CN101633900A - Method for preparing cycloamylose 4-alpha-glycosyl transferase production - Google Patents

Abstract

The invention provides a method for preparing cycloamylose 4-alpha-glycosyl transferase production, belonging to the technical field of enzyme engineering. The method uses Escherichia coli CGMCC No. 3093 as a parent strain to prepare pure enzyme 4-alpha-transferase through preparing a seed culture medium by jar fermentation, fermenting and culturing the culture medium in a fermentation cylinder, collecting strains, extracting crude enzyme by ultrasonication and purifying. The transferase prefers a temperature at 75 DEG C and pH of 7.5, and has the characteristics of high temperature resistance, high enzyme activity and the like, wherein, vitality of the 4-alpha-transferase which is primarily purified, frozen and dried can reach 7,000-10,000U/g. The 4-alpha-transferase can act on amylase to generate cycloamylose, and can modify amylopectin to generate slowly digestible starch. Therefore, the transferase can be widely applied in foods, pharmacy and other industries, and has enormous potential commercial value.

Description

Produce the preparation method of large cyclodextrin 4-alpha-glycosyl transferring enzyme

Technical field

Produce the preparation method of large cyclodextrin 4-alpha-glycosyl transferring enzyme, belong to technical field of enzyme engineering.

Background technology

Large cyclodextrin is to be different from common cyclodextrin, and the polymerization degree is from the 9 ring-type dextran that do not wait to hundreds of.Because large cyclodextrin has highly water-soluble, low-viscosity and characteristic such as do not bring back to life, and can be widely used in foodstuffs industry, chemical industry, the pharmaceutical industry.Large cyclodextrin mainly is that glycosyltransferase produces.Abroad the research of the glycosyltransferase that produces large cyclodextrin has been carried out decades, and domestic only in recent years the someone begin to study, obtaining huge achievement aspect the common cyclodextrin producing, but seldom for the achievement in research of producing the large cyclodextrin enzyme.The 4-alpha-glycosyl transferring enzyme of the product large cyclodextrin that the present invention relates to is genetic engineering bacterium (DH 5 α-TA deposit number: CGMCC No.3093) express generation that make up under professor's Piao of South Korea Seoul university help.Thick enzyme enzyme liquid is of light color, and dispense with decoloration is handled, and can handle by 70 ℃ of insulation 10min enzyme is carried out preliminary purification, improves the content of target enzyme.Separate from genetic engineering bacterium and extract the enzyme that obtains in the enzyme obtain and the environmental microorganism and compare, have higher resistance toheat, purification procedures is simple and easy to do, has established good basis for realizing large-scale industrial production.

Summary of the invention

The object of the present invention is to provide a kind of preparation method who produces large cyclodextrin 4-alpha-glycosyl transferring enzyme.

Technical scheme of the present invention: the bacterial strain of 4-alpha-glycosyl transferring enzyme is produced in a strain, its called after colon bacillus (Escherichia coli) of classifying, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No.3093.

A kind of preparation method who produces large cyclodextrin 4-alpha-glycosyl transferring enzyme, with colon bacillus (Escherichiacoli) CGMCC No.3093 is starting strain, prepare seed culture medium, ferment tank cultivation, collect thalline through shake flask fermentation, ultrasonic broken wall extracts thick enzyme, purifying obtains pure enzyme 4-alpha-glycosyl transferring enzyme, and step is:

1) single bacterium colony is selected: dip in the bacterium liquid that takes a morsel draw the LB flat board that contains 100 μ g/mL ampicillin natriums from genetically engineered bacterial classification CGMCC No.3093 to connect collarium, flat board was placed in the biochemical incubator 37 ℃ of constant temperature culture 12 hours;

2) seed culture: picking list colony inoculation contains in the triangular flask of LB substratum of ampicillin natrium 100 μ g/mL in 50mL, triangular flask is fixed on 37 ℃, 200 rev/mins shaking culture 8h make seed culture medium on the constant temperature shaking table;

3) fermentation culture: seed culture medium is seeded in the fermentor tank of the 3L substratum that contains ampicillin natrium 100 μ g/mL, 37 ℃ of attemperation, 200 rev/mins of stirring velocitys, pH are 7.5, air flow is 10L/h, inoculum size is 10%, add 10mL/L soya-bean oil as defoamer, with this understanding fermentation culture 7h;

