CN103627691A - Immobilized glutathione synthetase, preparation thereof and applications thereof - Google Patents
Immobilized glutathione synthetase, preparation thereof and applications thereof Download PDFInfo
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- CN103627691A CN103627691A CN201210306568.7A CN201210306568A CN103627691A CN 103627691 A CN103627691 A CN 103627691A CN 201210306568 A CN201210306568 A CN 201210306568A CN 103627691 A CN103627691 A CN 103627691A
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Abstract
The invention relates to an immobilized glutathione synthetase, preparation thereof and applications thereof. In particular, the immobilized glutathione synthetase having largely improved stability and reactivity is obtained by immobilization of a glutathione synthetase and a carrier. In particular, a long connecting component between the glutathione synthetase and the immobilization carrier can achieve an excellent immobilization effect. The immobilized glutathione synthetase has a long half-life period so that the enzyme productivity per unit is effectively enhanced. The immobilized glutathione synthetase can be used as a GSH biosynthesis system, thus simplifying production processes, increasing the production efficiency and simplifying purification technology.
Description
Technical field
The invention belongs to biological technical field; Particularly, the present invention relates to a kind of immobilization glutathione synthetase and preparation and application.
Background technology
Gsh (being called for short GSH), has another name called γ-GSH, is a kind of a kind of small molecules sulfhydryl compound being extensively present in animal, plant and microorganism cells, has the function that maintains redox equilibrium in born of the same parents.The important physiological function of GSH makes its be with a wide range of applications at aspects such as medical science, food, makeup [Meister A and Anderson M.E., Glutathione.Annu Rev Biochem, 1983.52:p.711-760; Wu, G. .Fang Y.Z, Yang S., Lupton J.R., and Turner N.D.Glutathione Metabolism and Its Implications for Health.J.Nutr., 2004.134:p.489-492.].
The production method of GSH is mainly chemical synthesis and biological process at present.Although, both there is certain weak point in chemical synthesis and extract GSH the yeast cell of GSH and realized industrialization in being rich in born of the same parents.Chemical synthesis is early applied to the production of GSH, but have complexity, consuming time etc. not enough [Wu Jianping, woods Jianping, Cen Peilin. the biosynthesizing .1999. of gsh].GSH is by gamma-glutamylcysteine synthetase (EC6.3.2.2 in vivo, GSH I) and GSH synthetic enzyme (EC6.3.2.3, GSH II) under the condition existing at ATP, catalysis Pidolidone, it is synthetic that Cys and glycine carry out sequential reaction.Biological process is produced gsh and is mainly comprised enzyme transforming process and two kinds of modes of fermentation method, and both all realize the synthetic of GSH by utilizing high active enzyme in cell to tie up under gentle condition; Its topmost difference is that enzyme transforming process need provide ATP, and fermentation rule can be utilized cell self metabolism, without ATP is additionally provided.The output of fermentative Production GSH is not high at present, and separation purifying technique is complicated, and the costs such as raw materials cost and energy consumption are all higher; With respect to fermentation method, the enzyme process reaction times is short, and reaction is simple, can reach the advantages such as greater concn and purification yield, is the direction of large-scale production GSH technical study.
Enzyme process synthesizes GSH, and highly active glutathione synthetase is one of key obtaining high density product.In the glutathione synthetase of having reported, except above-mentioned common GSH I and GSH II, also found to have the difunctional synthetic enzyme of highly active gsh, for example derive from the difunctional synthetic enzyme of gsh of streptococcus agalactiae, and from the gsh bifunctional enzyme of thermophilus streptococcus, the gsh bifunctional enzyme 12h of thermophilus streptococcus can produce 10.4g/L GSH, and the transformation efficiency of Cys reaches 85%.The discovery of these enzymes, the large-scale production of synthesizing GSH for enzyme process provides possibility.
The consumption of the synthetic ATP of being accompanied by of GSH (synthetic 1M GSH need consume 2M ATP), because ATP is expensive, directly adding ATP has significantly increased the cost of the synthetic GSH of enzyme process, and the existence of the ATP of high density and meta-bolites ADP thereof also can strongly inhibited GSH synthetic enzyme activity, therefore Production by Enzymes GSH adopts ATP regeneration at present, mainly adopts the mode of yeast cell regeneration.The ATP of yeast cell regeneration has at present developed into the state of the art that adopts fixed yeast cell regeneration, thereby realizes reusing of regeneration system rapidly.This has just proposed corresponding requirement for the recycling of GSH synthetic enzyme.
Immobilized enzyme (immobilized enzyme) is a kind of enzyme to be fixed on to the technology on carrier.Usually, through immobilized enzyme itself or water-soluble, just by method physics or chemistry, make enzyme be combined with water-insoluble macromolecular carrier or enzyme is embedded in wherein, the mobility of enzyme is reduced.Although the enzyme stability after fixing at present increases, easily separated from reactive system, be convenient to transportation and store, activity decreased (general is only below 30% of resolvase, is only even below 10%).
