CN109652352B - Genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and immobilization method - Google Patents

Abstract

The invention discloses a genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and an immobilization method. The invention provides a genetic engineering bacterium for efficiently immobilizing enterococcus faecium GADEscherichia coli(DH 5 alpha-LNSF 1), the engineering bacteria are deposited in the microorganism strain collection of Guangdong province at the date of 18 and 9 in 2018, and the deposit number is: GDMCC No.60445. The enterococcus faecium GAD immobilized enzyme can be efficiently produced by using the genetically engineered bacterium, and the method is to utilize modified chitin to affinity adsorb CBM-GAD fusion enzyme crude enzyme liquid, so as to obtain the enterococcus faecium GAD immobilized enzyme. The modified chitin used by the method has strong affinity and firm combination with CBM-GAD fusion enzyme, and the obtained GAD immobilized enzyme has high activity and stability, and solves the problems of difficult immobilization and half-aging of GADShort period and high cost; in addition, the immobilization method is quick, high in efficiency and greatly reduced in cost, and has a wide application prospect in the aspect of industrial production of enterococcus faecium GAD immobilized enzyme.

Description

Genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and immobilization method

Technical Field

The invention belongs to the technical field of food biology, and particularly relates to a genetically engineered bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and an immobilization method.

Background

Glutamate decarboxylase (glutamate decarboxylase, GAD, EC 4.1.1.15) specifically catalyzes the decarboxylation of L-glutamic acid (L-glutamic acid, L-Glu) alpha-carboxyl, and has important applications in the biosynthesis of gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) and the resolution of chiral substances DL-glutamic acid (DL-Glu) (Brazilian Journal of Microbiology,2012,43 (4): 1230-1241;Amino Acids,2016,48 (11): 2519-2531; chemical and biological engineering, 2013,30 (11): 55-56.). Because of the high safety of lactic acid bacteria for use in fermented foods, lactic acid bacteria GAD has received great attention, and microorganisms such as Lactobacillus brevis (Lactobacillus brevis) (LWT-Food Sci technology, 2016, 67:22-26), lactobacillus paracasei (Lactobacillus paracasei) (Food Microbiol 2005, 22:497-504), lactobacillus rhamnosus (Lactobacillus rhamnosus) (Braz J Microbiol 2013; 44:183-187), lactobacillus sake (Lactobacillus sakei) (J Microbiol Biotechnol 2015; 25:696-703), streptococcus salivarius thermophilus subspecies (Streptococcus salivarius ssp. Thermophilus) (Food science, 2011,32 (1): 162-167), pediococcus pentosaceus (Pediococcus pentosaceus) (Hubei agricultural science, 2010,49 (6): 1450-1453), enterococcus faecium (Enterococcus faecium) (Food science, 2018,39 (4): 90-98) and Lactobacillus plantarum (Lactobacillus plantarum) (Microbiol Biotechnol Lett,2015, 43:300-305) have GAD. However, since the wild strain GAD is mainly used for meeting the requirement of the microorganism on the acid-resistant mechanism, the expression level is generally low, the activity of GAD is low, and meanwhile, free enzyme is difficult to recover and cannot be recycled and continuously produced, the application cost is high, and the requirement of industrial production of GABA or DL-Glu is difficult to meet.

Because the immobilized enzyme is easy to separate from the reaction liquid and stabilize the activity of the enzyme, the enzyme can be reused conveniently, and the production cost is reduced, the immobilized enzyme is attracting attention in the field of industrial biocatalysis. Currently, GAD immobilization is mainly focused on two aspects: (1) immobilizing wild strain cells with GAD; for example, huang Junyong calcium alginate beads immobilized Lactobacillus brevis cells with GAD activity (doctor's university of Zhejiang paper, 2006). (2) Immobilization of recombinant GAD: expressing high-activity GAD by using genetically engineered bacteria, and immobilizing GAD crude enzyme; for example, zhu Fei studies on immobilization of Lactobacillus brevis GAD with polyvinyl alcohol-sodium alginate, ni-NTA (university of Zhejiang, shuo Shi thesis, 2011); lee et al expressed the E.coli GAD fused with a histidine tag, immobilized the recombinase with nickel chelating resin, and the immobilized enzyme remained 58% of its initial activity after 10 repeated uses (International Journal of Molecular Sciences,2013,14 (1): 1728-1739). However, the reported immobilization methods generally have the problems of higher cost, complicated operation, short half-life, low activity and the like of the immobilization carrier, and particularly, the immobilized enzyme prepared by many immobilization methods is not pure enzyme (such as immobilized cells) and cannot overcome the influence of downstream metabolic enzymes or hybrid proteins. Therefore, there is still a need to find an immobilization method which is convenient, stable, low in cost and high in enzyme purity.

The fusion enzyme expression vector is constructed by cloning the target enzyme gene and the affinity tag by adopting a genetic engineering technology, and is transformed into an expression system such as escherichia coli, yeast and the like for expression, and then the fusion enzyme is immobilized by utilizing the affinity vector, so that the fusion enzyme is a novel technology of efficient immobilized enzyme. If the fusion enzyme with the affinity tag is scientifically designed, constructed and efficiently expressed, an insoluble carrier with low price and rich sources is searched, and then the immobilized enzyme is prepared by utilizing the affinity tag of the fusion enzyme and the strong affinity of the insoluble carrier, so that the immobilized enzyme has good theoretical and practical significance.

Most cellulose degrading enzymes comprise three domains: cellulose Binding Domain (CBD), flexible linking region and catalytic domain (J Biol Chem,1992,267:6743-6749;J Bacteriol,1993,175:5762-5768;Bioresour Technol,2011,102:2910-2915). Cellulose is relatively inexpensive, chemically inert, has low non-specific affinity for most proteins, and can exist commercially in many different forms. Thus, by utilizing the property of a cellulose-binding module (CBM) of a CBD that it can bind specifically and irreversibly to cellulose, CBM can be developed as an affinity tag to construct fusion proteins, and immobilized enzymes can be prepared by affinity adsorption to cellulose (Int J Mol Sci,2012,13:358-368;Biotechnol Prog,2009,25:68-74;J Ind Microbiol Biotechnol,2008,35:1455-1463). Although the thought of application of CBM affinity tag on other enzymes can be theoretically consulted, because of great structure and gene difference of different enzymes, there is no direct replication method in the aspects of construction and efficient expression of CBM-fusion enzyme, and many factors such as whether CBM-fusion enzyme can be efficiently expressed, expression cost and safety still restrict the key and bottleneck of the technical development. As with the research and development of other genetically engineered bacteria, the construction and expression of CBM-fusion enzymes also requires scientific design and construction for individual cases to achieve beneficial effects.