4) collect thalline, ultrasonic broken wall extracts thick enzyme: above-mentioned fermented liquid is transferred in the 250mL Centrifuge Cup, and under 4000 rev/mins of conditions centrifugal 20 minutes, collecting precipitation was wet thallus;

Wet thallus is put into pH7.5,0.05mol/L, the 20mL Tris-HCl buffered soln of prior precooling, the ultrasonic 3s of 400W, interval 2s, ice-bath ultrasonic 10 minutes; To collect supernatant liquor through centrifugal 10 minutes of 4 ℃ of bacterium liquid, the 10000rpm of ultrasonic broken wall treatment, be crude enzyme liquid;

5) purifying of enzyme:,, collect supernatant liquor and be enzyme liquid through preliminary purification then in centrifugal 10 minutes of 4 ℃, 10000rpm with 70 ℃ of crude enzyme liquids insulation 10min;

With 50mM Tris-HCl pH7.5, the 300mM NaCl of 5 times of column volumes, the rinse damping fluid balance Ni-NTA affinity column of 20mM imidazoles;

Ni-NTA affinity column after will all injecting rinse through the enzyme liquid of preliminary purification;

Rinse damping fluid with 50 times of column volumes carries out wash-out earlier, remove the foreign protein that is not kept by affinity column, use 50mM Tris-HCl pH7.5, the 300mM NaCl of 2 times of column volumes, the elution buffer wash-out of 250mM imidazoles then, collect elutriant, include target protein;

The elutriant that will contain target protein is filled to the dialysis tubing of molecular weight 1000Da, put into deionization water-bath dialysis desalting behind the tying of dialysis tubing two ends, water-bath stirring at room 12h, change a deionized water every 4h, after desalination is finished dialysis tubing is put in the large beaker that contains polyoxyethylene glycol 20,000 and concentrates, collect concentrated solution behind the 4h and promptly get pure enzyme liquid.

Beneficial effect of the present invention: separate from genetic engineering bacterium and extract the enzyme that obtains in the enzyme obtain and the environmental microorganism and compare, have higher resistance toheat, purification procedures is simple and easy to do, has established good basis for realizing large-scale industrial production.The optimum temperuture of this enzyme is 75 ℃, and optimal pH is 7.5, and the vigor of the 4-alpha-glycosyl transferring enzyme that obtains through preliminary purification and lyophilize can reach 7000～10000U/g.4-alpha-glycosyl transferring enzyme can act on amylose starch and produce large cyclodextrin, can also modify the generation slow-digestion starch to amylopectin, change the rheological properties and the resistance of starch, thereby can promote starch in wider scope, to use on a large scale, this enzyme can be widely used in industries such as food, medicine, has huge potential commercial value.

The biological material specimens preservation: colon bacillus (Escherichia coli) is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, is called for short CGMCC, deposit number CGMCC No.3093, preservation date on June 8th, 2009.

Description of drawings

The electrophorogram of Fig. 1 4-alpha-glycosyl transferring enzyme.M represents standard molecular weight albumen among the figure; G1 represents to obtain target enzyme through behind the chromatography; G2 represents through the target enzyme after chromatography and the dialysis; G3 represents through the crude enzyme liquid after the heat treated; G4 represents crude enzyme liquid.

The HPLC figure of Fig. 2 glucose standard specimen, maltose standard specimen, maltotetrose mixture.No. 5 lines are glucose standard specimens among the figure, and No. 4 lines are maltose standard specimens, and 1,2, No. 3 line is the maltotetrose mixture

Fig. 3 is the HPLC figure of substrate reactions after product with glucose.

