CN108048441A - A kind of preparation method of hexokinase freeze-dried powder - Google Patents
A kind of preparation method of hexokinase freeze-dried powder Download PDFInfo
- Publication number
- CN108048441A CN108048441A CN201711301261.7A CN201711301261A CN108048441A CN 108048441 A CN108048441 A CN 108048441A CN 201711301261 A CN201711301261 A CN 201711301261A CN 108048441 A CN108048441 A CN 108048441A
- Authority
- CN
- China
- Prior art keywords
- hexokinase
- freeze
- dried powder
- enzyme solution
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01001—Hexokinase (2.7.1.1)
Abstract
The invention belongs to the preparations of enzyme preparation, particularly relate to a kind of preparation method of hexokinase freeze-dried powder.The pure enzyme solution of hexokinase is prepared according to the method in Publication No. CN106479992A；Sucrose, lactose, sorbierite are added in the pure enzyme solution of hexokinase as freeze drying protectant, protective agent is made fully to dissolve and is evenly distributed in enzyme solution；Pre-freeze is placed on freeze-drying in vacuum freeze drying agent and obtains white hexokinase freeze-dried powder.The present invention solve the problems, such as current hexokinase be directly freeze-dried caused by inactivation rate it is high, have many advantages, such as high, the opposite liquid hexokinase enzymatic property of hexokinase freeze-dried powder enzyme activity storage rate relatively stablize, meet in national standard for kit requirement and be more conducive to preserve.
Description
Technical field
The invention belongs to the preparations of enzyme preparation, particularly relate to a kind of preparation method of hexokinase freeze-dried powder.
Background technology
The application that serum glucose (GLU) detects in the diagnosis of diabetes, observation of curative effect and general clinical diagnosis is non-
Often extensively.Hexokinase method acts on that alpha-D-glucose is very capable, and Km value very littles are affine to substrate because its disturbing factor is few
Power is very strong, and it is short to be made into the finished product kit measurement time, is particularly suitable for fully-automatic analyzer, makes especially suitable for emergency treatment inspection
With, be identified measure serum glucose reference method.
For hexokinase, it is necessary to preserve for a long time during production and use, liquid condition lower 4 DEG C of holding times are short, and -20
DEG C freezing the adverse consequences such as can cause to assemble because of multigelation, modify, degrade, polluting.Hexokinase freeze-drying is by sample
It freezes at low temperature, then under vacuum, removes moisture removal using distillation so that the unit that sample is in drying regime is grasped
Make.The freeze drying protectant of addition can substitute water and protein binding, and the structure of albumen is made to exempt from destruction, ensure that hexokinase
Stability.Hexokinase freeze-dried powder has very extensive application prospect, but domestic hexokinase at present in biomedicine field
Vacuum Freezing ＆ Drying Technology it is still incomplete, hexokinase is directly freeze-dried, and inactivation rate is selected up to more than 99%
It is most important to select suitable hexokinase freeze drying protectant.
Hexokinase determines the quality of kit quality as the key enzyme in diagnostic kit preparation, enzymatic property,
The particularly stability of hexokinase directly affects the stability of diagnostic kit.In clinical chemistry vitro detection reagent (box)
Examination criteria in (GB/T 26124-2011) provide that stability is to examine the qualified or not important indicator of diagnostic kit, because
This, it is the key that prepare the diagnostic kit having good stability to prepare the high hexokinase of stability.
The domestic and international patent document situation of applicant's retrieval is as follows：
Applicant had not inquired the pertinent literature report on the preparation method of hexokinase freeze-dried powder.Publication No.
A kind of enzyme process creatine kinase checkout and diagnosis kit of stabilization is disclosed in the patent document of CN106932353A, be by R1 and
Two kinds of reagents of R2 complete the preparation of diagnostic kit, and wherein hexokinase is the component of reagent R1, but in the document not
It is related to the preparation of hexokinase freeze-dried powder.Disclosed in the patent document of Publication No. CN106868096A a kind of stability it is strong and
Hexokinase method glucose determination reagent at low cost, using hexokinase as primary raw material prepared a kind of stabilization low cost
Liquid hexokinase method detects serum glucose detection reagent, but the preparation of hexokinase freeze-dried powder is also not directed in the document.
