CN104212775A - Preparation method of nitrite reductase of lactobacillus casei subsp rhamnosus - Google Patents

Preparation method of nitrite reductase of lactobacillus casei subsp rhamnosus Download PDF

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CN104212775A
CN104212775A CN201410437877.7A CN201410437877A CN104212775A CN 104212775 A CN104212775 A CN 104212775A CN 201410437877 A CN201410437877 A CN 201410437877A CN 104212775 A CN104212775 A CN 104212775A
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nitrite reductase
nir
enzyme
nitrite
lactobacillus casei
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刘冬梅
陈浩
周劲松
张馨月
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South China University of Technology SCUT
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    • C12Y107/01—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with NAD+ or NADP+ as acceptor (1.7.1)
    • C12Y107/01004—Nitrite reductase [NAD(P)H] (1.7.1.4)

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Abstract

The invention discloses a preparation method of nitrite reductase of lactobacillus casei subsp rhamnosus. The method comprises the following steps: firstly, carrying inductive culture of nitrite reductase on lactobacillus casei subsp rhamnosus 6013; adding an enzyme protective agent to extract coarse nitrite reductase when the wall is broken; after carrying out vacuum-freezing and drying, finally preparing pure nitrite reductase sequentially through anion exchange chromatography and/or sephadex chromatography. The activity of NiR extracted from lactobacillus casei subsp rhamnosus is high and the condition for degrading nitrite reductase by NiR is simple. According to the disclosed condition, the enzyme activity loss of NiR is less, and the obtained NiR is high in purity and can be used for solving problem of nitrite in three fields such as vegetable fermentation, meat products and aquatic water.

Description

A kind of preparation method of nitrite reductase of lactobacillus casei subsp.rhamnosus
Technical field
The present invention relates to field of biological product, is a kind of preparation method of lactobacillus casei subsp.rhamnosus Lactobacillus casei subsp.rhamnosus6013 (referred to as LCR 6013) nitrite reductase enzyme.
Background technology
Nitrite is a kind of potential carcinogenic substance, very easily accumulate in vegetable fermentation process, bring potential food-safety problem to product, and Excess free enthalpy nitrite can bring out methemoglobinemia, the content therefore strictly controlling nitrite in food is extremely important.Much research shows that nitrite is the precursor of nitrosamine, and nitrosamine is a kind of strong carcinogen, can bring out the multiple canceration in Digestive tract, as cancer of the stomach, intestinal cancer and liver cancer.In view of food may exist the nitrite pollution that exceeds standard, potential food-safety problem containing nitrite in meat product, aquaculture system nitrite exceeds standard a series of problems such as causing potential food-safety problem, the method of seeking effectively control or degrading nitrite is imperative, except the microorganism adding edible safety, nitrite reductase (Nitrite reductase, referred to as NiR) also can be utilized to carry out the biological degradation of nitrite.But, at present good method also be there is no to the NiR how obtaining a large amount of edible safeties.NiR is a kind of oxydo-reductase, needs the participation of electron transit mediator in reaction process, and is subject to the suppression of oxygen.
In the method for degrading nitrite, microbial degradation method is the most frequently used method, mainly contain Rhodopseudomonas [Chen Ruifang, Xu Changan, Liu Limei, Deng. the nitrite degradation characteristic of a pseudomonas and the cleaning action to fishpond bed mud thereof, research report, 2011,35 (6): 30-33] and milk-acid bacteria (Zhang Xinyue, Liu Dongmei, Xu Xilin, Deng the approach of .LCR 6013 degrading nitrite and the Primary Location of nitrate reductase thereof, modern food science and technology, 2013,11 (29): 2627-2632) etc.Potential toxicity and risk is there is because pseudomonas falls nitrite for food, and from the lactobacterium casei of edible safety, extract nitrite reductase have no report, application number be 201110207974.3 Chinese invention patent disclose " a kind of production method of nitrite reductase from lactobacillus ", this method utilizes simple Mechanical Crushing and centrifugal method to collect thick enzyme, owing to containing a large amount of foreign proteins in crude enzyme liquid, make follow-up abstraction and purification work complicated, target product NiR cannot be obtained in a large number, in addition, medium component for producing enzyme in this method contains Tomato juice, due to the reason of tomato redness, make in final NiR inevitable with haematochrome, affect follow-up purification work and application.
Summary of the invention
The object of the invention is the preparation method providing NiR in a kind of lactobacterium casei, LCR 6013 thalline containing nitrite reductase is gone out by inducing culture, crude enzyme liquid is prepared again, finally by anion chromatography post and the pure nitrite reductase of sephadex chromatography column chromatography for separation by the method for a series of broken wall.
