CN105112394A - Separation and purification method of lyase capable of degrading aflatoxin B1 - Google Patents

Abstract

The invention discloses a separation and purification method of lyase capable of degrading aflatoxin B1, and belongs to the field of bioengineering. According to the method, the steps of ammonium sulfate fractional precipitation, DEAE FF anion exchange chromatography, Superdux 75 gel filtration chromatography and the like are preformed on bacillus amyloliquefaciens CGMCC NO.9021 fermentation liquor to obtain an active substance capable of degrading the aflatoxin B1; besides, mass spectrum identification and fluorescence chromatography are adopted for analyzing the active protein and the degradation mechanism thereof. It is determined that the active protein, capable of degrading the aflatoxin B1, in the bacillus amyloliquefaciens CGMCC NO.9021 is the lyase with the molecular weight being about 27 kDa, and toxicity is reduced by destroying the lactone ring structure of the aflatoxin B1. The method can guide and optimize a fermentation and purification process for industrially producing the aflatoxin B1 lyase from the bacillus amyloliquefaciens CGMCC NO.9021 and lays an important foundation for industrial production and application of the lyase.

Description

A kind of energy aflatoxin degradation B 1the separation purification method of lyase

Technical field

The present invention relates to a kind of separation purification method of lyase of energy degrading aflatoxin B 1, belong to bioengineering field.

Background technology

The aflatoxin strong toxicity secondary metabolite with a coumarin and a bifuran that to be a class produce primarily of flavus (Aspergillusflavus) and Aspergillus parasiticus (Aspergillusparasiticus).The aflatoxin identified at present has kind more than 20, and AFB ₁being modal in grain, is also that toxicity is maximum, is decided to be 1A class carcinogenic substance by the World Health Organization.

AFB ₁in grain, there is the health that animal and human body in serious threat, research be efficient, safe disposal AFB ₁become the focus of current concern.At various AFB ₁in minimizing technology, there is the nutritive substance composition of destruction grain due to Physical and chemical method or cause chemical residue and affect the deficiencies such as palatability, current biological detoxication is generally considered to the most promising method.Biological detoxication refers to that the toxophore on lps molecule is decomposed by microorganism or its meta-bolites and destroys, and produces the process of non-toxic degradation products simultaneously.Have been found that at nature and comprise bacterium, fungi (containing yeast) can adsorb the material of mycotoxins or secretion degradative fungi toxin at interior multiple-microorganism, and the detoxification mechanism of these microorganisms mainly contains: the Degradation of the enzyme contratoxin that the adsorption of somatic cells contratoxin and fermentation produce.

We screened 1 strain and not only can obviously suppress flavus grow early stage, and energy efficient degradation AFB ₁safe bacterial strain, at present preservation to China General Microbiological culture presevation administrative center, called after bacillus amyloliquefaciens CGMCCNO.9021.To bacillus amyloliquefaciens CGMCCNO.9021 degraded AFB ₁characteristic research find, AFB ₁degraded be not rely on thalline absorption, but the outer active substance of the born of the same parents produced due to its metabolism, this active substance is a kind of protein.But, and the AFB of great majority degraded at present ₁bacterial classification research is similar, and because target protein is indefinite, fermentation technology optimization work is difficult to carry out, and causes heavy industrialization application work progress slow.

Summary of the invention

For solving the problem, the invention provides a kind of energy aflatoxin degradation B ₁the separation purification method of lyase.The present invention obtains in bacillus amyloliquefaciens CGMCCNO.9021 fermented liquid by steps such as ammonium sulfate precipitation, DEAEFF anion-exchange chromatography and Superdux75 gel permeation chromatographies the AFB that degrades ₁active substance, by Mass Spectrometric Identification and this activated protein of fluorescence chromatogram technical Analysis and mechanism of degradation thereof.

Described method is from the fermented liquid of bacillus amyloliquefaciens CGMCCNO.9021, utilizes ammonium sulfate precipitation, DEAEFF anion-exchange chromatography and Superdux7510/300GL gel permeation chromatography to carry out separation and purification successively and obtains AFB ₁lyase.

