阅读说明：本技术 一种亲和层析法制备高纯度糜蛋白酶的工艺 (A kind of technique that affinity chromatography prepares high purity chymotrypsin ) 是由 叶昀 林克 范伟伟 于 2019-09-25 设计创作，主要内容包括：本发明公开了一种亲和层析法制备高纯度糜蛋白酶的工艺,其主要步骤包括：(1)取糜蛋白酶原,用去离子水溶解并调节pH值,加入高比活性的胰蛋白酶,低温酶解后的溶液离心得到酶解液；(2)制备低分子尿蛋白酶抑制剂亲和层析介质,然后装柱,并用0.1M Tris-Hcl,pH8.0缓冲液进行平衡,平衡后用酶解液上样；上样完毕后用平衡后的缓冲液冲柱,冲至流出液的OD<Sub>280</Sub>值小于0.1,然后换用0.1mol/L甲酸-0.05mol/L KCl,pH2.2的缓冲液洗脱,收集洗脱峰；(3)超滤浓缩,然后过滤除菌；(4)除菌后的酶液装冻干盘,真空冷冻干燥,得到成品。本发明开发出了新的亲和层析法,可以一步从糜蛋白酶原粗品纯化糜蛋白酶,操作方便,专一性更好,糜蛋白酶效价更高。(The invention discloses the techniques that a kind of affinity chromatography prepares high purity chymotrypsin, its key step includes: that (1) takes chymotrypsinogen, with deionized water dissolving and pH value is adjusted, the trypsase of high specific acitivity is added, the solution after low temperature enzymatic hydrolysis is centrifuged to obtain enzymolysis liquid；(2) low molecule Ulinastatin affinity chromatography medium is prepared, then fills column, and with 0.1M Tris-Hcl, pH8.0 buffer is balanced, and enzymolysis liquid loading is used after balance；Column is rushed with the buffer after balance after loading, is rushed to the OD of efflux 280 Then value uses the buffer elution of 0.1mol/L formic acid -0.05mol/L KCl, pH2.2 instead, collects eluting peak less than 0.1；(3) it is concentrated by ultrafiltration, then filtration sterilization；(4) enzyme solution after degerming fills lyophilized plate, and vacuum freeze drying obtains finished product.The present invention has developed new affinity chromatography, can be with a step from chymotrypsinogen purifying crude chymotrypsin, and easy to operate, specificity is more preferable, and chymotrypsin potency is higher.)

1. the technique that a kind of affinity chromatography prepares high purity chymotrypsin, which comprises the following steps:

(1) chymotrypsinogen is taken, with deionized water dissolving and pH value is adjusted to 7.5-8.5, the trypsase of high specific acitivity is added, 24-48h is digested in 2-8 DEG C of environment, the solution after enzymatic hydrolysis is centrifuged to obtain enzymolysis liquid；

(2) low molecule Ulinastatin affinity chromatography medium is prepared, then fills column, and with 0.1M Tris-Hcl, pH8.0 Buffer is balanced, and enzymolysis liquid loading, coutroi velocity 30-120cm/h are used after balance；With slow after balance after loading Fliud flushing rushes column, rushes to the OD of efflux₂₈₀Then value uses 0.1mol/L formic acid -0.05mol/L KCl instead less than 0.1, pH2.2's Buffer elution, flow velocity 50-120cm/h collect eluting peak；

(3) eluting peak collected is concentrated by ultrafiltration with ultrafiltration membrane system, then passes through film filtration sterilization；

(4) enzyme solution after degerming fills lyophilized plate, using Vacuum Freezing & Drying Technology that enzyme solution directly freezed is dry, obtains finished product.

2. the technique that affinity chromatography as claimed in claim 1 or 2 prepares high purity chymotrypsin, which is characterized in that step (1) in, chymotrypsin reason fresh food frozen ox pancreas or pig pancreas, which crushed, extracts albumen, salt fractionation, crystallization and filtration obtains.