The GAD from different sources has great difference in structure, even if the same genus of microorganism, the subunit composition and molecular weight of GAD from different strains are greatly different. For example, escherichia coli GAD contains 6 identical subunits and has a molecular weight of 53kDa (Biochemistry, 1970,9 (2): 226-232); streptococcus pneumoniae (Streptococcus pneumonia) GAD is similar in molecular weight (54 kDa) and sequence to mammalian GAD65 subunits (59% similar, 28% identical) (FEMS Microbiol Lett,1995,133 (1-2): 113-138); the molecular weight of the GAD subunit of Neurospora crassa (Neurospora crassa) is determined to be about 33kDa, with kinetic properties similar to E.coli GAD (The Journal Of Biological Chemistry,1991,266 (8): 5135-5139); the GAD molecular weight of the Bacillus perfringens (Clostridium perfringens) is 290kDa (Biochem J,1970, 118:135-141); lactobacillus brevis (Lactobacillus brevis) GAD has two subunits with a molecular weight of 60kDa (Biosci Biotech Biochem,1997,61 (7): 1168-1171); the GAD of lactococcus lactis subsp (Lactococcus lactis subsp. Lactis) has only one subunit with a molecular weight of about 54kDa (Microbiology, 1999, 145:1375-1380), however, although the GAD of lactococcus lactis (Lactococcus lactis) SYFS1.009 also has only one subunit, the subunit molecular weight is 65kDa (Xu Jianjun, doctor's article, university of Jiangnan, 2 months 2004), indicating that there is a certain difference between GADs of different subspecies even from the same microorganism. Therefore, the method of immobilizing microbial enzymes using CBM as an affinity tag, which has been reported, is not necessarily suitable for enterococcus faecium GAD, and it is necessary to conduct research on specific enzymes of specific microorganisms and to investigate suitable methods.

From the above analysis, it can be seen that, although the immobilization of CBM-fusion proteins is achieved by means of constructing and expressing CBM tagged fusion proteins theoretically utilizing the specific, irreversible binding properties of CBM to cellulose, there are a number of unpredictability in practice:

(1) Is the gadB gene cloned into the strain of interest? Is there a diversity of microbial gadB genes that can be expressed in different escherichia coli expression systems using the same technology?

(2) Is the constructed CBM-GAD fusion enzyme recombinant vector plasmid scientifically viable? Is it possible to efficiently express CBM-GAD fusion enzymes in e.coli expression systems?

(3) After fusion of the CBM with GAD, it will change the folding structure of GAD and thus the activity or nature of GAD?

(4) What are the inexpensive carriers available, of abundant origin and which bind strongly to CBM-GAD fusion enzymes? How does the support need to be modified? Whether or not the immobilized enzyme is stable and its half-life can meet the production requirements?

It follows that there is still a need in the art to address the above-described key issues depending on the specific microbial GAD situation.

In patent application CN201710510283.8, the inventors have isolated Enterococcus faecium LNSF2 with high GAD activity from kimchi and subjected to patent strain preservation of the wild strain, deposited in the microorganism strain collection in the cantonese province, with deposit number: GDMCC No.60203.

Disclosure of Invention

The invention aims to solve the technical problems that the wild strain GAD has low expression level, low activity, difficult recovery of free enzyme, difficult immobilization, short half-life, incapability of repeated utilization and continuous production, high cost and difficulty in meeting the industrial production requirements of GABA or DL-Glu, provides a genetically engineered bacterium for efficiently immobilizing the GAD of enterococcus faecium, and provides an immobilization method for efficiently producing the GAD immobilized enzyme of enterococcus faecium.

According to the invention, the cellulose binding module gene CBM3, enterococcus faecium GAD gene gadB and a self-constructed pRPOCB expression vector are used for constructing engineering bacteria capable of efficiently expressing enterococcus faecium GAD fusion enzyme (CBM-GAD) carrying a Cellulose Binding Module (CBM) together, the CBM-GAD is prepared by fermentation culture, and the MC-CBM-GAD insoluble immobilized enzyme can be obtained by carrying out specific affinity adsorption on the CBM and Modified Chitin (MC), so that the aim of efficiently immobilizing enterococcus faecium GAD is fulfilled, and the problems of difficult immobilization of GAD, short half-life and high cost are solved.

The first aim of the invention is to provide a genetically engineered bacterium for efficiently immobilizing enterococcus faecium GAD.

The second object of the present invention is to provide a method for constructing the above genetically engineered bacterium.

The third object of the invention is to provide the application of the genetically engineered bacterium in high-efficiency immobilization of enterococcus faecium GAD or preparation of enterococcus faecium GAD immobilized enzyme.

The fourth object of the invention is to provide a method for efficiently producing enterococcus faecium GAD immobilized enzyme by using the genetically engineered bacterium.

The fifth object of the present invention is to provide a method for preparing a modified chitin insoluble carrier of enterococcus faecium GAD immobilized enzyme.

The above object of the present invention is achieved by the following technical scheme:

the invention uses stress-induced promoter P _rpoS Recombinant construction of pRPOCB-efagadB plasmid by GAD gene gadB and T7 terminator sequences of CBM coding sequences CBM3 and Enterococcus faecium LNSF2 of cellulose binding module, and E.coli DH conversionE.coli (DH 5 alpha-LNSF 1) bacterial cells which are used for efficiently expressing CBM-GAD fusion enzyme are cultured and collected, crude enzyme liquid of the CBM-GAD fusion enzyme is extracted by ultrasonic crushing in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution under ice water bath, and then the crude enzyme liquid is mixed with filtered modified chitin for affinity adsorption, thus obtaining the chitin-CBM-GAD immobilized enzyme.

The promoter Prpos (743 bp) in pRPOCB plasmid constructed in the invention contains the 5' -untranslated region of the RpoS transcriptional regulatory system, which plays an important role in the response of bacteria to stress conditions; the promoter Prpos is independent of any additional inducer, and can effectively drive the expression of the exogenous protein in the logarithmic growth phase; the use of an inducer-independent promoter to drive protein expression is beneficial to cost reduction; CBM is an effective protein separation label, can be specifically adsorbed with modified chitin, and is used for affinity adsorption of recombinant protein in the invention, so as to obtain enterococcus faecium GAD immobilized enzyme.

The invention provides a method for constructing a genetic engineering bacterium for efficiently immobilizing enterococcus faecium GAD, which comprises the following steps:

s1, cloning GAD gene gadB of Enterococcus faecium LNSF;

s2, constructing an escherichia coli pRPOCB expression vector containing a stress inducible promoter Prpos, a cellulose binding module CBM coding sequence CBM3 and a T7 terminator sequence;

s3, constructing a recombinant plasmid pRPOCB-efagadB by adopting enterococcus faecium gadB gene and pRPOCB expression vector;

s4, converting the recombinant plasmid pRPOCB-efagadB into competent cells of Escherichia coli to construct engineering bacteria Escherichia coli (DH 5 alpha-LNSF 1) for expressing CBM-GAD fusion enzyme;

wherein CBM is a cellulose binding module and GAD is glutamate decarboxylase.

