CN107326052A - A kind of method that glutamate decarboxylase activity is improved with D101 macroporous absorbent resins - Google Patents
A kind of method that glutamate decarboxylase activity is improved with D101 macroporous absorbent resins Download PDFInfo
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Abstract
The present invention relates to a kind of method that VREF glutamate decarboxylase activity is improved with D101 macroporous absorbent resins, belong to biological technical field；The present invention is the enzyme activity promoter using D101 macroporous absorbent resins as VREF glutamate decarboxylase, according to D101 macroporous absorbent resin quality: substrate solution volume: VREF bacteria suspension or glutamate decarboxylase resolvase liquid product build D101 macroporous absorbent resin glutamate decarboxylase composite catalyst systems for 1: 1: 1 ratio mixing, when stirring at low speed reacts the h of 24 h~36 in 80 r/min, the reaction of 37~43 DEG C of water bath chaders or agitator tank, the yield of gamma aminobutyric acid can improve 18.54%~149.11%.D101 macroporous absorbent resins are outer except the activity for significantly improving glutamate decarboxylase, and its exchange adsorption effect to gamma aminobutyric acid is also the process of gamma aminobutyric acid purifying, simplifies downstream extraction purifying process, reduction production cost；Method is easy, environmental protection.
Description
Technical field
The present invention relates to a kind of method that glutamate decarboxylase activity is improved with D101 macroporous absorbent resins, belong to biological skill
Art field.
Background technology
γ-aminobutyric acid (γ-Aminobutyric acid, GABA) is a kind of nonprotein group with 4 carbon atoms
Be the main inhibitory neurotransmitter of mammalian central nervous system into amino acid, with diuresis, reducing blood lipid, anti-diabetic,
Anti-oxidant, anti-inflammatory, anticancer, hypotensive, calmness and improve sleep etc. effect, it has also become the medicine received much concern and health products into
Point.Due to that can form 2-Pyrrolidone by lactamization, therefore γ-aminobutyric acid still produces biological plastics polyamides
The important source material of amine 4.
Microbial growth is fast, produces γ-aminobutyric acid without limitation of time and space with it, therefore pass through microorganism
Glutamate decarboxylase (glutamate decarboxylase, GAD, EC4.1.1.15) production γ-aminobutyric acid receives much concern.
Glutamate decarboxylase is a kind of lyases dependent on phosphopyridoxal pyridoxal phosphate (pyridoxal 5 '-phosphate, PLP), is present in
In cytoplasm, be organism catalysis Pidolidone (L-glutamic acid, L-Glu) occur α-carboxyl decarboxylation generation γ-
Unique enzyme of aminobutyric acid.Because the substrate (Pidolidone) and product (γ-aminobutyric acid) of glutamic acid decarboxylase enzyme effect are
Small molecule, can pass through cell membrane, therefore directly can produce γ-aminobutyric acid using cell conversion process, reduce endocellular enzyme
Extraction cost, simplifies production technology.Existing document passes through saliva chain coccus thermophilous subspecies, Pediococcus pentosaceus, VREF, short and small
Lactobacillus, Bacillus coli cells conversion method prepare the report of γ-aminobutyric acid.
Lu Zhaoxin, Yang Sheng are remote etc. to disclose a kind of gamma-amino in Chinese patent (patent No. ZL 200510040758.9)
The production method of butyric acid, it is, for strain, to make with saliva chain coccus thermophilous subspecies (Streptococcus thermophilus)
For glutamic acid, glutamate, the material containing glutamic acid or glutamate, α-carboxyl of glutamic acid is set to occur decarboxylation, from
And generate γ-aminobutyric acid.
Wu Tianxiang etc. discloses solid state fermentation in Chinese patent (publication number CN101240301) and prepares γ-aminobutyric acid
Method, comprise the following steps：Go out monascus specie by raw material screening of fermented bean curd first；Then monascus MP1104 strains are put
In cultivating 7d on slant medium, make actication of culture；Then fermented bacterium is transferred in culture medium, in 30 DEG C of temperature, rotating speed 150
By activated spawn shaking table culture 2d under r/min, fermentation seed, preferably solid state fermentation conditionses and culture medium are prepared；Finally by strain
In previous step culture production γ-aminobutyric acid is carried out under preferred culture medium and condition of culture.This method is using rice as hair
Ferment raw material, using monascus as strain, with edible safety, can directly eat as health food；The gamma-amino of this method
Butyric acid yield and purity are high, and under optimal conditions of fermentation and culture medium, γ-aminobutyric acid yield can by initial 0.21mg/g
0.35mg/g is reached, the purity of final products can reach 45%.
Jiao's celebrating just waits a kind of enzyme of γ-aminobutyric acid disclosed in Chinese patent (patent No. ZL 200410064813.3)
Method conversion preparation method, two kinds of mixing acidic amino acids of the preparation method Pidolidone and L-Aspartic acid, will as raw material
The somatic cells of Escherichia Escherichia coli AS1.505 with high vigor Pidolidone decarboxylase are with containing L-
The conversion fluid of glutamic acid and L-Aspartic acid mixture is mixed, and is carried out enzymatic reaction at 28-45 DEG C, is then used isoelectric point crystallizing method
Or isoelectric point crystallizing separates converted product with the method that ion exchange resin is combined, obtain high-purity γ-aminobutyric acid and
L-Aspartic acid.This method solve the problem that two kinds of acidic mixed amino acid are efficiently separated, obtained the higher γ of added value-
Aminobutyric acid, and with low in raw material price, easy to operate, transformation time is short, the low advantage of production cost.