Fig. 4 is the HPLC figure of substrate reactions after product with maltose.Qualitative according to retention time, be to have generated Fructus Hordei Germinatus seven sugar, MALTOHAXAOASE, maltopentaose, maltotetrose, trisaccharide maltose and glucose behind the substrate reactions with maltose

Fig. 5 maltotetrose mixture is the HPLC figure of substrate reactions after product.Qualitative according to retention time, be to have generated Fructus Hordei Germinatus seven sugar, MALTOHAXAOASE, maltopentaose, maltotetrose, trisaccharide maltose and glucose behind the substrate reactions with the maltotetrose mixture

Embodiment

The preparation and the purifying of embodiment 1,4-alpha-glycosyl transferring enzyme

1, the preparation of enzyme

1) single bacterium colony is selected: dip in the bacterium liquid that takes a morsel draw the LB flat board that contains 100 μ g/mL ampicillin natriums from the genetically engineered bacterial classification to connect collarium, flat board was placed in the biochemical incubator 37 ℃ of constant temperature culture 12 hours;

2) seed culture: picking list colony inoculation contains in the triangular flask of LB substratum of ampicillin natrium 100 μ g/mL in 50mL, triangular flask is fixed on 37 ℃, 200 rev/mins shaking culture 8h make seed culture medium on the constant temperature shaking table;

3) fermentation culture: seed culture medium is seeded in the fermentor tank of the 3L substratum that contains ampicillin natrium 100 μ g/mL, 37 ℃ of attemperation, 200 rev/mins of stirring velocitys, pH are 7.5, air flow is 10L/h, inoculum size is 10%, add 10mL/L soya-bean oil as defoamer, with this understanding fermentation culture 7h;

4) collect thalline, ultrasonic broken wall extracts thick enzyme: above-mentioned fermented liquid is transferred in the 250mL Centrifuge Cup, and under 4000 rev/mins of conditions centrifugal 20 minutes, collecting precipitation was wet thallus;

Wet thallus is put into pH7.5,0.05mol/L, the 20mL Tris-HCl buffered soln of prior precooling, the ultrasonic 3s of 400W, interval 2s, ice-bath ultrasonic 10 minutes;

To collect supernatant liquor through centrifugal 10 minutes of 4 ℃ of bacterium liquid, the 10000rpm of ultrasonic broken wall treatment, be crude enzyme liquid;

2, the evaluation of the purifying of enzyme and purity

(1) purifying of enzyme

1),, collects supernatant liquor and be enzyme liquid through preliminary purification then in centrifugal 10 minutes of 4 ℃, 10000rpm with 70 ℃ of crude enzyme liquids insulation 10min;

2) with 50mM Tris-HCl pH7.5, the 300mM NaCl of 5 times of column volumes, the rinse damping fluid balance Ni-NTA affinity column of 20mM imidazoles;

3) the Ni-NTA affinity column after will all injecting rinse through the enzyme liquid of preliminary purification;

4) elder generation carries out wash-out with the rinse damping fluid of 50 times of column volumes, remove the foreign protein that is not kept by affinity column, use 50mM Tris-HCl pH7.5, the 300mM NaCl of 2 times of column volumes, the elution buffer wash-out of 250mM imidazoles then, collect elutriant, include target protein;

5) elutriant that will contain target protein is filled to the dialysis tubing of molecular weight 1000Da, put into deionization water-bath dialysis desalting behind the tying of dialysis tubing two ends, water-bath stirring at room 12h, change a deionized water every 4h, after desalination is finished dialysis tubing is put in the large beaker that contains polyoxyethylene glycol 20,000 and concentrates, collect concentrated solution behind the 4h and promptly get pure enzyme liquid.

(2) evaluation of enzyme purity

1) preparing gel: make vertical board slot (can not leak glue) with two electrophoresis sheet glass, the vertical placement.The separation gel solution for preparing is poured into, is added dropwise to deionized water, treat that gel is assembled after, pour out deionized water, blot with thieving paper, pour concentrated glue into, insert comb again.Press table 1 preparation 10% separation gel and 3% and concentrate glue.

Table 1

	Separation gel storage liquid (ml)	Separation gel damping fluid (ml)	Concentrate glue storage liquid (ml)	Concentrate glue damping fluid (ml)	??10％ ??SDS(ml)	??1％TEMED ??(ml)	Double distilled water (ml)	??10％AP ??(ml)
									10% separation gel	??6.66	??2.50	??-	??-	??0.20	??2.00	??8.54	??0.10
3% concentrates glue	??-	??-	??3.0	??1.25	??0.10	??2.00	??4.60	??0.05

2) go up sample: sample thief 20 μ L add sample buffer in 1/1 ratio in centrifuge tube respectively, heat 3～5min in the boiling water bath, take out stand-by.Draw 8 μ L standard protein samples and test sample injection sample cell respectively with microsyringe.After point sample finished, (2～3mA/em), holding current was stablized constant, moves to from the bottom of the separation gel during 1～2cm when tetrabromophenol sulfonphthalein, can stop electrophoresis to 10mA to regulate the electrophoresis apparatus electric current.