Further, in a preferred embodiment, the E.coli is Escherichia coli DH alpha.

The engineering bacteria Escherichia coli (DH 5 alpha-LNSF 1) is deposited in the microorganism strain collection of Guangdong province on the 9 th month 18 th year of 2018, and the deposit number is: GDMCC No.60445, the preservation address is: guangzhou city first middle road No. 100 college No. 59 building 5.

The engineering bacteria Escherichia coli (DH 5 alpha-LNSF 1) are: comprising promoter P inducible by stress _rpoS pRPOCB-efagadB plasmid is formed by recombination of CBM coding sequences CBM3, enterococcus faecium LNSF glutamate decarboxylase gene galB and T7 terminator sequences.

The wild strain Enterococcus faecium LNSF is isolated from kimchi, and has been deposited in the microorganism strain collection of Guangdong province at 19 th month 6 in 2017, and has a deposit number of: GDMCC No.60203, the preservation address is: guangzhou city first middle road No. 100 college No. 59 building 5; the strain is used as a GAD gene GAdB source strain; e.coli DH5 alpha is adopted as an expression system of gene cloning and cellulose binding module-glutamate decarboxylase (CBM-GAD) fusion enzyme; the constructed engineering bacterium Escherichia coli (DH 5 alpha-LNSF 1) carries pRPOCB-efagadB plasmid, and the total length of pRPOCB-efagadB is 5130bp; the cellulose binding module gene cbm3 is inserted before the galB gene, and the total length of the fusion gene is 1887bp; the length of the gadB gene is 1401bp, the number of amino acids is 466aa, and the theoretical molecular weight is about 53710D; the CBM-GAD fusion enzyme has a full length of 628aa and a theoretical molecular weight of about 71522D.

As a preferred embodiment, the method for constructing the genetically engineered bacterium for the efficient immobilization of enterococcus faecium GAD comprises the following steps:

s1. GAD Gene gadB cloning of Enterococcus fascium LNSF 2: amplifying and obtaining a gadB gene from Enterococcus faecium LNSF2 genome DNA by using efa-1 and efa-2 as primers, purifying, connecting with pMD19-T Simple Vector, constructing recombinant plasmid pMD-efagadB, transforming escherichia coli, and sequencing;

s2, constructing an escherichia coli plasmid pRPOCB: constructing a pUC57 plasmid, a stress inducible promoter Prpos, a cellulose binding module CBM coding sequence CBM3 and a T7 terminator sequence to obtain a pRPOCB plasmid;

s3, constructing an expression vector pRPOCB-efagadB: the pRPOCB is used as a template, primers Info-22 and Info-27 are designed, and a linearization vector pRPOCB is prepared through PCR amplification; designing primers Info-13 and Info-24, amplifying a galB gene by taking pMD-EfagadB as a template, purifying, and assembling a linearization vector pRPOCB with the pure galB gene to obtain a recombinant plasmid pRPOCB-efaagadb;

s4, constructing engineering bacteria Escherichia coli (DH 5 alpha-LNSF 1): transforming recombinant plasmid pRPOCB-efagadB into E.coli DH5 alpha competent cells to construct engineering bacteria Escherichia coli (DH 5 alpha-LNSF 1) capable of expressing CBM-GAD fusion enzyme;

wherein CBM is a cellulose binding module and GAD is glutamate decarboxylase.

Correspondingly, the application of the genetically engineered bacterium Escherichia coli (DH 5 alpha-LNSF 1) in the efficient immobilization of enterococcus faecium GAD or the preparation of enterococcus faecium GAD immobilized enzyme is also within the protection scope of the invention.

The invention also provides a method for efficiently producing enterococcus faecium GAD immobilized enzyme by using the genetically engineered bacterium, which comprises the following steps:

s11, crushing Escherichia coli (DH 5 alpha-LNSF 1) thalli in a buffer solution under the condition of ice-water bath, and extracting a CBM-GAD fusion enzyme crude enzyme solution;

s12, mixing and stirring the CBM-GAD fusion enzyme crude enzyme solution and modified chitin, performing affinity adsorption to form chitin-CBM-GAD, filtering the liquid, and separating to obtain a solid substance, namely enterococcus faecium GAD immobilized enzyme;

the CBM is a cellulose binding module and the GAD is glutamate decarboxylase.

Preferably, the modified chitin in the step S12 is chitin modified by concentrated phosphoric acid or modified by NaOH/urea mixed solution.

More preferably, the modified chitin in step S12 is chitin modified by concentrated phosphoric acid.

The preparation method of the concentrated phosphoric acid modified chitin comprises the following steps: dissolving chitin in concentrated phosphoric acid in ice bath until the mixture becomes transparent, diluting with ice water to precipitate chitin, collecting chitin precipitate, and adding distilled water or Na ₂ CO ₃ Washing the chitin precipitate to neutrality to obtain concentrated phosphoric acid modified chitin.

Preferably, the buffer solution in step S11 is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution. The disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is used as the buffer solution for extracting the CBM-GAD fusion enzyme crude enzyme solution, so that the cost is low, the safety is high, and the defect of the Tris-HCl buffer solution is overcome.

Preferably, the concentration of the buffer solution in the step S11 is 10-100 mmol/L.

More preferably, the concentration of the buffer in step S11 is 20mmol/L. The influence of the concentration of the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution on the extraction of the CBM-GAD fusion enzyme crude enzyme solution and the affinity adsorption capacity of the CBM-GAD fusion enzyme crude enzyme solution and the modified chitin is researched, and when the concentration of the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is 20mmol/L, the ionic strength is proper, and the affinity adsorption capacity of the extracted fusion enzyme crude enzyme solution on the modified chitin is better than that of the Tris-HCl buffer solution.

Preferably, the pH of the buffer solution in step S11 is 7-8.

More preferably, the pH of the buffer in step S11 is 8.

Preferably, the crushing method in step S11 is as follows: ultrasonic disruption was performed in an ice water bath.

Preferably, in the step S12, the ratio of the volume (mL) of the crude enzyme solution of the CBM-GAD fusion enzyme to the mass (g) of the filtered chitin is 3-5:1.

More preferably, the ratio of the volume (mL) of the crude enzyme solution of the CBM-GAD fusion enzyme to the mass (g) of the filtered chitin in the step S12 is 4:1.