The happy grade of plum discloses a kind of biosynthesis γ-ammonia in Chinese patent (patent No. ZL 200510049187.5)
The method of base butyric acid, it is characterised in that:Deposit number is CGMCC NO.1306 Lactobacillus brevis (Lactobacillus
Brevis), after being activated through agar slant culture-medium, transfer in GYP seed culture mediums or MRS seed culture mediums, cultivate 10~30h
Afterwards, it is inoculated in 0.5%~5% inoculum concentration in GYP or MRS fermentation mediums, the quiescent culture 48h at 25 DEG C~35 DEG C
~120h, produces the zymotic fluid of mycetome, and thalline, which is centrifuged, to be collected；Thalline after centrifugation is washed with sterilized deionization again
Wash, take 0.25~2g wet thallus, be suspended in 15~50mL citric acid-disodium hydrogen phosphate buffer system, L-sodium
Content is 5 mmol/L~60mmol/L, reacts 1~10h, and reaction solution centrifugation produces the solution containing γ-aminobutyric acid.Plum is happy
PH fermenting and producing γ-aminobutyric acids are controlled Deng one kind is also disclosed in Chinese patent (patent No. ZL 200510049187.5)
Method.It is characterized in that:Deposit number is CGMCC NO.1306 Lactobacillus brevis (Lactobacillus brevis), warp
After agar slant culture-medium activation, transfer in GYP seed culture mediums, after 25~35h of culture, connect with 5~10% inoculum concentration
Plant in fermentation tank, fermentation tank liquid amount is 1~3L, speed of agitator is 50~150r/min, the quiescent culture at 30 DEG C, to it
PH control fermentations being carried out, fermented and cultured about 25~40h treats that thalli growth enters stationary phase, after pH value is gone up, continuous stream adds 1~
3mol/L hydrochloric acid, fermentation medium pH controls continue to cultivate about 40~60h, produced containing γ-aminobutyric acid 5.0~5.6
Zymotic fluid.
Guo Xiaofeng etc. discloses one kind in Chinese patent (patent publication No. CN101102683) and is related to containing gamma-amino
The production method of the food of butyric acid, it includes making yeast or its processed material act on sugar and/or glycometabolism intermediate, or makees
For sugar or glycometabolism intermediate and glutamic acid or its salt, wherein, above-mentioned yeast has in the presence of sugar or glycometabolism intermediate
The ability of γ-aminobutyric acid is produced by fermentation reaction.
Jiang Donghua etc. discloses a kind of highly producing gamma-aminobutyric acid in Chinese patent (patent publication No. CN101302480)
Red monascus Mr-5 bacterial strains and its screening technique and purposes.The high yield GABA red monascus (Monascus of the invention
Ruber Mr-5) bacterial strain, its deposit number is：CCTCC NO：M208043, be preservation：In China typical culture collection
The heart.It there is further disclosed herein the screening technique and purposes of above-mentioned red monascus Mr-5 bacterial strains and for γ-aminobutyric acid
The method of synthesis, the gamma-amino fourth for containing 6~9g/L in the zymotic fluid obtained by the method for γ-aminobutyric acid is synthesized using bioanalysis
Acid.
Cui Xiaojun etc. discloses a kind of γ-ammonia of biosynthesis in Chinese patent (patent publication No. CN101311273)
The method of base butyric acid preparation, the product contains by weight percentage：5% to 60% γ-aminobutyric acid.Above-mentioned biosynthesis
The preparation method of gamma-aminobutyric acid preparation carry out according to the following steps：Nisin strain is inoculated into 250mL by grape first
Sugar, corn starch, defatted soybean meal powder, monosodium glutamate composition fermentation seed culture medium, formed zymotic fluid, by zymotic fluid introduce at a high speed from
Centrifugation forms clear liquid in scheming, by clear liquid at 40 DEG C, adds 250mg/L chitosans, stirring flocculation, by the hair to be filtered through flocculation
Zymotic fluid obtains cleaner liquid by flame filter press, and cleaner liquid carries out ion exchange through cation resin exchange bed, treats ion exchange
After resin saturation, deionized water elution all elutes glutamic acid, then extracts γ-aminobutyric acid with ammoniacal liquor elution.
Cao Yusheng etc. discloses a kind of highly producing gamma-aminobutyric acid in Chinese patent (patent publication No. CN101333508)
Lactobacillus brevis, its feature and process step are：Lactobacillus brevis (Lactobacillus brevis) are identified as, country
Culture presevation number： Lactobacillus brevis CCTCCM 208054.The Lactobacillus brevis of MRS agar slants will be preserved in,
Transfer in MRS fluid nutrient mediums, it is activated after, MRSG fluid nutrient mediums are inoculated in 2-5% inoculum concentration, in 25-30 DEG C of training
The γ-aminobutyric acid supported in 60-90h, zymotic fluid reaches 50-145mmol/L.Also in Chinese patent, (patent is disclosed Cao Yusheng etc.
