阅读说明：本技术 一种利用沙雷氏菌提纯碱性金属蛋白酶的工艺方法 (Process method for purifying alkaline metalloprotease by using serratia ) 是由 张国基 张希兰 汤燕雯 赵甜 于 2019-10-16 设计创作，主要内容包括：本发明公开了一种利用沙雷氏菌提纯碱性金属蛋白酶的工艺方法,包括以下步骤：将黏质沙雷氏菌发酵培养,获得粗酶液；用硫酸铵沉淀粗酶液后,用缓冲液溶解、透析；用无菌的去离子水分别将Q Sephacryl Fast Flow和Sephacryl S100 HR溶胀、脱气；在预制Q Sephacryl Fast Flow色谱柱上样,用Tris-HCl平衡液冲洗,用不同浓度的NaCl进行梯度洗脱,用超滤管,缓冲液进行脱盐,用不同浓度的NaCl进行梯度洗脱,用超滤管脱盐、浓缩；上样至Sephacryl S100 HR柱,用洗脱液清洗,收集洗脱峰,用超滤管浓缩,即得到碱性金属蛋白酶。从黏质沙雷氏菌的分泌蛋白中分离抗病毒活性的蛋白质类物质,筛选具有良好抗病毒效果、性质稳定的蛋白,丰富抗病毒活性物质的种类,为植物病毒病防治药剂的研究提供理论依据。(The invention discloses a process method for purifying alkaline metalloprotease by using serratia, which comprises the following steps: fermenting and culturing serratia marcescens to obtain crude enzyme liquid; precipitating the crude enzyme solution with ammonium sulfate, dissolving with buffer solution, and dialyzing; respectively swelling and degassing Q Sephacryl Fast Flow and Sephacryl S100HR by using sterile deionized water; loading a sample on a prefabricated Q Sephacryl Fast Flow chromatographic column, washing with a Tris-HCl equilibrium solution, performing gradient elution with NaCl of different concentrations, desalting with an ultrafiltration tube and a buffer solution, performing gradient elution with NaCl of different concentrations, desalting with an ultrafiltration tube, and concentrating; and (3) loading the sample to a Sephacryl S100HR column, washing with eluent, collecting an elution peak, and concentrating by using an ultrafiltration tube to obtain the alkaline metalloprotease. Protein substances with antiviral activity are separated from secretory protein of serratia marcescens, protein with good antiviral effect and stable property is screened, the types of antiviral active substances are enriched, and a theoretical basis is provided for research of plant virus disease prevention and treatment medicaments.)

1.A process method for purifying alkaline metalloprotease by using Serratia is characterized by comprising the following steps:

respectively swelling and degassing Q Sephacryl Fast Flow and Sephacryl S100HR with sterile deionized water;

loading a sample on a prefabricated Q Sephacryl Fast Flow chromatographic column, washing with a Tris-HCl equilibrium solution with pH of 4.0-9.0 and 20mmol/L, performing gradient elution with NaCl of different concentrations, desalting with a PBS buffer solution with pH of 7.2 and 10mmol/L by using an ultrafiltration tube, performing gradient elution with NaCl of different concentrations, desalting with the ultrafiltration tube, and concentrating;

fermenting and culturing serratia marcescens to obtain crude enzyme liquid;

precipitating the crude enzyme solution with ammonium sulfate, dissolving with 10mmol/L PBS buffer solution with pH of 7.2, and dialyzing;

loading to Sephacryl S100HR column, washing with PBS eluent of pH7.2 and 10mmol/L, collecting eluate peak, and concentrating with ultrafiltration tube to obtain alkaline metalloprotease.

2. The process of claim 1, wherein the Tris-HCl equilibrium solution has a pH of 5.0.

3. The process of claim 1, wherein the concentration of the NaCl eluate is 0.2 mol/L.

4. The process of claim 1, wherein the ultrafiltration tube has a maximum cut-off of 3000 Da.

5. The process of claim 1, wherein the crude enzyme solution is prepared by the steps of:

and (3) carrying out shake culture on the serratia marcescens bacterial liquid in a shaking table at 120rpm at a constant temperature of 28 ℃ for 72h, taking out the serratia marcescens bacterial liquid, centrifuging the serratia marcescens bacterial liquid at 6000 Xg for 10min, and collecting supernatant to obtain crude enzyme liquid.

