CN105754892B - A method for separation and purification of microbial transglutaminase - Google Patents

Abstract

The present invention discloses a kind of isolation and purification method of new glutamine of microbe transaminase, the new strain number that Natural Selection is obtained is the luxuriant source streptomycete fermentation liquid of CGMCC No.10804 after alcohol precipitation, anion-exchange chromatography is carried out, using obtaining the glutamine of microbe transaminase after desalination, freeze-drying.Obtained glutamine transaminage Chun Du≤90%, endotoxin Shui Ping≤0.02EU/mL, specific enzyme activity is between 25-30U/mg, Hui Shou Shuai≤70%.Present invention process is simple; it is at low cost; the enzyme activity rate of recovery is high; obtained glutamine transaminage purity is high; and the glutamine of microbe transaminase property that this strain fermentation obtains is stablized; it is convenient for industrialized production, provides a practicable method to meet the large-scale production of MTGase of pharmaceutical grade.

Description

A method for separation and purification of microbial transglutaminase

technical field

The invention belongs to the field of separation and purification of biotechnology, in particular to a technology for the separation and purification of transglutaminase produced by a new bacterial strain.

Background technique

Transglutamine transaminase (Transglu _t ami _na se, TGase for short, EC2.3.2.13) is a transferase that catalyzes acyl transfer, which can catalyze the intramolecular and intermolecular ε-(γ-glutaminyl) lysine covalent bonds Formation, thereby catalyzing the intramolecular and intermolecular crosslinking of proteins, thereby changing the properties and functions of proteins.

Transglutaminase widely exists in plants, animals and microorganisms. TGase was first obtained by scholars in the liver of guinea pigs, which has been used as a source of commercial TGase in Europe for a long time. The complicated separation and purification process of TGase with scarce resources and animal sources leads to very high price of the enzyme, so that it is impossible to apply it in industrial scale food processing. So some scholars began to work on obtaining this enzyme from microorganisms. Later, Ando et al. first discovered microbial transglutaminase (MTGase) from Streptomyces verticillium culture medium, thereby realizing large-scale fermentation production. At the same time, transglutaminase derived from microorganisms has many advantages over transglutaminase derived from animals and plants, such as transglutaminase derived from microorganisms is a calcium-independent enzyme with low substrate specificity. These properties make MTGase widely used in various fields.

Since Ando et al. obtained transglutaminase from the Streptomyces verticillium culture medium in 1989, experts and scholars have successively discovered other Streptomyces producing TGase, such as Streptomyces hygroscopicus and Streptomyces maoyuan. The properties of MTGase from different Streptomyces genus and even the MTGase secreted by Streptomyces from different species of the same genus are not completely the same. Their thermal stability and pH stability are very different, which affects the later application. Not all of the Streptomyces producing MTGase that have been screened at present can be transformed into industrial production. Therefore, it is extremely urgent to screen the streptomyces with high enzyme production efficiency and high MTGase stability. And the MTGase stability produced by the Streptomyces Maoyuan screened from nature mentioned in the present invention is all higher than previous findings.

At present, MTGase derived from microorganisms has been used in industrial production. MTGase produced by microbial fermentation can be used in food processing after simple alcohol precipitation. But the MTGase purity that this method obtains is low, and impurity content is many and component is complicated. Studies have found that MTGase has great application prospects in the field of biopharmaceuticals. For example, MTG can catalyze the crosslinking of type I collagen to form high temperature resistant glue; it can also crosslink gelatin to form a good porous network for hemostasis and wound adhesion (Chinese patent: 200780051215.4, 200980131973.6; US patent: 8133484 ; European Patent: WO2012017415, EP2303344); Therefore, microbial transglutaminase can be used as a cross-linking agent for potential medical hemostatic materials in surgical operations such as hemostasis and wound adhesion. But, the purity that is applied to the MTGase of food field does not meet the requirement of pharmaceutical grade. At the same time, the existing traditional separation and purification process is complicated, the cost is high, the yield, purity and specific activity of the enzyme in the preparation process are not high, and it still contains a variety of miscellaneous proteins, which is difficult to meet the requirements of being used as a medical material. The invention overcomes the existing problems and obtains a simple and low-cost purification process.