Number CN101333548) in disclose a kind of method that utilization Lactobacillus brevis prepares γ-aminobutyric acid, its process step
For：1. after Lactobacillus brevis is activated using MRS fluid nutrient mediums, MRSG fermentation mediums, 34 are inoculated in 5% inoculum concentration
DEG C culture 40-60h, 4 DEG C are collected by centrifugation thalline；2. 2 rear overhangs are washed in paddy containing 10-100mmol/L using sterile saline
In propylhomoserin sodium, pH 5.2 acetate buffer solution, 34 DEG C of 1~8h of reaction are the solution containing γ-aminobutyric acid after centrifugation.
Zhao Jinglian etc. is in digest (bioengineering journal, 1989,5 (2)：Reported in 124-128) with calcium alginate embedded
Immobilized cell is made in Bacillus coli cells by method, is carried out intermittent reaction with 1% glutamic acid solution, is continuously stirred formula reaction and connect
Continuous pillar reaction production γ-aminobutyric acid.Intermittent reaction 5h conversion ratios have reached 100%；Formula reaction is continuously stirred in triangular flask
Carried out in reactor, substrate solution and output-response liquid are inputted with 6mL/h flow velocity, conversion ratio is up to 85%；Continuous pillar reaction
Carried out in device, coutroi velocity 12mL/h, conversion ratio is up to 95%.
Your equality of chapter is in digest (Changsha Institute of Electric Power Engineering journal (natural science edition), 1998,13 (4)：Reported in 433-435)
Immobilized cell is made in Bacillus coli cells with calcium alginate embedded method, extracts useless after glutamic acid to rear road sodium glutamate mother liquid
Liquid carries out conversion production γ-aminobutyric acid, obtains alpha-aminobutyric acid content and has reached 98.94%, yield is 49.65%.
Kono I etc. are in digest (Biosci.Biotechnol.Biochem., 2000,64 (3):Described in 617-619)
The change of γ-aminobutyric acid in being made to Koji, alpha-aminobutyric acid content has reached 120 μ g/g.
Wang JJ etc. are in digest (J Ind Microbiol Biotechnol, 2003,30：Profit is reported in 669-676)
Solid fermentation is carried out with Monascus purpureus NTU 601, alpha-aminobutyric acid content has reached 5004mg/kg.
Su YC etc. are in digest (J Ind Microbiol Biotechnol, 2003,30 (1)：Report and adopt in 41-46)
Solid fermentation is carried out with Monascus purpureus CCRC31615, alpha-aminobutyric acid content has reached 1200mg/kg.
Nomura M etc. are in digest (J Dairy Sci., 1998,81:Described in 1486-1491) from production cheese
One plant of Lactococcus lactis 01-7 is separated in bacterial strain, for cheese production, the content of the γ-aminobutyric acid of cheese
383mg/kg is reached.
Perhaps build up the Army to report in its Ph.D. Dissertation (Southern Yangtze University, 2 months 2004) and screened from lactic acid bacteria
The Lactococcus lactis bacterial strains of highly producing gamma-aminobutyric acid, 25L tanks fermentation 72h, the γ-aminobutyric acid of zymotic fluid reaches
250 mg/100mL.
Liu Qing etc. is in digest (amino acid and living resources, 2004,26 (1)：Also to highly producing gamma-aminobutyric acid in 40-43)
Lactic acid bacteria is screened and fermentation condition is reported that the γ-aminobutyric acid in zymotic fluid reaches 3.1g/L.
Yokoyama S etc. are in digest (Journal of Bioscience and Bioengineering, 2002,93
(1)：Report and vinasse are fermented in 95-97) using Lactobacillus brevis IFO-12005, gamma-amino fourth
The content of acid has reached 10.18 mmol/L, and preferable gamma-amino fourth is obtained by centrifugation, flocculation, decolouring and deodorization processing
Acid solution, available for Food fortification γ-aminobutyric acid.
Love generation height of delaying waits and is reported in digest (food と science, 2001, No.8,81-85) and use Lactobacillus
Plantarum utilizes the culture medium fermenting and producing γ-aminobutyric acid containing rice bran extract, and 5% has been reached in dry powder content.
Komatsuzaki N etc. are in digest (Food Microbiology, 2005,22:Reported in 497-504) from day
It is isolated to Lactobacillus paracasei in this traditional fermented food to produce for γ-aminobutyric acid, gamma-amino fourth
Acid concentration has reached 302 mmol/L.
Takahashi T etc. are in digest (Journal of Bioscience and Bioengineering, 2004,97
(6):412-418) in report screened Saccharomyces cerevisiae UT-1 γ-aminobutyric acid transaminase and
γ-aminobutyric acid concentration reaches respectively in succinic semialdehyde dehydrogenase defective mutant bacterial strain GAB7-1 and GAB7-2, its zymotic fluid
0.4mmol/L and 0.42 mmol/L have been arrived, 2.0 and 2.1 times have been respectively increased compared with wild strain.
Ijsseldijk etc. discloses to utilize in United States Patent (USP) (United States Patent, US5472718A) to be contained
The Yoghourt for having lactobacillus bulgaricus and streptococcus thermophilus is added in milk, produces cheese, the cheese obtained has larger
Cellular structure, quality is substantially improved, and detects micro γ-aminobutyric acid wherein.
Yang Sheng is far waited discloses a kind of substep control fermentation bar in Chinese patent (patent No. ZL200910114018.3)
The method that part submerged fermentation produces γ-aminobutyric acid, it is, for strain, to lead to VREF (Enterococcus faecium)
PH value and temperature conditionss that substep controls submerged fermentation are crossed, glutamic acid or sodium glutamate, fermenting and producing γ-ammonia are added by external source
γ-aminobutyric acid concentration reaches 10~20g/L in base butyric acid, karusen.