Described method, in one embodiment of the invention, comprising: (1) ammonium sulfate precipitation: centrifugal after bacillus amyloliquefaciens CGMCCNO.9021 fermentation liquor 0-40% ammonium sulfate precipitation, is then redissolved by the albumen of precipitation, dialysis; (2) protein solution after dialysis, after filtering with microporous membrane, is splined on DEAEFF anion-exchange column, rinses with phosphate buffered saline buffer, then carries out permanent gradient elution with the phosphate buffered saline buffer containing 1mol/LNaCl, collects and have degraded AFB ₁active component; (3) previous step is collected the active ingredient obtained and be splined on Superdux7510/300GL gel permeation chromatography post, carry out wash-out with phosphate buffered saline buffer, collect and there is degraded AFB ₁active component.

Described step (2), in one embodiment of the invention, middle collection be the protein solution at second absorbing proteins peak.

Described step (3), in one embodiment of the invention, collection be the protein solution at the 3rd absorbing proteins peak.

Described ammonium sulfate precipitation in one embodiment of the invention, is use 20-40% ammonium sulfate precipitation.

Described redissolution in one embodiment of the invention, is the phosphate buffered saline buffer using 20mmol/L, pH7.0.

Described phosphate buffered saline buffer, in one embodiment of the invention, concentration is 10-50mmol/L, pH6.8-7.4.

Described phosphate buffered saline buffer, in one embodiment of the invention, concentration is 20mmol/L, pH7.0.

Described method, in one embodiment of the invention, specifically comprises:

(1) bacillus amyloliquefaciens CGMCC-9021 fermentation liquor 40% ammonium sulfate precipitation, then in 4 DEG C, the centrifugal 10min of 10000r/min, redissolves the phosphate buffered saline buffer of protein precipitation with 20mmol/L, pH7.0, dialysed overnight;

(2) protein solution after dialysis is by 0.22 μm of filtering with microporous membrane, be splined on HiPrep16/10DEAEFF ion exchange column, rinse with the phosphate buffered saline buffer of 20mmol/L, pH7.0, then carry out permanent gradient elution with the phosphate buffered saline buffer (20mmol/L, pH7.0) containing 1mol/LNaCl, collect second absorbing proteins peak;

(3) protein solution that previous step is collected is splined on Superdux7510/300GL gel permeation chromatography post, carries out wash-out with the phosphate buffered saline buffer of 20mmol/L, pH7.0, collect the 3rd absorbing proteins peak.

Present invention also offers a kind of AFB ₁the authentication method of lyase.

Described authentication method is: collect in each step of separation and purification and have degraded AFB ₁active composition carries out Tricine-SDS-PAGE analysis, analyze band total in each swimming lane, total band (i.e. target stripe) is selected manually to cut glue, carry out Mass Spectrometric Identification (MALDI-TOF/TOF), in NCBInr database, carry out search coupling with Mascot software to the mass-spectrometric data of protein, Mascot score value is greater than 59 can assert that identification is effective.

Present invention also offers a kind of AFB ₁the aflatoxin mechanism of degradation analytical procedure of lyase.

Described analytical procedure is by AFB ₁standardized solution and degraded AFB ₁active substance solution add in brown reaction flask, the reaction of 37 DEG C of lucifuges.The long-pending trichloromethane of triploid is adopted to carry out liquid-phase extraction AFB ₁, the methanol solution concussion adding 50% is afterwards dissolved, under the excitation wavelength of 365nm, measure fluorescence intensity, infers mechanism of degradation by the change of fluorescence intensity.

Beneficial effect of the present invention: the AFB that electrophoretically pure bacillus amyloliquefaciens CGMCCNO.9021 originates has been arrived in successful separation and purification ₁lyase, activated protein is that molecular weight is about 27kDa.Lyase of the present invention is by destroying AFB ₁lactonic ring structure reduce toxicity, the inventive method can instruct optimization solution bacillus amyloliquefaciens CGMCCNO.9021 industrialization produce AFB ₁the fermentation of lyase and purifying process, establish the important foundation of its suitability for industrialized production application.