The present invention can be achieved through the following technical solutions:
A preparation method for the nitrite reductase of lactobacillus casei subsp.rhamnosus, comprises the steps and processing condition:
(1) lyophilized powder of lactobacillus casei subsp.rhamnosus (Lactobacillus casei subsp.rhamnosus) 6013 is placed in MRS substratum, after 30 DEG C ~ 40 DEG C static gas wave refrigerator 18h ~ 36h, makes seed liquor; Described lactobacillus casei subsp.rhamnosus, purchased from Chinese industrial Microbiological Culture Collection administrative center, is numbered CICC6013;
(2) with 1% ~ 10% volume percent, above-mentioned seed liquor is seeded in inducing culture A, in 30 DEG C ~ 40 DEG C static inducing culture 18h ~ 24h, obtains induction working stock culture; Described inducing culture A formula is: containing inductor NaNO 2the MRS substratum of 5 μ g/mL ~ 20 μ g/mL;
(3) with 5% ~ 15% volume percent, induction working stock culture is seeded in inducing culture B, in 30 DEG C ~ 40 DEG C static inducing culture 24h ~ 36h, must containing the nutrient solution of nitrite reductase; Described inducing culture B formula is: containing inductor NaNO 2the MRS substratum of 10 μ g/mL ~ 50 μ g/mL;
(4) nutrient solution containing nitrite reductase is carried out centrifugal, with centrifugal after physiological saline cleaning, must containing the thalline of nitrite reductase;
(5) add and carry enzyme buffer liquid, the cell concentration containing nitrite reductase is made to be 150mg/mL ~ 400mg/mL, add N,O-Diacetylmuramidase (purchased from Sigma-Aldrich, Shanghai, article No.: 62971-50G-F, enzyme is lived: 70000U/mg) 10mg/L, and broken wall 1h ~ 5h after mixing, the supernatant liquor of broken wall solution after centrifugal is nitrite reductase crude enzyme liquid.
The concentration of step (5) described N,O-Diacetylmuramidase is 10mg/mL ~ 50mg/mL.
Carry described in step (5) containing trypsin inhibitor and dithiothreitol (DTT) in enzyme buffer liquid, its concentration is respectively 2 μ g/mL and 2.0 μm ol/mL ~ 20.0, μ g/mL ~ 10 μm ol/mL.
Described nitrite reductase crude enzyme liquid, through vacuum lyophilization, obtains the thick enzyme powder of nitrite reductase; Described nitrite reductase thick enzyme powder also passes through following purification procedures: be dissolved in by thick enzyme powder in HEPES damping fluid and obtain thick enzyme powder liquid, more successively through anion-exchange chromatography and/or sephadex chromatography purifying, finally obtained pure nitrite reductase.
Described anion-exchange chromatography adopts high flow rate ionic sepharose (DEAE Sepharose Fast Flow) anion-exchange chromatography post, and described sephadex chromatography adopts dextrane gel (Sephadex G-150) chromatography column.
Described anion-exchange chromatography is by the thick enzyme powder liquid loading Zhiyin ion exchange column that dissolves in HEPES damping fluid, first with HEPES damping fluid, protein is washed till baseline, the NaCl solution adding 0mM ~ 600mM again in above-mentioned HEPES damping fluid carries out gradient elution, fraction collection, the nitrite reductase enzyme measuring each elution peak is lived, merge the component with nitrite reductase, and concentrate with PEG 20000.
Described sephadex chromatography is that carry out wash-out with Tris-HCl damping fluid, fraction collection, the nitrite reductase enzyme measuring each elution peak is lived, and merges the component having nitrite reductase enzyme and live by sample loading on sephadex chromatography post.
Described HEPES buffer concentration is 20mM ~ 80mM, pH is 7.0 ~ 8.6; Described Tris-HCl buffer concentration is 20mM ~ 80mM, pH is 7.8 ~ 8.6.
In described anion-exchange chromatography and sephadex chromatography, the flow velocity of wash-out is respectively 0.5mL/min ~ 1.5mL/min and 0.1mL/min ~ 0.6mL/min.
In described thick enzyme powder liquid, the concentration of thick enzyme powder is 3 ~ 20mg/mL.
Compared with prior art, tool has the following advantages in the present invention:
(1) the product NiR of the lactobacillus casei subsp.rhamnosus Lactobacillus casei subsp rhamnosus6013 used in the present invention stablizes, and required substratum wide material sources, production cost is low, has good commercial promise.
(2) the NiR vigor prepared by is high, and the condition of NiR degrading nitrite is simple, and the suitableeest enzyme catalysis temperature is 37 DEG C, and optimal pH is 7.2, the highest containing activity in 150mM/L NaCl solution, have stable NiR enzyme and live in 30 DEG C ~ 40 DEG C.