Yang Sheng is far waited and a kind of biosynthesis gamma-amino is disclosed in Chinese patent (patent No. ZL200910192105.0)
The method of butyric acid, it be with Pediococcus pentosaceus (Pediococcus pentosaceus) be strain, by fermentation engineering,
The glutamic acid or sodium glutamate that cell conversion process and enzyme engineering technology add external source are converted, and produce γ-ammonia of high content
Base butyric acid (conversion fluid alpha-aminobutyric acid content up to 1~30%).
Yang Sheng far wait disclosed in Chinese patent (patent No. 200910114016.4) a kind of cell conversion process production γ-
The method of aminobutyric acid, it is the paddy ammonia added with the cytosis of Lactococcus lactis (Lactococcus lactis) in external source
Acid or sodium glutamate, produce the γ-aminobutyric acid of high content, or L. lactis cells are fixed using sodium alginate
Change, glutamic acid is converted by immobilized cell technology or sodium glutamate produces γ-aminobutyric acid.
It is due to Review on Microbial Glutamate Decarboxylase although more with the public technology of Microbe synthesis γ-aminobutyric acid
Activity it is universal not high, γ-aminobutyric acid yield is relatively low, and cost is high, it is difficult to meet industrial production demand.Glutamate decarboxylase
It is the key enzyme of biosynthesis γ-aminobutyric acid, its activity height is directly related with the yield of γ-aminobutyric acid.Except paddy ammonia
Outside the structure and microbial metabolism yield of enzyme of acid decarboxylase, the external reaction condition of catalytic reaction is also influence glutamate decarboxylase
The key factor of activity.Therefore, how to improve Review on Microbial Glutamate Decarboxylase vigor, improve high vigor glutamate decarboxylase cell
Yield and by improving cell transformation reaction system or process conditions, improve the yield of γ-aminobutyric acid, reduction is produced into
This, will be the key issue of urgent need to resolve from now on.
The content of the invention
VREF glutamate decarboxylase is improved by D101 macroporous absorbent resins it is an object of the invention to provide one kind
The method of activity of conversion, by using D101 macroporous absorbent resins as cocatalyst, by D101 macroporous absorbent resins, ammonia containing paddy
The common catalytic body of VREF cell, buffer solution and reaction substrate Pidolidone or the sodium salt of Pidolidone one composition of acid decarboxylase
System, final realize improves VREF cell glutamate decarboxylase activity of conversion, improves the purpose of γ-aminobutyric acid yield, solution
Not the problem of certainly biosynthesis γ-aminobutyric acid cost can be in any more.
D101 macroporous absorbent resins have selection characterization of adsorption to material, and post processing is easy, cheap and easy to get, do not pollute ring
Border, not etching apparatus, can be easily separated and reclaim, reusable, be widely used in isolating and purifying for material.But, as auxiliary
Co-catalyst is applied in biological enzyme reaction system and not yet reported.The present invention is in the screening to a variety of different resins, reaction
On the Research foundation such as condition and its mechanism of action, using D101 macroporous absorbent resins as cocatalyst with VREF cell or
Free glutamic acid decarboxylase is catalyzed Pidolidone or the sodium decarboxylation of Pidolidone one synthesis γ-aminobutyric acid, D101 macroporous absorptions altogether
Resin can significantly improve the activity of conversion of VREF glutamate decarboxylase, improve the yield of γ-aminobutyric acid, reduction production
Cost.
Used VREF (Enterococcus faecium) strain isolation with GAD activity of the present invention is certainly
Pickles, carry out patent culture presevation on June 19th, 2017, and its deposit number is：GDMCC NO.60203, preservation title
For：Enterococcus faecium LNSF2, depositary institution is：Guangdong Province's Culture Collection, preservation address is：
5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100.
In order to realize the purpose of the present invention, inventor is first to pH value to the cell glutamic acid of VREF GDMCC 60203
The influence of decarboxylase activity of conversion is studied, and as a result (accompanying drawing 1) shows the optimal reaction pH value of cell glutamate decarboxylase
For 4.4, when pH value is when between 4.2~4.6, cell glutamate decarboxylase activity of conversion is stronger；When the pH value of reaction system is big
When 4.4, cell glutamate decarboxylase activity declines rapidly.Consider after glutamic acid decarboxylase base generation γ-aminobutyric acid, can cause
Reaction system pH is raised, from can reduce cell glutamate decarboxylase activity, therefore in order to reduce control difficulty and the reduction of pH value
Regulation reaction pH sour consumption, selects 4.2~pH4.6 of pH as the pH value of cell transformation reaction system.
In order to investigate whether D101 macroporous absorbent resins have an impact to conversion reaction buffer system pH value, inventor presses resin
Quality: buffer solution volume adds the suction of D101 macropores for 1: 2 ratio in pH 4.2,0.2mol/L acetic acid-sodium acetate buffer solutions
Attached resin, then determines the pH value of buffer solution, and as a result (accompanying drawing 2) is added after resin, and the pH value of buffer solution is varied less, by pH
4.2 are raised to 4.31 ± 0.06,40 DEG C of insulation 1h, do not have difference when pH of cushioning fluid is with just adding D101 macroporous absorbent resins.Plus
Enter reactions of the pH of buffer system after D101 macroporous absorbent resins still in the glutamate decarboxylases of VREF GDMCC 60203 suitably
In the range of 4.2~pH4.6 of pH value.
