CN105624130B

CN105624130B - S-adenosylmethionine synthetase preparation, preparation method and application thereof

Info

Publication number: CN105624130B
Application number: CN201610102727.XA
Authority: CN
Inventors: 龚俊; 高长文; 高秋峰; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2016-02-24
Filing date: 2016-02-24
Publication date: 2021-01-26
Anticipated expiration: 2036-02-24
Also published as: CN105624130A

Abstract

The present application relates to S-adenosylmethionine synthetase formulations, methods of preparation and uses thereof. The preparation method disclosed by the invention utilizes a genetic engineering technology to construct the S-adenosylmethionine synthetase gene into a prokaryotic expression vector and convert escherichia coli to construct a recombinant host cell; high-yield thalli are obtained by fed-batch fermentation; then purifying S-adenosylmethionine synthetase by affinity chromatography, and the chromatographic purity of the obtained protein is above 90%. The application also relates to the use of S-adenosylmethionine synthetase in the preparation of homocysteine diagnostic reagents.

Description

S-adenosylmethionine synthetase preparation, preparation method and application thereof

Technical Field

The present disclosure relates to the field of biochemistry; in particular to an S-adenosylmethionine synthetase enzyme preparation and a production method and application thereof.

Background

S-adenosylmethionine synthetase (hereinafter referred to as MAT) (EC 2.5.1.6) is a very important enzyme in organisms. In the presence of Mg²⁺And K⁺In the presence of this enzyme, it catalyzes the reaction of L-Met with ATP to form S-adenosylmethionine (SAM). SAM is an important intermediate metabolite in organisms and participates in various biochemical reactions in the bodies. S-adenosylmethionine synthetase is an important starting enzyme.

The existing production method of S-adenosylmethionine synthetase mainly adopts yeast system (see 1-4), including Pichia pastoris and Saccharomyces cerevisiae. The disadvantages of this method are mainly the relatively complex production process, low yield and high cost compared with the prokaryotic expression system. This ultimately leads to increased production costs of MAT. Therefore, there remains a need in the art for a more cost effective method for producing MAT of high purity.

In the current clinical examination, the detection of Homocysteine (HCY) is dominated by the enzyme cycling method. The detection kit of the prior art has two types: a cycloenzyme method which is methyltransferase and hydrolase; the other is cystathionine cycling enzyme method. The recycling enzyme method is simple and easy to implement, and has been widely adopted clinically.

In the prior art, detection reagents for detecting HCY by an enzyme cycling method include, but are not limited to, those reported in the following documents: CN 104140996A; CN 104630324A; CN 104198726A; zhang heribao et al, evaluation of homocysteine assay kit by the cycloenzymic method, J. China Med. 2006; evaluation of a kit for determining serum homocysteine by a circulating enzyme method, Chinese medical guidelines, 2013; wangning et al, kit evaluation for determining homocysteine by a cyclen method, labeled immunoassay and clinical, 2014.

However, cystathionine cycler has a drawback in that it is susceptible to interference by cystathionine endogenous to the human body. The circulating enzyme method of methyltransferase and hydrolase is not interfered by human endogenous cystathionine, but still has the defects of poor stability and the like. Therefore, there is still a need in the art to provide a more improved detection reagent for the cycloenzyme method.

Disclosure of Invention

According to an aspect of the present disclosure, there is provided an enzyme preparation containing not less than 90% by mass of S-adenosylmethionine synthetase; for example, S-adenosylmethionine synthetase having a purity of not less than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%.

In the context of the present application, purity characterizes the degree to which a substance contains impurities, the less the impurities, the higher the purity. Determination of protein purity allows for the use of any method known to those skilled in the art (including present and future assays), including but not limited to electrophoresis, chromatography. In some embodiments, the purity of the MAT enzyme preparation herein is a purity determined by electrophoresis. Specifically, in the electrophoresis method, a sample is applied to a gel to be subjected to electrophoresis, the gel is then stained, the staining intensity of a band representing MAT and the staining intensity of all bands are compared, and the purity of MAT in an enzyme preparation is determined from the obtained ratio.

In other embodiments, the purity of the MAT enzyme preparation of the present application is a purity determined by chromatography. For example, a sample is applied to a column, a signal is detected by ultraviolet light as components flow out of the column, the area of a chromatographic peak representing MAT is compared with the area of all chromatographic peaks, and the amount of MAT in the enzyme preparation is determined from the ratio obtained.