Contents of the invention

The purpose of the present invention is to provide a microbial transglutaminase purification method with low cost, high recovery rate and high purity, which is suitable for industrial production and meets the requirements of pharmaceutical grade, so as to solve the problem of complicated purification process of transglutaminase at present. The problems of high cost, low recovery rate and low purity. The microorganism transglutaminase obtained by separating and purifying by the method of the present invention meets the requirements of pharmaceutical grade.

In the method of the present invention, the number of the bacteria used in the General Microorganism Center of China Microbial Strain Preservation Management Committee is CGMCC No.10804, which is the Streptomyces Maoyuan obtained by natural screening, and the use of Streptomyces Maoyuan to produce microbial glutamine transamination Enzyme (MTGase) is a common method at present, and the characteristic of this bacterial strain is that its enzyme-producing efficiency is high, and the MTGase produced is compared with the MTGase produced by existing bacterial strains, and stability is high, as shown in Figure 2, accords with Pharmaceutical grade requirements.

The present invention proposes a kind of Maoyuan Streptomyces sp. (Streptomyces sp.) bacterial strain, is a strain Streptomyces sp. strain that obtains from natural screening in soil, and mycelia morphology is as shown in Figure 1. After comparison of the 16S rDNA sequence, it was found that it was different from other Streptomyces strains reported in the past, and the phylogenetic tree based on the 16S rDNA sequence of the strain proposed by the present invention and related strains constructed by the neighbor-joining method is shown in Figure 5. The strain proposed in this patent is an unknown Streptomyces between Streptomyces mobaraensis NBRC13819, Streptomyces mobaraensis NRRL B-3729 and Streptomyces sp.J1S1. The strains in the black box represent the Streptomyces proposed by the present invention, which can be speculated to be new wild strains .

It is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, with the preservation date being May 13, 2015, and the preservation number is CGMCC No.10804. The sequencing result of the 16S rDNA of Streptomyces Maoyuan is shown in SEQ ID NO.1.

The present invention also proposes a method for cultivating the Streptomyces Maoyuan: culturing the Streptomyces Maoyuan in the seed medium for 24-48h, the culture temperature is 30°C, and the shaking table speed is 200r/min, then, Then transfer to the fermentation medium and cultivate for 45-52 hours to obtain the fermentation broth of Streptomyces Maoyuan; wherein, the fermentation broth contains MTGase, a secondary metabolite of Streptomyces Maoyuan.

Specifically, the cultivation method of Streptomyces Maoyuan is as follows: the strain obtained by screening is cultivated in the seed medium for 24-48h, and the composition of the seed medium is (percentage by weight): 1.5-2.5% of glycerol, yeast extract 0.3-0.8%, fish meal peptone 2.1-2.7%, MgSO ₄ ·7H ₂ O 0.15-0.4%, K ₂ HPO ₄ 0.15-2.3%, pH 7.4, culture temperature 30°C, shaker speed 200r/min. Then, it was transferred to the fermentation medium and cultivated for 45-52 hours to obtain a large amount of secondary metabolite MTGase of Streptomyces Maoyuan. Wherein, the composition of the fermentation medium is (percentage by weight): glycerin 1.5-2.5%, yeast extract 0.3-0.8%, fish powder peptone 2.1-2.7%, MgSO ₄ ·7H ₂ O 0.15-0.4%, K ₂ HPO ₄ 0.15-2.3%, accelerator 1-2%, pH 7.4, culture temperature 30°C, shaker speed 200r/min.