(3) the method disclosed in the present can make the enzyme of NiR live loss less, and the NiR purity obtained is high, and NiR can solve the problem containing nitrite in 3 fields such as vegetable fermentation, meat product and aquaculture water.
Accompanying drawing explanation
Fig. 1 is the protein curve figure (wherein there is NiR activity at the 3rd peak) after LCR 6013 crude enzyme liquid anion-exchange chromatography post DEAE Sepharose Fast Flow is separated.
Fig. 2 is the protein curve figure after the protein sephadex chromatography post Sepharose G-150 at LCR 6013 crude enzyme liquid the 3rd peak after anion column is separated is separated.
Embodiment
For better understanding the present invention, below in conjunction with embodiment the present invention done and describe in detail further, but the scope of protection of present invention being not limited to the scope that embodiment represents.
Embodiment 1
(1) lyophilized powder of lactobacillus casei subsp.rhamnosus is placed in MRS substratum, after 37 DEG C of static gas wave refrigerator 24h, makes seed liquor; Described lactobacillus casei subsp.rhamnosus, purchased from Chinese industrial Microbiological Culture Collection administrative center, is numbered CICC6013, called after Lactobacillus casei subsp.rhamnosus6013 (referred to as LCR 6013).
(2) with 5% volume percent, LCR 6013 seed liquor is seeded in inducing culture A, in 37 DEG C of static inducing culture 24h, obtains induction working stock culture.In mass body volume concentrations, described inducing culture A formula is: containing inductor NaNO 2the MRS substratum of 10 μ g/mL.
(3) with 10% volume percent, induction working stock culture is seeded in inducing culture B, in 37 DEG C of static inducing culture 36h, must containing the nutrient solution of NiR.In mass body volume concentrations, described inducing culture B formula is: containing inductor NaNO 2the MRS substratum of 15 μ g/mL.
(4) nutrient solution containing NiR is carried out centrifugal, centrifugal after cleaning 2 times with physiological saline, must LCR 6013 thalline of NiR be contained and weigh.
(5) add and carry enzyme buffer liquid containing trypsin inhibitor 2 μ g/mL and dithiothreitol (DTT) 5.0 μm of ol/mL, LCR 6013 cell concentration is made to be 150mg/mL, add N,O-Diacetylmuramidase (purchased from Sigma-Aldrich, Shanghai, article No.: 62971-50G-F, enzyme is lived: 70000U/mg) 10mg/L, broken wall 2h after mixing, the supernatant liquor of broken wall solution after centrifugal is LCR 6013 NiR crude enzyme liquid, and its NiR enzyme is lived as 165U.
(6) LCR 6013 NiR crude enzyme liquid is carried out vacuum lyophilization, obtain the thick enzyme powder of LCR 6013 NiR.
(7) with concentration 3mg/mL, thick for LCR 6013 NiR enzyme powder is dissolved in the 50mM HEPES damping fluid of pH7.8, for the thick enzyme powder solution of LCR 6013, chromatography is carried out again through high flow rate ionic sepharose DEAE Sepharose Fast Flow anion-exchange chromatography post, anion-exchange chromatography first balances with identical HEPES damping fluid, then by thick for LCR 6013 enzyme powder solution loading Zhiyin ion exchange column, the NaCl solution adding 0mM ~ 250mM again in above-mentioned HEPES damping fluid carries out gradient elution, elution speed is 1.0mL/min, fraction collection, the NiR enzyme measuring each elution peak is lived, merge the component with NiR, and concentrate with PEG 20000, as shown in Figure 1, the NaCl wash-out concentration at three peaks is respectively 0mM, 50mM, 250mM to elution curve, and there is the activity of NiR at the 3rd peak.
(8) dextrane gel SephadexG-150 chromatography column first balances with the 20mM Tris-HCl damping fluid of pH7.8, again after anion chromatography the nitrite reductase component loading of gained to sephadex chromatography post, wash-out is carried out with identical Tris-HCl damping fluid, elution speed is 0.5mL/min, elution curve as shown in Figure 2, fraction collection, the NiR enzyme measuring often pipe in elution peak lives (12 ~ 15 pipes have activity), merge the component that there is NiR enzyme and live, obtain the pure enzyme liquid of NiR, its NiR enzyme is lived as 2510U.