In some embodiments, the amino acid sequence of the S-adenosylmethionine synthetase is set forth in SEQ ID NO 1.

MAKHLFTSESVSEGHPDKIADQISDAVLDAILEQDPKARVACETYVKTGMVLVGGEITTSAWVDIEEITRNTVREIGYVHSDMGFDANSCAVLSAIGKQSPDINQGVDRADPLEQGAGDQGLMFGYATNETDVLMPAPITYAHRLVQRQAEVRKNGTLPWLRPDAKSQVTFQYDDGKIVGIDAVVLSTQHSEEIDQKSLQEAVMEEIIKPILPAEWLTSATKFFINPTGRFVIGGPMGDCGLTGRKIIVDTYGGMARHGGGAFSGKDPSKVDRSAAYAARYVAKNIVAAGLADRCEIQVSYAIGVAEPTSIMVETFGTEKVPSEQLTLLVREFFDLRPYGLIQMLDLLHPIYKETAAYGHFGREHFPWEKTDKAQLLRDAAGLK(SEQ ID NO.1)。

The enzyme preparation of the present disclosure has an enzyme activity of not less than 95% of the initial enzyme activity (i.e., the enzyme activity at the beginning of the 37 ℃ accelerated stability test) under the 7-day 37 ℃ accelerated stability test.

In some embodiments, the enzyme formulations of the present disclosure allow for preparation into liquid or dry powder form; for example, for convenience of storage and transportation, it can be made into lyophilized powder.

According to an aspect of the present disclosure, there is provided an expression vector for S-adenosylmethionine synthetase, comprising the nucleotide shown in SEQ ID NO.2 and a pET vector. The pET vector contains a strong T7 promoter and is not recognized by the E.coli RNA polymerase. In the absence of an inducer, there was little expression. In the presence of an inducer (such as IPTG or Lac), the target gene can be expressed at a high level. The carrier contains antibiotic markers (such as Kan and Amp markers) and fluorescence markers, which are commonly used detection markers and are beneficial to screening of recombinant plasmids. In some embodiments, the pET vector is the pET41a vector. The multiple cloning site of the vector also contains the cutting sites of various restriction enzymes. The circular plasmid is cut by enzyme to obtain linear plasmid, which has the same cohesive end with the gene segment cut by the same enzyme, and the two are connected to obtain recombinant plasmid.

SEQ ID NO.2 is shown below:

ATGGCAAAACACCTTTTTACGTCCGAGTCCGTCTCTGAAGGGCATCCTGACAAAATTGCTGACCAAATTTCTGATGCCGTTTTAGACGCGATCCTCGAACAGGATCCGAAAGCACGCGTTGCTTGCGAAACCTACGTAAAAACCGGCATGGTTTTAGTTGGCGGCGAAATCACCACCAGCGCCTGGGTAGACATCGAAGAGATCACCCGTAACACCGTTCGCGAAATTGGCTATGTGCATTCCGACATGGGCTTTGACGCTAACTCCTGTGCGGTTCTGAGCGCTATCGGCAAACAGTCTCCTGACATCAACCAGGGCGTTGACCGTGCCGATCCGCTGGAACAGGGCGCGGGTGACCAGGGTCTGATGTTTGGCTACGCAACTAATGAAACCGACGTGCTGATGCCAGCACCTATCACCTATGCACACCGTCTGGTACAGCGTCAGGCTGAAGTGCGTAAAAACGGCACTCTGCCGTGGCTGCGCCCGGACGCGAAAAGCCAGGTGACTTTTCAGTATGACGACGGCAAAATCGTTGGTATCGATGCTGTCGTGCTTTCCACTCAGCACTCTGAAGAGATCGACCAGAAATCGCTGCAAGAAGCGGTAATGGAAGAGATCATCAAGCCAATTCTGCCCGCTGAATGGCTGACTTCTGCCACCAAATTCTTCATCAACCCGACCGGTCGTTTCGTTATCGGTGGCCCAATGGGTGACTGCGGTCTGACTGGTCGTAAAATTATCGTTGATACCTACGGCGGCATGGCGCGTCACGGTGGCGGTGCATTCTCTGGTAAAGATCCATCAAAAGTGGACCGTTCCGCAGCCTACGCAGCACGTTATGTCGCGAAAAACATCGTTGCTGCTGGCCTGGCCGATCGTTGTGAAATTCAGGTTTCCTACGCAATCGGCGTGGCTGAACCGACCTCCATCATGGTAGAAACTTTCGGTACTGAGAAAGTGCCTTCTGAACAACTGACCCTGCTGGTACGTGAGTTCTTCGACCTGCGCCCATACGGTCTGATTCAGATGCTGGATCTGCTGCACCCGATCTACAAAGAAACCGCAGCATACGGTCACTTTGGTCGTGAACATTTCCCGTGGGAAAAAACCGACAAAGCGCAGCTGCTGCGCGATGCTGCCGGTCTGAAG。

