; So in the reaction system; Creatine is excessive and investigate the transformation efficiency of Triphosaden), adopt method two to detect the content of phosphocreatine.The result sees Fig. 3 A.
The investigation of pH in embodiment 5 conversion reactions
As proenzyme, in every liter transformation system, add enzyme amount 300U/L with the lysate of the zymophyte somatocyte of the foregoing description 2 preparation or embodiment 3 preparations respectively; Substrate creatine 5g/L, Triphosaden 3.5g/L, anhydrous magnesium sulfate 50mmol/L; Invert point is 37 ℃, transformation time 0.5h.To the influence of Triphosaden transformation efficiency, adopt method two to detect the content of phosphocreatine when investigating conversion pH value 9-11.The result sees Fig. 3 B.
The investigation in reaction times in embodiment 6 conversion reactions
As proenzyme, in every liter transformation system, add enzyme amount 300U/L with the lysate of the zymophyte somatocyte of the foregoing description 2 preparation or embodiment 3 preparations respectively; Substrate creatine 5g/L, Triphosaden 3.5g/L, anhydrous magnesium sulfate 50mmol/L; Invert point is 37 ℃, and transforming the pH value is 9.To the influence of Triphosaden transformation efficiency, adopt method two to detect the content of phosphocreatine when investigating transformation time 0.5-3h.The result sees Fig. 3 C.
The investigation of the input amount of substrate Triphosaden in embodiment 7 conversion reactions
As proenzyme, in every liter transformation system, add enzyme amount 300U/L with the lysate of the zymophyte somatocyte of the foregoing description 2 preparation or embodiment 3 preparations respectively; Substrate creatine 15g/L, anhydrous magnesium sulfate 50mmol/L, invert point is 37 ℃; Transforming the pH value is 9, transformation time 3h.To the influence of Triphosaden transformation efficiency, adopt method two to detect the content of phosphocreatine when investigating Triphosaden 3.5-10g/L.The result sees Fig. 3 D.
The investigation of creatine input amount in embodiment 8 conversion reactions
As proenzyme, in every liter transformation system, add enzyme amount 300U/L with the lysate of the zymophyte somatocyte of the foregoing description 2 preparation or embodiment 3 preparations respectively; Triphosaden consumption 7.5g/L, anhydrous magnesium sulfate 50mmol/L, invert point is 37 ℃; Transforming the pH value is 9, transformation time 3h.To the influence of Triphosaden transformation efficiency, adopt method two to detect the content of phosphocreatine when investigating creatine input amount 5-15g/L.The result sees Fig. 3 E.
The investigation of enzyme amount in embodiment 9 conversion reactions
As proenzyme, in every liter transformation system, add substrate creatine 10g/L with the lysate of the zymophyte somatocyte of the foregoing description 2 preparation or embodiment 3 preparations respectively; Triphosaden 7.5g/L, anhydrous magnesium sulfate 50mmol/L, invert point is 37 ℃; Transform pH value 9, transformation time 3h.When being 300-900U/L, investigation enzyme addition, adopt method two to detect the content of phosphocreatine to the influence of Triphosaden transformation efficiency.The result sees Fig. 3 F.
The investigation of the concentration of anhydrous magnesium sulfate in embodiment 10 conversion reactions
As proenzyme, in every liter transformation system, add enzyme amount 450U/L with the lysate of the zymophyte somatocyte of the foregoing description 2 preparation or embodiment 3 preparations respectively; Substrate creatine consumption is 10g/L, Triphosaden consumption 7.5g/L, and invert point is 37 ℃; Transforming the pH value is 9, transformation time 3h.When being 32.5-95mmol/L, the concentration of investigation anhydrous magnesium sulfate, adopt method two to detect the content of phosphocreatine to the influence of Triphosaden transformation efficiency.The result sees Fig. 3 G.