Take Sodium Nitrite as substrate, adopt the NiR enzyme of spectrophotometry enzyme liquid to live.Concrete measurement and calculation method is as follows: the content measuring nitrite with hydrochloric acid-naphthalene-ethylenediamine method, getting 1mL testing sample joins in 25mL colorimetric cylinder, adding distil water is settled to 20mL, add 0.4% Sulphanilic Acid of 2mL more respectively, shake up, leave standstill 5min, finally add 0.2% hydrochloride naphthodiamide of 1mL, with distilled water constant volume 25mL, shake up, leave standstill 15min, by its absorbancy at 538nm of spectrophotometric determination, after this absorbancy substitutes into typical curve equation, can calculate the nitrite content in testing sample, unit is μ g/L.
NiR reaction system (cumulative volume is 1mL): in the 50mM HEPES buffered soln of the pH7.4 of 600 μ L, add the 500 μ g/mL nitrite (making nitrite concentration be 100 μ g/mL) of 200 μ L, add the pure enzyme liquid of NiR of the above-mentioned preparation of 200 μ L again, at 30 DEG C after standing and reacting 20h, go out enzyme, last assaying reaction system nitrite.The Mei Huo unit of NiR: be a Ge Meihuo unit (U) at nanogram (ng) number of reaction system every milligram of protein degradation Sodium Nitrite per hour.Enzyme calculation formula alive is as follows: Activity (U)=m/ (M × t), and wherein m is the quality of the Sodium Nitrite of degraded, and unit is ng; M is the quality of zymoprotein in reactant, and unit is mg; T is the reaction times, and unit is h.
Embodiment 2
(1) lyophilized powder of lactobacillus casei subsp.rhamnosus is placed in MRS substratum, after 30 DEG C of static gas wave refrigerator 18h, makes LCR 6013 seed liquor; Described lactobacillus casei subsp.rhamnosus, purchased from Chinese industrial Microbiological Culture Collection administrative center, is numbered CICC6013, called after Lactobacillus casei subsp.rhamnosus6013 (referred to as LCR 6013).
(2) with 1% volume percent, LCR 6013 seed liquor is seeded in inducing culture A, in 30 DEG C of static inducing culture 18h, obtains induction working stock culture.In mass body volume concentrations, described inducing culture A formula is: containing inductor NaNO 2the MRS substratum of 5 μ g/mL.
(3) with 5% volume percent, induction working stock culture is seeded in inducing culture B, in 30 DEG C of static inducing culture 24h, must containing the nutrient solution of NiR.In mass body volume concentrations, described inducing culture B formula is: containing inductor NaNO 2the MRS substratum of 10 μ g/mL.
(4) nutrient solution containing NiR is carried out centrifugal, centrifugal after cleaning 2 times with physiological saline, must LCR 6013 thalline of NiR be contained and weigh.
(5) add and carry enzyme buffer liquid containing trypsin inhibitor 3 μ g/mL and dithiothreitol (DTT) concentration 2.0 μm of ol/mL, LCR 6013 cell concentration is made to be 250mg/mL, add N,O-Diacetylmuramidase 50mg/L, broken wall 1h after mixing, the supernatant liquor of broken wall solution after centrifugal is LCR 6013 NiR crude enzyme liquid, and its NiR enzyme is lived as 151U.
(6) LCR 6013 crude enzyme liquid is carried out vacuum lyophilization, obtain the thick enzyme powder of LCR 6013 NiR.
(7) be that thick for LCR 6013 NiR enzyme powder is dissolved in the 20mMHEPES damping fluid of pH7.4 by 8mg/mL with concentration, for the thick enzyme powder solution of LCR 6013, chromatography is carried out again through high flow rate ionic sepharose DEAE Sepharose Fast Flow anion-exchange chromatography post, anion-exchange chromatography post first balances with identical HEPES damping fluid, then by thick for LCR 6013 enzyme powder solution, on loading Zhiyin ion exchange column, the NaCl solution adding 0mM ~ 400mM again in above-mentioned HEPES damping fluid carries out gradient elution, elution speed is 0.5mL/min, fraction collection, the NiR enzyme measuring each elution peak is lived, the NaCl wash-out concentration at three peaks is respectively 0mM, 100mM, 400mM, there is the activity of NiR at 3rd peak, merge the component with NiR, and concentrate with PEG 20000.
(8) dextrane gel SephadexG-150 chromatography column first balances with the 60mM Tris-HCl damping fluid of pH8.0, again after anion chromatography the nitrite reductase component loading of gained to sephadex chromatography post, wash-out is carried out with identical Tris-HCl damping fluid, elution speed is 0.1mL/min, fraction collection, the NiR enzyme measuring often pipe in elution peak is lived, and merges the component having NiR enzyme and live, and its NiR enzyme is lived as 2420U.