The separation and purification method of microbial transglutaminase of the present invention comprises the following steps:

1. Take the supernatant of Streptomyces Maoyuan fermentation broth, treat it with pre-cooled absolute ethanol 1:1, let it stand for 1 hour, centrifuge at 8000r/mim for 15 minutes, collect the precipitate, and obtain the crude extract of MTGase;

2. Dissolve the crude extract obtained in step 1 in phosphate buffer, and perform anion exchange chromatography with an AKTA avant 25 purifier at room temperature. The flow rate of the sample and the elution buffer is 1mL/min, and the first elution peak that appears during the elution process is collected by linear elution;

3. Desalt the sample recovered in step 2. The selected desalting column model is Hiprep 26/10Desalting, and the flow rate of sample and ultrapure water is 10mL/min;

4. Freeze-dry the sample recovered in step 3 to obtain high-purity microbial transglutaminase at last.

All buffer solutions and samples in the purification step of the present invention are sterilized by filtering through a 0.22nm filter membrane.

In the step 2, anion exchange chromatography is used for purification. Compared with the existing purification methods, the method is novel and the steps are simple. Only one step of main anion exchange chromatography is required to obtain MTGase that meets the requirements of pharmaceutical grade purity, and then Salt-free and impurity-free high-quality MTGase can be obtained by desalting and freeze-drying methods, and the powder form after freeze-drying is more suitable for later application, storage and transportation. The equilibrium buffer used is 15-25mM pH6.5-8.0 sodium phosphate buffer, the elution buffer is 15-25mM pH6.5-8.0 sodium phosphate buffer+1M sodium chloride, and the elution method is linear Elution, 0-20% elution buffer, the elution volume is 25 column volumes, the concentration of the sample is 0.4-0.5mg/mL, the unit enzyme activity is 5-6U/mL, and the total protein loaded is 10- 15mg, the total enzyme activity is 100-300U.

The solution used for desalting in step 3 is ultrapure water, and the sample loading volume is less than or equal to 30% of the column volume.

During the freeze-drying process in step 4, the first sublimation temperature is -10°C, and the second sublimation temperature is 4°C.

In the present invention, the recovery rate of MTGase finally obtained by the method of the present invention is ≧70%, the specific enzyme activity is between 25-30U/mg, the endotoxin content is ≦0.02EU/mL, and the purity is ≧90%. The invention has the advantages of simple process, low cost, high recovery rate of enzyme activity, and high purity of the obtained transglutaminase, and the microbial transglutaminase obtained by fermentation of the bacterial strain is stable in property, which is convenient for industrial production. Production provides a practical approach.

The invention proposes the application of Maoyuan Streptomyces for producing microbial transglutaminase.

The present invention proposes the microbial transglutaminase obtained by the above purification and separation method. The present invention also proposes the medical use of the microbial transglutaminase obtained by the above method. The MTGase finally obtained after purification by the present invention can be used together with gelatin for wound hemostasis and medical adhesive, and can also be used for biomodification of pharmaceutical proteins and the like.

To sum up, the beneficial effects of the present invention are: (1) The MTGase produced by the strain obtained by natural screening is stable in nature, and the loss of enzyme activity due to its own stability is less affected during post-treatment of the fermentation broth. (2) The isoelectric point of MTGase is about 8.0, and MTGase is theoretically positively charged when pH<8.0, and the ion-exchange chromatography method used in the existing scheme is all cation-exchange chromatography, and the purification mentioned in the present invention The method is to use anion exchange chromatography under the condition of pH6.5-8.0. This is because the bacterial strain proposed in the present invention can carry a large amount of negative charges on the surface under the condition of pH6.5-8.0, thereby combining with the anion column, thus breaking our conventional purification method and purification principle, and providing a new solution for MTGase purification and other Protein purification provides a new idea. (3) The MTMase purification methods in the existing schemes are all purified by cation exchange chromatography or gel filtration chromatography under the condition of pH≦7.0, and the one-step purification method cannot obtain MTGase with a purity higher than 20U/mg, and simultaneously obtain The loss of MTGase is relatively large, the steps are cumbersome, and cannot be applied to industrial production. In the purification scheme proposed in the present invention, MTGase with a specific enzyme activity greater than 25 U/mg can be obtained in one step by anion exchange chromatography under the condition of pH 6.5-8.0, and the final recovery rate can reach more than 70%, that is, adopt the present invention The purity of the enzyme obtained by the purification method is high, and the recovery rate is high. The method is simple, low in cost, can be scaled up industrially, and is suitable for industrialized production.