3) dyeing: after electrophoresis finishes, take out gel slab, immerse in the coomassie brilliant blue staining liquid, dyeing 30min.

4) decolouring: outwell staining fluid, behind the adding destainer 24h, can see protein band clearly.

Electrophorogram as shown in Figure 1.

M represents standard molecular weight albumen among the figure; G1 represents to obtain target enzyme through behind the chromatography; G2 represents through the target enzyme after chromatography and the dialysis; G3 represents through the crude enzyme liquid after the heat treated; G4 represents crude enzyme liquid.

From figure as can be seen, target enzyme can be removed the thermo-labile foreign protein of part through preliminary heat treated, and the enzyme purity that obtains behind affinity column chromatography is very high.

3, the character of enzyme: the optimum temperuture of this enzyme is 70 ℃, optimal pH 7.5, and through the SDS-PAGE electrophoretic analysis, the molecular weight of this pure enzyme is about 57000Da.

Can 4, the commentaries on classics glycosyl activity of enzyme: whether 4-alpha-glycosyl transferring enzyme has the ability of changeing glycosyl, determined 4-alpha-glycosyl transferring enzyme act on amylose starch and generated large cyclodextrin.Adopt the HPLC method substrate and substrate-enzymolysis product to be analyzed result such as Fig. 2, Fig. 3, Fig. 4, shown in Figure 5.

Fig. 2: the HPLC figure of glucose standard specimen, maltose standard specimen, maltotetrose mixture.No. 5 lines are glucose standard specimens among the figure, and No. 4 lines are maltose standard specimens, and 1,2, No. 3 line is the maltotetrose mixture.

The component of each retention time correspondence of table 2

Numbering	Retention time (min)	Sample
			??1	7.312	Maltopentaose
??2	7.844	Maltotetrose
			??3	8.578	Trisaccharide maltose
??4	9.745	Maltose
			??5	11.971	Glucose

Fig. 3: with glucose is the HPLC figure of substrate reactions after product.Qualitative according to retention time, be that the substrate reactions product does not change with glucose.

Table 3 glucose is the retention time and the peak area of substrate reactions after product correspondence

	Retention time (min)	Peak area	Per-cent (%)
				Glucose	11.971	??11563400	??100

Fig. 4: with maltose is the HPLC figure of substrate reactions after product.Qualitative according to retention time, be to have generated Fructus Hordei Germinatus seven sugar, MALTOHAXAOASE, maltopentaose, maltotetrose, trisaccharide maltose and glucose behind the substrate reactions with maltose.

Table 4 maltose is the retention time and the peak area of each component correspondence behind the substrate reactions

Numbering	Retention time (min)	Peak area	Per-cent (%)
				1 Fructus Hordei Germinatus, seven sugar	6.365	??1315361	??11.8167
2 MALTOHAXAOASE	6.817	??493566	??4.4340
				3 maltopentaoses	7.312	??620647	??5.5757
4 maltotetroses	7.844	??1007393	??9.0501
				5 trisaccharide maltoses	8.578	??1370583	??12.3128
6 maltose	9.745	??4512636	??40.5399
				7 glucose	11.971	??1811147	??16.2707

Fig. 5: with the maltotetrose mixture is the HPLC figure of substrate reactions after product.Qualitative according to retention time, be to have generated Fructus Hordei Germinatus seven sugar, MALTOHAXAOASE, maltopentaose, maltotetrose, trisaccharide maltose, maltose and glucose behind the substrate reactions with the maltotetrose mixture.