6. The process for purifying alkaline metalloprotease according to claim 5, wherein said Serratia marcescens bacterial liquid is prepared by the steps of:

and (3) taking a serratia marcescens single colony, putting the serratia marcescens single colony into an LB liquid culture medium, and performing shake culture at 28 ℃ and 120rpm for 16 h.

7. The process of claim 1, wherein the ammonium sulfate precipitation of the crude enzyme solution comprises the following steps:

(1) slowly adding ammonium sulfate into the crude enzyme solution, slowly stirring until the final concentration of ammonium sulfate reaches 45%, and standing for 16 h;

(2) then, high-speed freezing and centrifuging for 30min, discarding the precipitate, and collecting the supernatant;

(3) slowly adding ammonium sulfate into the supernatant until the final concentration reaches 85%, and standing for 16 h;

(4) and then the mixture is frozen and centrifuged at high speed for 30min again, the supernatant is discarded, and the precipitate is collected.

8. The process for purifying alkaline metalloprotease according to claim 7, wherein ammonium sulfate is added to said step (1) on ice.

9. The process of claim 7, wherein the crude enzyme solution is treated at 4 deg.C.

10. The process of claim 7, wherein the conditions of high speed freezing and centrifugation are 11000 Xg at 4 ℃.

Technical Field

The invention relates to the technical field of microbial ecology, in particular to a process method for purifying alkaline metalloprotease by using serratia.

Background

Serratia marcescens also produces a variety of extracellular enzymes including sericise, chitinase, metalloproteases, lipases, thiol proteases, nucleases, etc. (Gerc et al 2014; Hover et al 2016; Khanet al 2017; Singhetal 2016). The serratia marcescens extracellular protein has research and application in the fields of medicine, industry and the like, but has few researches in agriculture, and has no application of related products in the market. Serratia marcescens is a gram-negative bacterium widely existing in natural habitat and can live in soil, water and other environments. In soil, some strains are plant growth-promoting rhizobacteria (PGPR). For example, Serratia marcescens-90-166 isolated from soil can induce systemic resistance in plants by quorum sensing mechanisms and can enhance plant infestation by several plant pathogens, including cucumber mosaic virus.

The economic losses due to tobacco mosaic virus are as high as billions of dollars per year. At present, modes such as breeding of antiviral varieties, control of virus-carrying media, cross protection and the like are mainly adopted in the treatment of virus diseases, and few effective chemical pesticides aiming at plant virus diseases need to be solved by agricultural scientific researchers. Tobacco Mosaic Virus (TMV) is a pathogen of tobacco mosaic disease and the like, has a wide host range, can infect at least 125 plants of 9 families, such as cruciferae, solanaceae, cucurbitaceae and the like, has high occurrence frequency, is quick in prevalence, is serious in harm and difficult to treat, cannot be effectively controlled once a plant is infected, is very easy to cause the prevalence of the virus disease, and causes serious economic loss.

It has now been found that there are many bacteria and fungi and their metabolites that have a certain capacity to inactivate tobacco mosaic virus virions, but there is no good way to specifically isolate the antivirally active proteinaceous material therefrom. It has been studied that plant viruses lose their infectivity rapidly after they become contaminated with bacteria, and some researchers have been working on the antiviral activity of microorganisms and their metabolites. In view of the above problems, it is necessary to provide further solutions.

Disclosure of Invention

The invention aims to provide a process method for purifying alkaline metalloprotease by using serratia, which overcomes the defects in the prior art.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a process method for purifying alkaline metalloprotease by using Serratia comprises the following steps:

precipitating the crude enzyme solution with ammonium sulfate, dissolving with 10mmol/L PBS buffer solution with pH of 7.2, and dialyzing;

respectively swelling and degassing Q Sephacryl Fast Flow and Sephacryl S100HR with sterile deionized water;

loading a sample on a prefabricated Q Sephacryl Fast Flow chromatographic column, washing with a Tris-HCl equilibrium solution with pH of 4.0-9.0 and 20mmol/L, performing gradient elution with NaCl of different concentrations, desalting with a PBS buffer solution with pH of 7.2 and 10mmol/L by using an ultrafiltration tube, performing gradient elution with NaCl of different concentrations, desalting with the ultrafiltration tube, and concentrating;

fermenting and culturing serratia marcescens to obtain crude enzyme liquid;

loading to Sephacryl S100HR column, washing with PBS eluent of pH7.2 and 10mmol/L, collecting eluate peak, and concentrating with ultrafiltration tube to obtain alkaline metalloprotease.