Domestic relatively fewer to the research of hexokinase at present, applicant is disclosed in the patent document of Publication No. CN106479992A
The preparation method of diagnosis hexokinase is to have prepared liquid hexokinase using microbial strains production, but is not directed to
The preparation of hexokinase dry powder.
The content of the invention
One of the objects of the present invention is to provide a kind of preparation method of hexokinase freeze-dried powder, prepared hexokinases
Freeze-dried powder inactivation rate is low and property is stablized, beneficial to preservation.
The overall technology of the present invention is conceived：
A kind of preparation method of hexokinase freeze-dried powder, is made of following processing step：
A, the pure enzyme of hexokinase that enzyme activity is 200-300U/ml is prepared according to the method in Publication No. CN106479992A
Liquid；
B, 3%-5% (m/v) sucrose, 1%-2% (m/v) breasts are added in the pure enzyme solution of hexokinase prepared in step
Sugar, 6%-8% (m/v) sorbierites are placed in 4 DEG C of preservation 4-5h, protective agent are made fully to dissolve and in enzyme solution as freeze drying protectant
In be evenly distributed；
C, the pre-freeze 12-14h under conditions of pre-freezing temperature is -20 DEG C, pre-freeze are placed on freezing in vacuum freeze drying agent
Dry 16-20h, obtains white hexokinase freeze-dried powder, in -20 DEG C of preservations.
The country has no explanation for the detection method of hexokinase enzyme activity in national standard and industry specifications at present, to test
The enzyme activity of the hexokinase freeze-dried powder prepared by the present invention is demonstrate,proved, applicant carries out enzyme with reference to the assay method of Sigma hexokinases
Detection living, is as follows：
Assay method：The TEA-HCL of addition 1ml0.05mol/L respectively, the D-Glucose of 1ml0.555mol/L,
The MgCl of the ATP of 0.1ml0.019mol/L, 0.2ml0.1mol/L2, the NADP+ of 0.2ml0.014mol/L, 0.02ml125U/
The glucose-6-phosphate dehydrogenase (G6PD) of mL is to developmental tube and blank tube.Mixing reads at 340nm and inhales after 25 DEG C of water-bath 1min
Light value A1.Then developmental tube adds in 0.05mL enzyme solutions to be measured, and blank tube adds in 0.05mL ddH2O, in 25 DEG C of the reaction was continued 5min,
Light absorption value A2 is read afterwards.
Enzyme activity calculation formula (U/mL)=6.22 ÷ 0.05 of (Δ A developmental tubes-Δ A blank tubes) × 2.57 × Df ÷
In formula
2.57：Reaction solution total volume；
Df：Extension rate；
6.22：Absorptivitys of the β-NADPH at 340nm；
0.05：Cuvette volume.
Substantive distinguishing features and significant technological progress acquired by the present invention are：
The present invention is logical to screen freeze drying protectant, optimum organization, has finally obtained primary yeast production hexokinase and has frozen
Dry powder, the hexokinase freeze-dried powder enzyme activity storage rate prepared by method using the present invention reach as high as 85%, prepared oneself
Sugared kinases freeze-dried powder is relatively stablized with respect to liquid hexokinase enzymatic property, meets for the requirement of kit in national standard, more sharp
In preservation.Solve the problems, such as that hexokinase enzyme activity loss in storage and transportational process is serious.For hexokinase application
Great convenience condition is provided in biomedicine field.
Specific embodiment
The invention will be further described with reference to embodiments, but not as a limitation of the invention.The guarantor of the present invention
Shield scope is subject to the content of claim record, and any equivalent technical elements made according to specification are replaced, all without departing from
The protection category of the present invention.