in some embodiments, the nucleotide sequence set forth in SEQ ID NO.2 is inserted between the NdeI and XhoI sites of the plasmid pET41 a.

According to an aspect of the present disclosure, there is provided an expression host comprising an expression vector for S-adenosylmethionine synthetase according to the present disclosure; wherein the host is Escherichia coli, including but not limited to JM109(DE3), BL21(DE3), DH5 alpha or TOP 10. In some embodiments, it is E.coli BL21(DE 3).

According to an aspect of the present disclosure, there is provided a method for producing S-adenosylmethionine synthetase, comprising the steps of:

1) providing a polynucleotide encoding an S-adenosylmethionine synthetase, wherein the amino acid sequence of said S-adenosylmethionine synthetase is SEQ ID NO: 1; preferably, the sequence of the polynucleotide comprises SEQ ID NO 2;

2) inserting the polynucleotide between NdeI and XhoI restriction sites of pET41a plasmid to construct expression vector;

3) transforming the expression vector into escherichia coli BL21(DE3) to obtain an expression host;

4) performing fed-batch fermentation on the expression host;

5) collecting thalli obtained by fermentation;

6) crushing the thallus and collecting the supernatant;

7) purifying said S-adenosylmethionine synthetase from said supernatant by affinity chromatography;

8) obtaining S-adenosylmethionine synthetase.

There are various ways of transferring foreign nucleic acids into host cells, such as transformation, transduction, or transfection. Transformation is an important means for introducing a plasmid or a viral vector into a host cell in genetic engineering, and chemical transformation by a calcium chloride method, preferably electric shock transformation, and high-voltage electric shock transformation are common. In some embodiments, the expression vector is introduced into the host cell escherichia coli BL21(DE3) using calcium chloride chemical transformation according to experimental conditions, creating an expression host capable of stably expressing S-adenosylmethionine synthetase.

The medium of E.coli usually contains the carbon source, nitrogen source, energy source, growth factors, inorganic salts and water necessary for its growth, i.e., six nutrients necessary for the growth of microorganisms. Common media are LB liquid medium, LB solid medium (10 g tryptone, 5g yeast extract, 10g NaCl and 15g agarose per liter, pH 7.0), broth medium (10 g tryptone, 3g beef extract and 5g NaCl per liter, pH7.4), TB medium (12 g tryptone, 24g yeast extract, 4ml glycerol, 17mmol KH per liter, medium)₂PO₄And 72mmolK K₂HPO₄) M9 Medium (0.5 g NH/l Medium)₄Cl、3.0g Na₂HPO₄、1.5g KH₂PO₄1mmol MgSO4, 0.1% w/v glucose and 50mmol CaCl₂pH7.4), and the like. In some embodiments, the medium used in the construction of the expression vector and transformation of E.coli is preferably LB medium. To facilitate screening of positive clones, selection may be performed based on a selection marker carried by the expression vector, such as by supplementing the medium with an antibiotic, such as, but not limited to, kanamycin.

In some embodiments, the initial medium used for fermentation in step 4) comprises:

the pH of the initial medium is 6 to 8, preferably 6.8 to 7.2.

In some embodiments, the expression host is fermented in a fed-batch fermentation. In a particular embodiment, the inoculation ratio for the fermentation is 10⁸L to 10⁹Per liter, i.e.10 per liter of medium⁸To 10⁹Bacteria.

In some embodiments, IPTG is added to induce expression vector for expression when glucose is depleted in the initial medium and the dissolved oxygen DO value is approaching 40%; and was cultured by adding glycerol at a rate of 10 ml/h/L.