Accompanying drawing explanation

Fig. 1: ammonium sulfate precipitation each several part is to AFB ₁degradation;

Fig. 2: HiPrep16/10DEAEFF column chromatography wash-out collection of illustrative plates;

Fig. 3: Superdux7510/300GL column chromatography wash-out collection of illustrative plates;

Fig. 4: Tricine-SDS-PAGE collection of illustrative plates; Wherein: M is standard specimen, 1 is supernatant liquor, and 2 is 40% ammonium sulfate precipitation solution, and 3 is L1 (chromatography of ions peak 1), and 4 is L2 (chromatography of ions peak 2), and 5 is N3 (gel chromatography peak 3);

Fig. 5: the mass spectrometry results of protein; Note: the MALDI-TOF that (a) is band 1 analyzes, the MALDI-TOF-TOF that (b) ~ (f) is band 1 analyzes;

Fig. 6: bacillus amyloliquefaciens lyase is to AFB ₁the impact of fluorescent characteristic;

Fig. 7: bacillus amyloliquefaciens lyase is to AFB ₁the possible mechanism of action.

Embodiment

Embodiment 1: AFB ₁the separation of lyase, purifying, qualification

(1) bacillus amyloliquefaciens CGMCCNO.9021 fermentation liquor saturated ammonium sulphate fractionation precipitation, measures various piece protein precipitation to AFB ₁degradation effect, result is as shown in Figure 1.Finally determine that saturated ammonium sulphate interval is for 0-40%.After fermentation liquor 40% saturated ammonium sulphate, 4 DEG C, the centrifugal 10min of 10000r/min, redissolves each several part protein precipitation 20mmol/L phosphate buffered saline buffer (pH7.0), dialysed overnight.

(2) protein solution after dialysis is by 0.22 μm of filtering with microporous membrane, be splined on HiPrep16/10DEAEFF ion exchange column, rinse with phosphate buffered saline buffer (20mmol/L, pH7.0), then permanent gradient elution is carried out with the phosphate buffered saline buffer (20mmol/L, pH7.0) containing 1mol/LNaCl, collect 2 absorbing proteins peaks, as shown in Figure 2, difference called after L1 and L2.Measure L1, L2 to AFB ₁degraded situation.Wherein, L2 has higher degraded AFB ₁activity, its degradation rate is the degradation rate of 80.3%, L1 is 17.0%.Therefore, the active substance of degraded AFB1 mainly concentrates on second chromatographic peak, collects second chromatographic peak, and measures its protein concentration, volume, be stored in 4 DEG C with for subsequent use.

(3) by Superdux7510/300GL gel filtration chromatography, L2 chromatographic peak is separated into 4 chromatographic peaks, respectively called after N1, N2, N3, N4, result as shown in Figure 3, and measures it to AFB ₁degradation effect, wherein N3 has obvious degradation AFB ₁activity, degradation rate is 32.4%.Collect the 3rd chromatographic peak, and its protein concentration, volume are measured, be stored in 4 DEG C with for subsequent use.

(4) collect in each step of separation and purification and there is degraded AFB ₁active composition carries out Tricine-SDS-PAGE analysis, and electrophoresis result as shown in Figure 4, can find out in 5 swimming lanes all have a total band, and N3 only has this band itself, illustrates that it may be the AFB that degrades from Tricine-SDS-PAGE collection of illustrative plates ₁active substance, and molecular weight about 27kDa.

(5) protein band 1 in Tricine-SDS-PAGE swimming lane 5 is dug carry out enzyme a little and cut, then carried out tandem mass spectrum (MALDI-TOF/TOF) to analyze and albumen database (NCBInr) retrieval, matching result is in table 1, and Fig. 5 is shown in mass spectroscopy.This protein site qualification result is lyticenzymeL27 [Bacillussubtilis] (accession number is gi|3986320), its coupling must be divided into 494, protein molecular weight (Mr) is 27479, and isoelectric points of proteins (pI) is 6.65.

The peptide sequence data mated in table 1 mass spectroscopy

Table1TheinformationofmatchpeptidesequencebyMassSpectrometryanalysis

Note: accession number: gi|3986320, molecular weight: 27479, lyticenzymeL27 [Bacillussubtilis].

