CN104073479A

CN104073479A - Method for purifying alliinase by double-water-phase separation

Info

Publication number: CN104073479A
Application number: CN201410298149.2A
Authority: CN
Inventors: 崔波; 姜秀敏; 檀琮萍; 卢艳敏
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2014-06-27
Filing date: 2014-06-27
Publication date: 2014-10-01

Abstract

The invention belongs to the field of deep processing of garlic, and particularly discloses a method for purifying alliinase by double-water-phase separation. The method comprises the following steps: (1) peeling and precooling garlic squamous bulbs, immersing in a precooled extract, homogenizing, centrifuging, and discarding garlic slag, thereby obtaining the supernate crude enzyme solution; (2) dissolving ammonium sulfate, sodium dihydrogen phosphate or trisodium citrate in a Britton-Robinson buffer solution, oscillating to completely dissolve, adding a PEG (polyethylene glycol) solution, evenly mixing by oscillation, and standing for phase splitting, thereby obtaining a double-water-phase extractant; (3) adding the crude enzyme solution into the double-water-phase extractant, oscillating, centrifuging, and standing for phase splitting; and (4) ultrafiltering the lower phase of the enriched alliinase, and carrying out vacuum freeze drying on the trapped fluid to obtain the alliinase solid. The method is simple to operate, has the advantages of simple apparatuses, time saving, low cost, and higher alliinase purification multiple and enzyme activity recovery rate than the prior art, and can easily implement scale-up production.

Description

A kind of method of double water-phase separation and purification allinase

(1) technical field

The invention belongs to garlic intensive processing field, particularly a kind of method of double water-phase separation and purification allinase.

(2) background technology

Allinase is a kind of endogenous enzyme being present in garlic, full name Alliinase, account for greatly the 10-12% of soluble proteins in garlic, formed by two identical subunits, each subunit is made up of 448 amino acid, allinase and its natural substrate alliin are positioned at the different sites of garlic, allinase is arranged in vacuole tissue, substrate is arranged in tenuigenin, in the time that garlic is broken, alliin contacts the thiosulfinate of producing and has anticancer with allinase, anti-oxidant, the effects such as atherosclerosis, can prepare the production of thiosulfinate for garlic capsule with allinase and alliin, therefore the separation and purification of allinase is very important for the production of thiosulfinate.

Allinase purifying mainly adopts a series of protein purification technology at present, as saltout, affinity chromatography, gel-filtration etc., although these methods can make allinase reach certain purifying, but there is shortcomings, as complex operation, condition harshness, sepn process easily makes protein denaturation, reduces the rate of recovery of enzyme work and enzyme.Allinase enzyme in the time that temperature is greater than 30 DEG C is lived and can be reduced, so purifying allinase is to the having relatively high expectations of environment, finds a kind of method of applicable allinase purifying particularly important.The invention provides a kind of double water-phase method, the separation purification method that this kind method is simple to operate, operational condition is gentle, be easy to amplification, consumed energy is little, be the major subjects of current people's research and development, carry out in this way purifying allinase and can reach the requirement that enzyme is lived.

(3) summary of the invention

The present invention, in order to make up the deficiencies in the prior art, provides one to reduce production costs, and improves the enzyme rate of recovery alive, improves the method for the bioactive double water-phase separation and purification of product allinase.

The present invention is achieved through the following technical solutions:

A method for double water-phase separation and purification allinase, taking garlic bulb as raw material, comprises the steps:

(1) garlic bulb is removed the peel to precooling, add the precooling extracting solution of 4 DEG C to soak, homogenate in the tissue refiner of precooling, centrifugal, discards garlic slag, and supernatant liquor is crude enzyme liquid;

(2) ammonium sulfate, SODIUM PHOSPHATE, MONOBASIC or trisodium citrate are dissolved in Bloomsbury smooth-Robinson, Robert damping fluid in, concussion then adds PEG solution after dissolving completely, concussion mixes, and leaves standstill phase-splitting, obtains double water-phase extraction agent;

(3) crude enzyme liquid is placed in to double water-phase extraction agent, after concussion mixes, centrifugal standing phase-splitting;

(4) the lower phase ultrafiltration to enrichment allinase, to trapped fluid vacuum lyophilization, obtains allinase solid.

More excellent technical scheme of the present invention is:

In step (1), the pH of precooling extracting solution is 7.0, the sodium-chlor that the glycerine that the EDTA that contains 0.05mol/L SODIUM PHOSPHATE, MONOBASIC, 0.05mol/L potassium primary phosphate, 5mmol/L, the PLP of 25 μ mol/L, volume ratio are 10% and weight ratio are 1%, the use of garlic bulb and precooling extracting solution is than being 1g:50mL, and soak time is 20-40min.

In step (2), double water-phase extraction agent is ammonium sulfate/PEG system, and the molecular weight of PEG is 1000-3000, and the mass concentration of ammonium sulfate is 10-18%(w/w), the mass concentration of PEG is 7.5-17.5%(w/w).

In step (3), the pH of double water-phase extraction agent is 2.0-5.0, and crude enzyme liquid add-on is 3-6%(v/v).