The present invention is further to D101 macroporous absorbent resins to being dissolved in pH 4.2,0.2mol/L acetic acid-sodium acetate buffer solutions
Pidolidone and the adsorption capacity of γ-aminobutyric acid be tested, when by resin quality and 0.2mol/L Pidolidones-
0.2mol/L γ-aminobutyric acids mixed solution (being dissolved in 0.2mol/L acetic acid-sodium acetate buffer solutions, pH 4.2) volume is 1: 2 mixed
Close 5min, as a result (accompanying drawing 3) D101 macroporous absorbent resins have adsorption capacity to Pidolidone and γ-aminobutyric acid, when with
Resin quality: 70% ethanol solution that effluent volume is 1: 20 is eluted, Pidolidone and γ-aminobutyric acid in eluent
Respectively (37.45 ± 0.11) mmol/L and (27.51 ± 0.15) mmol/L.
In order to avoid resin influences the concentration of conversion reaction substrate to the suction-operated of Pidolidone, the present invention will be adopted respectively
The D101 macroporous absorption trees balanced with the pH 4.2 of the Pidolidone containing 0.2mol/L, 0.2mol/L acetic acid-sodium acetate buffer solutions
Fat and unbalanced D101 macroporous absorbent resins press resin quality respectively: the sodium of 0.3mol/L Pidolidones one (monosodium glutamate) solution is (molten
In 0.2mol/L acetic acid-sodium acetate buffer solutions, pH 4.2) volume: the bacteria suspension volumes of VREF GDMCC 60203 are 1: 1: 1
Ratio mixing, then in 80r/min, (40 ± 3) DEG C water bath chader reaction 24h, filtered through gauze collects filtrate (note respectively
For conversion fluid, similarly hereinafter) and resin, then resin is eluted and (is designated as eluent, similarly hereinafter), merge conversion fluid and eluent, knot
Really the test group of (accompanying drawing 4) addition D101 macroporous absorbent resins is remarkably improved the glutamic acid of VREF GDMCC 60203 and taken off
The activity of conversion of carboxylic acid, the D101 macroporous absorbent resins through the acetic acid-sodium acetate buffer solution balance containing Pidolidone are compared with lack of equilibrium
D101 macroporous absorbent resins it is more notable to the facilitation of the activity of conversion of glutamate decarboxylase, conversion reaction 24h, relative to
Without the control group of resin, 41.87% and 18.54% has been respectively increased in γ-aminobutyric acid yield.As a result show selection with containing
PH 4.2, the D101 macroporous absorbent resins and dung of 0.2mol/L acetic acid-sodium acetate buffer solutions balance of 0.2mol/L Pidolidones
It is more preferable that the cell constructions of enterococcus GDMCC 60203 are combined conversion reaction system effect.
The addition of D101 macroporous absorbent resins is the important ginseng for building resin-cell glutamate decarboxylase activity of conversion
Number, the present invention adds the resin of Different adding amount in the transformation system of same liquid volume, as a result (accompanying drawing 5), with resin
Addition increase, the yield of γ-aminobutyric acid also increases, when the addition of test system resin and the ratio of total liquid volume reach
During to 1: 2, γ-aminobutyric acid yield adds 48.66% compared with the control group without resin, continues to increase resin content,
The yield and rate of growth of γ-aminobutyric acid tend towards stability.Therefore, the addition of D101 macroporous absorbent resins with transformation system
The ratio of total liquid volume is more suitable when being 1: 2.
In order to understand the differential responses time production that D101 macroporous absorbent resins-cell glutamate decarboxylase is combined transformation system
Thing generates situation and system pH situation of change, and the present invention is by with pH4.2, the 0.2mol/L of the Pidolidone containing 0.2mol/L
The D101 macroporous absorbent resin quality: the sodium of 0.3mol/L Pidolidones one (monosodium glutamate) that acetic acid-sodium acetate buffer is fully balanced
Solution (being dissolved in 0.2mol/L acetic acid-sodium acetate buffer solutions, pH 4.2) volume: the bacteria suspension volumes of VREF GDMCC 60203
Mixed for 1: 1: 1 ratio, then in 80r/min, (40 ± 3) DEG C water bath chader reaction different time, while to be not added with tree
Fat is used as control, and the pH value of tracking and measuring different time conversion reaction solution by same operation, when as a result (accompanying drawing 6) is with reaction
Between extend, the γ-aminobutyric acid yield of test group is gradually higher than control group, the gamma-amino fourth of conversion reaction 24h, 1L test group
Acid yield has reached (171.43 ± 8.08) mmol, and 59.63% is improved compared with control group [(107.39 ± 11.12) mmol].
D101 macroporous absorbent resins are to controlling the pH value of transformation system to have preferable effect, conversion reaction 24h, test group conversion fluid pH
Value is only raised to 4.85 by 4.2, and the conversion fluid pH value of control group has been raised to 5.25, and test group γ-aminobutyric acid is relatively tested
Group yield is high, and alkalinization is stronger, illustrates that D101 macroporous absorbent resins have preferable effect to regulation transformation system pH value.But
It is that, because yield is higher, alkalinization also can be bigger, after test group reaction 9h, system pH has been increasingly detrimental to paddy
The catalytic reaction of propylhomoserin decarboxylase is, it is necessary to aid in the pH value of outer acid adding control system.Accompanying drawing 6 is displayed that simultaneously, reacts more than 24h
When, the effect of D101 macroporous absorbent resins can be more protrusion.