(6) by 25 μ LAFB ₁standardized solution, the degraded AFB that 975 μ L are aseptic ₁active substance solution add in brown reaction flask, make AFB in mixing solutions ₁concentration be 1000ng/mL, 37 DEG C of lucifuges react 0 day, 1 day, 3 days.The trichloromethane that triploid amasss in reaction solution carries out liquid-phase extraction AFB ₁, the methanol aqueous solution concussion adding 1.5mL50% is afterwards dissolved, and measures the AFB under differential responses condition under the excitation wavelength of 365nm ₁fluorescence intensity.AFB ₁after the different time process of bacillus amyloliquefaciens lyase, its fluorescent characteristic there occurs obvious change, along with the increase in reaction times, and AFB ₁fluorescent characteristic constantly reduce, namely also the chemical structure of its degraded product there occurs change, and fluorescent characteristic disappears, as shown in Figure 6.Infer that bacillus amyloliquefaciens lyase is to AFB ₁action site may be lactonic ring structure, as shown in Figure 7.

Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (10)

1. an energy aflatoxin degradation B ₁the separation purification method of lyase, it is characterized in that, described method is from the fermented liquid of bacillus amyloliquefaciens CGMCCNO.9021, and utilizing ammonium sulfate precipitation, DEAEFF anion-exchange chromatography and Superdux7510/300GL gel permeation chromatography successively, separation and purification obtains.

2. method according to claim 1, it is characterized in that, described method comprises: (1) ammonium sulfate precipitation: centrifugal after bacillus amyloliquefaciens CGMCCNO.9021 fermentation liquor 0-40% ammonium sulfate precipitation, is then redissolved by the albumen of precipitation, dialysis; (2) protein solution after dialysis, after filtering with microporous membrane, is splined on DEAEFF anion-exchange column, rinses with phosphate buffered saline buffer, then carries out permanent gradient elution with the phosphate buffered saline buffer containing 1mol/LNaCl, collects and have degraded AFB ₁active component; (3) previous step is collected the active ingredient obtained and be splined on Superdux7510/300GL gel permeation chromatography post, carry out wash-out with phosphate buffered saline buffer, collect and there is degraded AFB ₁active component.

3. method according to claim 2, is characterized in that, the concentration of described phosphate buffered saline buffer is 10-50mmol/L, pH6.8-7.4.

4. method according to claim 1, is characterized in that, described ammonium sulfate precipitation uses 20-40% ammonium sulfate precipitation.

5. method according to claim 2, is characterized in that, the concentration of described phosphate buffered saline buffer is 20mmol/L, pH7.0.

6. method according to claim 2, is characterized in that, described redissolution is the phosphate buffered saline buffer using 20mmol/L, pH7.0.

7. method according to claim 2, is characterized in that, what collect in described step (2) is the protein solution at second absorbing proteins peak.

8. method according to claim 1, is characterized in that, described method specifically comprises:

(1) bacillus amyloliquefaciens CGMCCNO.9021 fermentation liquor 40% ammonium sulfate precipitation, then in 4 DEG C, the centrifugal 10min of 10000r/min, redissolves the phosphate buffered saline buffer of protein precipitation with 20mmol/L, pH7.0, dialysed overnight;

(2) protein solution after dialysis is by 0.22 μm of filtering with microporous membrane, be splined on HiPrep16/10DEAEFF ion exchange column, rinse with the phosphate buffered saline buffer of 20mmol/L, pH7.0, then carry out permanent gradient elution with the phosphate buffered saline buffer of 20mmol/L, pH7.0 containing 1mol/LNaCl, collect second absorbing proteins peak;

(3) protein solution that previous step is collected is splined on Superdux7510/300GL gel permeation chromatography post, carries out wash-out with the phosphate buffered saline buffer of 20mmol/L, pH7.0, collect the 3rd absorbing proteins peak.

9. the lyase that obtains of the arbitrary described method of claim 1-8.

10. the application of lyase described in claim 9 in aflatoxin degradation.