Description of drawings

Fig. 1 is the observation of the growth morphology of the bacterial strain proposed by the present invention on the ISP medium.

Fig. 2 is that the MTGase (experimental group) produced by Streptomyces Maoyuan proposed by the present invention and the MTGase (control group 1: 40587, control group 2: 40847) produced by Maoyuan Streptomyces in existing reports are enzyme stability at different temperatures Comparison. A is the result of different MTGase stability at 70°C, B is the result of different MTGase stability at 60°C, C is the result of different MTGase stability at 50°C, D is the result of different MTGase stability at 40°C result.

Figure 3 is an anion exchange chromatography chromatogram. The breakthrough peak represents the impurity protein peak that is not bound to the anion chromatography column; the elution peak 1 represents the target protein peak; the elution peak 2 represents the impurity protein peak that is firmly bound to the anion chromatography column.

Fig. 4 is the electrophoresis diagram of microbial transglutaminase during the purification process. M represents the protein standard maker; lane 1 represents the electrophoresis of fermentation broth; lane 2 represents the electrophoresis of alcohol precipitated products; lane 3 represents the electrophoresis of the target peak after anion exchange chromatography; lane 4 represents the electrophoresis of MTGase after desalting; lane 5 represents the freeze-drying Post-MTGase electropherogram.

Fig. 5 is a phylogenetic tree based on the 16S rDNA sequences of the strains proposed by the present invention and related strains constructed by the neighbor-joining method.

Detailed ways

In conjunction with the following specific examples and accompanying drawings, the present invention is described in further detail, and the protection content of the present invention is not limited to following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are included in the present invention, and are protected by the appended claims. The process, conditions, reagents, experimental methods, etc. of implementing the present invention, except the content specifically mentioned below, are general knowledge and common knowledge in the art, and the present invention has no special limitation content.

Embodiment 1 alcohol precipitation obtains MTGase crude extract

(1) Centrifuge 8,000g of the fermented broth at 4°C for 10 minutes to remove the precipitate; slowly adjust the pH to 5.0 with phosphoric acid, and centrifuge at 8,000g for 10 minutes at 4°C to remove the precipitate; Add, place in an ice box for 1 hour, centrifuge at 8000g for 10 minutes at 4°C, and remove the supernatant.

(2) SDS-PAGE detection results are shown in lane 2 in Figure 4, the molecular weight of MTGase is about 38KDa, and from lane 2 it can be seen that the crude extract obtained by alcohol precipitation contains a large amount of foreign proteins. The total enzyme activity of the fermentation broth before alcohol precipitation was 2760.6U, and the total enzyme activity after alcohol precipitation was 2484.5U. The specific results are shown in Table 1. The enzyme activity assay results showed that the total enzyme activity recovery rate in this step was 90%.

The detection of embodiment 2MTGase crude extract temperature stability

(1) The MTGase (experimental group) obtained by alcohol precipitation in Example 1 is lyophilized and dissolved in the phosphate buffer of pH6.0 with 3U/mL, and two arbitrary two MTGases (control group) that have been reported at present are selected simultaneously. 1: 40587 and the control group 2: 40847 (both from Taixing Dongsheng Food Technology Co., Ltd.) were used as controls, and were also dissolved in phosphate buffer at pH 6.0 at 3 U/mL.

(2) Place the three MTGases in water baths at 40°C, 50°C, 60°C, and 70°C respectively, store the MTGase at the time intervals shown in Figure 2, and measure the enzyme activity of the MTGase after a certain period of time . A is the result of different MTGase stability at 70°C, B is the result of different MTGase stability at 60°C, C is the result of different MTGase stability at 50°C, D is the result of different MTGase stability at 40°C result.