The pH value of transformation system is to influence the essential condition of glutamate decarboxylase, in order to preferably control the pH of transformation system
Value, while preventing moment local overacidification and causing glutamate decarboxylase to inactivate, the present invention uses 0.1mol/L~0.5mol/L
The pH value of transformation system is adjusted HCl, controls the pH value of transformation system in the range of 4.2~4.6, as a result both plus with containing
PH 4.2, the D101 macroporous absorbent resins of 0.2mol/L acetic acid-sodium acetate buffer solutions balance and the control ph of Pidolidone exist
Every liter of γ-aminobutyric acid yield of test group in the range of 4.2~4.6 has reached (267.52 ± 16.04) mmol, more only adds tree
Test group (171.43 ± 8.08) mmol of fat but non-control ph improves 56.05%, is relatively not added with resin but control ph exists
Test group (209.11 ± 13.63) mmol in the range of 4.2~4.6 improves 27.93%, relative to both without resin or not
Control group (107.39 ± 11.12) mmol of control ph improves 149.11%.
Pidolidone is the substrate of glutamic acid decarboxylase enzymic catalytic reaction, and conversion fluid middle reaches can be caused after Pidolidone is adsorbed
From concentration of substrate reduction, so as to influence the rate of catalysis reaction of glutamate decarboxylase, therefore be used as the D101 of cocatalyst
Macroporous absorbent resin first should be balanced using Pidolidone, but equilibrium process should be consistent with the pH value of enzymic catalytic reaction system,
Otherwise the pH value of enzymic catalytic reaction system can be influenceed, the result of study based on accompanying drawing 3 and accompanying drawing 4, the present invention is using containing 0.2mol/
PH4.2,0.2mol/L acetic acid-sodium acetate buffer solution of L L- glutamic acid are balanced to D101 macroporous absorbent resins, can be simultaneously
The problem of solution Pidolidone and pH value are balanced.Accompanying drawing 3 is proved in pH 4.2,0.2mol/L acetic acid-sodium acetate buffer solution bodies
In system, D101 macroporous absorbent resins also have very strong energy of adsorption to the product γ-aminobutyric acid of glutamic acid catalytic reaction
Power, when Pidolidone produces γ-aminobutyric acid under the catalytic action of glutamate decarboxylase, γ-aminobutyric acid can be big with D101
Pidolidone or H that macroporous adsorbent resin is adsorbed when balancing+Generation competitive adsorption, the Pidolidone or H of release+It can supplement
The substrate of enzyme reaction or the pH value for adjusting reaction system, while removing the γ-aminobutyric acid dissociated in reaction solution in time, are reduced
The feedback inhibition of γ-aminobutyric acid, accelerates the speed that γ-aminobutyric acid leaves glutamate decarboxylase activity center, improves paddy ammonia
The catalytic activity of acid decarboxylase, is metabolized while γ-aminobutyric acid can also be reduced by downstream enzyme, increase γ-aminobutyric acid
Yield, accompanying drawing 6 fully demonstrates this result.
Research process example described above for this patent, but it is not limited only to the studies above content.Through use pH 4.2~
The acetic acid-sodium acetate buffer solution of different pH in the range of pH 4.6 substitutes above-mentioned pH 4.2, the 0.2mol/L acetic acid-second being related to
Sour sodium buffer solution is studied, and as a result D101 macroporous absorbent resins are remarkably improved the activity of conversion of glutamate decarboxylase, though
The output increased amplitude of right γ-aminobutyric acid, but also in the range of the amplification 18.54%~149.11% that example is obtained；
Reaction substrate is prepared using the sodium of Pidolidone one (monosodium glutamate) or Pidolidone, and effect is consistent, substrate Pidolidone solution
Effect of the concentration between 0.2mol/L~0.3mol/L is also equal.
The present invention by the cells of VREF GDMCC 60203 by ultrasonic disruption, using 4.2~pH of pH 4.6,0.2
Mol/L acetic acid-sodium acetate buffer solutions extract glutamate decarboxylase, with its free glutamic acid decarboxylase enzyme liquid and D101 macroporous absorptions
Resin carries out catalytic reaction altogether, and as a result D101 macroporous absorbent resins can also remarkably promote the activity of glutamate decarboxylase, gamma-amino
The output increased amplitude of butyric acid is also between 18.54%~149.11%.
The technical scheme is that：
By pH 4.2~pH 4.6 of the D101 macroporous absorbent resins for having regenerated or having pre-processed Pidolidone containing 0.2mol/L,
0.2mol/L acetic acid-sodium acetate buffer solutions are fully balanced, then by D101 macroporous absorbent resin quality: 0.2mol/L~
The sodium of 0.3mol/L Pidolidones one (monosodium glutamate) or Pidolidone solution (are dissolved in 0.2mol/L acetic acid-sodium acetate buffer solutions, pH
4.2~pH 4.6) volume: VREF bacteria suspension (or glutamate decarboxylase enzyme liquid) volume mixes for 1: 1: 1 ratio, in
Stirring at low speed is reacted in 80r/min, 37 DEG C~43 DEG C water bath chader reactions or agitator tank, is passed through during conversion reaction
The pH value of 0.1mol/L~0.5mol/L HCl regulation transformation systems passes through filtering, tree after 4.2~4.6, reaction 24h~36h
The process such as fat elution and centrifugation can obtain γ-aminobutyric acid mother liquor.