In addition, as a preferred embodiment, the method for efficiently producing enterococcus faecium GAD immobilized enzyme by using the genetically engineered bacterium comprises the following steps:

expression of CBM-GAD fusion enzyme: inoculating Escherichia coli (DH 5 alpha-LNSF 1) into a culture medium, and culturing in a shaking incubator to efficiently express CBM-GAD fusion enzyme;

s12, extracting a CBM-GAD fusion enzyme crude enzyme solution: centrifugally collecting thalli by fermenting fermentation liquor with a culture medium, stirring, dispersing and washing thalli, centrifugally collecting thalli again, adding disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution according to the ratio of the weight (g) of wet thalli to the volume (mL) of the buffer solution of 1:15, ultrasonically crushing cells under the condition of ice-water bath, centrifuging, and taking supernatant to obtain CBM-GAD fusion enzyme crude enzyme liquid;

s13, preparing enterococcus faecium GAD immobilized enzyme: soaking the modified chitin in water, centrifuging or filtering to remove residual water, mixing the volume (mL) of the crude enzyme solution of the CBM-GAD fusion enzyme and the mass (g) of the filtered modified chitin at a ratio of 4:1, intermittently stirring to ensure that the CBM-GAD fusion enzyme and the modified chitin are fully subjected to affinity adsorption to form chitin-CBM-GAD, centrifuging or filtering to remove the liquid, and separating to obtain a solid substance, namely the enterococcus faecium GAD immobilized enzyme.

As a preferred embodiment, the preparation method of the concentrated phosphoric acid modified chitin comprises the following steps: slowly adding pre-frozen concentrated phosphoric acid at-7deg.C into chitin under stirring, and ice-bathing, continuously stirring or intermittently stirring until the mixture becomes transparent; adding ice water for dilution, fully precipitating the chitin, and centrifuging or filtering to collect chitin precipitate; adding ice water, stirring, resuspending and washing chitin, repeating for 2 times to remove phosphoric acid; then re-suspending the chitin with distilled water under stirring, and using Na ₂ CO ₃ The pH of the suspension is regulated to be close to neutral by the solution, and the liquid is filtered; washing the chitin precipitate with distilled water for 4 times to obtain concentrated phosphoric acid modified chitin; the concentrated phosphoric acid is commercial concentrated phosphoric acid, and the concentration of the phosphoric acid is more than 85%.

Compared with the prior art, the invention has the following beneficial effects:

(1) The genetically engineered bacterium Escherichia coli (DH 5 alpha-LNSF 1) constructed by the invention is based on the excellent gadB gene of a wild strain Enterococcus faecium LNSF2 with high GAD activity, and the adopted promoter Prpos comprises a 5' -untranslated region of an RpoS transcription regulation system, does not depend on any additional inducer, can effectively drive the expression of exogenous proteins in the logarithmic phase, has high CBM-GAD fusion enzyme expression and strong GAD activity, and can reduce the production cost of the CBM-GAD fusion enzyme.

(2) The buffer solution used in the extraction of the CBM-GAD fusion enzyme has low cost and high safety, and overcomes the defects of high cost and low safety of Tris-HCl buffer solution; meanwhile, the obtained enterococcus faecium GAD immobilized enzyme has high activity.

(3) The insoluble carrier modified chitin selected by the invention has larger particle size than modified cellulose, better liquid fluidity and overcomes the defect of poor liquid fluidity of the modified cellulose; CBM is generally used as a cellulose binding tag, however, the present invention shows that CBM has stronger affinity for modified chitin than cellulose and stronger binding; the protein amount of the modified chitin adsorbed CBM-GAD fusion enzyme reaches (626.98 +/-61.74) mug/g, the activity of the finally obtained enterococcus faecium GAD immobilized enzyme reaches (5809.46 +/-208.99) U/g, and the immobilization rate is more than 98.13%.

(4) The construction design of the engineering bacteria is scientific and reasonable, the strong specific affinity of CBM to chitin is organically combined, and the modified chitin and the CBM-GAD fusion enzyme crude enzyme liquid are fully mixed at normal temperature and normal pressure, so that the CBM-GAD and the chitin are subjected to specific adsorption to form insoluble chitin-CBM-GAD, and the enterococcus faecium GAD immobilized enzyme can be obtained; in addition, the enterococcus faecium GAD immobilized enzyme is prepared by using the modified chitin and CBM-GAD fusion enzyme, and has the advantages of high immobilization efficiency, strong activity, stable performance and long half life of the obtained enterococcus faecium GAD immobilized enzyme, and the immobilization method is quick and greatly reduces the cost, thereby solving the problems of difficult immobilization of GAD, short half life, high cost and the like.

Drawings

FIG. 1 is a schematic representation of the design route for enterococcus faecium GAD immobilization; wherein, 1: cellulose binding module coding sequence cbm3,2: enterococcus faecium LNSF2,3: enterococcus faecium LNSF2 GAD gene gadB,4: prpoc b expression vector, 5: pRPOCB-efagadB recombinant plasmid, 6: escherichia coli DH5 a, 7: CBM-GAD fusion enzyme, 8: modified chitin, 9: enterococcus faecium GAD immobilized enzyme.

FIG. 2 is an electrophoretogram of GAD gene gadB of PCR amplification Enterococcus faecium LNSF 2; wherein, lane M is a DNA molecular weight standard Marker, and lane 1 is GAD gene gadB of Enterococcus faecium LNSF2 obtained by PCR amplification.

FIG. 3 is a schematic diagram of the structure of recombinant plasmid pRPOCB-efagadB.

FIG. 4 is a graph showing the effect of different buffers on crude CBM-GAD fusion enzyme liquid extraction and affinity adsorption capacity of the crude CBM-GAD fusion enzyme liquid to modified chitin; wherein PBS represents disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, tris-HCl represents Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer, and GAD represents glutamate decarboxylase.

FIG. 5 is a graph showing the results of a modified chitin immobilized enterococcus faecium GAD stability test; wherein GABA represents gamma-aminobutyric acid and GAD represents glutamate decarboxylase.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

The basic route of the embodiment of the invention is as follows: the wild strain Enterococcus faecium LNSF is taken as a GAD gene galB donor, a cellulose binding module gene CBM3, a galB gene and a pRPOCB expression vector which is constructed by self are recombined to construct pRPOCB-efagadB recombinant plasmid, E.coli DH5 alpha is transformed to construct engineering bacteria Escherichia coli (DH 5 alpha-LNSF 1), CBM-GAD fusion enzyme is cultured and expressed, and the CBM-GAD fusion enzyme is immobilized by modified chitin in a specific way, so that MC-CBM-GAD immobilized enzyme is obtained.

Based on the basic route of the embodiment of the invention, the designed genetic engineering bacterium for efficiently immobilizing enterococcus faecium glutamate decarboxylase and the immobilization method are shown in the schematic diagram of the invention route in figure 1.

In the research of the invention, the GAdB electrophoresis chart obtained by carrying out PCR amplification on the GAD gene GAdB of Enterococcus faecium GDMCC No.60203 is shown in figure 2, the size of the GAdB gene is about 1.4kb, the length of the sequenced GAdB gene is 1401bp, and the nucleotide sequence is shown in SEQ ID NO:7, preparing a base material; the nucleotide sequence of the constructed recombinant plasmid pRPOCB-efagadB is shown as SEQ ID NO:12, a schematic structural diagram is shown in fig. 3; e.coli DH5 alpha competent cells are transformed by recombinant plasmid pRPOCB-efagadB to construct engineering bacteria Escherichia coli (DH 5 alpha-LNSF 1) capable of efficiently expressing CBM-GAD fusion enzyme, and GAD activity of CBM-GAD expressed by fermentation of LB culture medium reaches (5920.17 +/-212.97) IU/L. The construction method of engineering bacteria Escherichia coli (DH 5. Alpha. -LNSF 1) is described in example 1.