Also exist and utilize immobilized cell to prepare the technique of GSH at present, still due to cell itself, need the consumption of energy, and the restriction of growth cycle, therefore can not recycle for a long time, enzyme is lived also lower.In addition, utilize in vitro Pidolidone, Cys and glycine carry out that sequential reaction is synthetic has proposed very high requirement to the activity of enzyme.For above-mentioned reasons, this area does not also have a kind of immobilization glutathione synthetase at present, therefore, in the urgent need to breaking through the bottleneck of Production by Enzymes GSH, develops corresponding immobilized enzyme for industrial production GSH.
Summary of the invention
Object of the present invention is just to provide a kind of immobilization glutathione synthetase and preparation and application.
In a first aspect of the present invention, a kind of immobilized glutathione synthetase is provided, described immobilized glutathione synthetase comprises:
(1) fixation support; With
(2) be fixed on the glutathione synthetase of described carrier.
In another preference, described glutathione synthetase is fixed on fixation support by being selected from the mode of lower group: absorption, embedding, covalent attachment, crosslinked or its combination.
In another preference, the mass ratio of described fixation support and described glutathione synthetase is: 1: 1-100:1(is preferably 10:1-50:1).
In another preference, described fixation support comprises the matrix of fixation support, and the connect elements being connected with described matrix.
In another preference, the matrix of described fixation support is microballoon or high molecular polymer.
In another preference, the structure of described immobilized glutathione synthetase is as follows:
Z-(L-E)n
In formula,
Z represents fixation support matrix;
E represents glutathione synthetase;
L represents the connect elements between E and Z;
N is more than or equal to 1 positive integer.
In another preference, n is the positive integer that is selected from 1-100000.
In another preference, the main chain of described connect elements has 4-50 atom that is selected from C, N or O.
In another preference, the main chain of described connect elements has the atom that 5-30 (preferably 4-20, preferably 4-10) is selected from C, N or O.
In another preference, the main chain of described connect elements by or substantially by following group, formed :-CH
2-,-C (R) H-,-CO-,-NH-,-NR-,-O-, wherein R represents branched group, as OH, amino, C1-C4 alkyl, halogen.
In another preference, described immobilized glutathione synthetase has following characteristic:
(a) the immobilized glutathione synthetase of enzymic activity >=0.5U/g, preferably >=immobilized glutathione synthetase of 2U/g;
(b) half-life characteristics >=10 day, preferably >=15 days, more preferably >=20 days.
In another preference, described fixation support is organic carrier.
In another preference, described organic carrier is selected from lower group: ester group fixation support, amino fixation support, carboxyl fixation support, cyano group fixation support or its combination.
In another preference, described organic carrier is amino fixation support or epoxy group(ing) fixation support.
In another preference, described glutathione synthetase is by being fixed on amino fixation support in covalently bound mode.
In another preference, described glutathione synthetase is by being fixed on epoxy group(ing) fixation support in covalently bound mode.
In another preference, described amino fixation support is selected from lower group:
the EC-HA of EC series or EC-EA, Seplite LX-1000HA or Relizyme
tMhA403 or the EA403 of series; Preferred EC-HA and HA403.
In another preference, described epoxy group(ing) fixation support is selected from lower group:
the EC-EP of EC series or EC-HFA, Seplite LX-1000EP or Relizyme
tMeP403 or the HFA403 of series; Preferred EC-HFA and HFA403.
In another preference, described glutathione synthetase is selected from lower group: gamma-glutamylcysteine synthetase (EC6.3.2.2, GSH I), GSH synthetic enzyme (EC6.3.2.3, GSH II) or the difunctional synthetic enzyme of gsh or its combination.
In another preference, described glutathione synthetase is the combination of gamma-glutamylcysteine synthetase (EC6.3.2.2, GSH I) and GSH synthetic enzyme (EC6.3.2.3, GSH II).
In another preference, gamma-glutamylcysteine synthetase (EC6.3.2.2, GSH I) is 1:1 ~ 5:1 with the ratio of GSH synthetic enzyme (EC6.3.2.3, GSH II).
In another preference, described gamma-glutamylcysteine synthetase (EC6.3.2.2, GSH I) and GSH synthetic enzyme (EC6.3.2.3, GSH II) derive from intestinal bacteria (Escherichia coli).
In another preference, described glutathione synthetase is the difunctional synthetic enzyme of gsh.
In another preference, the difunctional synthetic enzyme of described gsh is for deriving from the difunctional synthetic enzyme of gsh of streptococcus agalactiae (Streptococcus agalactiae) or thermophilus streptococcus (Streptococcus thermophilus).
In another preference, described glutathione synthetase is the combination of the difunctional synthetic enzyme of gsh and gamma-glutamylcysteine synthetase (EC6.3.2.2, GSH I); Or glutathione synthetase is the combination of the difunctional synthetic enzyme of gsh and GSH synthetic enzyme (EC6.3.2.3, GSH II).In another preference, the ratio of the difunctional synthetic enzyme of gsh and gamma-glutamylcysteine synthetase (EC6.3.2.2, GSH I) is 1:1 ~ 1:3.
In another preference, the ratio of the difunctional synthetic enzyme of gsh and GSH synthetic enzyme (EC6.3.2.3, GSH II) is 1:1 ~ 4:1.
In a second aspect of the present invention, a kind of method of preparing immobilized glutathione synthetase described in first aspect present invention is provided, comprise step: glutathione synthetase is fixed on described fixation support, thereby forms immobilized glutathione synthetase.