As shown in Figure 2, at 70°C, the stability of MTGase described in the present invention was 23% higher than that of 40587 of control group 1 when placed for 75s, and 19% higher than that of 40847 of control group 2; at 60°C Under the condition of 50 minutes, the stability of MTGase described in the present invention is 22% higher than 40587, and 25% higher than 40847 of control group 2; The stability is 18% higher than that of 40587 of control group 1, and 21% higher than that of 40847 of control group 2; at 40°C, the stability of MTGase described in the present invention is 14% higher than that of 40587 of control group 1 when left for 4 days %, which is 15% higher than the 40847 of the control group 2. In summary, under certain temperature conditions, the stability of MTGase described in the present invention is all higher than other two kinds of enzyme powders.

Example 3 Anion-exchange chromatography method obtains high-purity MTGase after purification

(1) The purification instrument selected is from General Corporation of the United States avant 25, the chromatographic column used for purification is a prepacked anion exchange column Hiprap Q Sepharose FF (1 mL) purchased from General Company of the United States.

(2) Appropriate amounts of 20 mM sodium dihydrogen phosphate and 20 mM disodium hydrogen phosphate were prepared respectively, and the two buffer solutions were mixed in proportion to finally prepare a phosphate buffer solution with a pH of 6.5, and the conductance was about 2.5 mS/cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use.

(3) On the basis of the equilibrium buffer, add 1M sodium chloride, fully dissolve and then adjust the pH to 6.5 with phosphoric acid, and the conductance is about 75.0mS/cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in 4°C refrigerator for later use.

(4) Dissolve the MTGase crude extract obtained by alcohol precipitation in 20mM phosphate buffer solution of pH 6.5, centrifuge at 8000r/min 4°C for 10min after fully dissolving, then filter the enzyme solution with a 0.22um filter membrane, and then in Degas in the ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use. The protein concentration of the prepared sample was 0.36mg/mL, and the unit enzyme activity was 4.81U/mL.

(5) Equilibrate the anion chromatographic column with 20CV equilibration buffer first, take 30mL of the enzyme solution prepared in advance to load the sample, and then use 10CV equilibration buffer to elute the protein that is not bound to the chromatographic column or is not firmly bound , the breakthrough peak shown in Figure 3, and then eluted with 25CV of the elution buffer in a linear elution method. At this time, the sodium chloride ion strength increased from 0 to 0.2M, and the elution peak obtained was shown in the figure 3 shows the elution peak 1, and finally use 1M sodium chloride to elute the impurity protein that is firmly bound to the chromatographic column, and the obtained elution peak is the elution peak 2 shown in Figure 3. The flow rate during all experiments was 1 mL/min. The collected elution peak 1 is the purified MTGase.

(6) SDS-PAGE test results are shown in lane 3 in Figure 4, the purity of the purified sample is 92% through grayscale scanning, and the total enzyme activity recovery rate of all samples obtained after alcohol precipitation is 89.6% after this step , The specific enzyme activity is 26.34U/mg.

Example 4 Anion-exchange chromatography method obtains high-purity MTGase after purification

(1) The purification instrument selected is from General Corporation of the United States avant 25, the chromatographic column used for purification is a prepacked anion exchange column Hiprap Q Sepharose FF (1 mL) purchased from General Company of the United States.

(2) Appropriate amounts of 20 mM sodium dihydrogen phosphate and 20 mM disodium hydrogen phosphate were prepared respectively, and the two buffer solutions were mixed in proportion to finally prepare a phosphate buffer solution with a pH of 7.0, and the conductance was about 2.5 mS/cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use.

(3) Add 1M sodium chloride on the basis of the equilibrium buffer, and after fully dissolving, adjust the pH to 7.0 with phosphoric acid, and the conductance is about 75.0mS/cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in 4°C refrigerator for later use.

(4) Dissolve the MTGase crude extract obtained by alcohol precipitation in 20mM phosphate buffer solution with pH 7.0, centrifuge at 8000r/min 4°C for 10min after fully dissolving, then filter the enzyme solution with a 0.22um filter membrane, and then in Degas in the ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use. The protein concentration of the prepared sample was 0.42mg/mL, and the unit enzyme activity was 5.09U/mL.