In the research of the invention, 50mmol/L Tris-HCl buffer solution, 100mmol/L PBS buffer solution, 50mmol/L PBS buffer solution and 20mmol/L PBS buffer solution are respectively adopted as extracting solutions to extract CBM-GAD fusion enzyme crude enzyme solution, and then the CBM-GAD fusion enzyme crude enzyme solution and phosphoric acid modified chitin are respectively subjected to affinity adsorption to prepare immobilized enzymes, and the GAD activity of each immobilized enzyme is measured after full washing, so that the result is shown in figure 4. FIG. 4 shows that CBM-GAD fusion enzymes extracted by each buffer solution can be subjected to affinity immobilization with chitin to a certain extent, and the enzyme activity of immobilized enzymes is not greatly different through continuous 5 batches of enzyme activity measurement, so that each immobilized enzyme is stable, but the effect of 20mmol/L PBS buffer solution as an extracting solution is optimal, the affinity adsorption capacity of the CBM-GAD fusion enzyme with the chitin is maximum, the GAD activity reaches (5809.46 +/-208.99) U/g, the immobilization rate is more than 98.13 percent, and the effect is better than that of 50mmol/L Tris-HCl buffer solution; the effect difference of the PBS buffer solution of 50mmol/L and 100mmol/L is not large, and the activity of immobilized enzyme is relatively low; the result shows that the buffer solution ion strength has a larger influence on the affinity adsorption performance of the chitin and the CBM-GAD, and the high ion strength is not beneficial to immobilization of the chitin on enterococcus faecium GAD.

Enterococcus faecium GAD immobilized enzyme, MC-CBM-GAD, was prepared according to the procedure described in example 4 below, and the effect of the immobilized enzyme MC-CBM-GAD in the biosynthesis of GABA was tested using 2 hours of catalytic reaction as the production time per batch, as shown in FIG. 5. As can be seen from FIG. 5, the immobilized enzyme MC-CBM-GAD still maintains more than 50% of activity by repeating the production of 22 batches; the immobilized enzyme MC-CBM-GAD chitin immobilized enzyme can maintain more than 43% activity after repeated production and use of 25 batches.

Therefore, the invention utilizes the modified chitin and CBM-GAD fusion enzyme to prepare the enterococcus faecium GAD immobilized enzyme, which has high immobilization efficiency, high activity, stable performance and long half-life of the obtained GAD immobilized enzyme, and the immobilization method is quick and greatly reduces the cost, thereby solving the problems of difficult immobilization of GAD, short half-life, high cost and the like.

Specific embodiments are exemplified as follows:

EXAMPLE 1 construction of genetically engineered bacterium Escherichia coli (DH 5. Alpha. -LNSF 1)

S1, inoculating Enterococcus faecium LNSF to a PSB or MRS culture medium, standing at 37 ℃ for 12 hours, taking 5mL of culture solution, centrifuging at 8500r/min, collecting bacterial precipitate, discarding supernatant, extracting genome DNA according to a description of a century bacteria genome extraction kit, and storing at-20 ℃ for later use; primers efa-1 and efa-2 were designed, and the biological sequences of primers efa-1 and efa-2 are shown in Table 1, and were synthesized by the company Shanghai, inc. of the engineering biological engineering; using efa-1 and efa-2 as primers and Enterococcus faecium LNSF2 genome DNA as a template, and performing PCR reaction by using full-scale gold TransStart Fast Pfu DNA polymerase;

the PCR reaction conditions were: 95 ℃ for 2min; cycling for 33 times at 95 ℃ for 20s,50 ℃ for 30s and 72 ℃ for 55 s; 72 ℃ for 5min;

after the PCR reaction is finished, 1 mu L Taq DNA Polymerase is added according to the volume of 100 mu L, the reaction is carried out for 30min at 72 ℃, the electrophoresis result of the PCR product is shown in FIG. 2, and the length of the product is about 1400bp; then directly purifying the gadB gene by using a purification kit, connecting the gene with a pMD19-T Simple Vector overnight, constructing a recombinant plasmid pMD-efagadB, transforming E.coli DH5 alpha competent cells, and sequencing the competent cells by a division of biological engineering (Shanghai), wherein the nucleotide sequence of the gadB gene is shown as SEQ ID NO:7, the length of the gadB gene is 1401bp;

s2, designing a stress inducible promoter Prpos and cbm3 (derived from a family 3 cellulose binding domain of Clostridium thermocellum) coding sequence and a T7 terminator sequence, and entrusting the synthesis of gold Style biotechnology Co., ltd; the pUC57 plasmid is firstly digested with NdeI/HindII, and then Prpos, cbm3 and T7 are inserted into pUC57 to splice and construct an escherichia coli plasmid pRPOCB; wherein, the nucleotide sequences of pUC57 plasmid, stress inducible promoter Prpos, CBM coding sequence CBM3 and T7 terminator are respectively shown in SEQ ID NO: 8-SEQ ID NO: 11;

s3, designing and entrusting a primer Inpu-22 and an Inpu-27 synthesized by a biological engineering (Shanghai) limited company, wherein sequences of the primer Inpu-22 and the primer Inpu-27 are shown in a table 1, and performing PCR reaction by using pRPOCB as a template and using full-scale gold TransStart Fast Pfu DNA polymerase;

the PCR reaction conditions were: 95 ℃ for 2min;95℃20s,54℃30s,72℃1min 45s, and the cycle is 33 times, 72℃5min;

after the PCR reaction is finished, electrophoresis is used for cutting gel, a DNA gel recovery kit is used for recovering a linearization carrier pRPOCB, DNA strips are cut off, and a target fragment is recovered and purified; designing and entrusting the primers Info-13 and Info-24 synthesized by the biological engineering (Shanghai) limited company, wherein the sequences of the primers Info-13 and Info-24 are shown in a table 1, amplifying the gadB gene by taking pMD-efagadB as a template, performing electrophoresis gel cutting, and recovering and purifying the gadB gene; after purification, the galB gene and the linearization vector pRPOCB are spliced together by using an In-Fusion HD Cloning Kit gene cloning kit, wherein the reaction system is 1 mu L of an In-fusion enzyme 2 mu L, gadB gene and 2 mu L, ddH of the linearization vector ₂ O5. Mu.L, 10. Mu.L in total, centrifuged for 30s and ligated at 50℃for 15min to assemble recombinant plasmid pRPOCB-efagadB having the nucleotide sequence shown in SEQ ID NO:12, the structural schematic diagram of which is shown in fig. 3;

s4, converting the recombinant plasmid pRPOCB-efagadB into E.coli DH5 alpha competent cells to construct engineering bacteria Escherichia coli (DH 5 alpha-LNSF 1) capable of efficiently expressing CBM-GAD fusion enzyme;