In some embodiments, after fermentation is complete, the cells are disrupted, including but not limited to, sonication, high pressure homogenization disruption, repeated freeze-thaw disruption. Lysozyme and/or nuclease with certain concentration can be added during the crushing, which is helpful for enhancing the crushing efficiency of the thalli. When the cells are broken, a certain amount of heat is generated to heat the bacterial liquid, and the higher temperature has a certain influence on the activity and stability of the protein, and the breaking is preferably carried out at a lower temperature (such as ice bath). And centrifuging and filtering the crushed thallus, and removing cell debris to obtain a bacterial liquid supernatant containing the target protein S-adenosylmethionine synthetase.

The purification can be carried out in a variety of ways, and commonly used methods are column chromatography, including but not limited to metal chelate chromatography, ion exchange chromatography, gel filtration, hydrophobic chromatography, and reverse phase chromatography. In some embodiments, the supernatant of the bacterial liquid is subjected to Ni affinity chromatography to obtain S-adenosylmethionine synthetase with higher purity, and preferably, the S-adenosylmethionine synthetase is desalted to obtain the final target protein.

There are various ways of protein identification, such as SDS-PAGE gel electrophoresis analysis, Western Blot, HPLC-MS analysis, ELISA, Coomassie brilliant blue method, diffusion analysis, sedimentation analysis, Kjeldahl method, biuret method, Lowry method, UV absorption method, etc. Among them, SDS-PAGE gel electrophoresis can determine the presence, concentration and purity of a target protein from the molecular weight of a known protein, and is one of the most common methods in laboratories at present. In some embodiments, the S-adenosylmethionine synthetase is identified in the final product by SDS-PAGE gel electrophoresis.

According to yet another aspect of the present disclosure, there is provided use of an enzyme preparation of the present disclosure in the manufacture of a homocysteine diagnostic device; the diagnostic device is preferably a diagnostic kit.

According to yet another aspect of the present disclosure, there is provided a use of the S-adenosylmethionine synthetase of the present disclosure in the preparation of homocysteine diagnostic devices; the diagnostic device is preferably a diagnostic kit.

In some embodiments, the enzyme preparations of the present disclosure are directly incorporated into reagents formulated into homocysteine diagnostic kits.

According to yet another aspect of the present disclosure, there is provided a diagnostic reagent comprising an enzyme preparation according to the present disclosure. In a specific embodiment, the diagnostic reagent is a homocysteine diagnostic reagent, wherein the diagnostic reagent further comprises homocysteine methyltransferase, and the content of S-adenosylmethionine synthetase is lower than the content of homocysteine methyltransferase. In specific embodiments, the S-adenosylmethionine synthetase and the homocysteine methyltransferase are in the same or different containers.

According to still another aspect of the present disclosure, there is provided a homocysteine diagnostic kit comprising: a first reagent, a second reagent, a third reagent, and optionally a calibrator and/or a quality control,

wherein,

the first reagent comprises:

the second reagent comprises:

homocysteine methyltransferase 4 to 6KU/L, preferably 5.0KU/L

Glutamate dehydrogenase from 8 to 15KU/L, preferably 10 KU/L;

the third reagent comprises:

s-adenosyl homocysteine hydrolase 2 to 5KU/L, preferably 3.0KU/L

Adenosine deaminase 3 to 8KU/L, preferably 5.0 KU/L;

the calibrator comprises: homocysteine in an amount of 0 to 100. mu. mol/L (preferably 0 to 60. mu. mol/L), and human serum matrix;

the quality control product comprises:

5 to 45 mu mol/L homocysteine, and human serum matrix.

In a specific embodiment, there is provided a homocysteine diagnostic kit comprising: a first reagent, a second reagent, a third reagent, and optionally a calibrator and/or a quality control material, wherein

The first reagent comprises:

the second reagent comprises:

homocysteine methyltransferase 5.0KU/L

Glutamate dehydrogenase 10 KU/L;

the third reagent comprises:

s-adenosyl homocysteine hydrolase 3.0KU/L

Adenosine deaminase 5.0 KU/L.

Drawings

FIG. 1: SDS-PAGE gel electrophoretic analysis of S-adenosylmethionine synthetase.

Detailed Description

The disclosure is further illustrated with reference to specific examples. The following provides specific materials and sources thereof used in embodiments of the present disclosure. However, it should be understood that these are exemplary only and are not intended to limit the present disclosure, and that materials that are the same as or similar to the type, model, quality, nature, or function of the reagents and instruments described below may be used in the practice of the present disclosure.