(5) Equilibrate the anion chromatographic column with 20CV equilibration buffer first, take 30mL of the enzyme solution prepared in advance to load the sample, and then use 10CV equilibration buffer to elute the protein that is not bound to the chromatographic column or is not firmly bound , the breakthrough peak shown in Figure 3, and then eluted with 25CV of the elution buffer in a linear elution method. At this time, the sodium chloride ion strength increased from 0 to 0.2M, and the elution peak obtained was shown in the figure 3 shows the elution peak 1, and finally use 1M sodium chloride to elute the impurity protein that is firmly bound to the chromatographic column, and the obtained elution peak is the elution peak 2 shown in Figure 3. The flow rate during all experiments was 1 mL/min. The collected elution peak 1 is the purified MTGase.

(6) SDS-PAGE test results are shown in lane 3 in Figure 4, the purity of the purified sample is 90% through grayscale scanning, and the total enzyme activity recovery rate of all samples obtained after alcohol precipitation is 92.0% after this step , the specific enzyme activity is 28.61U/mg, and the specific results are shown in Table 1.

Example 5 Anion-exchange chromatography method obtains high-purity MTGase after purification

(1) The purification instrument selected is from General Corporation of the United States avant 25, the chromatographic column used for purification is a prepacked anion exchange column Hiprap Q Sepharose FF (1 mL) purchased from General Company of the United States.

(2) Appropriate amounts of 20 mM sodium dihydrogen phosphate and 20 mM disodium hydrogen phosphate were prepared respectively, and the two buffer solutions were mixed in proportion to finally prepare a phosphate buffer solution with a pH of 7.5, and the conductance was about 2.5 mS/cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use.

(3) On the basis of the equilibrium buffer, add 1M sodium chloride, fully dissolve and then adjust the pH to 7.5 with phosphoric acid, and the conductance is about 75.0mS/cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in 4°C refrigerator for later use.

(4) Dissolve the MTGase crude extract obtained by alcohol precipitation in 20mM phosphate buffer solution with pH 7.5, centrifuge at 8000r/min 4°C for 10min after fully dissolving, then filter the enzyme solution with a 0.22um filter membrane, and then in Degas in the ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use. The protein concentration of the prepared sample was 0.4mg/mL, and the unit enzyme activity was 5.19U/mL.

(5) Equilibrate the anion chromatographic column with 20CV equilibration buffer first, take 30mL of the enzyme solution prepared in advance to load the sample, and then use 10CV equilibration buffer to elute the protein that is not bound to the chromatographic column or is not firmly bound , the breakthrough peak shown in Figure 3, and then eluted with 25CV of the elution buffer in a linear elution method. At this time, the sodium chloride ion strength increased from 0 to 0.2M, and the elution peak obtained was shown in the figure 3 shows the elution peak 1, and finally use 1M sodium chloride to elute the impurity protein that is firmly bound to the chromatographic column, and the obtained elution peak is the elution peak 2 shown in Figure 3. The flow rate during all experiments was 1 mL/min. The collected elution peak 1 is the purified MTGase.

(6) SDS-PAGE test results are shown in lane 3 in Figure 4, the purity of the purified sample is 93% through grayscale scanning, and the total enzyme activity recovery rate of all samples obtained after alcohol precipitation is 91.4% after this step , The specific enzyme activity is 28.21U/mg.

Example 6 Anion-exchange chromatography method obtains high-purity MTGase after purification

(1) The purification instrument selected is from General Corporation of the United States avant 25, the chromatographic column used for purification is a prepacked anion exchange column Hiprap Q Sepharose FF (1 mL) purchased from General Company of the United States.

(2) Appropriate amounts of 20 mM sodium dihydrogen phosphate and 20 mM disodium hydrogen phosphate were prepared respectively, and the two buffer solutions were mixed in proportion to finally prepare a phosphate buffer solution with a pH of 8.0, and the conductance was about 2.5 mS/cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use.

(3) Add 1M sodium chloride on the basis of the equilibrium buffer, and after fully dissolving, adjust the pH to 8.0 with phosphoric acid, and the conductance is about 75.0mS/cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in 4°C refrigerator for later use.