Table 5 maltotetrose mixture is the retention time and the peak area of each component correspondence behind the substrate reactions

Numbering	Retention time (min)	Peak area	Per-cent (%)
				1 MALTOHAXAOASE, seven sugar	6.423	??8204452	??43.5856
2 maltopentaoses	7.276	??2045276	??10.8654
				3 maltotetroses	7.836	??2426201	??12.8890
4 trisaccharide maltoses	8.581	??2477391	??13.1610
				5 maltose	9.736	??2094668	??11.1278
6 glucose	11.974	??1575791	??8.3713

As seen from the figure, this enzyme does not change the glycosyl activity to glucose, and the least action substrate is a maltose.4-alpha-glycosyl transferring enzyme can act on maltose and the maltotetrose mixture generates Fructus Hordei Germinatus seven sugar, MALTOHAXAOASE, maltopentaose, maltotetrose, trisaccharide maltose, maltose and glucose etc., and this shows that this enzyme has good commentaries on classics glycosyl activity.

Enzyme purification principle among the present invention: this enzyme has certain thermotolerance, and 70 ℃ of heat treated can make a part of heat labile protein denaturation, can realize separating of 4-alpha-glycosyl transferring enzyme and metaprotein by centrifugal; In addition, 4-alpha-glycosyl transferring enzyme has histidine-tagged, can with Ni in the filler ²⁺Fully combination, when enzyme liquid process Ni-NTA affinity column, having histidine-tagged 4-alpha-glycosyl transferring enzyme will be trapped, and other foreign proteins will so just can be realized that 4-alpha-glycosyl transferring enzyme separates with foreign protein by wash-out, adopt energy and Ni again ²⁺Bonded elutriant wash-out can be collected 4-alpha-glycosyl transferring enzyme.

The agent prescription of using in the electrophoresis process:

(1) separation gel damping fluid (Tris-HCl pH of buffer 8.9): get 1mol/L hydrochloric acid 48mL, Tris36.3g is with being settled to 100mL after the deionized water dissolving.

(2) concentrate glue damping fluid (Tris-HCl pH of buffer 6.7): get 1mol/L hydrochloric acid 48mL, Tris5.98g is with being settled to 100mL after the deionized water dissolving.

(3) 30% separation gels storage liquid: claim acrylamide (Acr) 30g and N, N '-methylene diacrylamide (Bis) 0.8g is dissolved in the redistilled water, is settled to 100mL, filters in the rearmounted brown reagent bottle 4 ℃ of preservations.

(4) 10% concentrate glue storage liquid: claim Acr 10g and Bis 0.5g, be dissolved in the redistilled water, be settled to 100mL at last, filter in the rearmounted brown reagent bottle 4 ℃ of storages.

(5) 10%SDS solution: SDS easily separates out crystallization at low temperature, and low-grade fever before using is dissolved it fully.

(6)1％TEMED；

(7) 10% ammonium persulphates (AP): matching while using.

(8) electrophoretic buffer (Tris-glycine buffer pH8.3): take by weighing Tris 6.0g, glycine 28.8g, SDS 1.0g is with being settled to 1L after the deionized water dissolving.

(9) sample buffer: get SDS 100mg, mercaptoethanol 0.1mL, glycerine 1mL, tetrabromophenol sulfonphthalein 2mg, 0.2mol/L, pH7.2 phosphoric acid buffer 0.5mL adds redistilled water to 10mL.

(10) staining fluid: 0.25g Xylene Brilliant Cyanine G G-250 adds 454mL 50% methanol solution and 46mL glacial acetic acid and gets final product.

(11) destainer: 75mL glacial acetic acid, 875mL redistilled water and 50mL methyl alcohol mixing.

(12) separation gel preparation: press table 1 and prepare 20mL 10% separation gel, with elongated dropper coagulant liquid is added in the slit between long and short sheet glass about 8cm height behind the mixing, get a little distilled water with the 1mL syringe, slowly inject along long sheet glass wooden partition, about 3-4mm height is to carry out water seal.Behind about 30min, gel the different boundary line of specific refractory power occurs with the water seal interlayer, then represents the complete polymerization of gel.The distilled water of seal of anhydrating that inclines is inhaled with the filter paper bar and is removed excessive moisture.

