阅读说明：本技术 己糖激酶喷干粉的制备方法 (Preparation method of hexokinase spray-dried powder ) 是由 刘春卯 曹倩荣 郑翔 秦艳梅 马清河 于 2019-11-12 设计创作，主要内容包括：本发明属于酶制剂的制备,特别是指一种己糖激酶喷干粉的制备方法。按照公开号为CN106479992A中的方法制备己糖激酶纯酶液；在己糖激酶纯酶液中加入山梨醇、硫酸钠作为喷干保护剂,置于4℃保存3h,使保护剂充分溶解并在酶液中分布均匀；以进风口80℃、出风口65℃、进样速度为12转/分钟的条件对加有保护剂的酶液喷雾干燥得到白色己糖激酶喷干粉。本发明解决了目前己糖激酶直接进行喷雾干燥导致的失活率高的问题,具有己糖激酶喷干粉酶活保存率高、相对液态己糖激酶酶性质较稳定、符合国标中对于试剂盒的使用要求且更利于保存等优点。(The invention belongs to preparation of enzyme preparations, and particularly relates to a preparation method of hexokinase spray-dried powder. Preparing a hexokinase pure enzyme solution according to the method disclosed in the publication number CN 106479992A; adding sorbitol and sodium sulfate into pure hexokinase enzyme solution as spray-drying protective agent, and storing at 4 deg.C for 3 hr to make the protective agent fully dissolved and uniformly distributed in the enzyme solution; and carrying out spray drying on the enzyme solution added with the protective agent under the conditions that an air inlet is 80 ℃, an air outlet is 65 ℃ and the sample injection speed is 12 r/min to obtain white hexokinase spray dry powder. The invention solves the problem of high inactivation rate caused by directly carrying out spray drying on the hexokinase at present, and has the advantages of high enzyme activity preservation rate of the hexokinase spray-dried powder, relatively stable property of the liquid hexokinase, accordance with the use requirements of the kit in the national standard, more favorable preservation and the like.)

1. The preparation method of the hexokinase spray dry powder is characterized by comprising the following process steps:

A. preparing hexokinase pure enzyme solution with the enzyme activity of 200-300U/ml according to the method disclosed in the publication number CN 106479992;

B. adding 2-5% (m/v) sorbitol and 1-2% (m/v) sodium sulfate as spray-drying protective agents into the pure hexokinase enzyme solution prepared in the step A, and storing at 4 ℃ for 1-3h to ensure that the protective agents are fully dissolved and uniformly distributed in the enzyme solution;

C. and C, spray-drying the material prepared in the step B under the conditions that the air inlet is 70-80 ℃, the air outlet is 55-65 ℃ and the sample injection speed is 12 r/min to obtain white hexokinase spray-dried powder, and storing at low temperature.

Technical Field

The invention belongs to preparation of enzyme preparations, and particularly relates to a preparation method of hexokinase spray-dried powder.

Background

Hexokinase is the first enzyme of biological glycolytic metabolic pathway, catalyzes glucose phosphorylation to generate glucose-6-phosphate, and the glucose-6-phosphate continuously participates in reaction to complete sugar metabolism. The hexokinase method blood sugar detection kit prepared according to the principle has the advantages of strong specificity and high accuracy, is suitable for a full-automatic analyzer, is particularly suitable for emergency examination, and is also an internationally recommended glucose determination method for determining the blood sugar content by using the hexokinase method.

During the production and use processes of hexokinase, the hexokinase needs to be stored for a long time, the storage time at 4 ℃ in a liquid state is short, and the adverse effects of aggregation, modification, degradation, pollution and the like caused by repeated freeze thawing can be realized during freezing and storage at-20 ℃. Spray drying is to spray enzyme solution into droplets with a diameter of only tens of micrometers by a spray device, disperse the droplets in hot air flow, and evaporate water rapidly to obtain a powdery dried enzyme preparation. The added spray-drying protective agent can replace water to be combined with protein, so that the structure of the protein is prevented from being damaged, and the stability of hexokinase is ensured. The dry powder of hexokinase has very wide application prospect in the field of biomedicine, but the spray drying technology of hexokinase in China at present is not complete enough, and the inactivation rate reaches more than 99 percent when the hexokinase is directly spray dried, so that the selection of a proper hexokinase spray drying protective agent is very important.