3. the technique that affinity chromatography as claimed in claim 1 or 2 prepares high purity chymotrypsin, which is characterized in that step (1) trypsase in is derived from bovine trypsin or Porcine trypsin, and the potency of trypsase is greater than 3000u/mg.

4. the technique that affinity chromatography as claimed in claim 1 or 2 prepares high purity chymotrypsin, which is characterized in that step (1) weight ratio of chymotrypsinogen and deionized water is 1:8-13 in.

5. the technique that affinity chromatography as described in claim 1 or 4 prepares high purity chymotrypsin, which is characterized in that step (1) weight for the trypsase being added in is the 0.004-0.01% of chymotrypsinogen.

6. the technique that affinity chromatography as described in claim 1 prepares high purity chymotrypsin, which is characterized in that step (2) Middle affinity chromatography medium the preparation method comprises the following steps: using GE Health Sepharose 4B gel, under alkaline condition with low point Sub- Ulinastatin obtains after reacting 24 hours.

7. the technique that affinity chromatography as described in claim 1 prepares high purity chymotrypsin, which is characterized in that step (3) It is middle that the ultrafiltration membrane system for using combined closure system 10000 is concentrated by ultrafiltration.

8. the technique that affinity chromatography as described in claim 1 prepares high purity chymotrypsin, which is characterized in that step (3) Middle concentrate passes through 0.25-0.35 μm of membrane filtration degerming.

9. the technique that affinity chromatography as described in claim 1 prepares high purity chymotrypsin, which is characterized in that the enzyme of finished product Vigor is higher than 1750u/mg.

Technical field

The present invention relates to bio-chemistry separations and technical field of purification, prepare more particularly to the affinity chromatography of medicinal trypsase Method more particularly to a kind of technique that new affinity chromatography prepares high purity chymotrypsin.

Background technique

Chymotrypsin be a kind of protease for being isolated and purified from ox pancreas or pig pancreas it in pharmacology and clinically Function mainly include two aspects that one, anti-inflammatory: have good therapeutic effect to various inflammation, inflammatory edema, hemotoncus, ulcer and thrombus etc.. Two, anticancer: can promote anticancer drug to permeate to lesion, chemotherapeutics is promoted to play a role, and have been reported that on anticancer synergia effectively Rate is more than 50%, and when local bolus use acts on stronger.Large dosage of use can treat kinds of tumors, as breast cancer, cervical carcinoma, Gastric cancer etc., and without any side effects or adverse reaction.

Chinese patent CN200610117613.9 provides the high purity chymotrypsin of a whole set of process stabilizing, quality assurance Preparation method.The production technology mainly includes two parts, first is that it is thick to prepare chymotrypsinogen by fresh food frozen ox pancreas or pig pancreas Product, second is that by arrowhead protease inhibitors affinity column prepare high purity chymotrypsin (potency reach 1400u/mg with On).For over ten years, being widely used with chymotrypsin, requirement of the high-end customer to quality is also higher and higher, chymotrypsin effect Valence will reach 1600u/mg or more.

Therefore, those skilled in the art is dedicated to developing a kind of new affinity chromatography --- the suppression of low molecule Urine proteins enzyme Preparation affinity column --- to prepare high purity chymotrypsin, the affinity chromatography specificity is more preferable, and chymotrypsin potency is more It is high.

Summary of the invention

In view of the above drawbacks of the prior art, the technical problem to be solved by the present invention is to gruel eggs made from the prior art White enzyme potency is unable to satisfy high demand, it is desirable to provide a kind of new preparation process.