TABLE 1 primers for constructing genetically engineered bacteria Escherichia coli (DH 5. Alpha. -LNSF 1)

The PSB culture medium in the step S1 comprises the following components: 15g/L peptone, 10g/L beef extract, 12.5g/L sucrose, 6.0g/L, L sodium acetate, 10g/L monosodium glutamate, 1.0g/L Tween 80 and pH of 6.8-7.0;

the MRS culture medium in the step S1 comprises the following components: 15g/L peptone, 12.5g/L beef extract, 12.5g/L sucrose, 2.0g/L diammonium citrate, 5.0g/L, K sodium acetate ₂ HPO ₄ 2.0g/L、CaCl ₂ 2.0g/L、Tween 80 1.0mL/L、pH 6.8～7.0。

EXAMPLE 2 preparation of phosphate-modified chitin immobilized insoluble Carrier

SS1, adding distilled water with the mass of 3 times of that of the chitin into the chitin, fully mixing to prepare chitin slurry, rapidly stirring and slowly adding concentrated phosphoric acid with the mass of 50 times of that of the chitin and pre-frozen at-7 ℃ under ice bath conditions, stirring until the mixture becomes transparent, and carrying out ice bath for 1h (stirring from time to time);

SS2, adding ice water with the mass 500 times that of the chitin into the solution obtained in the step SS1 for 4 times under rapid stirring, standing for 30min to fully precipitate the chitin, centrifuging at 4 ℃ for 15min at 8500r/min, and collecting the chitin precipitate;

SS3, adding ice water according to 20 times of the mass of the chitin sediment, stirring and re-suspending, centrifuging at 4 ℃ and 8500r/min for 15min, collecting the chitin sediment, repeating for 2 times to wash out phosphoric acid;

SS4 adding distilled water 20 times of the mass of chitin precipitate, stirring, and suspending with 2mol/L Na ₂ CO ₃ Adjusting the pH to be close to neutral, neutralizing residual phosphoric acid, and filtering to collect chitin;

SS5 re-suspending and washing chitin with distilled water with mass 20 times of that of chitin precipitate, and repeatedly washing for 4 times to obtain phosphoric acid Modified Chitin (MC), and storing in distilled water at 4deg.C;

the concentrated phosphoric acid in the step SS1 is commercial concentrated phosphoric acid, and the concentration of the phosphoric acid is more than 85%.

EXAMPLE 3 preparation of modified chitin-immobilized insoluble Carrier by NaOH/Urea Mixed solution

SS1, adding 50 times of NaOH/urea mixed solution into chitin, stirring and mixing uniformly, freezing at-30 ℃ for 4 hours, thawing at room temperature and rapidly stirring, then freezing at-30 ℃ for 4 hours, thawing at room temperature and rapidly stirring, and repeating the freeze thawing until the chitin is dissolved into a transparent state;

SS2, adding ice water with the mass 500 times of that of the chitin into the solution obtained in the step SS1 while stirring, standing for 30min to fully precipitate the chitin, centrifuging at 4 ℃ for 15min at 8500r/min, removing the supernatant, and collecting the chitin precipitate;

SS3, repeatedly washing the chitin precipitate with distilled water 20 times of the chitin precipitate for 4 times to remove NaOH and urea;

SS4 adding 20 times of distilled water into the chitin precipitate, stirring and suspending again, adjusting pH to be close to neutral by 1mol/LHCl, neutralizing the residual NaOH/urea mixed solution, stirring and mixing uniformly, and removing liquid by suction filtration;

SS5 re-suspending and washing chitin with distilled water with mass 20 times of that of chitin precipitate, and repeatedly washing for 4 times to obtain Modified Chitin (MC) with NaOH/urea mixed solution, and storing in distilled water at 4deg.C;

the composition of the NaOH/urea mixed aqueous solution in the step SS1 is 110g/L NaOH and 40g/L urea.

EXAMPLE 4 Rapid production of enterococcus faecium GAD immobilized enzyme

S11, inoculating the Escherichia coli (DH 5 alpha-LNSF 1) constructed in the embodiment 1 into an LB culture medium, and carrying out shake culture at 37 ℃ and 120r/min for 24 hours to efficiently express CBM-GAD fusion enzyme;

s12, centrifuging Escherichia coli (DH 5 alpha-LNSF 1) fermentation liquor at 4 ℃ for 10min, collecting thalli, adding normal saline, stirring, dispersing and washing the thalli, centrifuging again, collecting thalli, adding disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with the pH of 8.0 and 20mmol/L according to the ratio of the weight (g) of wet thalli to the volume (mL) of the buffer of 1:15, performing 400W ultrasonic cell disruption in an ice-water bath (working for 5s, cooling for 5s and 50min in the whole course), centrifuging the cell disruption liquor at 8500r/min and 4 ℃ for 15min, and taking supernatant to obtain CBM-GAD fusion enzyme crude enzyme liquid;

s13, centrifuging or filtering the phosphoric acid Modified Chitin (MC) obtained in the embodiment 2 to remove residual water, mixing the crude enzyme solution of the CBM-GAD fusion enzyme and the filtered MC according to the volume to mass ratio of 4:1, intermittently stirring for 30min to enable the CBM-GAD fusion enzyme and the modified chitin to fully carry out affinity adsorption to form MC-CBM-GAD, and centrifuging or filtering to collect MC-CBM-GAD solid matters, namely enterococcus faecium GAD immobilized enzyme;

wherein, the composition of the LB medium in the step S11 is as follows: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of NaCl, 10g/L of monosodium L-glutamate and pH value of 6.0; subpackaging in triangular bottles with 250mL specifications, sterilizing at 121deg.C for 20min; immediately after cooling and before use, 240. Mu.L of 50mg/mL ampicillin was added and shaken well.

EXAMPLE 5 rapid production of enterococcus faecium GAD immobilized enzyme

This example the procedure for the rapid production of enterococcus faecium GAD immobilized enzyme according to example 4 was followed, steps S11 and S12 being identical to example 4, step S13 being as follows:

s13, centrifuging or filtering the NaOH/urea mixed solution Modified Chitin (MC) obtained in the embodiment 3 to remove residual water, mixing the crude enzyme solution of the CBM-GAD fusion enzyme and the filtered MC according to the volume to mass ratio of 4:1, intermittently stirring for 30min to enable the CBM-GAD fusion enzyme and the modified chitin to fully carry out affinity adsorption to form MC-CBM-GAD, and centrifuging or filtering to collect MC-CBM-GAD solid matters, namely enterococcus faecium GAD immobilized enzyme.