Embodiment 1
A kind of preparation method of hexokinase freeze-dried powder, is made of following processing step：
A, the pure enzyme solution of hexokinase that enzyme activity is 200U/ml is prepared according to the method in Publication No. CN106479992A；
B, 3% (m/v) sucrose, 1% (m/v) lactose, 6% (m/ are added in the pure enzyme solution of hexokinase prepared in step
V) sorbierite is placed in 4 DEG C of preservation 4-5h, protective agent is made fully to dissolve and is evenly distributed in enzyme solution as freeze drying protectant；
C, the pre-freeze 12-14h under conditions of pre-freezing temperature is -20 DEG C, pre-freeze are placed on freezing in vacuum freeze drying agent
Dry 16-20h, obtains white hexokinase freeze-dried powder, in -20 DEG C of preservations.
Embodiment 2
The present embodiment and embodiment 1 difference lies in：
A kind of preparation method of hexokinase freeze-dried powder, is made of following processing step：
A, the pure enzyme solution of hexokinase that enzyme activity is 300U/ml is prepared according to the method in Publication No. CN106479992A；
B, 5% (m/v) sucrose, 2% (m/v) lactose, 8% (m/ are added in the pure enzyme solution of hexokinase prepared in step
V) sorbierite is placed in 4 DEG C of preservation 4-5h, protective agent is made fully to dissolve and is evenly distributed in enzyme solution as freeze drying protectant；
Remaining content is the same as embodiment 1.
Embodiment 3
The present embodiment and embodiment 1 difference lies in：
A kind of preparation method of hexokinase freeze-dried powder, is made of following processing step：
A, the pure enzyme solution of hexokinase that enzyme activity is 250U/ml is prepared according to the method in Publication No. CN106479992A；
B, 4% (m/v) sucrose, 1.5% (m/v) lactose, 7% are added in the pure enzyme solution of hexokinase prepared in step
(m/v) sorbierite is placed in 4 DEG C of preservation 4-5h, protective agent is made fully to dissolve and is evenly distributed in enzyme solution as freeze drying protectant.
Remaining content is the same as embodiment 1.
Embodiment 4
The present embodiment and embodiment 1 difference lies in：
A kind of preparation method of hexokinase freeze-dried powder, is made of following processing step：
A, the pure enzyme solution of hexokinase that enzyme activity is 220U/ml is prepared according to the method in Publication No. CN106479992A；
B, in the pure enzyme solution of hexokinase prepared in step add in 3.5% (m/v) sucrose, 1.2% (m/v) lactose,
6.5% (m/v) sorbierite is placed in 4 DEG C of preservation 4-5h, protective agent is made fully to dissolve and is distributed in enzyme solution as freeze drying protectant
Uniformly.
Remaining content is the same as embodiment 1.
Embodiment 5
The present embodiment and embodiment 1 difference lies in：
A kind of preparation method of hexokinase freeze-dried powder, is made of following processing step：
A, the pure enzyme solution of hexokinase that enzyme activity is 280U/ml is prepared according to the method in Publication No. CN106479992A；
B, in the pure enzyme solution of hexokinase prepared in step add in 3.8% (m/v) sucrose, 1.8% (m/v) lactose,
6.8% (m/v) sorbierite is placed in 4 DEG C of preservation 4-5h, protective agent is made fully to dissolve and is distributed in enzyme solution as freeze drying protectant
Uniformly.
Remaining content is the same as embodiment 1.