To achieve the above object, the present invention provides the technique that a kind of affinity chromatography prepares high purity chymotrypsin, packets Include following steps:

(1) chymotrypsinogen is taken, with deionized water dissolving and pH value is adjusted to 7.5-8.5, the pancreas egg of high specific acitivity is added White enzyme digests 24-48h in 2-8 DEG C of environment, and the solution after enzymatic hydrolysis is centrifuged to obtain enzymolysis liquid；

(2) low molecule Ulinastatin affinity chromatography medium is prepared, then fills column, and with 0.1M Tris-Hcl, PH8.0 buffer is balanced, and enzymolysis liquid loading, coutroi velocity 30-120cm/h are used after balance；With balance after loading Buffer afterwards rushes column, rushes the OD280 value to efflux less than 0.1, then uses 0.1mol/L formic acid -0.05mol/L KCl instead, The buffer of pH2.2 elutes, flow velocity 50-120cm/h, collects eluting peak；

(3) eluting peak collected is concentrated by ultrafiltration with ultrafiltration membrane system, then passes through film filtration sterilization；

(4) enzyme solution after degerming fills lyophilized plate, using Vacuum Freezing & Drying Technology that enzyme solution directly freezed is dry, obtains into Product.

Further, in step (1), chymotrypsin reason fresh food frozen ox pancreas or pig pancreas are crushed, extract albumen, classification It saltouts, crystallization and filtration obtains.

Specifically, following step is passed through in the acquisition of chymotrypsinogen:

(a) fresh food frozen ox pancreas or pig pancreas are chosen, contains 25mmol/L according to raw material weight 2-3 times of volume of addition after weighing CaCl₂Pre-cooling Tris-Hcl buffer, with high-speed organization beveller by low speed to grinding is carried out at high speed, obtain uniform thick Slurry；

(b) thick slurry is stood overnight in 2-8 DEG C of freezer, and next day will slightly starch in high speed freezing centrifuge by 4 DEG C, 10000- 15000rpm is centrifuged 40-60min, takes supernatant, handles after the residue pack after separation according to environmental requirement, supernatant is in 2-8 Removal fat etc. is filtered with filter paper after standing overnight in DEG C freezer, obtained clear liquid is extracting solution；

(c) ammonium sulfate is supplemented in extracting solution to 60% saturation degree, is stood overnight in 2-8 DEG C of freezer, next day draws Clear liquid discards, and collects bottom sediment with centrifugal process；

(d) above-mentioned precipitating is dissolved with the ice water of 1-4 times of weight, adds the saturated ammonium sulfate solution of 0.1-1 times of weight, adjusted PH to 4.5-6.5 is saved, heat preservation, which stands 24-48h, at 20-30 DEG C makes chymotrypsinogen sufficient crystallising, and chymotrypsin is obtained by filtration It is former.

Further, the trypsase in step (1) is derived from bovine trypsin or Porcine trypsin, and the effect of trypsase Valence is greater than 3000u/mg.

Further, the weight ratio of chymotrypsinogen and deionized water is 1:8-13 in step (1).

Further, the weight for the trypsase being added in step (1) is the 0.004-0.01% of chymotrypsinogen.

Further, in step (2) affinity chromatography medium the preparation method comprises the following steps: using GE Health Sepharose 4B Gel, (pH 8.0-8.5) is obtained after reacting 24 hours with low molecule Ulinastatin under alkaline condition.

Further, the ultrafiltration membrane system for using combined closure system 10000 is concentrated by ultrafiltration in step (3).

Further, concentrate passes through 0.25-0.35 μm of membrane filtration degerming in step (3).

Further, the enzyme activity of finished product is higher than 1750u/mg.

The present invention has developed new affinity chromatography --- low molecule Ulinastatin affinity column, for making Standby high purity chymotrypsin, can be easy to operate with a step from chymotrypsinogen purifying crude chymotrypsin, and compared to existing Arrowhead protease inhibitors affinity chromatography, specificity is more preferable, and chymotrypsin potency is higher, meets 1600u/mg's or more Demand.

It is described further below with reference to technical effect of the embodiment to design of the invention, specific structure and generation, To fully understand the purposes, features and effects of the present invention.

Specific embodiment