阅读说明：本技术 葡萄糖氧化酶冻干粉的制备方法 (Preparation method of glucose oxidase freeze-dried powder ) 是由 罗同阳 高庆华 董聪 王玥 王庆庆 马清河 秦艳梅 于 2019-11-13 设计创作，主要内容包括：本发明属于酶制剂的制备,特别是指葡萄糖氧化酶冻干粉的制备方法。按照公开号CN107937360A中的方法制备含有葡萄糖氧化酶的发酵液；将含有葡萄糖氧化酶的发酵液高速离心去除发酵液中的菌体,将离心后上清液用无水乙醇纯化后用磷酸盐缓冲液稀释至所需酶活；加入PEG2000作为冻干保护剂使其充分溶解并在酶液中分布均匀；预冻后置于真空冷冻干燥剂内冷冻干燥得到淡黄色葡萄糖氧化酶冻干粉。本发明解决了目前葡萄糖氧化酶直接进行冷冻干燥导致的酶活回收率低的问题,具有葡萄糖氧化酶冻干粉酶活回收率高、易于保存和运输、为葡萄糖氧化酶应用于生物医药、饲料及食品加工等领域提供了便利等优点。(The invention belongs to preparation of enzyme preparations, and particularly relates to a preparation method of glucose oxidase freeze-dried powder. Preparing a fermentation broth containing glucose oxidase according to the method of publication No. CN 107937360A; centrifuging the fermentation liquor containing glucose oxidase at high speed to remove thalli in the fermentation liquor, purifying the centrifuged supernatant with absolute ethyl alcohol, and diluting with phosphate buffer solution to obtain the required enzyme activity; adding PEG2000 as freeze-drying protective agent to make it fully dissolved and uniformly distributed in enzyme solution; and after pre-freezing, placing the mixture in a vacuum freezing drying agent for freeze-drying to obtain the faint yellow glucose oxidase freeze-dried powder. The invention solves the problem of low enzyme activity recovery rate caused by direct freeze drying of the existing glucose oxidase, and has the advantages of high enzyme activity recovery rate of glucose oxidase freeze-dried powder, easy storage and transportation, convenience for applying the glucose oxidase to the fields of biological medicine, feed, food processing and the like.)

1. The preparation method of the glucose oxidase freeze-dried powder is characterized by comprising the following process steps:

A. preparing a fermentation broth containing glucose oxidase according to the method of publication No. CN 107937360A;

B. b, centrifuging the fermentation liquor containing the glucose oxidase prepared in the step A at a high speed to remove thalli in the fermentation liquor; purifying the centrifuged supernatant with absolute ethyl alcohol, and diluting with phosphate buffer solution until the enzyme activity is required;

C. adding PEG2000 with the volume mass ratio of 2-6% into the pure enzyme solution prepared in the step B as a freeze-drying protective agent, and uniformly mixing to ensure that the protective agent is fully dissolved and uniformly distributed in the enzyme solution;

D. pre-freezing at-20 deg.C for 12-14h, freeze-drying in vacuum freeze-drying machine for 16-20h to obtain yellowish glucose oxidase lyophilized powder, and storing at low temperature.

2. The preparation method of glucose oxidase lyophilized powder of claim 1, wherein the conditions for removing the bacteria in the fermentation broth in step B are as follows: centrifuging at 4 deg.C and 6000-.

3. The preparation method of glucose oxidase lyophilized powder according to claim 1 or 2, characterized in that the process conditions for purifying with absolute ethanol in step B are as follows: adding absolute ethyl alcohol into the supernatant after centrifugation until the final concentration of the ethyl alcohol in the system is 20-25%, standing overnight at 4 ℃, and centrifuging for 20-30 minutes at 6000-; taking the supernatant, adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the system is 60-65%, standing overnight at 4 ℃, centrifuging for 20-30 minutes at 6000-8000 rpm, and diluting the obtained precipitate to the required enzyme activity by using a phosphate buffer solution with the concentration of 0.1-0.15mol/L, pH-7.1-7.5.