(13) preparation of concentrated glue: press table 1 preparation 10mL 3% and concentrate glue, to concentrate glue with elongated dropper behind the mixing and be added to own polymeric separation gel top, until the about 0.5cm of the short sheet glass upper limb of distance place, gently the sample cell template is inserted in the concentrated glue, avoid bringing into bubble.Gel polymerisation behind about 30min is placed 20-30min again.Treat that gel solidifies, carefully take out the sample cell template, inhale with fillet filter paper and go redundant moisture in the sample groove, the pH8.3Tris-glycine buffer is poured in the upper and lower storage tank, should not have more than the about 0.5cm of short slab, can prepare application of sample.

The HPLC separation condition: sugared post, 85 ℃ of column temperatures, moving phase is ultrapure water, flow velocity 0.4mL/min.

Claims (2)

1, the bacterial strain of 4-alpha-glycosyl transferring enzyme is produced in a strain, and its called after colon bacillus (Escherichiacoli) of classifying has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No.3093.

2, a kind of preparation method who produces large cyclodextrin 4-alpha-glycosyl transferring enzyme, it is characterized in that, CGMCC No.3093 is a starting strain with colon bacillus (Escherichia coli), prepare seed culture medium, ferment tank through shake flask fermentation and cultivate, collect that thalline, ultrasonic broken wall extract thick enzyme, purifying obtains pure enzyme 4-alpha-glycosyl transferring enzyme, step is:

1) single bacterium colony is selected: dip in the bacterium liquid that takes a morsel draw the LB flat board that contains 100 μ g/mL ampicillin natriums from genetically engineered bacterial classification CGMCC No.3093 to connect collarium, flat board was placed in the biochemical incubator 37 ℃ of constant temperature culture 12 hours;

2) seed culture: picking list colony inoculation contains in the triangular flask of LB substratum of ampicillin natrium 100 μ g/mL in 50mL, triangular flask is fixed on 37 ℃, 200 rev/mins shaking culture 8h make seed culture medium on the constant temperature shaking table;

3) fermentation culture: seed culture medium is seeded in the fermentor tank of the 3L substratum that contains ampicillin natrium 100 μ g/mL, 37 ℃ of attemperation, 200 rev/mins of stirring velocitys, pH are 7.5, air flow is 10L/h, inoculum size is 10%, add 10mL/L soya-bean oil as defoamer, with this understanding fermentation culture 7h;

4) collect thalline, ultrasonic broken wall extracts thick enzyme: above-mentioned fermented liquid is transferred in the 250mL Centrifuge Cup, and under 4000 rev/mins of conditions centrifugal 20 minutes, collecting precipitation was wet thallus;

Wet thallus is put into pH 7.5,0.05mol/L, the 20mL Tris-HCl buffered soln of prior precooling, the ultrasonic 3s of 400W, interval 2s, ice-bath ultrasonic 10 minutes; To collect supernatant liquor through centrifugal 10 minutes of 4 ℃ of bacterium liquid, the 10000rpm of ultrasonic broken wall treatment, be crude enzyme liquid;

5) purifying of enzyme

With 70 ℃ of crude enzyme liquids insulation 10min,, collect supernatant liquor and be enzyme liquid through preliminary purification then in centrifugal 10 minutes of 4 ℃, 10000rpm;

With 50mM Tris-HCl pH 7.5, the 300mM NaCl of 5 times of column volumes, the rinse damping fluid balance Ni-NTA affinity column of 20mM imidazoles;

Ni-NTA affinity column after will all injecting rinse through the enzyme liquid of preliminary purification;

Rinse damping fluid with 50 times of column volumes carries out wash-out earlier, remove the foreign protein that is not kept by affinity column, use 50mM Tris-HCl pH 7.5, the 300mM NaCl of 2 times of column volumes, the elution buffer wash-out of 250mM imidazoles then, collect elutriant, include target protein;

The elutriant that will contain target protein is filled to the dialysis tubing of molecular weight 1000Da, put into deionization water-bath dialysis desalting behind the tying of dialysis tubing two ends, water-bath stirring at room 12h, change a deionized water every 4h, after desalination is finished dialysis tubing is put in the large beaker that contains polyoxyethylene glycol 20,000 and concentrates, collect concentrated solution behind the 4h and promptly get pure enzyme liquid.

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