Embodiment 3
(1) lyophilized powder of lactobacillus casei subsp.rhamnosus is placed in MRS substratum, after 40 DEG C of static gas wave refrigerator 20h, makes seed liquor; Described lactobacillus casei subsp.rhamnosus, purchased from Chinese industrial Microbiological Culture Collection administrative center, is numbered CICC6013, called after Lactobacillus casei subsp.rhamnosus6013 (referred to as LCR 6013).
(2) with 3% volume percent, LCR 6013 seed liquor is seeded in inducing culture A, in 40 DEG C of static inducing culture 20h, obtains induction working stock culture.In mass body volume concentrations, described inducing culture A formula is: containing inductor NaNO 2the MRS substratum of 20 μ g/mL.
(3) with 15% volume percent, induction working stock culture is seeded in inducing culture B, in 40 DEG C of static inducing culture 24h, must containing the nutrient solution of NiR.In mass body volume concentrations, described inducing culture B formula is: containing inductor NaNO 2the MRS substratum of 25 μ g/mL.
(4) nutrient solution containing NiR is carried out centrifugal, centrifugal after cleaning 2 times with physiological saline, must LCR 6013 thalline of NiR be contained and weigh.
(5) add and carry enzyme buffer liquid containing trypsin inhibitor 5 μ g/mL and dithiothreitol (DTT) concentration 8.0 μm of ol/mL, LCR 6013 cell concentration is made to be 200mg/mL, add N,O-Diacetylmuramidase 20mg/L, broken wall 3h after mixing, the supernatant liquor of broken wall solution after centrifugal is LCR 6013 NiR crude enzyme liquid, and its NiR enzyme is lived as 148U.
(6) LCR 6013 NiR crude enzyme liquid is carried out vacuum lyophilization, obtain the thick enzyme powder of LCR 6013 NiR.(7) be that thick for LCR 6013 NiR enzyme powder is dissolved in the 80mMHEPES damping fluid of pH8.0 by 5mg/mL with concentration, for the thick enzyme powder solution of LCR 6013, chromatography is carried out again through high flow rate ionic sepharose DEAE Sepharose Fast Flow anion-exchange chromatography post, anion-exchange chromatography post first balances with identical HEPES damping fluid, then by thick for LCR 6013 enzyme powder solution, on loading Zhiyin ion exchange column, the NaCl solution adding 0mM ~ 500mM again in above-mentioned HEPES damping fluid carries out gradient elution, elution speed is 1.5mL/min, fraction collection, the NiR enzyme measuring each elution peak is lived, the NaCl wash-out concentration at three peaks is respectively 0mM, 150mM, 500mM, there is the activity of NiR at 3rd peak, merge the component with NiR, and concentrate with PEG 20000.
(8) sephadex chromatography post SephadexG-150 first balances with the 80mM Tris-HCl damping fluid of pH8.6, again by the nitrite reductase component loading of gained after anion chromatography to sephadex chromatography post, wash-out is carried out with identical Tris-HCl damping fluid, elution speed is 0.6mL/min, fraction collection, the NiR enzyme measuring often pipe in elution peak is lived, and merge and have NiR enzyme component alive, its NiR enzyme is lived as 2351U.
Embodiment 4
(1) lyophilized powder of lactobacillus casei subsp.rhamnosus is placed in MRS substratum, after 37 DEG C of static gas wave refrigerator 36h, makes seed liquor; Described lactobacillus casei subsp.rhamnosus, purchased from Chinese industrial Microbiological Culture Collection administrative center, is numbered CICC6013, called after Lactobacillus casei subsp.rhamnosus6013 (referred to as LCR 6013).
(2) with 10% volume percent, LCR 6013 seed liquor is seeded in inducing culture A, in 37 DEG C of static inducing culture 22h, obtains induction working stock culture.In mass body volume concentrations, described inducing culture A formula is: containing inductor NaNO 2the MRS substratum of 15 μ g/mL.
(3) with 12% volume percent, induction working stock culture is seeded in inducing culture B, in 37 DEG C of static inducing culture 30h, must containing the nutrient solution of NiR.In mass body volume concentrations, described inducing culture B formula is: containing inductor NaNO 2the MRS substratum of 40 μ g/mL.
(4) nutrient solution containing NiR is carried out centrifugal, centrifugal after cleaning 2 times with physiological saline, must LCR 6013 thalline of NiR be contained and weigh.
(5) add and carry enzyme buffer liquid containing trypsin inhibitor 6 μ g/mL and dithiothreitol (DTT) 15.0 μm of ol/mL, LCR 6013 cell concentration is made to be 300mg/mL, add N,O-Diacetylmuramidase 15mg/L, broken wall 4h after mixing, the supernatant liquor of broken wall solution after centrifugal is LCR 6013 NiR crude enzyme liquid, and its NiR enzyme is lived as 132U.