In the preparation of the hexokinase method blood sugar kit, solid hexokinase is used more, and the solid hexokinase has more stable property than liquid hexokinase and is also beneficial to storage. Hexokinase is used as a key enzyme in the preparation of a diagnostic kit, the enzyme property of the hexokinase determines the quality of the kit, particularly the stability of the hexokinase, and the stability of the diagnostic kit is directly influenced. In the detection standard of the clinical chemistry in-vitro detection reagent (kit) (GB/T26124-2011), the stability is an important index for detecting whether the diagnostic kit is qualified or not, so that the preparation of the liquid hexokinase into the solid hexokinase can be better applied to the preparation of the kit.

Patent documents retrieved by the applicant include:

the related patents at home and abroad are consulted, and the related patents about the preparation method of the hexokinase spray dry powder are not inquired. Patent document CN106932353A discloses a stable creatine kinase assay diagnostic kit, which is prepared by two reagents, R1 and R2, wherein hexokinase is a component of reagent R1, but the patent document does not mention the preparation of hexokinase spray-dried powder; patent document CN110038523A discloses a method for preparing an affinity chromatography medium for isolating and purifying hexokinase, and does not mention the preparation of a hexokinase spray-dried powder. Currently, research on hexokinase in China is relatively rare, and a method for preparing hexokinase for diagnosis is disclosed in patent document No. CN106479992A, which is a method for preparing liquid hexokinase by using a microorganism strain, but does not relate to the preparation of hexokinase dry powder. Patent document CN108048441A discloses a method for preparing a hexokinase lyophilized powder, and the patent document is to prepare the hexokinase lyophilized powder by using a lyophilization method.

Disclosure of Invention

The invention aims to provide a preparation method of hexokinase spray-dried powder, and compared with the product prepared by the existing method, the prepared hexokinase spray-dried powder has low inactivation rate and stable property.

The overall technical concept of the invention is as follows:

the preparation method of the hexokinase spray-dried powder comprises the following process steps:

A. preparing hexokinase pure enzyme solution with the enzyme activity of 200-300U/ml according to the method disclosed in the publication number CN 106479992;

B. adding 2-5% (m/v) sorbitol and 1-2% (m/v) sodium sulfate as spray-drying protective agents into the pure hexokinase enzyme solution prepared in the step A, and storing at 4 ℃ for 1-3h to ensure that the protective agents are fully dissolved and uniformly distributed in the enzyme solution;

C. and C, spray-drying the material prepared in the step B under the conditions that the air inlet is 70-80 ℃, the air outlet is 55-65 ℃ and the sample injection speed is 12 r/min to obtain white hexokinase spray-dried powder, and storing at low temperature.

At present, the domestic detection method for the enzymatic activity of hexokinase is not described in national standards and industrial technical specifications, and in order to verify the enzymatic activity of the hexokinase spray dry powder prepared by the invention, the applicant refers to a Sigma hexokinase determination method for detecting the enzymatic activity, and the specific steps are as follows:

the determination method comprises the following steps:0.05mol/L of TEA-HCL1ml, 0.555mol/L of D-glucose 1ml, 0.019mol/L of ATP0.1ml, and 0.1mol/L of MgCl were added₂0.2mL, 0.014mol/L NADP +0.2mL, 125U/mL glucose-6-phosphate dehydrogenase 0.02mL to test tubes and blank tubes. Mixing, placing in 25 deg.C water bath for 1min, and reading light absorption value A at 340nm₁. Then 0.05mL of enzyme solution to be tested is added into the test tube, and ddH is added into the blank tube₂O0.05mL, continue the reaction at 25 ℃ for 5min, and read the absorbance A2.

Enzyme activity calculation formula (U/mL) ═ delta A test tube-delta A blank tube (2.57 XDf ÷ 6.22 ÷ 0.05 ÷

In the formula

2.57: the total volume of the reaction solution;

df: dilution times;

6.22: absorbance coefficient of β -NADPH at 340 nm;

0.05: cuvette volume.

The invention achieves the substantive characteristics and obvious technical progress that:

the spray-drying protective agent for producing hexokinase by yeast is obtained finally by screening and optimally combining the spray-drying protective agent, and the preservation rate of the enzyme activity after spray-drying can reach 50 percent (the enzyme activity loss rate after spray-drying treatment without adding the protective agent is up to 99 percent). The hexokinase spray-dried powder prepared by the method meets the use requirements of the kit, and improves the stability of the diagnostic kit.

Detailed Description

The present invention is further described with reference to the following examples, but the present invention is not limited thereto. The protection scope of the present invention is subject to the content of the claims, and any equivalent technical means made according to the specification can be substituted without departing from the protection scope of the present invention.