Visible from the result, be cofactor with anhydrous magnesium sulfate 32.5-95mmol/L, at 30-45 ℃, the pH value is 9-11, enzyme amount 300-900U/L, reaction 0.5-3h can conversion of substrate creatine and Triphosaden production phosphocreatine.Simultaneously, through analyzing experimental result, take actually operating and cost into consideration, preferred conversion condition is: creatine 10g/L, Triphosaden 7.5g/L, anhydrous magnesium sulfate 65mmol/L, enzyme amount 450U/L, 37 ℃ of temperature of reaction, pH value 9, reaction times 3h.Under this optimum condition, carry out confirmatory experiment, the transformation efficiency of Triphosaden is 92% (see figure 4).
Claims (5)
1. a microbial enzyme method transforms the method for creatine and Triphosaden production phosphocreatine; It is characterized in that this method comprises: fermentation thalline or lysate with the gene engineering recombinant bacterium of expressing Tn are the enzyme source, are substrate with creatine and Triphosaden, and the add-on of substrate creatine is 5 to 15g/L; The add-on of Triphosaden is 2.5 to 10g/L; The addition in enzyme source is 300 to 900U/L, under room temperature to 45 ℃, and pH9 to 11; Through enzymatic conversion method reaction 0.5h to 5h, produce phosphocreatine.
2. method according to claim 1; It is characterized in that: the gene engineering recombinant bacterium of described expression Tn is e. coli bl21 (DE3)-pET21a (+)-CPK; Expression vector is pET21a (+)-CPK recombinant expression vector, and expressing in the reorganization somatic cells has highly active Tn.
3. method according to claim 1 is characterized in that also comprising the cofactor anhydrous magnesium sulfate in the enzymatic reaction liquid, and the add-on of anhydrous magnesium sulfate is 32.5 to 95mmol/L.
4. method according to claim 3 is characterized in that the consumption of said anhydrous magnesium sulfate is preferably 65mmol/L.
5. according to each described method in the claim 1 to 4; It is characterized in that: in the enzymatic reaction liquid, the consumption of substrate creatine is preferably 10g/L, and the consumption of substrate Triphosaden is preferably 7.5g/L; The addition in enzyme source is preferably 450U/L; Preferred invert point is 37 ℃, preferably transforms pH9, preferred transformation time 3h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108315367A (en) * | 2018-03-19 | 2018-07-24 | 郑州四维健康管理有限公司 | Method for producing creatine phosphate by two-step enzymolysis method |
| US10822581B2 (en) | 2015-03-13 | 2020-11-03 | Geneharbor (Hong Kong) Biotechnologies Limited | Immobilized reaction device and a method for carrying out a reaction by utilizing the immobilization technology |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1478899A (en) * | 2002-08-27 | 2004-03-03 | 中国科学院生物物理研究所 | Production method of creatine phosphate |
| CN102533880A (en) * | 2010-12-31 | 2012-07-04 | 北京百川飞虹生物科技有限公司 | Bioengineering method for synthesizing sodium phosphocreatine |
-
2012
- 2012-08-23 CN CN2012103018258A patent/CN102808006A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1478899A (en) * | 2002-08-27 | 2004-03-03 | 中国科学院生物物理研究所 | Production method of creatine phosphate |
| CN102533880A (en) * | 2010-12-31 | 2012-07-04 | 北京百川飞虹生物科技有限公司 | Bioengineering method for synthesizing sodium phosphocreatine |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10822581B2 (en) | 2015-03-13 | 2020-11-03 | Geneharbor (Hong Kong) Biotechnologies Limited | Immobilized reaction device and a method for carrying out a reaction by utilizing the immobilization technology |
| 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 |
| 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 |
| 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 |
| CN108315367A (en) * | 2018-03-19 | 2018-07-24 | 郑州四维健康管理有限公司 | Method for producing creatine phosphate by two-step enzymolysis method |
| CN108315367B (en) * | 2018-03-19 | 2021-05-11 | 郑州四维健康管理有限公司 | Method for producing creatine phosphate by two-step enzymolysis method |
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Application publication date: 20121205 |