In another preference, before glutathione synthetase is fixed on described carrier, also comprise the step that carrier is activated.
In a third aspect of the present invention, the purposes of immobilized glutathione synthetase described in first aspect is provided, it is used to produce gsh GSH.
In a fourth aspect of the present invention, a kind of method of producing gsh GSH is provided, comprise step:
(1) immobilized glutathione synthetase described in first aspect is contacted with reaction substrate, obtain the reaction mixture that contains GSH;
(2) separated GSH in the reaction mixture from (1).
In another preference, described reaction substrate comprises glycine, halfcystine and L-glutamic acid.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, at this, tire out and state no longer one by one.
Accompanying drawing explanation
Following accompanying drawing is used for illustrating specific embodiment of the invention scheme, and be not used in, limits the scope of the invention being defined by claims.
Fig. 1 is the structural representation of fixation support; A is that long side chain carrier EC-HFA, B are long side chain carrier EC-HA, and C is short-side chain carrier EC-EP.
Fig. 2 is the beta stability line figure of immobilized enzyme EC-HA-SaGSH.
Fig. 3 is the beta stability line figure of immobilized enzyme EC-EP-STH.
Fig. 4 is gsh bifunctional enzyme (Sa-GSH) and the immobilized comparison of different fixation supports.
Fig. 5 is the beta stability line figure of gsh bifunctional enzyme (Sa-GSH) solution enzyme.
Embodiment
The inventor through extensive and deep research, is surprised to find that first, after glutathione synthetase and being fixed of carrier, obtains the immobilization glutathione synthetase that stability improves greatly; Especially, between fixation support and glutathione synthetase, longer connect elements can obtain particularly preferred immobilization effect.Immobilization glutathione synthetase long half time of the present invention, the productivity of the unit's of making enzyme effectively improves.With immobilization glutathione synthetase of the present invention, as GSH biosynthesis system, production process is simplified, production efficiency improves, and has simplified purifying technique.
GSH
As used herein, term " GSH " or " gsh " or " γ-GSH ", can Alternate.GSH is a kind of a kind of small molecules sulfhydryl compound being extensively present in animal, plant and microorganism cells, has the function that maintains redox equilibrium in born of the same parents.
The main Physiological Function of GSH is to remove free radical, anti-oxidant, anti-ageing waiting for a long time.Many free radicals meeting damaging cells films that body intracellular metabolic produces, invasion and attack life macromolecule, promotes body aging, and induced tumor or atherosclerotic generation, GSH can eliminate free radical, can play strong provide protection.In the structure of GSH, contain an easy oxidized deoxidation of active sulfydryl-SH, this specific structure just becomes main free-radical scavengers in body.GSH not only removes human free radical, can also improve body immunity, promotes the function that blood cell is manufactured Protective substances, and Protective substances refers to protect health to exempt from infected material, reduces the total content of inflammation material in body.
Glutathione synthetase
As used herein, term " glutathione synthetase ", " GSH synthetic enzyme " or " gamma-glutamylcysteine synthetase " can Alternates, all refer to the zymoprotein of synthetic GSH.
Glutathione synthetase mainly comprises single functional enzyme and bifunctional enzyme.In vivo, GSH is under the condition being existed at ATP by gamma-glutamylcysteine synthetase (EC6.3.2.2, GSH I) and GSH synthetic enzyme (EC6.3.2.3, GSH II), catalysis Pidolidone, it is synthetic that Cys and glycine carry out sequential reaction.
Except GSH I and GSH II, the difunctional synthetic enzyme of highly active gsh, include, but is not limited to: derive from the difunctional synthetic enzyme of gsh of streptococcus agalactiae (Streptococcus agalactiae), and from gsh bifunctional enzyme of thermophilus streptococcus (Streptococcus thermophilus) etc.
In a preference, the streptococcus agalactiae (Streptococcus agalactiae) that produces the difunctional synthetic enzyme of gsh includes, but is not limited to: enterococcus faecalis (Enterococcus faecalis), honeycomb honeybee coccus (Melissococcus plutonius), cud methane tyrothricin (Methanobrevibacter ruminantium), pasteurella multocida (Pasteurella multocida), clostridium perfringens (Clostridium perfringens) or listerisa monocytogenes in mjme (Listeria monocytogenes).
In a preference, the thermophilus streptococcus (Streptococcus thermophilus) of producing the difunctional synthetic enzyme of gsh includes, but is not limited to: enterococcus faecalis (Enterococcus faecalis), honeycomb honeybee coccus (Melissococcus plutonius), cud methane tyrothricin (Methanobrevibacter ruminantium), pasteurella multocida (Pasteurella multocida), clostridium perfringens (Clostridium perfringens) or listerisa monocytogenes in mjme (Listeria monocytogenes).
Those of ordinary skill in the art can use ordinary method to prepare glutathione synthetase, as commercially available purchase, separated from bacterial strain, full chemosynthesis or external synthetic by molecular biology.