(6) LCR 6013 NiR crude enzyme liquid is carried out vacuum lyophilization, obtain the thick enzyme powder of LCR 6013 NiR.
(7) be that thick for LCR 6013 NiR enzyme powder is dissolved in the 40mMHEPES damping fluid of pH8.6 by 10mg/mL with concentration, for the thick enzyme powder solution of LCR 6013, chromatography is carried out again through high flow rate ionic sepharose DEAE Sepharose Fast Flow anion-exchange chromatography post, anion-exchange chromatography post first balances with identical HEPES damping fluid, then by thick for LCR 6013 enzyme powder solution, on loading Zhiyin ion exchange column, the NaCl solution adding 0mM ~ 600mM again in above-mentioned HEPES damping fluid carries out gradient elution, elution speed is 1.2mL/min, fraction collection, the NiR enzyme measuring each elution peak is lived, the NaCl wash-out concentration at three peaks is respectively 0mM, 125mM, 600mM, there is the activity of NiR at 3rd peak, merge the component with NiR, and concentrate with PEG 20000.
(8) dextrane gel SephadexG-150 chromatography column first balances with the 40mM Tris-HCl damping fluid of pH8.4, again by the nitrite reductase component loading of gained after anion chromatography to sephadex chromatography post, wash-out is carried out with identical Tris-HCl damping fluid, elution speed is 0.4mL/min, fraction collection, the NiR enzyme measuring often pipe in elution peak is lived, and merges the component having NiR enzyme and live, and its NiR enzyme is lived as 2025U.
Embodiment 5
(1) lyophilized powder of lactobacillus casei subsp.rhamnosus is placed in MRS substratum, after 37 DEG C of static gas wave refrigerator 24h, makes seed liquor; Described lactobacillus casei subsp.rhamnosus, purchased from Chinese industrial Microbiological Culture Collection administrative center, is numbered CICC6013, called after Lactobacillus casei subsp.rhamnosus6013 (referred to as LCR 6013).
(2) with 5% volume percent, LCR 6013 seed liquor is seeded in inducing culture A, in 37 DEG C of static inducing culture 24h, obtains induction working stock culture.In mass body volume concentrations, described inducing culture A formula is: containing inductor NaNO 2the MRS substratum of 10 μ g/mL.
(3) with 12% volume percent, induction working stock culture is seeded in inducing culture B, in 37 DEG C of static inducing culture 36h, must containing the nutrient solution of NiR.In mass body volume concentrations, described inducing culture B formula is: containing inductor NaNO 2the MRS substratum of 20 μ g/mL.
(4) nutrient solution containing NiR is carried out centrifugal, centrifugal after cleaning 2 times with physiological saline, must LCR 6013 thalline of NiR be contained and weigh.
(5) what add trypsin inhibitor 8 μ g/mL and dithiothreitol (DTT) 10.0 μm of ol/mL carries enzyme buffer liquid, LCR 6013 cell concentration is made to be 400mg/mL, add N,O-Diacetylmuramidase 50mg/L, broken wall 1h after mixing, the supernatant liquor of broken wall solution after centrifugal is LCR 6013 NiR crude enzyme liquid, and its NiR enzyme is lived as 143U.
(6) LCR 6013 NiR crude enzyme liquid is carried out vacuum lyophilization, obtain the thick enzyme powder of LCR 6013 NiR.
(7) with concentration 15mg/mL, thick for LCR 6013 NiR enzyme powder is dissolved in the 60mM HEPES damping fluid of pH7.0, for the thick enzyme powder solution of LCR 6013, chromatography is carried out again through high flow rate ionic sepharose DEAE Sepharose Fast Flow anion-exchange chromatography post, anion-exchange chromatography post first balances with identical HEPES damping fluid, then by thick for LCR 6013 enzyme powder solution, on loading Zhiyin ion exchange column, the NaCl solution adding 0mM ~ 450mM again in above-mentioned HEPES damping fluid carries out gradient elution, elution speed is 1.5mL/min, fraction collection, the NiR enzyme measuring each elution peak is lived, the NaCl wash-out concentration at three peaks is respectively 0mM, 200mM, 450mM, there is the activity of NiR at 3rd peak, merge the component with NiR, and concentrate with PEG 20000.
(8) dextrane gel SephadexG-150 chromatography column first balances with the 50mM Tris-HCl damping fluid of pH7.8, again after anion chromatography the nitrite reductase component loading of gained to sephadex chromatography post, wash-out is carried out with identical Tris-HCl damping fluid, elution speed is 0.3mL/min, fraction collection, fraction collection, the NiR enzyme measuring often pipe in elution peak is lived, merge the component having NiR enzyme and live, its NiR enzyme is lived as 2158U.