Preferably, the Tris-HCl equilibration solution has a pH of 5.0.

Preferably, the concentration of the NaCl eluent is 0.2 mol/L.

Preferably, the ultrafiltration tube has a maximum molecular weight cut-off of 3000 Da.

Preferably, the preparation method of the crude enzyme solution comprises the following steps:

and (3) carrying out shake culture on the serratia marcescens bacterial liquid in a shaking table at 120rpm at a constant temperature of 28 ℃ for 72h, taking out the serratia marcescens bacterial liquid, centrifuging the serratia marcescens bacterial liquid at 6000 Xg for 10min, and collecting supernatant to obtain crude enzyme liquid.

Preferably, the preparation method of the serratia marcescens bacterial liquid comprises the following steps:

and (3) taking a serratia marcescens single colony, putting the serratia marcescens single colony into an LB liquid culture medium, and performing shake culture at 28 ℃ and 120rpm for 16 h.

Preferably, the ammonium sulfate precipitation of the crude enzyme solution comprises the following steps:

(1) slowly adding ammonium sulfate into the crude enzyme solution, slowly stirring until the final concentration of ammonium sulfate reaches 45%, and standing for 16 h;

(2) then, high-speed freezing and centrifuging for 30min, discarding the precipitate, and collecting the supernatant;

(3) slowly adding ammonium sulfate into the supernatant until the final concentration reaches 85%, and standing for 16 h;

(4) and then the mixture is frozen and centrifuged at high speed for 30min again, the supernatant is discarded, and the precipitate is collected.

Preferably, the step (1) is carried out with addition of ammonium sulphate on ice.

Preferably, the ammonium sulfate precipitation crude enzyme solution is operated at 4 ℃.

Preferably, the conditions of the high-speed refrigerated centrifugation are 11000 Xg at 4 ℃.

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

the invention discloses a process method for purifying alkaline metalloprotease by using serratia, which separates protein substances with antiviral activity from secretory protein of serratia marcescens, screens protein with good antiviral effect and stable property, enriches the types of antiviral active substances and provides a theoretical basis for the research of plant virus disease prevention and treatment medicaments.

Drawings

FIG. 1 shows the chromatographic separation and purification results, using Q Sephacryl Fast Flow chromatographic column, using NaCl solutions of different concentrations to gradient elute the obtained components and their antiviral effects;

FIG. 2 shows the chromatographic separation and purification results of the different protein fractions obtained by Sephacryl S100HR exclusion chromatography and their antiviral activity;

FIG. 3 shows SDS-PAGE electrophoresis detection of eluted proteins, wherein a)1, 2, 4, 5 represent 15, 16, 17, 18 tubes of eluents, respectively, and b) 1 represents a single band obtained by the second exclusion chromatography;

FIG. 4 shows that tandem mass spectrometry identifies 7 peptide fragments;

FIG. 5 shows an alignment of the amino acid sequences of SAMP and three homologous proteins, 1sat (S. marcocens), 5d7w (Serratia sp. Fs14) and 1srp (Serratia sp. E-15), in which the same amino acid residues are shown in grey and different amino acid residues are marked with different colours;

FIG. 6 is a block diagram of SAMP predicted by SWISS-MODEL;

FIG. 7 shows SAMP structure prediction and matching to a reference protein;

FIG. 8 is a three-dimensional block diagram of the N-terminal hydrolysis domain of SAMP;

FIG. 9 is a three-dimensional block diagram of the C-terminal domain of SAMP;

FIG. 10 is a graph of the effect of pH on SAMP enzyme activity and enzyme stability;

FIG. 11 is a graph showing the effect of temperature on SAMP enzyme activity and enzyme stability.

Detailed Description

The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended.

The plant material used in this experiment was a Sansheng cigarette (Nicotiana tabacum cv. SunsamnN) with a blight marker gene; burkhamiana (Nicotiana benthamiana);

TMV virions; TMV-GFP (TMV 30B).

A process method for purifying alkaline metalloprotease by using Serratia comprises the following steps:

fermenting and culturing serratia marcescens to obtain crude enzyme liquid;

1. chromatography column prefabrication

Q Sephacryl Fast Flow and Sephacryl S100HR were swollen and degassed with sterile deionized water, respectively. Sealing the sample outlet of the chromatographic column, slowly pouring the swelled filler into the chromatographic column along the wall (the chromatographic column is vertically placed, bubbles are avoided during the process), standing for a period of time, opening the sample outlet to allow the sterile water to flow out, and washing 1-2 column volumes by using a balance buffer solution. And (5) placing the prefabricated chromatographic column in a chromatographic cabinet for temporary storage.