From bacterial strain (preferably streptococcus agalactiae and thermophilus streptococcus), the cultivation of separating glutathione synthetic enzyme and described bacterial strain is that those of ordinary skill in the art is known, except producing bacterium difference, in other conditions and prior art, the method for fermentative production and purifying protein is basic identical.The fermentation condition of bacterial strain of the present invention is close with general bacterium, in the substratum of carbonaceous sources, nitrogenous source and trace element, in pH5.0-9.0 (preferably pH7.0-7.5) and 20-45 ℃ (preferably 25-40 ℃) fermentation.In the present invention, " mycelium ", " fermented liquid " or " nutrient solution " can, by cultivating bacterial strain of the present invention under the condition being applicable to growth, make it grow to certain mycelium concentration and obtain.For cultivating the nutrition source of the substratum of bacterial strain of the present invention, have no particular limits.Those skilled in the art can select suitable carbon source, nitrogenous source and other nutrition sources according to known technology.For example, carbon source can be starch, dextrin, glucose, fructose, sucrose, glycerine, inositol, N.F,USP MANNITOL etc.Nitrogenous source can be peptone, soyflour, soybean cake powder, meat extract, protein powder, wheat skin, rice sugar, yeast powder, corn steep liquor, ammonium salt and other organism or inorganic nitrogen-containing compound.In addition, in substratum, also can suitably add some inorganic salts, as metal-salts such as sodium-chlor, phosphoric acid salt (as potassium primary phosphate and dipotassium hydrogen phosphate etc.), manganous sulfate, ammonium sulfate, magnesium sulfate, calcium carbonate.Conventionally can adopt various known conventional mediums, as LB nutrient agar, nutrient agar, glucose yeast cream nutrient agar and ox meat extract nutrient agar etc. carry out inclined-plane solid culture and carry out preliminary preservation under 4 ℃ of environment this bacterial strain.Yet, it will be appreciated by those skilled in the art that the present invention is not limited to these concrete culture medium prescriptions of enumerating herein.
By the external synthetic and separating glutathione synthetic enzyme of molecular biology, be that those of ordinary skill in the art is known.The present invention, so that the Glutatione synthetase gene of different sources is carried out to construction of genetic engineering, utilizes different being fixed of fixation support, or two enzymes are fixed altogether.
According to Glutatione synthetase gene sequence, the art personnel can make coding nucleic acid of the present invention with various currently known methodss easily.These methods are such as but not limited to PCR, DNA synthetic etc., and concrete method can be referring to J. Pehanorm Brooker, < < molecular cloning experiment guide > >.As one embodiment of the present invention, can build nucleic acid sequence encoding of the present invention by PCR method.
Comprising coding for glutathion synthetase gene sequence can expression regulation sequence operability be connected.Described " operability is connected " or " being operationally connected in " refer to a kind of like this situation, and some part of linear DNA sequence can regulate or control the activity of same linear DNA sequence other parts.For example, if the transcribing of promotor control sequence, it is exactly to be operationally connected in encoding sequence so.
Those skilled in the art can select suitable expression vector according to host cell, for expressing glutathione synthetase.According to the restriction enzyme mapping of known unloaded expression vector, those skilled in the art can shear and splicing by Restriction Enzyme according to ordinary method, and Glutatione synthetase gene sequence is inserted to suitable restriction site, make recombinant expression vector.
Described coding for glutathion synthetase gene sequence is imported to the multiple known technology that host cell can adopt this area, such as but not limited to: calcium phosphate precipitation, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, alkalimetal ion method.About cultivation and the expression of host cell can be referring to Olander RM Dev Biol Stand 1996; 86:338.Can, by cell and residue in centrifugal removal suspension, collect clear liquid.Can identify by agarose gel electrophoresis technology.
The character that can be basic homogeneous by the above-mentioned zymoprotein purifying preparing, for example, be single band on SDS-PAGE electrophoresis.For example, when recombinant protein is secreting, expressing, can adopt commercial ultra-filtration membrane to carry out separated described albumen, the product such as the company such as Millipore, Pellicon, first will express supernatant and concentrate.The method that concentrated solution can adopt gel chromatography is purifying in addition further, or adopts the method purifying of ion exchange chromatography.Such as anion-exchange chromatography (DEAE etc.) or cation-exchange chromatography.Gel matrix can be the matrix that agarose, dextran, polymeric amide etc. are usually used in protein purification.Q-or SP-group are comparatively desirable ion-exchange groups.Finally, available hydroxyapatite adsorption chromatography also, metal chelate chromatography, the methods such as hydrophobic interaction chromatography and RPLC (RP-HPLC) are to the further refining purifying of above-mentioned purified product.Above-mentioned all purification steps can utilize different combinations, finally make purity of protein reach basic homogeneous.
Fixation support
The present invention relates to a kind of fixedly fixation support of glutathione synthetase that is particularly suitable for, described fixation support comprises the matrix of fixation support, and the connect elements being connected with described matrix.
Be used for the fixedly preferred organic carrier of carrier of glutathione synthetase.Described organic carrier is selected from lower group: ester group fixation support, amino fixation support, carboxyl fixation support, cyano group fixation support.
Preferably, the main chain of described connect elements has 4-50 atom that is selected from C, N or O.
In another preference, the main chain of described connect elements has 5-30 atom that is selected from C, N or O.
In another preference, the main chain of described connect elements by or substantially by following group, formed :-CH
2-,-C (R) H-,-CO-,-NH-,-NR-,-O-, wherein R represents branched group, as OH, amino, C1-C4 alkyl, halogen.