The foregoing detailed description of the preferred embodiments has been presented to facilitate an understanding of the invention, but the invention is not limited to the embodiments described above, i.e. it is not intended that the invention must be practiced in dependence upon them. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.

Sequence listing

<110> Ling nan academy of teachers and students

<120> genetically engineered bacterium for efficient immobilization of enterococcus faecium glutamate decarboxylase and immobilization method

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgttatacg gaaaagataa tcaag 25

<210> 2

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

ttagtgagta aagccgtacg ttttc 25

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gaattcctcg agggctcttc cag 23

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ggatcccggt tctttacccc aaacc 25

<210> 5

<211> 52

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gtttggggta aagaaccggg atccatgtta tacggaaaag ataatcaaga ag 52

<210> 6

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gccctcgagg aattcttagt gagtaaagcc gtacgttttc ac 42

<210> 7

<211> 1401

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atgttatacg gaaaagataa tcaagaagaa aaaaactatt tggaaccaat ttttggctct 60

gcaagtgagg atgttgactt gcctaaatat aagttaaaca aagaatccat tgaaccacga 120

attgcttatc aattagtaca agacgagatg ttggatgaag gaaatgcgcg attaaactta 180

gctacttttt gtcaaacgta tatggaacct gaagcagtga aattgatgac ccaaacgtta 240

gaaaaaaatg caattgataa atcagaatac ccacgaacaa cggaaattga aaaccgctgt 300

gtaaatatga ttgctgattt atggcatgct ccaaataatg aaaaattcat gggaacttca 360

acgatcggct cttcagaagc ctgcatgctg ggtggtatgg ccatgaaatt tgcttggcgt 420

aaacgtgctg aaaaattagg tcttgatatt caagcaaaaa aacctaacct ggtgatctct 480

tctggttacc aagtttgttg ggaaaaattc tgtgtatatt gggatgtgga actgagagaa 540

gtcccaatgg atgaaaaaca tatgtcaatt aatctagata ctgtcatgga ttatgtggat 600

gagtacacaa ttggtattgt aggtattatg ggtattactt acactggtcg ttatgatgat 660

atcaagggtc tgaatgattt agttgaagct cacaataaac aaactgacta taaagtatac 720

attcatgttg acgctgcatc gggtggcttt tatgcaccat ttactgaacc tgatctagtt 780

tgggattttc aattgaaaaa tgttatctca attaattctt caggtcacaa atatggtttg 840

gtatatccag gtgtgggttg ggtcttatgg cgtgaccaac aatacttacc agaagaatta 900

gtatttaaag ttagttactt aggtggagaa atgccaacta tggctatcaa cttctctcat 960

agtgcagcac aactaattgg gcaatactac aactttgtac gctatggctt tgatggttat 1020

cgtgatattc accaaagaac tcatgatgtt gctgtttatt tagccaaaga gatcgaaaaa 1080

actggtattt ttgaaatcat taatgatgga tcagaattgc cagttgtgtg ctataagctg 1140

aaagaagatc ccaatcgcga atggacacta tatgatttat ctgatcgtct gttaatgaag 1200

ggatggcaag tcccagccta cccattacct aaagacttgg atcaattaat tattcaacgc 1260

ttagttgttc gagcagactt tggaatgaac atggctggtg attatgtaca agatatgaac 1320

caagcaattg aagagttgaa taaagctcat attgtttatc ataaaaaaca ggatgtgaaa 1380

acgtacggct ttactcacta a 1401

<210> 8

<211> 2710

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420

tgcatctaga tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg 480

taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 540

atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 600

ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 660

taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 720

tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 780

aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 840

aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 900

ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 960

acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 1020

ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 1080

tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1140

tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1200

gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1260

agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1320

tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1380

agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1440

tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1500

acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1560

tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1620

agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1680

tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1740

acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1800

tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1860

ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1920

agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1980

tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 2040

acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 2100

agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2160

actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2220

tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2280

gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2340

ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2400

tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2460

aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2520

tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2580

tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2640

gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 2700

ccctttcgtc 2710

<210> 9

<211> 743

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ccatacgcgc tgaacgttgg tcagaccttg caggtgggta atgcttccgg tacgccaatc 60

actggcggaa atgccattac ccaggccgac gcagcagagc aaggagttgt gatcaagcct 120

gcacaaaatt ccaccgttgc tgttgcgtcg caaccgacaa ttacgtattc tgagtcttcg 180

ggtgaacaga gtgctaacaa aatgttgccg aacaacaagc caactgcgac cacggtcaca 240

gcgcctgtaa cggtaccaac agcaagcaca accgagccga ctgtcagcag tacatcaacc 300

agtacgccta tctccacctg gcgctggccg actgagggca aagtgatcga aacctttggc 360

gcttctgagg ggggcaacaa ggggattgat atcgcaggca gcaaaggaca ggcaattatc 420

gcgaccgcag atggccgcgt tgtttatgct ggtaacgcgc tgcgcggcta cggtaatctg 480

attatcatca aacataatga tgattacctg agtgcctacg cccataacga cacaatgctg 540

gtccgggaac aacaagaagt taaggcgggg caaaaaatag cgaccatggg tagcaccgga 600

accagttcaa cacgcttgca ttttgaaatt cgttacaagg ggaaatccgt aaacccgctg 660

cgttatttgc cgcagcgata aatcggcgga accaggcttt tgcttgaatg ttccgtcaag 720

ggatcacggg taggagccac ctt 743

<210> 10

<211> 480

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

atgccggtgt ctggcaacct gaaagtggaa ttttacaaca gcaatccgag cgatacgacc 60

aatagcatca acccgcagtt taaagtgacc aacacgggta gctctgcaat tgatctgtct 120

aaactgaccc tgcgttatta ctatacggtt gatggccaga aagaccaaac cttttggtgc 180

gaccatgcgg ccattatcgg ctctaacggt agttacaatg gtatcacctc gaatgtcaaa 240

ggcacgttcg tgaaaatgag ttcctcaacc aacaatgccg atacgtatct ggaaattagc 300

tttaccggcg gtacgctgga accgggtgca cacgtccaga tccaaggccg tttcgctaaa 360

aacgattggt caaattacac ccagtccaac gactattcat ttaaatcggc gagccagttc 420

gttgaatggg atcaagtcac cgcctacctg aatggcgtgc tggtttgggg taaagaaccg 480

<210> 11

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ccccttgggg cctctaaacg ggtcttgagg ggttttttg 39