The present invention method be：
A kind of method for improving glutamate decarboxylase activity with D101 macroporous absorbent resins of the present invention mainly includes following
Step：
1. the D101 macroporous absorbent resins for having regenerated or having pre-processed fully are balanced with substrate solution；
2. the D101 macroporous absorbent resin quality: substrate solution volume: VREF bacteria suspension or free paddy 1. balanced by step
Propylhomoserin cocarboxylase liquid product mixes three for 1: 1: 1 ratio；
3. by step, 2. mixture reacts or in 37 DEG C~43 DEG C stirring at low speed in 80r/min, 37 DEG C~43 DEG C water bath chaders
Or reaction 24h~36h is stood, the pH value of transformation system is adjusted during conversion reaction by 0.1mol/L~0.5mol/L HCl
4.2~4.6；
4. the mixture after step 3. conversion reaction can be obtained into gamma-amino by processes such as filtering, resin elution and centrifugations
Butyric acid mother liquor.
1. and 2. the substrate is Pidolidone or the sodium of Pidolidone one to above-mentioned steps；The concentration of the substrate solution is
0.2 mol/L~0.3mol/L, preparation method is that substrate is dissolved in into 0.2mol/L acetic acid-sodium acetate buffer solutions, regulation solution
PH is 4.2~4.6.
Compared with prior art, the present invention has the advantages that：
The D101 macroporous absorbent resins selected in the method that the present invention is provided can control conversion by adsorbing γ-aminobutyric acid
The H dissociated in liquid+And γ-aminobutyric acid, glutamate decarboxylase pH value in reaction can be maintained to a certain extent, reduce regulation acid
Amount；Meanwhile, after D101 macroporous absorbent resins absorption γ-aminobutyric acid, it is possible to reduce the feedback inhibition and downstream enzyme of product
Metabolism, and can by reduce conversion fluid dissociate γ-aminobutyric acid concentration and accelerate γ-aminobutyric acid and leave glutamate decarboxylase
The speed in activated centre, promotes the catalytic rate of glutamate decarboxylase.
The present invention by D101 macroporous absorbent resins by adding VREF cell or the free enzymatic conversion of glutamate decarboxylase
System, significantly improves the glutamate decarboxylase activity of VREF, and the yield of γ-aminobutyric acid can improve 18.54%~
149.11%, cost can be significantly reduced.In addition to the activity for promoting VREF glutamate decarboxylase, D101 macroporous absorbent resins
Suction-operated to γ-aminobutyric acid is also a kind of process of γ-aminobutyric acid purifying, can simplify downstream extraction purifying work
Skill, reduces production cost.In addition, the post processing of D101 macroporous absorbent resins is easy, and it is cheap and easy to get, it can be easily separated and reclaim, can weigh
Use again, free from environmental pollution, not etching apparatus.
Brief description of the drawings
Fig. 1 is influence of the pH value to the cell glutamate decarboxylase activity of conversion of VREF GDMCC 60203；
Fig. 2 is influence of the D101 macroporous absorbent resins to conversion reaction buffer system pH value；
Fig. 3 is D101 macroporous absorbent resins to Pidolidone and the adsorption capacity of γ-aminobutyric acid；
Fig. 4 is that D101 macroporous absorbent resins prepared by different balance methods are de- to the cell glutamic acid of VREF GDMCC 60203
The influence of carboxylic acid activity of conversion；
Fig. 5 lives for the addition of D101 macroporous absorbent resins to the cell glutamic acid decarboxylase enzymatic conversions of VREF GDMCC 60203
The influence of property.
Fig. 6 is differential responses time D101 macroporous absorbent resins to pH value of reaction system and VREF GDMCC 60203
The influence of cell glutamate decarboxylase activity of conversion；
GABA is english abbreviation in Fig. 5 and Fig. 6 legend, represents γ-aminobutyric acid；
C.K. is english abbreviation in Fig. 6 legend, represents control group；D101 resins represent the examination of addition D101 macroporous absorbent resins
Test group.
Embodiment
Embodiment of the present invention is with the cells of VREF GDMCC 60203 or the glutamic acid decarboxylase enzymatic conversion L- of free form
Exemplified by glutamic acid production γ-aminobutyric acid, the method for illustrating to improve glutamate decarboxylase activity using D101 macroporous absorbent resins,
But VREF GDMCC 60203 is not limited only to, D101 macroporous absorbent resins are thin to other microorganisms containing glutamate decarboxylase
The activity of conversion of born of the same parents or glutamate decarboxylase resolvase equally has facilitation.