In the present invention, described ester group fixation support refers to that the active group fixing with glutathione synthetase is ester group.
In the present invention, described amino fixation support refers to that the active group fixing with glutathione synthetase is for amino.
In the present invention, described cyano group fixation support refers to that the active group fixing with glutathione synthetase is cyano group.
In the present invention, described carboxyl fixation support refers to that the active group fixing with glutathione synthetase is carboxyl.
The preferred amino fixation support of organic carrier or epoxy group(ing) fixation support.Described amino fixation support is selected from lower group:
the EC-HA of EC series or EC-EA, Seplite LX-1000HA or Relizyme
tMhA403 or the EA403 of series.Described epoxy group(ing) fixation support is selected from lower group:
the EC-EP of EC series or EC-HFA, Seplite LX-1000EP or Relizyme
tMeP403 or the HFA403 of series.
The present invention particularly preferably
the carrier of EC series.The matrix of examples of such carriers is that highly porous polymethyl acrylic acid is spherical, have stable physics and chemistry character, and the rate of expansion in highly concentrated solution and usual solvents is low, has excellent mechanical osmotic stability.
the structure of several carriers of EC series is in Table 1.
Table 1
* side chain lengths is by the C in the main chain being connected with carrier matrix, N, O atomicity, except the amino and epoxy group(ing) of end.Do not calculate the side chain of main chain.
Glutathione synthetase
The invention provides a kind of immobilized glutathione synthetase, described immobilized glutathione synthetase comprises: fixation support; With the glutathione synthetase that is fixed on described carrier.
In another preference, the mass ratio of described fixation support and described glutathione synthetase is: 1: 1-100:1(is preferably 10:1-50:1).
Described glutathione synthetase is fixed on fixation support by being selected from the mode of lower group: absorption, embedding, covalent attachment, crosslinked or its combination.
The preferred fixation support matrix in this aspect is microballoon or high molecular polymer.
Preferably, described fixation support comprises the matrix of fixation support, and the connect elements being connected with described matrix.
In a preference of the present invention, the structure of described immobilized glutathione synthetase is as follows:
Z-(L-E)n
In formula, Z represents fixation support matrix; E represents glutathione synthetase; L represents the connect elements between E and Z; N is more than or equal to 1 positive integer.
In another preference, n is the positive integer that is selected from 1-100000.
Preferably, described immobilized glutathione synthetase has following characteristic:
(a) the immobilized glutathione synthetase of enzymic activity >=0.5U/g, preferably >=immobilized glutathione synthetase of 2U/g;
(b) half-life characteristics >=10 day, preferably >=15 days, more preferably >=20 days.
In the present invention, although be to represent the structure of described immobilized glutathione synthetase with Z-(L-E) n, it should be understood that, not each connect elements on carrier matrix is combined with a glutathione synthetase, some connect elements may be combined with glutathione synthetase, or may have more than 2 or 2 connect elements to be combined with same glutathione synthetase yet.Therefore Z-(L-E) n is only schematic diagram, is not completely real structure.
Fixing of glutathione synthetase
The present invention also provides the preparation method of immobilization glutathione synthetase, comprises step: glutathione synthetase is fixed on described carrier, thereby forms described immobilized glutathione synthetase.Before glutathione synthetase is fixed on described carrier, can also comprise the activation step of carrier.
The method of immobilization gsh provided by the invention, by the glutathione synthetase of different sources and different being fixed of fixation support, or fixes altogether to two enzymes.
According to a preferred embodiment of the present invention, the difunctional synthase gene of gsh is cloned into expression vector, import in intestinal bacteria, realize the expression of this enzyme.By crude enzyme liquid and fixation support, be fixed in proportion.This immobilized enzyme can effectively be realized the bio-transformation of gsh.
According to another preferred embodiment of the present invention, gamma-glutamylcysteine synthetase and Glutatione synthetase gene are cloned into respectively to expression vector and import in intestinal bacteria, realize the expression of enzyme, form the catalyst system of glutathione synthetase.Crude enzyme liquid and fixation support with containing certain proportion enzyme amount, be fixed in proportion.This immobilized enzyme can effectively be realized the bio-transformation of gsh.
Application
Immobilized glutathione synthetase of the present invention is used to produce GSH.
The present invention also provides the method for producing GSH, comprises step: immobilized glutathione synthetase of the present invention is contacted with reaction substrate, obtain the reaction mixture that contains GSH; Separated GSH from above-mentioned mixed solution.
In another preference, described substrate contains glycine, halfcystine and L-glutamic acid.
Major advantage of the present invention is:
(1) immobilization glutathione synthetase of the present invention can repeatedly be used, and stability is high, effectively improves the productivity of unit enzyme;
(2) with immobilization glutathione synthetase of the present invention, as the simplification of GSH biosynthesis system production process, production efficiency, improve, simplified purifying technique.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The immobilization of the gsh bifunctional enzyme (Sa-GSH) in streptococcus agalactiae source
The preparation of the gsh bifunctional enzyme crude enzyme liquid in 1.1 streptococcus agalactiae sources
1.1.1 preparation
The expression bacterial classification of the gsh bifunctional enzyme (Sa-GSH) in streptococcus agalactiae source builds, cultivates and abduction delivering with reference to method described in Blythe E.Janowiak and Owen W.Griffith.Glutathione synthesis in Streptococcus agalactiae.Journal of Biological Chemistry.2005.280:11829-39..