(4) Dissolve the MTGase crude extract obtained by alcohol precipitation in 20mM phosphate buffer solution with pH 8.0, centrifuge at 8000r/min 4°C for 10min after fully dissolving, then filter the enzyme solution with a 0.22um filter membrane, and then in Degas in the ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use. The protein concentration of the prepared sample was 0.42mg/mL, and the unit enzyme activity was 5.09U/mL.

(5) Equilibrate the anion chromatographic column with 20CV equilibration buffer first, take 30mL of the enzyme solution prepared in advance to load the sample, and then use 10CV equilibration buffer to elute the protein that is not bound to the chromatographic column or is not firmly bound , the breakthrough peak shown in Figure 3, and then eluted with 25CV of the elution buffer in a linear elution method. At this time, the sodium chloride ion strength increased from 0 to 0.2M, and the elution peak obtained was shown in the figure 3 shows the elution peak 1, and finally use 1M sodium chloride to elute the impurity protein that is firmly bound to the chromatographic column, and the obtained elution peak is the elution peak 2 shown in Figure 3. The flow rate during all experiments was 1 mL/min. The collected elution peak 1 is the purified MTGase.

(6) SDS-PAGE test results are shown in lane 3 in Figure 4, the purity of the purified sample is 87% through grayscale scanning, and the total enzyme activity recovery rate of all samples obtained after alcohol precipitation is 94.7% after this step , The specific enzyme activity is 27.81U/mg.

Obtain high-purity salt-free MTGase after desalting in embodiment 7

(1) The purification instrument selected is from General Corporation of the United States Avant 25, desalting column is a prepacked column Hiprep 26/10 Desalting purchased from General Corporation of the United States.

(2) First balance the desalting column with ultrapure water at a flow rate of 10mL/min, then directly load the sample obtained by anion exchange chromatography in Example 5, the total volume of the sample is 12mL, and the total enzyme activity of the sample is 100U, the total protein loaded is 5mg.

(3) Collect the samples corresponding to the protein peaks. The SDS-PAGE test results are shown in lane 4 in Figure 4. The purity of the desalted samples is 92% through grayscale scanning. After this step, all samples obtained after anion exchange chromatography The recovery rate of the total enzyme activity was 95%, and the specific results are shown in Table 1.

The recovery rate of transglutaminase in the separation and purification process of the present invention is as shown in Table 1:

Table 1 MTGase purification results

Note: alcohol precipitation is the result in Example 1; anion exchange chromatography is the result in Example 4; desalination is the result in Example 7.

In summary, the separation and purification method of a new microbial transglutaminase proposed by the present invention has obvious beneficial effects. First of all, it can be seen from Example 2 that the stability of MTGase proposed in the present invention is significantly higher than that of other types of MTGase, and the stability at different temperatures is 10-20% higher, and the property is stable. It has high stability in application and meets the requirements of pharmaceutical grade. Secondly, through Examples 2-6, it can be found that under the condition of pH 6.5-8.0, the method of anion exchange chromatography can obtain MTGase with a specific activity greater than 25U/mg in one step, and finally obtain MTGase that meets the requirements of pharmaceutical grade after a series of treatments. The final recovery rate of the solid enzyme powder can reach more than 70%, which has obvious advantages compared with the existing literature reports. In existing bibliographical reports, the recovery rate of the higher MTGase obtained by different purification schemes is all below 70%, and this result is the result before desalting and freeze-drying, and the method mentioned in the present invention simply obtains high The recovery rate of pure MTGase can reach more than 80%. Even after desalination and freeze-drying, the final recovery rate of dried MTGase enzyme powder can reach more than 70%. The recovery rate is high, the method is simple, the cost is low, and it can be scaled up industrially, suitable for industrialization Production. Finally, the isoelectric point of MTGase is 8.0, the ion exchange chromatography method used in the existing scheme is all cation exchange chromatography, and the purification method mentioned in the present invention is to use anion exchange layer under the condition of pH6.5-8.0 Analysis breaks our conventional purification method and purification principle, and provides a new idea for MTGase purification and other protein purification.