Applicant using Sigma hexokinases assay method to the hexokinase freeze-dried powder that is prepared in embodiment 1-5 into
The detection of row enzyme activity, result are as follows：
| Enzyme activity (U/ml) | Inactivation rate (%) | |
| Hexokinase freeze-dried powder prepared by embodiment 1 | 162 | 19 |
| Hexokinase freeze-dried powder prepared by embodiment 2 | 246 | 18 |
| Hexokinase freeze-dried powder prepared by embodiment 3 | 213 | 15 |
| Hexokinase freeze-dried powder prepared by embodiment 4 | 183 | 17 |
| Hexokinase freeze-dried powder prepared by embodiment 5 | 235 | 16 |
Applicant it should be noted that：
Sigma official websites have hexokinase solid-state enzyme on sale at present, and product article No. H4502, unit enzyme activity is than the present invention
In obtained hexokinase freeze-dried powder it is high, applicant thinks main reason is that ferment bacterial strain used and the skill that handles enzyme solution
Art means difference is related.Applicant utilizes production hexokinase yeast strain in the patent document of Publication No. CN106479992A
(production hexokinase bacterium source：The saccharomyces cerevisiae of Angel Yeast Co., Ltd's production, product standard code are GB/
T20886) fermented purification obtains the pure enzyme solution of hexokinase, carries out freeze-drying and prepares hexokinase freeze-dried powder, compared to directly right
The pure enzyme solution of hexokinase is freeze-dried, and hexokinase freeze-dried powder enzyme activity inactivation rate is reduced to 15%-19% by 99%.
Claims (1)
1. a kind of preparation method of hexokinase freeze-dried powder, it is characterised in that be made of following processing step：
A, the pure enzyme solution of hexokinase that enzyme activity is 200-300U/ml is prepared according to the method in Publication No. CN106479992A；
B, in the pure enzyme solution of hexokinase prepared in step add in 3%-5% (m/v) sucrose, 1%-2% (m/v) lactose,
6%-8% (m/v) sorbierites are placed in 4 DEG C of preservation 4-5h, protective agent are made fully to dissolve and divide in enzyme solution as freeze drying protectant
Cloth is uniform；
C, the pre-freeze 12-14h under conditions of pre-freezing temperature is -20 DEG C, pre-freeze are placed on freeze-drying in vacuum freeze drying agent
16-20h obtains white hexokinase freeze-dried powder, in -20 DEG C of preservations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711301261.7A CN108048441B (en) | 2017-12-10 | 2017-12-10 | Preparation method of hexokinase freeze-dried powder |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711301261.7A CN108048441B (en) | 2017-12-10 | 2017-12-10 | Preparation method of hexokinase freeze-dried powder |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108048441A true CN108048441A (en) | 2018-05-18 |
| CN108048441B CN108048441B (en) | 2021-03-16 |
Family
ID=62123272
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711301261.7A Active CN108048441B (en) | 2017-12-10 | 2017-12-10 | Preparation method of hexokinase freeze-dried powder |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108048441B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669752A (en) * | 2019-11-12 | 2020-01-10 | 河北省微生物研究所 | Preparation method of hexokinase spray-dried powder |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5599190A (en) * | 1979-01-25 | 1980-07-28 | Mitsubishi Petrochem Co Ltd | Preparation of heat-resistant hexokinase |
| CN101129361A (en) * | 2007-07-17 | 2008-02-27 | 山东华诺生物科技有限公司 | Sirolimus lipidosome freeze-dried acanthopanax powder and technique of preparing the same |
| CN101214263A (en) * | 2007-12-26 | 2008-07-09 | 浙江大学宁波理工学院 | Method for preparing freeze-dried powder containing bacillus natto and nattokinase |
| CN101361717A (en) * | 2007-08-08 | 2009-02-11 | 北京赛生药业有限公司 | Batroxobin freeze-dry powder injection and preparation method thereof |
| JP4245229B2 (en) * | 1998-06-23 | 2009-03-25 | 旭化成ファーマ株式会社 | Stable hexokinase and process for producing the same |
| CN104198717A (en) * | 2014-08-30 | 2014-12-10 | 中国科学院苏州生物医学工程技术研究所 | Freeze-drying concentrated glucose detection reagent microsphere and preparation method thereof |