After the thalline fermenting is centrifugal, weigh, with the broken born of the same parents of squeezing after 0.02M potassium phosphate buffer (pH8.0 ± 0.2) suspendible of 1:4 (mass volume ratio), centrifugal, obtain crude enzyme liquid.
1.1.2 crude enzyme liquid determination of activity
5ml reaction system is: 0.1M PBS (pH7.4), 40mM Mg
2+, 40mM ATP, 120mM glycine, 80mM Cys, 120mM Pidolidone, 4M KOH adjusts pH 6.6, adds after the above-mentioned crude enzyme liquid of 1ml, 30 ℃ of reaction 30min, by 10% hydrochloric acid termination reaction, the growing amount of GSH in HPLC detection reaction system.
The HPLC detection method of GSH is as follows: get 6.8g KH
2pO
4with 2.2g sodium heptanesulfonate, add water and be settled to 1L, then use H
3pO
4regulating PH is 3.0, is damping fluid.Proportion of mobile phase (Buffer:MEOH=97:3), chromatographic condition is: sample size 5 μ L, flow velocity 1.0ml/min, acquisition time 15min, C18 reversed-phase column-SB series, 30 ℃ of column temperatures, detect wavelength 210nm.
Measurement result shows, the activity of described crude enzyme liquid gsh bifunctional enzyme, and activity can reach 21.5U/g wet thallus.
1.2 immobilization
The fixation support that the present embodiment is used is in Table 2, and structural representation is shown in Fig. 1:
Table 2
Note: described in table, long side chain and short-side chain are comparatively speaking.
1.2.1 amino fixation support
Amino fixation support activation method is: 10g carrier is immersed in to 40ml 0.1M potassium phosphate buffer (pH4.2-4.5) 150rpm, jolting 15min under 20-25 ℃ of condition; Abandon supernatant, with 0.1M potassium phosphate buffer (pH 8.0 ± 0.2), clean 2 times; Standing, remove supernatant; Carrier is immersed in to 40ml 0.02M potassium phosphate buffer (pH 8.0 ± 0.2) again, 150rpm, jolting 5min at 20-25 ℃; Remove supernatant, with 40ml containing 0.02M potassium phosphate buffer (pH 8.0 ± 0.2) the submergence carrier of 2% glutaraldehyde, 150rpm, jolting 60min at 20-25 ℃; Standing, remove supernatant; With 0.02M potassium phosphate buffer (pH 8.0 ± 0.2), cleaning carrier drains for twice.
Amino fixation support and the difunctional synthetic enzyme of gsh fixing, take EC-HA-SaGSH as example, and the preparation method of immobilized enzyme is as follows: by the wet carrier of 10g with 40ml enzyme liquid (approximately containing 0.6g albumen) under 20-25 ℃ of condition, 150rpm jolting.After 1min, stop jolting, checking and adjusting pH is 8.0 ± 0.1, continues jolting 18h, measures supernatant enzyme and lives and protein concentration; Remove supernatant, with 40ml 0.02M potassium phosphate buffer (pH 8.0 ± 0.2) jolting 1-2min under 25 ℃ of conditions; Remove supernatant, potassium phosphate buffer (pH 8.0 ± 0.2) 20-25 ℃ with 0.02M containing 0.5MNaCl, 150rpm jolting 45min; Remove supernatant, again use 0.02M potassium phosphate buffer (pH 8.0 ± 0.2) to clean, with vacuum pump, drain, measure immobilized enzyme enzyme and live.
The fixing preparation with EC-HA-SaGSH of other amino fixation supports and the difunctional synthetic enzyme of gsh.
1.2.2 epoxies fixation support
Epoxies fixation support, without activation, can be directly used in the fixing of enzyme.Fixing of such carrier and the difunctional synthetic enzyme of gsh, take EC-EP as example, and the preparation method of immobilized enzyme is as follows: by the wet carrier of 10g and 40ml enzyme liquid (approximately containing 0.6g albumen), be placed in 1.25M potassium phosphate buffer (pH 8.0 ± 0.2), under 20-25 ℃ of condition, 150rpm jolting; After 1min, stop jolting, checking and adjusting pH is 8.0 ± 0.1, continues jolting 18h; Stop jolting, at the same temperature standing 20-24h; Measuring supernatant enzyme lives and protein concentration; Remove supernatant, with 40ml 0.02M potassium phosphate buffer (pH 8.0 ± 0.2) jolting 1-2min under 20-25 ℃ of condition; Remove supernatant, again with same buffer jolting washing 45min; Remove supernatant, again use 40ml 0.02M potassium phosphate buffer (pH 8.0 ± 0.2) to clean, with vacuum pump, drain, measure immobilized enzyme enzyme and live.
The fixing preparation with EC-EP-SaGSH of other epoxies fixation supports and the difunctional synthetic enzyme of gsh.
1.2.3 activity of the immobilized enzyme is measured
The enzyme detection method alive of immobilization Sa-GSH is: in 10ml reaction system, contain 0.1M PBS (pH 7.4), 40mM Mg
2+, 30mM ATP, 120mM glycine, 80mM Cys, 120mM Pidolidone, 4M KOH adjusts pH 6.6, adds 1.5g immobilization Sa-GSH.30 ℃ of reaction 30min, the growing amount of GSH in HPLC detection reaction system.Enzyme work is defined as, and per minute generates the amount of the immobilized enzyme of 1 μ mol GSH.