<210> 12

<211> 5130

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatggt cgacccatac gcgctgaacg ttggtcagac cttgcaggtg ggtaatgctt 240

ccggtacgcc aatcactggc ggaaatgcca ttacccaggc cgacgcagca gagcaaggag 300

ttgtgatcaa gcctgcacaa aattccaccg ttgctgttgc gtcgcaaccg acaattacgt 360

attctgagtc ttcgggtgaa cagagtgcta acaaaatgtt gccgaacaac aagccaactg 420

cgaccacggt cacagcgcct gtaacggtac caacagcaag cacaaccgag ccgactgtca 480

gcagtacatc aaccagtacg cctatctcca cctggcgctg gccgactgag ggcaaagtga 540

tcgaaacctt tggcgcttct gaggggggca acaaggggat tgatatcgca ggcagcaaag 600

gacaggcaat tatcgcgacc gcagatggcc gcgttgttta tgctggtaac gcgctgcgcg 660

gctacggtaa tctgattatc atcaaacata atgatgatta cctgagtgcc tacgcccata 720

acgacacaat gctggtccgg gaacaacaag aagttaaggc ggggcaaaaa atagcgacca 780

tgggtagcac cggaaccagt tcaacacgct tgcattttga aattcgttac aaggggaaat 840

ccgtaaaccc gctgcgttat ttgccgcagc gataaatcgg cggaaccagg cttttgcttg 900

aatgttccgt caagggatca cgggtaggag ccaccttatg ccggtgtctg gcaacctgaa 960

agtggaattt tacaacagca atccgagcga tacgaccaat agcatcaacc cgcagtttaa 1020

agtgaccaac acgggtagct ctgcaattga tctgtctaaa ctgaccctgc gttattacta 1080

tacggttgat ggccagaaag accaaacctt ttggtgcgac catgcggcca ttatcggctc 1140

taacggtagt tacaatggta tcacctcgaa tgtcaaaggc acgttcgtga aaatgagttc 1200

ctcaaccaac aatgccgata cgtatctgga aattagcttt accggcggta cgctggaacc 1260

gggtgcacac gtccagatcc aaggccgttt cgctaaaaac gattggtcaa attacaccca 1320

gtccaacgac tattcattta aatcggcgag ccagttcgtt gaatgggatc aagtcaccgc 1380

ctacctgaat ggcgtgctgg tttggggtaa agaaccggga tccatgttat acggaaaaga 1440

taatcaagaa gaaaaaaact atttggaacc aatttttggc tctgcaagtg aggatgttga 1500

cttgcctaaa tataagttaa acaaagaatc cattgaacca cgaattgctt atcaattagt 1560

acaagacgag atgttggatg aaggaaatgc gcgattaaac ttagctactt tttgtcaaac 1620

gtatatggaa cctgaagcag tgaaattgat gacccaaacg ttagaaaaaa atgcaattga 1680

taaatcagaa tacccacgaa caacggaaat tgaaaaccgc tgtgtaaata tgattgctga 1740

tttatggcat gctccaaata atgaaaaatt catgggaact tcaacgatcg gctcttcaga 1800

agcctgcatg ctgggtggta tggccatgaa atttgcttgg cgtaaacgtg ctgaaaaatt 1860

aggtcttgat attcaagcaa aaaaacctaa cctggtgatc tcttctggtt accaagtttg 1920

ttgggaaaaa ttctgtgtat attgggatgt ggaactgaga gaagtcccaa tggatgaaaa 1980

acatatgtca attaatctag atactgtcat ggattatgtg gatgagtaca caattggtat 2040

tgtaggtatt atgggtatta cttacactgg tcgttatgat gatatcaagg gtctgaatga 2100

tttagttgaa gctcacaata aacaaactga ctataaagta tacattcatg ttgacgctgc 2160

atcgggtggc ttttatgcac catttactga acctgatcta gtttgggatt ttcaattgaa 2220

aaatgttatc tcaattaatt cttcaggtca caaatatggt ttggtatatc caggtgtggg 2280

ttgggtctta tggcgtgacc aacaatactt accagaagaa ttagtattta aagttagtta 2340

cttaggtgga gaaatgccaa ctatggctat caacttctct catagtgcag cacaactaat 2400

tgggcaatac tacaactttg tacgctatgg ctttgatggt tatcgtgata ttcaccaaag 2460

aactcatgat gttgctgttt atttagccaa agagatcgaa aaaactggta tttttgaaat 2520

cattaatgat ggatcagaat tgccagttgt gtgctataag ctgaaagaag atcccaatcg 2580

cgaatggaca ctatatgatt tatctgatcg tctgttaatg aagggatggc aagtcccagc 2640

ctacccatta cctaaagact tggatcaatt aattattcaa cgcttagttg ttcgagcaga 2700

ctttggaatg aacatggctg gtgattatgt acaagatatg aaccaagcaa ttgaagagtt 2760

gaataaagct catattgttt atcataaaaa acaggatgtg aaaacgtacg gctttactca 2820

ctaagaattc ctcgagggct cttccagatc tccccttggg gcctctaaac gggtcttgag 2880

gggttttttg aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 2940

cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 3000

aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 3060

acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 3120

ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 3180

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 3240

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 3300

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 3360

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 3420

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 3480

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 3540

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 3600

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 3660

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 3720

agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 3780

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 3840

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 3900

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 3960

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 4020

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 4080

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 4140

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 4200

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 4260

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 4320

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 4380

ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 4440

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 4500

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 4560

cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 4620

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 4680

cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 4740

aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 4800

aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 4860

gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 4920

gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 4980

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 5040

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 5100

aaaataggcg tatcacgagg ccctttcgtc 5130

Claims (2)

1. A method for efficiently producing enterococcus faecium GAD immobilized enzyme by using escherichia coli genetic engineering bacteria DH5 alpha-LNSF 1 is characterized by comprising the following steps:

s1, crushing escherichia coli genetic engineering bacteria DH5 alpha-LNSF 1 in a buffer solution under the ice water bath condition, and extracting a CBM-GAD fusion enzyme crude enzyme solution; the escherichia coli genetically engineered bacterium DH5 alpha-LNSF 1 is preserved in the microorganism strain collection of Guangdong province at the date of 2018, 9 and 18, and the preservation number is as follows: GDMCC No.60445; the buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution; the concentration of the buffer solution is 20mmol/L, and the pH value is 8;

s2, mixing and stirring the CBM-GAD fusion enzyme crude enzyme solution and modified chitin, performing affinity adsorption to form chitin-CBM-GAD, filtering the liquid, and separating the solid to obtain enterococcus faecium GAD immobilized enzyme;

the CBM is a cellulose binding module, and the GAD is glutamate decarboxylase;

the volume-mass ratio of the crude enzyme liquid of the CBM-GAD fusion enzyme to the modified chitin is 3-5:1, and the unit is mL/g;

the modified chitin is chitin modified by concentrated phosphoric acid;

the preparation method of the concentrated phosphoric acid modified chitin comprises the following steps: dissolving chitin in concentrated phosphoric acid in ice bath until the mixture becomes transparent, diluting with ice water to precipitate chitin, collecting chitin precipitate, and adding distilled water or Na ₂ CO ₃ Washing the chitin precipitate to neutrality to obtain concentrated phosphoric acid modified chitin.

2. An enterococcus faecium GAD immobilized enzyme produced by the method of claim 1.