Claims (6)

1. a kind of cyclopentadienyl source streptomycete (Streptoverticillium mobaraense) bacterial strain, which is characterized in that be from soil One plant of new streptomycete bacterial strain that middle Natural Selection obtains is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life Object center, the deposit date is on May 13rd, 2015, deposit number was CGMCC No.10804.

2. a kind of luxuriant source streptomycete (Streptoverticillium mobaraense) bacterial strain paddy ammonia as described in claim 1 The isolation and purification method of amide transaminase, which is characterized in that the described method comprises the following steps:

(1) the luxuriant source streptomycete fermentation liquid supernatant is taken, is handled with the dehydrated alcohol 1:1 of pre-cooling, ice chest is placed in and places 1h, 8000g, 4 DEG C of centrifugation 10min collect precipitating, obtain the crude extract of MTGase；

(2) step (1) resulting crude extract is dissolved in the phosphate buffer of pH 6.5-8.0, after completely dissolution 8000r/min 4 DEG C of centrifugation 10min, are then filtered with 0.22 μm of filter membrane, and then deaerate 20-30min, use AKTAavant 25 at room temperature Purification system carries out anion-exchange chromatography, the purification column model anion-exchange column Hitrap Q Sepharose of selection Fast Flow 1mL, the flow velocity of equilibration buffer, sample and elution buffer are 1mL/min, with the mode of linear elution Collect first eluting peak occurred in elution process；

Wherein, the specific steps that first eluting peak occurred in elution process is collected with the mode of linear elution are as follows:

First anion chromatographic column is balanced with the equilibration buffer of 20CV, then with the equilibration buffer of 10CV will not with color Spectrum column combines or gets off in conjunction with unstable albumen wash-out, then is washed with the elution buffer of 25CV in the method for linear elution De-, sodium chloride ionic strength increases to 0.2M from 0 at this time, finally with the miscellaneous egg of the sodium chloride elution and chromatographic column firm connection of 1M White, collecting first eluting peak is MTGase after purification；

(3) sample that step (2) recycling obtains is subjected to desalination, the desalting column model Hiprep 26/10 of selection again The flow velocity of Desalting, sample and ultrapure water is 10mL/min；

(4) sample that step (3) recycling obtains is freeze-dried, finally obtains the luxuriant source streptomycete glutamine of high-purity Transaminase.

3. the isolation and purification method of cyclopentadienyl source according to claim 2 streptomycete glutamine transaminage, which is characterized in that institute 0.22 μm of film filtration sterilization will be passed through by stating sample all in method and used buffer.

4. the isolation and purification method of cyclopentadienyl source according to claim 2 streptomycete glutamine transaminage, which is characterized in that institute State the sodium phosphate buffer for the 15-25mM that the equilibration buffer in step (2) is pH6.5-8.0, elution buffer pH6.5- Sodium phosphate buffer+1M the sodium chloride of 8.0 15-25mM, elution process are linear elution, 0-20% elution buffer, elution 25 column volumes, the concentration of sample are 0.4-0.5mg/mL, and unit enzyme activity is 5-6U/mL, and the total protein of loading is 10-15mg, Total enzyme activity is 100-300U；

Wherein, the preparation method of the equilibration buffer are as follows: prepare the sodium dihydrogen phosphate of 20mM and the phosphoric acid hydrogen two of 20mM respectively Two kinds of buffers are mixed the sodium phosphate buffer for being finally formulated as the 15-25mM of the pH6.5-8.0 by sodium in proportion.

5. the isolation and purification method of cyclopentadienyl source according to claim 2 streptomycete glutamine transaminage, which is characterized in that institute Stating solution used in desalination in step (3) is ultrapure water, and sample loading volume is less than or equal to the 30% of column volume.

6. the isolation and purification method of cyclopentadienyl source according to claim 2 streptomycete glutamine transaminage, which is characterized in that institute It states in the freezing dry process in step (4), first time sublimation temperature is -10 DEG C, and second of sublimation temperature is 4 DEG C.