Embodiment 6
(1) lyophilized powder of lactobacillus casei subsp.rhamnosus is placed in MRS substratum, after 35 DEG C of static gas wave refrigerator 30h, makes seed liquor; Described lactobacillus casei subsp.rhamnosus, purchased from Chinese industrial Microbiological Culture Collection administrative center, is numbered CICC6013, called after Lactobacillus casei subsp.rhamnosus6013 (referred to as LCR 6013).
(2) with 8% volume percent, LCR 6013 seed liquor is seeded in inducing culture A, in 37 DEG C of static inducing culture 24h, obtains induction working stock culture.In mass body volume concentrations, described inducing culture A formula is: containing inductor NaNO 2the MRS substratum of 12 μ g/mL.
(3) with 8% volume percent, induction working stock culture is seeded in inducing culture B, in 37 DEG C of static inducing culture 36h, must containing the nutrient solution of NiR.In mass body volume concentrations, described inducing culture B formula is: containing inductor NaNO 2the MRS substratum of 50 μ g/mL.
(4) will carry out centrifugal containing NiR nutrient solution, centrifugal after cleaning 2 times with physiological saline, must LCR 6013 thalline of NiR be contained and weigh.
(5) add and carry enzyme buffer liquid containing trypsin inhibitor 10 μ g/mL and dithiothreitol (DTT) 20.0 μm of ol/mL, LCR 6013 cell concentration is made to be 250mg/mL, add N,O-Diacetylmuramidase 30mg/L, broken wall 5h after mixing, the supernatant liquor of broken wall solution after centrifugal is LCR 6013 NiR crude enzyme liquid, and its NiR enzyme is lived as 159U.
(6) LCR 6013 NiR crude enzyme liquid is carried out vacuum lyophilization, obtain the thick enzyme powder of LCR 6013 NiR.(7) be that thick for LCR 6013 NiR enzyme powder is dissolved in the 30mM HEPES damping fluid of pH7.8 by 20mg/mL with concentration, for the thick enzyme powder solution of LCR 6013, chromatography is carried out again through high flow rate ionic sepharose DEAE Sepharose Fast Flow anion-exchange chromatography post, anion-exchange chromatography post first balances with identical HEPES damping fluid, then by thick for LCR 6013 enzyme powder solution, on loading Zhiyin ion exchange column, the NaCl solution adding 0mM ~ 550mM again in above-mentioned HEPES damping fluid carries out gradient elution, elution speed is 1.0mL/min, fraction collection, the NiR enzyme measuring each elution peak is lived, the NaCl wash-out concentration at three peaks is respectively 0mM, 220mM, 550mM, there is the activity of NiR at 3rd peak, merge the component with NiR, and concentrate with PEG 20000.
(8) dextrane gel SephadexG-150 chromatography column first balances with the 30mM Tris-HCl damping fluid of pH8.2, again after anion chromatography the nitrite reductase component loading of gained to sephadex chromatography post, wash-out is carried out again with identical Tris-HCl damping fluid, elution speed is 0.5mL/min, fraction collection, the NiR enzyme measuring often pipe in elution peak is lived, and merges the component having NiR enzyme and live, and its NiR enzyme is lived as 2310U.

Claims (10)

1. a preparation method for the nitrite reductase of lactobacillus casei subsp.rhamnosus, is characterized in that, comprises the steps and processing condition:
(1) lyophilized powder of lactobacillus casei subsp.rhamnosus (Lactobacillus casei subsp.rhamnosus) 6013 is placed in MRS substratum, after 30 DEG C ~ 40 DEG C static gas wave refrigerator 18h ~ 36h, makes seed liquor;
(2) with 1% ~ 10% volume percent, above-mentioned seed liquor is seeded in inducing culture A, in 30 DEG C ~ 40 DEG C static inducing culture 18h ~ 24h, obtains induction working stock culture; Described inducing culture A formula is: containing inductor NaNO 2the MRS substratum of 5 μ g/mL ~ 20 μ g/mL;
(3) with 5% ~ 15% volume percent, induction working stock culture is seeded in inducing culture B, in 30 DEG C ~ 40 DEG C static inducing culture 24h ~ 36h, must containing the nutrient solution of nitrite reductase; Described inducing culture B formula is: containing inductor NaNO 2the MRS substratum of 10 μ g/mL ~ 50 μ g/mL;
(4) nutrient solution containing nitrite reductase is carried out centrifugal, with centrifugal after physiological saline cleaning, must containing the thalline of nitrite reductase;
(5) add and carry enzyme buffer liquid, make the cell concentration containing nitrite reductase be 150mg/mL ~ 400mg/mL, add N,O-Diacetylmuramidase, broken wall 1h ~ 5h after mixing, the supernatant liquor of broken wall solution after centrifugal is nitrite reductase crude enzyme liquid.