Technical Field

The invention belongs to preparation of enzyme preparations, and particularly relates to a preparation method of glucose oxidase freeze-dried powder.

Background

Glucose oxidase has a wide application range, and in the pharmaceutical industry, because glucose oxidase has strict substrate specificity to beta-D-glucose, glucose oxidase is usually prepared into test paper or a kit for detecting blood glucose concentration and diagnosing diabetes. In the feed industry, the enzyme is used as a feed additive, is beneficial to the nutrient absorption of animals, and reduces the diarrhea rate. The glucose oxidase can also be used as food additive for food preservation, and has certain effect on protecting color of fruit wine.

The vacuum freeze-drying technology, which is referred to as freeze-drying technology for short, is widely applied to various fields of food, medicine, biology and the like, and is a drying method for freezing a sample into a solid in advance and then directly sublimating the solid to remove water from the frozen state without passing through a liquid state under the conditions of low temperature and low pressure. The glucose oxidase dry powder preparation has very wide application prospect in the field of biological medicine, but the glucose oxidase preparation products of domestic enterprises have a large gap compared with foreign enterprises at present. The glucose oxidase is used as a protein, is easy to cause inactivation and denaturation when meeting high temperature, is in a low-temperature state in the operation process of a freeze-drying technology, and is more suitable for preparing dry powder preparations of the glucose oxidase. Since lyophilization is a complex phase transition process, there are many factors that induce enzyme denaturation during freezing, freezing and thawing, drying and storage, and thus a protective agent needs to be added during lyophilization. The added freeze-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 the glucose oxidase is ensured. The glucose oxidase is not easy to deteriorate after freeze drying, can be stored for a long time, is easy to rehydrate and regenerate, and is beneficial to maintaining activity. Compared with spray drying, the recovery rate of the glucose oxidase preparation prepared by the vacuum freeze drying method is high, but the related technology is not complete enough, the glucose oxidase is directly subjected to freeze drying, and the enzyme activity recovery rate can only reach about 70%, so that the selection of a proper glucose oxidase freeze-drying protective agent is very important.

In the pharmaceutical industry, the dosage form commonly used for biotech products is solid, so glucose oxidase as a key enzyme is often made into a blood glucose test strip or a diagnostic kit in a solid form for use. In other application industries, the solid glucose oxidase is more convenient to transport and store and use than the liquid glucose oxidase.

The background literature retrieved by the applicant is as follows:

patent publication No. CN1060026A discloses a process for producing glucose oxidase cold-dried powder, which aims to freeze-dry glucose oxidase by a new purification process, and does not mention what kind of protective agent and addition amount are used; patent document No. CN101481680A discloses a method for producing glucose oxidase, which uses penicillium to prepare a glucose oxidase fermentation broth, and the fermentation broth is purified, desalted and lyophilized to prepare a solid preparation, but the patent does not mention what protecting agent and additive amount are used; patent document No. CN109825483A discloses a process for producing high-yield glucose oxidase, in which fermented pichia pastoris is used to produce liquid fermentation broth, and glucose oxidase powder is obtained by spray drying, and no mention is made of using freeze-drying method to prepare glucose oxidase dry powder.

Cloning of glucose oxidase Gene from Penicillium notatum and its research on enzymatic Properties (Biotechnology report 2016, 32(7): 152-. Cloning GOD gene from genome DNA of Penicillium notatum No.8312 strain by PCR technology, cloning the gene to shuttle vector pMD-AOX and expressing in Pichia pastoris X33, and analyzing the enzymatic properties of purified glucose oxidase. The result shows that the X33-GOD can highly express the active GOD, and the enzyme activity of the culture supernatant GOD can reach 496U/mL and the specific activity of 123.0U/mg under the conditions of 30 ℃ and pH 6.5; the optimum temperature of the recombinant expressed glucose oxidase is 40-45 ℃, the optimum pH is 6.0, and the stability research of the enzyme shows that the enzyme is stable in a pH range of 3.5-7.0 and at the temperature lower than 50 ℃. 1mmol/L Zn2+ has activating effect; ag + has a great inhibition effect on the enzyme activity. The high-yield Pichia pastoris engineering strain of GOD is constructed, and has higher fermentation enzyme activity and specific activity compared with Penicillium notatum GOD.