The results are shown in Table 3.
The carrier that table 3 is different and Sa-GSH prepare the enzyme of immobilized enzyme and live
| EC-HA-SaGSH | EC-HFA-SaGSH | EC-EP-SaGSH | EC-EA-SaGSH | |
| Enzyme (U/g) alive | 2.9 | 2.45 | 0.61 | 0.93 |
| Enzyme yield (%) alive | 33.7 | 28.5 | 7.1 | 10.8 |
The immobilization of the gsh bifunctional enzyme (STH) of thermophilus streptococcus
The expression strain of the gsh bifunctional enzyme of thermophilus streptococcus is with reference to Li W, Li ZM, Yang JH, method builds, cultivates and abduction delivering described in Ye Q.Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli.Journal of Biotechnology.2011.154:261-8..
The pretreatment process of the preparation of crude enzyme liquid, HPLC detection and fixation support is with embodiment 1.
In addition, the enzyme of the gsh bifunctional enzyme crude enzyme liquid of thermophilus streptococcus detection method alive is removed pH and is adjusted into 7.5, the mensuration that all the other are lived with the gsh bifunctional enzyme crude enzyme liquid enzymes in streptococcus agalactiae source in embodiment 1.
The preparation of immobilized enzyme adopts the amino described in embodiment 1 and epoxies fixation support equally, and corresponding method is fixed.Immobilized enzyme enzyme activity determination method is removed pH and is adjusted into 7.5, and all the other are with the method for measuring alive of immobilized enzyme in embodiment 1.
Result shows, EC-HA-STH, and the enzyme work of EC-EP-STH is respectively 2.33U/g, 1.15U/g, the enzyme yield alive of immobilized enzyme is respectively 44.4% and 21.8%.
Gamma-glutamylcysteine synthetase (GSH I) and the GSH synthetic enzyme (GSH II) in intestinal bacteria source are fixed altogether
Gamma-glutamylcysteine synthetase (GSH I) and the GSH synthetic enzyme (GSH II) in intestinal bacteria source are expressed bacterial classification, with reference to Shen LX, Wei DZ, Zhao ZF, Zhang SL, Wang EL.Cloning and expression of the genes of glutathione synthetases.Sheng Wu Gong Cheng Xue Bao.2001.17 (1): described in 98-100., method builds, cultivates and abduction delivering.
The pre-treatment of the preparation of crude enzyme liquid, HPLC detection and fixation support is with embodiment 1.
In addition, the measuring method that gamma-glutamylcysteine synthetase (GSH I) enzyme in intestinal bacteria source is lived removes pH and is adjusted into 7.5, GSH I and GSH II add respectively 0.2ml, 0.8ml, the mensuration that all the other are lived with the gsh bifunctional enzyme enzyme in streptococcus agalactiae source in embodiment 1.
In GSH synthetic enzyme (GSH II) enzyme activity determination, remove GSH I and GSH II and add respectively 0.9ml, 0.1ml, the mensuration that all the other are lived with above-mentioned GSH I enzyme.
The preparation of immobilized enzyme: by gamma-glutamylcysteine synthetase (GSH I), the GSH synthetic enzyme (GSH II) in intestinal bacteria source, ratio with 1:1, on amino carrier or epoxies carrier, fix altogether, immobilized method is with embodiment 1; Or, by gamma-glutamylcysteine synthetase (GSH I), GSH synthetic enzyme (GSH II), on amino carrier or epoxies carrier, be fixed respectively.The enzyme activity determination method of fixing immobilized enzyme is removed pH and is adjusted into 7.5 altogether, the mensuration that all the other are lived with the gsh bifunctional enzyme immobilized enzyme enzymes in streptococcus agalactiae source in embodiment 1; Fixedly time, in the enzyme activity determination of immobilization GSH I and immobilization GSH II, except adding respectively the crude enzyme liquid of 0.3ml GSH II and 0.3ml GSH I, all the other are with above-mentioned fixing enzyme activity determination method altogether respectively.
Result shows, GSH I and GSH II, with the ratio of 1:1, are fixed altogether on EC-HA, and its comprehensive enzyme work can reach 1.2U/g; GSH I and GSH II are fixed respectively on EC-HA, and enzyme work is respectively 2.3U/g, 5.9U/g, and the enzyme rate of recovery alive of immobilized enzyme is respectively 36.0%, 13.0%.
Embodiment 4
The stability test of immobilized enzyme EC-HA-SaGSH
Immobilized enzyme EC-HA-SaGSH to embodiment 1 preparation carries out stability test, this immobilized enzyme is placed in to temperature under 30 ℃ of conditions and bathes, and gets a certain amount of mensuration enzyme every day and lives, and investigates continuously 20 days.The enzyme work of immobilized enzyme first day of take is benchmark, investigates the enzyme retention rate of living.This immobilized enzyme enzyme activity determination method is with described in embodiment 1.
Result (Fig. 2) shows: this immobilized enzyme activity when the activity of the 15th day is 70%, the 20 day is still greater than 50%, as seen its transformation period very long, there is satisfactory stability.