Enzyme activity determination method is:

Taking the S-allyl-L-ceisteine that synthesizes as substrate, measure enzymic activity with acetone method: 1mL enzyme liquid adds under 1L substrate (adding 25 mol/LPLP in system) room temperature and reacts after 1min, add equivalent 10% trichoroacetic acid(TCA) termination reaction, add 2 of 1mL0.1%, 25 DEG C of constant temperature 5min of 4-dinitrophenylhydrazine, add again 5mL2.5mol/L sodium hydroxide to shake up after colour developing 10min, survey the absorbance value (OD) at wavelength 420nm place with 756 ultraviolet spectrophotometers.Under 25 DEG C of conditions, in the enzymatic reaction of alliin, per minute produces 1 mol pyruvic acid and is defined as a Ge Meihuo unit (U).

The present invention is simple to operate, and instrument is simple, on the time, saves, with low cost, allinase purification and the enzyme rate of recovery alive is all high than prior art, sepn process economy, environment gentleness, activity influence to allinase is less, can save the step of repeated precipitation, is easy to amplify scale production.

(4) embodiment

The preparation of crude enzyme liquid: take garlic bulb 100g, peeling, 4 DEG C of precoolings, add 4 DEG C of precooling extracting solutions (pH7.0,0.05mol/L SODIUM PHOSPHATE, MONOBASIC, 0.05mol/L potassium primary phosphate, the glycerine that volume ratio is 10%, 1% sodium-chlor, the EDTA of 5mmol/L, the PLP of 25 mol/L) lixiviate 30min, homogenate in the tissue refiner of precooling, the centrifugal 10min of 3000rpm, discards garlic slag, supernatant liquor is crude enzyme liquid, and the enzyme of measuring is wherein lived and protein content.

Embodiment 1:

Preparation massfraction is 7.5% (w/w) PEG3000/10% (w/w) ammonium sulfate, the double-aqueous phase system of pH=5.0, the allinase crude extract of interpolation 5% (v/v), concussion 15min makes to disperse completely mutually, centrifugal making separates mutually completely, records upper and lower phase volume.To lower remove and desalt by ultrafiltration after, vacuum lyophilization, obtaining enzyme purification multiple is 5.06, the enzyme rate of recovery of living is 92.2%.

Embodiment 2:

Preparation massfraction is 17.5% (w/w) PEG3000/18% (w/w) ammonium sulfate, the double-aqueous phase system of pH=5.0, the allinase crude extract of interpolation 5% (v/v), concussion 15min makes to disperse completely mutually, centrifugal making separates mutually completely, records upper and lower phase volume.To lower remove and desalt by ultrafiltration after, vacuum lyophilization, obtaining enzyme purification multiple is 5.58, the enzyme rate of recovery of living is 94.4%.

Embodiment 3:

Preparation massfraction is 15% (w/w) PEG3000/14% (w/w) ammonium sulfate, the double-aqueous phase system of pH=5.0, the allinase crude extract of interpolation 5% (v/v), concussion 15min makes to disperse completely mutually, centrifugal making separates mutually completely, records upper and lower phase volume.To lower remove and desalt by ultrafiltration after, vacuum lyophilization, obtaining enzyme purification multiple is 5.95, the enzyme rate of recovery of living is 96.2%.

Claims

1. the method for a double water-phase separation and purification allinase, taking garlic bulb as raw material, it is characterized by, comprise the steps: that garlic bulb removed the peel precooling by (1), add the precooling extracting solution of 4 DEG C to soak, homogenate in the tissue refiner of precooling, centrifugal, discards garlic slag, and supernatant liquor is crude enzyme liquid; (2) ammonium sulfate, SODIUM PHOSPHATE, MONOBASIC or trisodium citrate are dissolved in Bloomsbury smooth-Robinson, Robert damping fluid in, concussion then adds PEG solution after dissolving completely, concussion mixes, and leaves standstill phase-splitting, obtains double water-phase extraction agent; (3) crude enzyme liquid is placed in to double water-phase extraction agent, after concussion mixes, centrifugal standing phase-splitting; (4) the lower phase ultrafiltration to enrichment allinase, to trapped fluid vacuum lyophilization, obtains allinase solid.

2. the method for double water-phase separation and purification allinase according to claim 1, it is characterized in that: in step (1), the pH of precooling extracting solution is 7.0, the sodium-chlor that the glycerine that the EDTA that contains 0.05mol/L SODIUM PHOSPHATE, MONOBASIC, 0.05mol/L potassium primary phosphate, 5mmol/L, the PLP of 25 μ mol/L, volume ratio are 10% and weight ratio are 1%, the use of garlic bulb and precooling extracting solution is than being 1g:50mL, and soak time is 20-40min.

3. the method for double water-phase separation and purification allinase according to claim 1, is characterized in that: in step (2), double water-phase extraction agent is ammonium sulfate/PEG system.

4. the method for double water-phase separation and purification allinase according to claim 1, is characterized in that: in step (3), the pH of double water-phase extraction agent is 2.0-5.0, and crude enzyme liquid add-on is 3-6%(v/v).

5. the method for double water-phase separation and purification allinase according to claim 3, it is characterized in that: in step (2), the molecular weight of PEG is 1000-3000, and the mass concentration of ammonium sulfate is 10-18%(w/w), the mass concentration of PEG is 7.5-17.5%(w/w).