Embodiment 1
A kind of method for improving glutamate decarboxylase activity with D101 macroporous absorbent resins of the present invention comprises the following steps：
1. the sodium of Pidolidone one (monosodium glutamate) is dissolved in 0.2mol/L acetic acid-sodium acetate buffer solutions by, and pH is to 4.2~4.6 for regulation,
The sodium solution of 0.2mol/L~0.3mol/L Pidolidones one is prepared into, substrate solution is used as；
2. is pre-processed D101 macroporous absorbent resins according to resin specification with 95% ethanol and distilled water, then uses bottom
Thing solution equilibria；
3. the D101 macroporous absorbent resin quality: substrate solution volume: the bacterium of VREF GDMCC 60203 that 2. is balanced by step
The ratio that suspension volume is 1: 1: 1 mixes three, and mixture is that glutamate decarboxylase is combined conversion reaction system；
4. by step, 3. mixture reacts or in 37 DEG C~43 DEG C stirring at low speed in 80r/min, 37 DEG C~43 DEG C water bath chaders
Or reaction 24h~36h is stood, the pH value of transformation system is adjusted during conversion reaction by 0.1mol/L~0.5mol/L HCl
4.2~4.6；
5. collects filtrate (conversion reaction solution) and resin, then respectively by the mixture after step 4. conversion reaction by filtering
(eluent) is eluted to the γ-aminobutyric acid of resin adsorption using 70% ethanol solution, conversion reaction solution and will be washed respectively
De- liquid is centrifuged in 4 DEG C~35 DEG C, 4000r/min~10000r/min, and removing sediment can obtain containing γ-aminobutyric acid
Mother liquor.
Embodiment 2
A kind of method for improving glutamate decarboxylase activity with D101 macroporous absorbent resins of the present invention comprises the following steps：
1. Pidolidone is dissolved in 0.2mol/L acetic acid-sodium acetate buffer solutions by, and pH is to 4.2~4.6 for regulation, is prepared into 0.2
Mol/L~0.3mol/L Pidolidone solution, is used as substrate solution；
2. is pre-processed D101 macroporous absorbent resins according to resin specification with 95% ethanol and distilled water, then uses bottom
Thing solution equilibria；
3. the D101 macroporous absorbent resin quality: substrate solution volume: the bacterium of VREF GDMCC 60203 that 2. is balanced by step
The ratio that suspension volume is 1: 1: 1 mixes three, and mixture is that glutamate decarboxylase is combined conversion reaction system；
4. by step, 3. mixture reacts or in 37 DEG C~43 DEG C stirring at low speed in 80r/min, 37 DEG C~43 DEG C water bath chaders
Or reaction 24h~36h is stood, the pH value of transformation system is adjusted during conversion reaction by 0.1mol/L~0.5mol/L HCl
4.2~4.6；
5. collects filtrate (conversion reaction solution) and resin, then respectively by the mixture after step 4. conversion reaction by filtering
(eluent) is eluted to the γ-aminobutyric acid of resin adsorption using 70% ethanol solution, conversion reaction solution and will be washed respectively
De- liquid is centrifuged in 4 DEG C~35 DEG C, 4000r/min~10000r/min, and removing sediment can obtain containing γ-aminobutyric acid
Mother liquor.
Embodiment 3
The method that a kind of utilization D101 macroporous absorbent resins of the present invention improve glutamate decarboxylase activity includes following step
Suddenly：
1. the sodium of Pidolidone one (monosodium glutamate) is dissolved in 0.2mol/L acetic acid-sodium acetate buffer solutions by, and pH is to 4.2~4.6 for regulation,
The sodium solution of 0.2mol/L~0.3mol/L Pidolidones one is prepared into, substrate solution is used as；
2. is pre-processed D101 macroporous absorbent resins according to resin specification with 95% ethanol and distilled water, then uses bottom
Thing solution equilibria；
3. the D101 macroporous absorbent resin quality: substrate solution volume: the paddy of VREF GDMCC 60203 that 2. is balanced by step
Propylhomoserin decarboxylase resolvase liquid product mixes three for 1: 1: 1 ratio, and mixture is the compound conversion of glutamate decarboxylase
Reaction system；
4. by step, 3. mixture reacts or in 37 DEG C~43 DEG C stirring at low speed in 80r/min, 37 DEG C~43 DEG C water bath chaders
Or reaction 24h~36h is stood, the pH value of transformation system is adjusted during conversion reaction by 0.1mol/L~0.5mol/L HCl
4.2~4.6；
5. collects filtrate (conversion reaction solution) and resin, then respectively by the mixture after step 4. conversion reaction by filtering
(eluent) is eluted to the γ-aminobutyric acid of resin adsorption using 70% ethanol solution, conversion reaction solution and will be washed respectively
De- liquid is centrifuged in 4 DEG C~35 DEG C, 4000r/min~10000r/min, and removing sediment can obtain containing γ-aminobutyric acid
Mother liquor.
Claims (1)
1. it is a kind of with D101 macroporous absorbent resins improve glutamate decarboxylase activity method, it is characterised in that will regenerate or
The D101 macroporous absorbent resins of pretreatment are balanced with substrate solution, then by D101 macroporous absorbent resin quality: substrate solution body
Product: the bacteria suspensions of VREF GDMCC 60203 or glutamate decarboxylase resolvase liquid product mix for 1: 1: 1 ratio, in 80
Stirring at low speed reacts the h of 24 h~36 in r/min, the reaction of 37 DEG C~43 DEG C water bath chaders or agitator tank, during conversion reaction
The pH value of transformation system is controlled to be 4.2~4.6 by the mol/L HCl of 0.1 mol/L~0.5；The substrate isL- glutamic acid orL- monosodium glutamate；The concentration of the substrate solution is the mol/L of 0.2 mol/L~0.3, and preparation method is that substrate is dissolved in into 0.2
Mol/L acetic acid-sodium acetate buffer solutions, the pH of regulation solution is 4.2~4.6.
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