2. pH selection for anion exchange chromatography (Q Sephacryl FF)

First, 1mL of the column was loaded on a preparative Q Sephacryl FF column, 1 column volume was washed with an equilibration solution (corresponding pH), then proteins bound to the column were eluted in one portion with eluents containing 2mol/L NaCl at different pH (4.0, 5.0, 6.0, 7.0, 8.0 and 9.0), desalted with a Millipore ultrafiltration tube with a maximum molecular weight cut-off of 3000Da, pH7.2, 10mmol/L PBS buffer, and finally the proteins were concentrated to 2 mL. Binding to the "half-leaf method" measures the optimal pH range for binding of the protein of interest to the chromatographic column.

3. Isolation of Total protein Using Q Sephacryl Fast Flow

After washing 1-2 column volumes with equilibration solution, the desalted total protein was gently added to the column along the wall (without splashing the packing) and then eluted with a gradient of NaCl at different concentrations. The elution conditions were as follows:

elution was performed for 1 column volume per concentration of eluate, and each eluate was collected and desalted using a Millipore ultrafiltration tube with a molecular weight cut-off of 3000, concentrated and then assayed for TMV activity by the "half-leaf method".

4. Sephacryl S100HR isolation of protein fractions having anti-TMV Activity

The proteins having anti-TMV activity eluted from step 3 were pooled together and concentrated and applied to a Sephacryl S100HR column. Then washed with eluent (PBS,10mmol/L, pH 7.2).

The chromatographic conditions were as follows:

the elution peaks were collected, concentrated using an ultrafiltration tube with a Millipore molecular weight cut-off of 3000, and the inhibitory effect of each fraction on TMV was determined by the "half-leaf method".

5. Determination of protein concentration (BCA method): the concentration of the isolated protein was measured using a BCA kit (Solebao Biotechnology Co., Ltd.).

Wherein, the ammonium sulfate precipitation crude enzyme solution comprises the following steps:

the method comprises the following specific steps:

1. 3L of the bacterial solution was shake-cultured in a shaker for 72 hours (28 ℃,120rpm), taken out, centrifuged at 6000 Xg for 10min, and the supernatant was collected and the cells were discarded. The bacterial liquid preparation is that a single colony of the serratia marcescens is put into an LB liquid culture medium and is subjected to shake culture at 28 ℃ and 120rpm for 16 h.

2. The resulting mixture was filtered through a microfiltration membrane (pore size: 0.45 μm) to remove the remaining cells. Ammonium sulfate was slowly added to the supernatant of the broth and stirred slowly until the final concentration of ammonium sulfate reached 45%, which required an ice-wash procedure.

3. The supernatant was placed in a chromatography cabinet (4 ℃ C.), and allowed to stand for 16 hours.

4. The supernatant was slowly removed (without shaking), centrifuged in a high-speed refrigerated centrifuge (4 ℃ C., 11000 Xg) for 30min and the precipitate was discarded, and the supernatant was collected.

5. Ammonium sulfate was slowly added to the supernatant to a final concentration of 85%, and then placed in a chromatography cabinet and allowed to stand for 16 hours. (the whole process was carried out at 4 ℃ C.)

6. Centrifuging with a high-speed refrigerated centrifuge (4 deg.C, 11000 Xg) for 30min, and discarding the supernatant.

7. The precipitate was collected and dissolved in 100mL of phosphate buffer (PBS,10mmol/L, pH 7.2).

8. Desalting the crude extract of total protein: putting the total protein extract into a dialysis bag with molecular weight cutoff of 3kDa, and wiping to remove ammonium sulfate in the total protein solution by a dialysis desalting method. The dialyzate was 10mmol/L PBS buffer, pH 7.2.

The desalted protein solution was freeze-dried using a vacuum freeze dryer and then stored at-20 ℃.

Separating the obtained protein, and detecting the size of the protein by SDS-PAGE gel electrophoresis. The anti-TMV protein species were identified by tandem mass spectrometry (MS/MS), protein sequences were aligned by Bioedit software, and the amino acid sequences of the proteins were structurally predicted by SWISS-MODEL.