| CN106479992A (en) * | 2016-11-24 | 2017-03-08 | 河北省微生物研究所 | The preparation method of diagnosis hexokinase |
-
2017
- 2017-12-10 CN CN201711301261.7A patent/CN108048441B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5599190A (en) * | 1979-01-25 | 1980-07-28 | Mitsubishi Petrochem Co Ltd | Preparation of heat-resistant hexokinase |
| JP4245229B2 (en) * | 1998-06-23 | 2009-03-25 | 旭化成ファーマ株式会社 | Stable hexokinase and process for producing the same |
| CN101129361A (en) * | 2007-07-17 | 2008-02-27 | 山东华诺生物科技有限公司 | Sirolimus lipidosome freeze-dried acanthopanax powder and technique of preparing the same |
| CN101361717A (en) * | 2007-08-08 | 2009-02-11 | 北京赛生药业有限公司 | Batroxobin freeze-dry powder injection and preparation method thereof |
| CN101214263A (en) * | 2007-12-26 | 2008-07-09 | 浙江大学宁波理工学院 | Method for preparing freeze-dried powder containing bacillus natto and nattokinase |
| CN104198717A (en) * | 2014-08-30 | 2014-12-10 | 中国科学院苏州生物医学工程技术研究所 | Freeze-drying concentrated glucose detection reagent microsphere and preparation method thereof |
| CN106479992A (en) * | 2016-11-24 | 2017-03-08 | 河北省微生物研究所 | The preparation method of diagnosis hexokinase |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110669752A (en) * | 2019-11-12 | 2020-01-10 | 河北省微生物研究所 | Preparation method of hexokinase spray-dried powder |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108048441B (en) | 2021-03-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kawase et al. | Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S. coelicolor A3 (2) | |
| Pfaller et al. | Standardized susceptibility testing of fluconazole: an international collaborative study | |
| Hendrix et al. | Artifacts in the Analysis of Plant Tissues for Soluble Carbohydrates 1 | |
| CN103695380B (en) | Fructosyl amino acid oxidase, preparation method and the glycosylated albumin detection kit containing this enzyme | |
| Palmer et al. | Respiration and ripening of banana fruit slices | |
| WO2022068615A1 (en) | Formulation for improving activity and/or thermostability of superoxide dismutases and use thereof | |
| JP6230796B2 (en) | Freeze-dried bacteria sample and production method thereof | |
| CN108048441A (en) | A kind of preparation method of hexokinase freeze-dried powder | |
| Lineweaver | The energy of activation of enzyme reactions, and their velocity below 0° | |
| CN104483314A (en) | Method for improvingthermal stability of test strips for detecting peroxide value of cooking oil | |
| CN108130350A (en) | The preparation method of high-content trehalose | |
| CN102676474B (en) | Method for extracting pear sucrose invertin and method for measuring activity thereof | |
| CN114250196A (en) | Exosome amplification and storage method | |
| Harris et al. | Succinic and lactic dehydrogenase activity in myocardial homogenates from animals at high and low altitude | |
| CN112075629B (en) | Method for improving antioxidant activity of Withania somnifera extract by probiotic fermentation | |
| CN114945667A (en) | Nucleic acid and cell preservative compositions and methods of use | |
| CN102564979A (en) | Method for determining alcohol concentration by using enzyme cycling method and alcohol determination kit | |
| CN106479992A (en) | The preparation method of diagnosis hexokinase | |
| EP2961840B1 (en) | Preparation of stabilized catalase enzymes using polyvinyl alcohol | |
| Hall et al. | Metabolic changes in washed, isolated steles | |
| Sandegren et al. | CELL WALL DECOMPOSING ENZYMES OF BARLEY AND MALT I.—DETERMINATION AND STABILITY INVESTIGATIONS | |
| CN113528499A (en) | Buffer system capable of stably storing Hexokinase (HK) | |
| CN105400862A (en) | Glucose detection kit | |
| CN107583699A (en) | A kind of heparin tube examined for clinical medicine and preparation method thereof | |
| ES2650273T3 (en) | Method for alcoholic fermentation of honey must, with high sugar content |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: No. 2089, Wusi Middle Road, Baoding City, Hebei Province 071051 Patentee after: Hebei Institute of Microbiology Co.,Ltd. Address before: No. 2089, Wusi Middle Road, Jingxiu District, Baoding City, Hebei Province 071051 Patentee before: HEBEI Research Institute OF MICROBIOLOGY |