Examples

Example 1: providing a target sequence

The nucleotide sequence of S-adenosylmethionine synthetase published according to NCBI website (Genbank accession number CP007391.1) is shown as SEQ ID NO:2, respectively.

Designing a primer, and obtaining a gene fragment of S-adenosylmethionine synthetase by PCR amplification by taking an escherichia coli genome as a template;

the PCR reaction system is as follows:

ddH₂o35. mu.l; 10 × Buffer 5 μ l; dNTP 2. mu.l; template DNA 2. mu.l; 2.5 μ l of forward primer; reverse primer 2.5. mu.l; 1 μ l of Taq enzyme;

the PCR reaction conditions were as follows:

3.5min at 94 ℃; at 94 ℃ for 40s, at 55 ℃ for 40s, and at 72 ℃ for 70s, for 35 cycles; 10min at 72 ℃; maintaining at 4 ℃;

the primer sequence is as follows:

forward direction: GGGAATTCCATATGATGGCAAAACACCTTTTTAC (SEQ ID No. 3);

and (3) reversing: ATCCGCTCGAGCTTCAGACCGGCAGCATCGC (SEQ ID No. 4).

After taking the PCR product and carrying out 1% agarose gel electrophoresis, a product band is visible at 1200 bp. The PCR product was recovered using a DNA purification kit (purchased from Qiagen).

The PCR product is verified to be SEQ ID NO: 2.

example 2: construction of expression vectors

Example 1 the resulting PCR product was recovered and subjected to double digestion with NdeI and XhoI enzymes (purchased from New England Biolabs);

the vector plasmid pET41a (Novagen) is subjected to the same double enzyme digestion treatment;

connecting the plasmid after enzyme digestion and the gene at 16 ℃ overnight;

the next day, E.coli DH 5. alpha. was transformed and screened by plating Kan (kanamycin, 50mg/L) resistant LB plates;

and (4) sequencing the screened positive clones after plasmid extraction, and storing the plasmids with correct sequencing results for later use.

Example 3: transformation of Escherichia coli BL21(DE3)

The plasmid constructed in example 2 was mixed with chemically competent cells BL21(DE3) (purchased from Invitrogen) and then ice-cooled for 20 minutes; heat shock at 42 deg.c for 90 sec; SOC medium (purchased from Invitrogen) was added quickly and incubated at 37 ℃ for 1 hour before plating.

Example 4: fermentation culture of Escherichia coli BL21(DE3)

(1) Fermentation culture:

inoculating the recombinant host cell E.coli BL21(DE3) containing the expression vector prepared in example 3 into a seed culture medium;

culturing at 37 deg.C until OD600 reaches 3-5, inoculating to initial culture medium of fermentation at inoculation ratio of 10⁸L to 10⁹L, preferably 10⁸/L。

Culturing in 10L fermenter (purchased from Beijing Arugmentum) with stirring speed of 300rpm, regulating aeration, allowing natural growth under Dissolved Oxygen (DO) of more than 20%, and regulating pH of the culture medium to 6.8-7.2 by adding dropwise ammonia water or sulfuric acid;

when the DO value of the dissolved oxygen of the culture medium is reduced to 40% when the glucose in the initial culture medium is gradually exhausted, adjusting the temperature of the culture medium to reduce the temperature to 25 ℃, and simultaneously supplementing the feed liquid 1 according to the proportion of 100 ml/L.

(2) Induced culture

After the feed liquid 1 is supplemented, adding IPTG with the final concentration of 1mM for induction, supplementing the feed liquid 2 at the speed of 10ml/h/L, and regulating the pH value of the culture medium by ammonia water or sulfuric acid to still keep 6.8-7.2;

after 15h of induction at 25 ℃, the fermentation culture is finished;

escherichia coli was collected by centrifugation at 9000rpm for 10min at 4 ℃.