The enzymatic activity of alkaline metalloproteases is determined by the forskolin method.

When the pH value of Tris-HCl is lower than 5.0 (excluding 5.0), the protein bound on the chromatographic column has no obvious inhibition effect on TMV, when the pH value is increased to 5.0, the protein bound on the chromatographic column has obvious inhibition effect on TMV, and the inhibition efficiency reaches 86.30%; the inhibition efficiency of the eluted protein on TMV showed an increasing trend with increasing pH, but the difference was not significant. This result indicates that: at the pH value of 5.0, the antiviral active protein began to bind to the column in a large amount, and as the pH value of the buffer solution increased, the amount of the anti-TMV active protein bound to the column also showed an increasing tendency (see Table 1).

TABLE 1 ratio of anion exchange column bound protein to TMV passivation Effect under different pH conditions

Compared with

Table1Inactivation effect of proteins binding onto the

anion-exchange chromatography in Tris-HCl buffer(0.02

mol·L^-1)with different pH.

The principle of anion exchange chromatography is: when the isoelectric point (pI) of the protein is lower than the pH value of the environment (the pH value of a balance solution in a chromatographic column), the protein is negatively charged and can be bound on the chromatographic column; conversely, proteins are positively charged and cannot bind to the column. According to this principle, the higher the pH of the equilibration solution, the more proteins can bind to the column, and we therefore need to screen the appropriate pH. The results of the pH screening in this experiment show that: the optimum pH for the separation of active proteins by anion exchange chromatography may be selected to be 5.0, so that proteins having anti-TMV activity are retained to the maximum extent and the influence of foreign proteins is eliminated as much as possible.

When the pH value is 5.0, the eluent with the concentration of 0.2mol/L NaCl is used for washing, the inhibition effect of the obtained eluent on TMV is up to more than 80%, and the value of an elution product A280 is the highest, so that when the concentration of NaCl is up to 0.2mol/L, the amount of protein obtained by elution is the largest, and the protein has the inhibition effect on TMV.

After separation of total protein by Q Sephacryl Fast Flow, the resulting fractions eluted with anti-TMV activity were collected and concentrated using a 3000Da Millipore ultrafilter tube, followed by further separation using Sephacryl S100HR exclusion chromatography. The results show that from tube 14, the antiviral activity, protein content of the pools began to rise, and that the pools from tube 17 both reached the highest level; starting from tube 19, the inhibitory effect of the pool on TMV began to decrease until tube 20 had no significant anti-TMV activity; wherein the collected liquid in the 17 th tube has 90 percent of TMV inhibition effect.

Protein pools of different numbers separated by exclusion chromatography Sephacryl S100HR were subjected to SDS-PAGE. The results show that: the protein pools with anti-TMV effect all showed obvious protein bands, the protein bands in the pool of No. 15 tube were relatively miscellaneous, and the pools of No. 17 and 18 tubes showed relatively single protein bands. The collected liquid from tubes 16-18 is concentrated and then subjected to secondary exclusion chromatography, and the collected protein solution is subjected to SDS-PAGE detection, and the result shows that a single protein band can be detected.

By two chromatographic separations, we collected the protein with the highest inhibitory effect on TMV. SDS-PAGE gel detection shows that after Q Sephacryl FF and Sephacryl S100HR chromatographic separation of total protein, the bands of the antiviral activity protein on the PAGE gel are gradually reduced, and finally, after two times of Sephacryl S100HR separation, a single band is shown on the polyacrylamide gel.

And cutting off a single band presented by the separated protein components on an SDS-PAGE gel, and identifying by using a tandem mass spectrometry method. By comparison with polypeptides of known proteins in the Eubacteria database, 7 polypeptide fragments were obtained, all matched with alkaline metalloprotease (Protein View No. wp _015377659.1), and the 7 identified polypeptide fragments were: DATYFEDSR, TFENAGLELVR, TGDTVYGFNSNTDR, NHTFVNNQIHENEYGR, GEDKIDLTLFNTGSADGIR, DTFVYFAAEESTAAAPDWIR, and IIFTAWDAGGNDTFDFSGFGQNQR.

This protein was tentatively designated as an alkaline metalloprotease (SAMP) based on the results of tandem mass spectrometry.