The stability test of immobilized enzyme EC-EP-STH
Immobilized enzyme EC-EP-STH to embodiment 2 preparations carries out stability test, and this immobilized enzyme enzyme activity determination method is with described in embodiment 2, and study on the stability mode is with described in embodiment 4.
Result (Fig. 3) shows: this immobilized enzyme activity when the activity of the 15th day is greater than 70%, 20 day still approaches 60%, as seen its transformation period very long, there is satisfactory stability.
Embodiment 6
The immobilization comparison of different carriers and GSH synthetic enzyme
By the gsh bifunctional enzyme (Sa-GSH) in streptococcus agalactiae source respectively with long side chain carrier EC-HFA, EC-HA and short-side chain carrier EC-EP, being fixed of EC-EA, measure respectively the enzyme work of immobilized enzyme, and compare.Process for fixation, the mensuration that immobilized enzyme enzyme is lived is with described in embodiment 1.
Result as shown in Figure 4, (EC-HFA-SaGSH is about 2.45U/g to show to live enzyme after being fixed with long side chain carrier, EC-HA-SaGSH is about 2.9U/g) be far longer than the fixing enzyme of short-side chain carrier and live (EC-EP-SaGSH is about 0.6U/g, and EC-EA-SaGSH is about 0.9U/g).
This shows: adopt the carrier of relatively long side chain to be fixed GSH synthetic enzyme, be better than short-side chain carrier, the former initial enzyme is lived higher.In the situation that it all possesses good stability, adopt being fixed of carrier of long side chain, can obtain the GSH reaction solution containing greater concn.
Embodiment 7
The stability test of Sa-GSH solution enzyme
By the crude enzyme liquid of the gsh bifunctional enzyme (Sa-GSH) in streptococcus agalactiae source, be placed in temperature under 30 ℃ of conditions and bathe, at different point in time sampling, to measure enzyme and live, the preparation of crude enzyme liquid and enzyme activity determination method are with described in embodiment 1.The initial enzyme work of take is 100%, and result as shown in Figure 5.
Visible, under same condition, the transformation period of solution enzyme is only 25h left and right, far below the transformation period (approximately 20 days) of immobilized enzyme; After 48h, enzyme is complete deactivation.By after this enzyme immobilization, greatly improved its stability.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. an immobilized glutathione synthetase, is characterized in that, described immobilized glutathione synthetase comprises:
(1) fixation support; With
(2) be fixed on the glutathione synthetase of described carrier.
2. immobilized glutathione synthetase as claimed in claim 1, is characterized in that, described fixation support comprises the matrix of fixation support, and the connect elements being connected with described matrix.
3. immobilized glutathione synthetase as claimed in claim 2, is characterized in that, the structure of described immobilized glutathione synthetase is as follows:
Z-(L-E)n
In formula,
Z represents fixation support matrix;
E represents glutathione synthetase;
L represents the connect elements between E and Z;
N is more than or equal to 1 positive integer.
4. immobilized glutathione synthetase as claimed in claim 3, is characterized in that, the main chain of described connect elements has 4-50 atom that is selected from C, N or O.
5. the immobilized glutathione synthetase as described in as arbitrary in claim 1-4, is characterized in that, described immobilized glutathione synthetase has following characteristic:
(a) the immobilized glutathione synthetase of enzymic activity >=0.5U/g, preferably >=immobilized glutathione synthetase of 2U/g;
(b) half-life characteristics >=10 day, preferably >=15 days, more preferably >=20 days.
6. immobilized glutathione synthetase as claimed in claim 1, it is characterized in that, described glutathione synthetase is selected from lower group: gamma-glutamylcysteine synthetase (EC6.3.2.2, GSH I), GSH synthetic enzyme (EC6.3.2.3, GSH II) or the difunctional synthetic enzyme of gsh or its combination.
7. a method of preparing immobilized glutathione synthetase described in claim 1, is characterized in that, comprises step: glutathione synthetase is fixed on described fixation support, thereby forms immobilized glutathione synthetase.
8. method as claimed in claim 7, is characterized in that, before glutathione synthetase is fixed on described carrier, also comprises the step that carrier is activated.
9. the purposes of immobilized glutathione synthetase described in claim 1, is characterized in that, it is used to produce gsh GSH.
10. a method of producing gsh GSH, is characterized in that, comprises step:
(1) immobilized glutathione synthetase described in claim 1 is contacted with reaction substrate, obtain the reaction mixture that contains GSH;
(2) separated GSH in the reaction mixture from (1).
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| CN117292748A (en) * | 2023-09-25 | 2023-12-26 | 河南大学 | Enzyme activity optimization method for producing glutathione by enzyme method |
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| WO2016114618A1 (en) * | 2015-01-16 | 2016-07-21 | 서강대학교산학협력단 | Method for continuously producing glutathione using photosynthetic cell membrane vesicle |
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| CN106086126A (en) * | 2016-08-29 | 2016-11-09 | 开平牵牛生化制药有限公司 | A kind of method of Enzyme catalyzed synthesis glutathion |
| CN117292748A (en) * | 2023-09-25 | 2023-12-26 | 河南大学 | Enzyme activity optimization method for producing glutathione by enzyme method |
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