TABLE 1 culture Medium formulations used in the present disclosure

Example 5: crushing of thallus

Precooling for 1h by a high-pressure homogenizer, wherein heat is generated during homogenization, so that the temperature of a bacterial liquid is increased, and the activity and stability of protein are influenced, therefore, the temperature is preferably precooled to be below 4 ℃ by the high-pressure homogenizer before homogenization;

resuspend the cells with solution A (50mM PB, 300mM NaCl, 50mM imidazole, 0.5% Tween 20, pH 8.0);

homogenizing at 850bar under high pressure for 3 times to break cell and release S-adenosylmethionine synthetase. The addition of lysozyme and/or nuclease can improve the breaking efficiency of the thallus and make the target protein release more completely.

Example 6: purification of S-adenosylmethionine synthetase

The cell disruption solution obtained in example 5 was centrifuged at 18000rpm at 4 ℃ for 30min to remove most of cell debris, nucleic acid and foreign proteins;

centrifuging to obtain supernatant, filtering with 0.22 μm filter membrane, and collecting filtrate;

following equilibration of the Ni affinity column (ex GE Healthcare) according to the supplier's instructions, the collected filtrate was initially loaded;

after the sample loading is finished, leaching with 2 column volumes of A liquid to remove impurities which are not combined with the column material or have low binding force;

after leaching, eluting the impurity protein by using 30% B solution (50mM PB, 300mM NaCl, 500mM imidazole, pH 8.0);

eluting target protein by using 100% B liquid, and collecting eluent corresponding to a protein chromatographic peak;

with ddH of 5-10 column volumes₂O washing the desalting column (ex GE Healthcare) to remove ethanol;

equilibrating the desalting column with 5-10 column volumes of desalting buffer (50mmol/L Tris-Cl, 0.1mM EDTA, pH 8.0);

desalting, wherein the sample loading volume is 0.25-0.3 column volume, and collecting the product corresponding to the protein peak.

Example 7: preparation of enzyme preparations

The final collected product of example 6 is formulated into different dosage specifications, liquid or dry powder forms (e.g. lyophilized powder forms) according to market needs. For example, on the production scale of example 6, the product may be lyophilized and stored in a suitable container for marketing.

Example 8: existing preparation process of homocysteine kit

SAM is produced from the enzyme preparation prepared in example 7 by any method known in the art, for example but not limited to the method described in CN1483829, CN201010503181.1, CN03126834.X, and the SAM is synthesized from the precursors methionine and adenosine triphosphate by catalysis.

The produced SAM is prepared into a homocysteine kit according to YZB/nation 0591-2005 homocysteine detection kit.

Example 9: preparation of control homocysteine kit

Control kits were prepared according to the method of paragraphs 0016 to 0018 of CN 104630324A.

Example 10 preparation of homocysteine kit of the present application

The enzyme preparation prepared in example 7 was used to prepare a kit according to the following composition.

Reagent 1:

reagent 2:

homocysteine methyltransferase 5.0KU/L

Glutamate dehydrogenase 10 KU/L;

reagent 3:

s-adenosyl homocysteine hydrolase 3.0KU/L

Adenosine deaminase 5.0 KU/L;

calibration products:

homocysteine 0. mu. mol/L-60. mu. mol/L (5 gradients)

Human serum matrix.

Test example

Test example 1: determination of protein purity

Protein purity was verified by 12% SDS-PAGE gel electrophoresis. The protein of interest, S-adenosylmethionine synthetase band, was analyzed by SDS-PAGE gel electrophoresis (FIG. 1) at 46 kDa. After the affinity chromatography of example 6, the purity of the product can reach more than 90%, and the purities obtained by three repeated experiments are respectively: 95%, 95% and 97%.

Test example 2: BCA method for measuring protein concentration

The final product S-adenosylmethionine synthetase in example 6 was assayed by BCA method to a concentration of 17mg/ml or more.

By using the production method disclosed by the disclosure, the yield of the target protein is as follows: the weight of the bacteria is more than 2 percent of the wet weight of the bacteria, and the highest weight can reach 4 percent.

Test example 3: determination of enzyme Activity

In the presence of Mg²⁺In the presence of S-adenosylmethionine synthetase, the enzyme catalyzes the reaction of L-Met with ATP to produce S-adenosylmethionine (SAM), and thus can be identified by detecting the amount of SAM produced.

(1) Definition of enzyme activity:

in a standard reaction system (100mM Tris-Cl pH 8.0,20mM MgCl)₂10mM ATP, 10mM L-Met) for 20min to produce 1. mu. mol of phosphoric acid.