Disclosure of Invention

The invention aims to provide a preparation method of glucose oxidase freeze-dried powder, and the prepared glucose oxidase freeze-dried powder is low in inactivation rate, stable in property and beneficial to preservation.

The overall technical concept of the invention is as follows:

the preparation method of the glucose oxidase freeze-dried powder comprises the following process steps:

A. preparing a fermentation broth containing glucose oxidase according to the method of publication No. CN 107937360A;

B. b, centrifuging the fermentation liquor containing the glucose oxidase prepared in the step A at a high speed to remove thalli in the fermentation liquor; purifying the centrifuged supernatant with absolute ethyl alcohol, and diluting with phosphate buffer solution until the enzyme activity is required;

C. adding PEG2000 with the volume mass ratio of 2-6% into the pure enzyme solution prepared in the step B as a freeze-drying protective agent, and uniformly mixing to ensure that the protective agent is fully dissolved and uniformly distributed in the enzyme solution;

D. pre-freezing at-20 deg.C for 12-14h, freeze-drying in vacuum freeze-drying machine for 16-20h to obtain yellowish glucose oxidase lyophilized powder, and storing at low temperature.

The specific technical concept of the invention is as follows:

the conditions for removing the thalli in the fermentation liquor in the step B are as follows: centrifuging at 4 deg.C and 6000-.

The process conditions for purifying by adopting absolute ethyl alcohol in the step B are as follows: adding absolute ethyl alcohol into the supernatant after centrifugation until the final concentration of the ethyl alcohol in the system is 20-25%, standing overnight at 4 ℃, and centrifuging for 20-30 minutes at 6000-; taking the supernatant, adding absolute ethyl alcohol until the final concentration of the ethyl alcohol in the system is 60-65%, standing overnight at 4 ℃, centrifuging for 20-30 minutes at 6000-8000 rpm, and diluting the obtained precipitate to the required enzyme activity by using a phosphate buffer solution with the concentration of 0.1-0.15mol/L, pH-7.1-7.5.

At present, the domestic detection method for the enzyme activity of the glucose oxidase is not described in the national standard and the technical specification of the industry, and in order to verify the enzyme activity of the hexokinase freeze-dried powder prepared by the invention, the determination method in the invention is according to the published documents: cloning of glucose oxidase gene from Penicillium notatum and its enzymological characterization, Gaoqing, Humeirong, Wu aryltong, etc. [ J ] Biotechnology report, 2016, 32(7): 152-.

The specific determination method comprises the following steps: in a 3mL reaction system containing 0.5mol/L acetate buffer (pH5.1), 0.17mmol/L o-dianisidine solution, 1.72% glucose solution and 0.1mg/mL horseradish peroxidase, were mixed by shaking at 35 ℃ and 0.1mL glucose oxidase solution was added, and the absorbance value Δ A500 was recorded using a spectrophotometer at A ═ 500nm for 1 minute.

Enzyme activity calculation formula

X＝(ΔA500×3.1×Df)÷7.5÷0.1

In the formula

X: sample enzyme activity (U/mL);

3.1: a reaction volume;

df: dilution times;

7.5: extinction coefficient of gluconolactone at 500 nm;

0.1: enzyme solution volume was added.

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

after the pichia pastoris gene engineering bacteria produce glucose oxidase and are freeze-dried without adding any protective agent, the recovery rate of enzyme activity is about 70 percent. The invention screens the freeze-drying protective agent to finally obtain the glucose oxidase freeze-drying protective agent, and the recovery rate of enzyme activity can reach more than 90 percent after the freeze-drying by adopting the method of the invention. The glucose oxidase freeze-dried powder prepared by the invention has high enzyme activity recovery rate, is easy to store and transport, and provides convenience for the application of the glucose oxidase in the fields of biological medicine, feed, food processing and the like.

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.