Through the reported whole genome sequence of the Serratia marcescens, the nucleic acid sequences of alkaline metalloproteases of several Serratia marcescens are firstly obtained, the coding genes are found to be very conservative through sequence comparison, the nucleic acid sequence of the alkaline metalloprotease SAMP of Serratia marcescens-S3 is obtained through PCR amplification by designing specific primers (SAPM-F/SAMP-R), the SAMP gene is determined to be 1413bp in total through nucleic acid sequencing, and the putative protein is composed of 471 amino acids. The amino acid sequence of SAMP is compared with 3 homologous proteins with determined protein structures, the acquisition numbers of a PDB database are respectively 1sat (S.Marcescens; Baumann U.S. 1994), 5d7w (Serratia sp.Fs 14; Wu D.et al,2016) and 1srp (Serratia sp.E-15; Hamada K.et al, 1996), and the result shows that the amino acid sequence of SAMP has 99.79 percent of sequence identity with the metalloprotease 5d7w isolated from the Serratia sp.Fs14 strain and only 1 amino acid is different from 471; there were only 9 amino acid differences with 1sat and 1 srp.

The three-dimensional structure diagram of SAMP was predicted by the SWISS-MODEL website, and the result showed that the three-dimensional structure of SAMP is most similar to that of 5d7w, has an N-terminal connexin-hydrolyzing domain, and has a Zn²⁺Binding site and 7 Ca²⁺The matching site (Bienert S.et al, 2017; Guex N.et al, 2009).

SAMP was analyzed for structure by alignment with a homologous protein of known crystal structure (PDB:5d7w) and using the SPDBView-4.10 software. Model _01 represents SAMP and 5d7w.1.A represents a homologous protein of SAMP isolated from the strain Serratia sp.FS14. By structural analysis, SAMP is known to consist of 6 α -helices and 24 β -sheets. With reference to the structural analysis of 5d7w, SAMP can be divided into two large domain N-terminal hydrolysis domains (23-253) and a "parallel β -roll" C-terminal domain. The N-terminal hydrolysis domain mainly comprises 5 alpha-helical structures and 8 beta-folded sheets, and a plurality of irregular structural regions which jointly form a concave 'cave' structure, and the bottom of the concave 'cave' structure is combined with Zn²⁺Ions. SAMP protein backbone 176Five amino acid residues of histidine at position 177, glutamic acid at position 177, histidine at 180, 186 and glycine at position 216 (H176, E177, H180, H186, Y216) form the binding site for zinc ions. The entire recessed "cave" structure is the active site of SAMP.

The C-terminal domain is formed by 10 beta-sheets connected with each other and arranged in parallel to form an elongated "parallel beta-roll" structure (see FIG. 10). 7 Ca in the beta-roll domain²⁺Binding sites at ca.1 (R253, G255, T257, D285, G287, D290), ca.2(g.288, d.290, t.327, e.329), and ca.3(g.334, g.336, d.338, g.351), respectively; ca.4(n.343, a.345, n.347, g.360), ca.5(g.352, g.354, d.356, g.369); CA.6(G.370, A.371, G.372, D.374, D.400) and CA.7(G.361, G.362, G.363, D.365, D.383, D.390) Each Ca²⁺The binding site consists of 4-6 amino acid residues.

Enzyme activity tests show that the single protein component obtained by chromatographic separation has strong metalloprotease activity (see fig. 3-6); when the pH value of the reaction buffer solution is 8.0-9.0, the enzyme activity value is highest; when the reaction temperature reaches 40-50 ℃, the enzyme activity is highest. The stability experiment of the enzyme on the pH value proves that when the pH value of the reaction solution is lower than 5.0, the enzyme activity of alkaline metal protease (SAMP) is reduced to about 40 percent of the original enzyme activity; under alkaline conditions (pH is more than 7.0), the enzyme activity of SAMP is stable and is kept above 70%. The thermal stability experiment shows that: when the experimental temperature is 30-50 ℃, the SAMP activity is relatively stable and is kept above 80%; when the temperature is higher than 50 ℃ (60-70 ℃), the enzyme activity of SAMP is rapidly reduced to about 20%, and when the temperature is continuously increased to 80 ℃ or above, the enzyme activity is restored to about 40%, which indicates that SAMP has certain thermal stability.

In conclusion, the invention discloses a process method for purifying alkaline metalloprotease by using serratia, which separates protein substances with antiviral activity from secretory proteins of serratia marcescens, screens proteins with good antiviral effect and stable property, enriches the types of antiviral active substances, and provides theoretical basis for the research of plant virus disease prevention and treatment medicaments.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.