(2) Amount of solution required for reaction:

(3) enzyme activity determination:

diluting: a100. mu.l sample of MAT (final product obtained in example 6) was added to 900. mu.l of an enzyme diluent (50mM Tris-Cl pH 8.0,20mM KCl, 20% glycerol, 0.1% Brij), and the enzyme was diluted 10-fold;

reaction: adding 50 μ l (containing about 0.05mg enzyme) diluted MAT into 1.0ml standard reaction system, and reacting at 37 deg.C for 20min with enzyme diluent as negative control;

and (4) terminating: 1.0ml of ice-cold 10% HClO was added₄Terminating the reaction, and finishing the machine reading within 20 min;

reading value: the concentration of the phosphoric acid produced by the reaction was measured on a biochemical analyzer according to the instructions of a commercially available G-cell series inorganic phosphorus detection kit (cat # GS421E), and the activity of MAT enzyme was calculated.

And (3) calculating: according to the detection result of the biochemical analyzer, the enzyme activity unit content in each milliliter of protein sample is converted according to the following conversion formula.

X＝13.2225Y*A

X: enzyme activity unit (U/ml); y ═ (Y1-Y2)/5; y1: the sample to be detected is mg/dl; y2: negative control reading value mg/dl; a: dilution factor.

Test example 4 accelerated stability

(1) The determination method comprises the following steps:

the enzyme preparations of example 7 were collected in a volume of 1ml each, and left at 4 ℃ and 37 ℃ for 7 days, respectively, after which the enzyme activities were measured by the method described in example 3.

(2) The enzyme activity results are as follows:

after standing for 7 days at 37 ℃, the enzyme activity can still maintain more than 95% of the initial enzyme activity, which is detailed in table 2.

TABLE 2 enzyme Activity after standing at 4 ℃ and 37 ℃ for 7 days

Test example 5 comparison of the stability of the kits of the present application and of the control kits

The preparations prepared in example 9 and example 10 were allowed to stand at 4 ℃ and 37 ℃ for 7 days, respectively, and then were taken out to measure the enzyme activity according to the method of example 3.

TABLE 3 accelerated stability data (calibrated absorbance) of the kit of example 9 at 37 deg.C

TABLE 4 accelerated stability data (calibrated absorbance) at 37 ℃ for the example 10 kit

Reference to the literature

1. Studies on the production of adenosylmethionine by fermentation of recombinant Pichia pastoris [ J ]; an industrial microorganism; 2004, stage 04;

2. zhangjian, Lixinhua and Yuan-Yi; the progress of gene and structure research of adenosylmethionine synthetase [ J ]; an industrial microorganism; 03 2005;

3. zhang Yi, Li Yuan Guang, jin Jian, Yang Dong, Shen Gumin; a control strategy [ J ] of fermentation kinetics and specific growth rate of SAM producing strain Saccharomyces cerevisiae HYS 98; process engineering newspaper; 03 2005;

4. shenli, Yuanliang, Jiang Yongming, Zhangliang and Yuan-zhong; preliminary study on fermentation conditions of recombinant Pichia pastoris [ J ]; the journal of the Chinese pharmaceutical industry; 2004, stage 02;

5. industry Standard YY/T1258-.

Claims

1. A homocysteine detection kit comprising:

a first reagent,

A second reagent, and

a third reagent for the second reagent, wherein the third reagent comprises,

wherein the first reagent comprises:

the second reagent comprises:

homocysteine methyltransferase 4-6 KU/L

Glutamate dehydrogenase from 8 to 15 KU/L;

the third reagent comprises:

s-adenosyl homocysteine hydrolase 2 to 5KU/L

Adenosine deaminase 3 to 8 KU/L.

2. The homocysteine detection kit according to claim 1, further comprising calibrator and/or quality control.

3. The homocysteine detection kit according to claim 2, wherein said calibrator comprises:

0 to 100. mu. mol/L homocysteine, and

human serum matrix.

4. The homocysteine detection kit according to claim 2, wherein the quality control product comprises:

5 to 45 μmol/L homocysteine, and

human serum matrix.

5. The homocysteine detection kit according to any of claims 1-4 wherein:

the first reagent comprises:

the second reagent comprises:

homocysteine methyltransferase 5.0KU/L

Glutamate dehydrogenase 10 KU/L;

the third reagent comprises:

s-adenosyl homocysteine hydrolase 3.0KU/L

Adenosine deaminase 5.0 KU/L.