2. method according to claim 1, is characterized in that, the concentration of step (5) described N,O-Diacetylmuramidase is 10mg/mL ~ 50mg/mL.
3. method according to claim 1, is characterized in that, carry described in step (5) containing trypsin inhibitor and dithiothreitol (DTT) in enzyme buffer liquid, its concentration is respectively 2 μ g/mL and 2.0 μm ol/mL ~ 20.0, μ g/mL ~ 10 μm ol/mL.
4. the method according to claim 1 or 2 or 3, is characterized in that, described nitrite reductase crude enzyme liquid, through vacuum lyophilization, obtains the thick enzyme powder of nitrite reductase; Described nitrite reductase thick enzyme powder also passes through following purification procedures: be dissolved in by thick enzyme powder in HEPES damping fluid and obtain thick enzyme powder liquid, more successively through anion-exchange chromatography and/or sephadex chromatography, finally obtained pure nitrite reductase.
5. method according to claim 4, it is characterized in that, described anion-exchange chromatography adopts high flow rate ionic sepharose DEAE Sepharose Fast Flow anion-exchange chromatography post, and described sephadex chromatography adopts dextrane gel Sephadex G-150 chromatography column.
6. method according to claim 5, it is characterized in that, described anion-exchange chromatography is by the thick enzyme powder liquid loading Zhiyin ion exchange column that dissolves in HEPES damping fluid, first with HEPES damping fluid, protein is washed till baseline, the NaCl solution adding 0mM ~ 600mM again in above-mentioned HEPES damping fluid carries out gradient elution, fraction collection, the nitrite reductase enzyme measuring each elution peak is lived, merge the component with nitrite reductase, and concentrate with PEG 20000.
7. method according to claim 6, it is characterized in that, described sephadex chromatography is on sephadex chromatography post by sample loading, wash-out is carried out with Tris-HCl damping fluid, fraction collection, the nitrite reductase enzyme measuring each elution peak is lived, and merges the component having nitrite reductase enzyme and live.
8. method according to claim 7, is characterized in that, described HEPES buffer concentration is 20mM ~ 80mM, pH is 7.0 ~ 8.6; Described Tris-HCl buffer concentration is 20mM ~ 80mM, pH is 7.8 ~ 8.6.
9. method according to claim 8, is characterized in that, in described anion-exchange chromatography and sephadex chromatography, the flow velocity of wash-out is respectively 0.5mL/min ~ 1.5mL/min and 0.1mL/min ~ 0.6mL/min.
10. method according to claim 9, is characterized in that, in described thick enzyme powder liquid, the concentration of thick enzyme powder is 3 ~ 20mg/mL.
CN201410437877.7A 2014-08-29 2014-08-29 Preparation method of nitrite reductase of lactobacillus casei subsp rhamnosus Pending CN104212775A (en)

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CN106467907A (en) * 2016-10-11 2017-03-01 南京财经大学 A kind of method preparing nitrite reductase from Flammulina velutiper (Fr.) Sing
CN107353049A (en) * 2017-08-10 2017-11-17 黑龙江九穗谷农业科技发展有限公司 A kind of preparation method of multiple-effect biological bacterium enzyme
CN107381834A (en) * 2017-08-24 2017-11-24 华南理工大学 A kind of immobilised enzymes biological reaction apparatus and its application in processing breeding water body nitrite
CN110628684A (en) * 2019-10-25 2019-12-31 华中农业大学 Lactobacillus enrichment agent and application thereof in high-density fermentation of lactobacillus

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105039470A (en) * 2015-08-03 2015-11-11 华南理工大学 Nitrite-degrading electron donor protein obtained from lactobacillus casei and preparation method thereof and application
CN105039470B (en) * 2015-08-03 2018-10-09 华南理工大学 A kind of electron donor albumen of Lactobacillus casei degrading nitrite and its preparation method and application
CN106467907A (en) * 2016-10-11 2017-03-01 南京财经大学 A kind of method preparing nitrite reductase from Flammulina velutiper (Fr.) Sing
CN107353049A (en) * 2017-08-10 2017-11-17 黑龙江九穗谷农业科技发展有限公司 A kind of preparation method of multiple-effect biological bacterium enzyme
CN107381834A (en) * 2017-08-24 2017-11-24 华南理工大学 A kind of immobilised enzymes biological reaction apparatus and its application in processing breeding water body nitrite
CN110628684A (en) * 2019-10-25 2019-12-31 华中农业大学 Lactobacillus enrichment agent and application thereof in high-density fermentation of lactobacillus

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