CN113088505B

CN113088505B - Application of polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase

Info

Publication number: CN113088505B
Application number: CN202110278025.8A
Authority: CN
Inventors: 冉艳红; 邓进丰; 杨晓苹; 王超; 张欣; 李弘剑
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-03-15
Filing date: 2021-03-15
Publication date: 2023-05-26
Anticipated expiration: 2041-03-15
Also published as: CN113088505A

Abstract

The invention discloses an application of a polysaccharide lyase coding gene 04147 in preparing recombinant peach gum polysaccharide hydrolase, which is obtained by connecting an optimized polysaccharide lyase coding gene shown in SEQ ID NO. 3 to an expression vector pET28a with a6 XHis tag, and transforming the gene into escherichia coli; and then carrying out IPTG induction expression on the recombinant expression bacteria, purifying by nickel affinity chromatography to obtain recombinant peach gum polysaccharide hydrolase, and obtaining single-kind enzyme, wherein the recombinant peach gum polysaccharide hydrolase has good peach gum cleavage activity, can be used for preparing hydrolase of low molecular weight and small molecular weight peach gum, and has a large application prospect.

Description

Application of polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase

Technical Field

The invention relates to the technical field of peach gum polysaccharide lyase, in particular to application of a polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase.

Background

Peach gum is a rosaceous plant such as peach, plum, apricot, cherry and the like, and after mechanical injury or microbial disease is caused, the bark secretes a colloid which is mostly light red or light yellow transparent, is an acidic polysaccharide substance with viscosity, contains a small amount of protein and impurities, has unique medicine-food homology characteristics, is recorded in ancient medical books in China to be effective for treating bloody stranguria, diabetes, urolithiasis and diarrhea symptoms, and can be used for treating cystitis and type II diabetes by modern medicine. Has larger development and application values.

However, the natural peach gum has a complex structure, is difficult to dissolve, and can be processed and utilized only after degradation. The current hydrolysis method of peach gum polysaccharide mainly comprises a hot water leaching method, an acid-base hydrolysis method and a microwave ultrasonic method. However, these treatments are not directed hydrolysis of glycosidic bonds, and are difficult to control the degree of polymerization, composition, viscosity, quality, etc. of the polysaccharide of the hydrolyzed product. The biological enzyme degradation method can realize directional hydrolysis by utilizing the enzyme method with the specificity of glycosidic bonds to degrade polysaccharide, stably generate specific enzyme catalysis products, has the advantages of mild reaction conditions, environmental friendliness and the like, but no commercial peach gum hydrolase is reported at present. Although Chinese patent CN104651340A discloses peach gum polysaccharide lyase from microbacterium, the peach gum polysaccharide lyase is obtained by separating and purifying a culture obtained by fermenting and culturing the acclimatized microbacterium A5, and the obtained peach gum polysaccharide lyase is a composite product of microbial metabolism, has low purity and is not suitable for commercial preparation of peach gum hydrolase preparations.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provides application of a polysaccharide lyase coding gene 04147 or a coding protein thereof in degradation of peach gum.

The invention also aims at providing an application of the polysaccharide lyase coding gene 04147 in preparing recombinant peach gum polysaccharide hydrolase.

The invention also aims at providing a preparation method of the recombinant peach gum polysaccharide hydrolase.

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

microbacterium sp.China is a strain isolated from the environment by the inventor and having a function of degrading peach gum polysaccharide, and the inventor obtains genome DNA information by sequencing the whole genome of Microbacterium sp.China from the head in the early stage, and uploads the genome DNA information to NCBI database with accession number of CP027434. The DNA size of the Microbacterium sp.China genome is 3,478,219bp, and there are 3409 open reading frames in total. Comparing the polysaccharide lyase gene protein sequences (such as BAR87238.1, AIE37653.1, AHZ54600.1 and AFU 18128.1) of other bacillus sources (Bacillus thuringiensis serovar tolworthi; bacillus thuringiensis serovar kurstaki str. HD-1;Bacillus thuringiensis serovar kurstaki str.YBT-1520;Bacillus thuringiensis MC28) with a CAZy database, comparing the polysaccharide hydrolase gene with a domain carried by polysaccharide lyase by using pfam analysis, finding that orf04147 gene encoding protein has a beta_helix domain of the polysaccharide lyase, screening out a gene orf04147 (04147 for short) with a potential peach gum polysaccharide degrading function, and annotating the gene function as the polysaccharide lyase; furthermore, the inventor further proves that 04147 is remarkably and highly expressed in the process of degrading peach gum by comparing carbohydrate activity related enzyme coding genes which are differentially expressed by Microbacterium sp.China based on peach gum polysaccharide.

Therefore, the invention provides the application of the polysaccharide lyase coding gene 04147 shown in SEQ ID NO. 1 or the polysaccharide lyase shown in SEQ ID NO. 2 in degrading peach gum.

Also provides the application of the polysaccharide lyase coding gene 04147 shown in SEQ ID NO. 1 in preparing recombinant peach gum polysaccharide hydrolase.

A preparation method of recombinant peach gum polysaccharide hydrolase comprises connecting an optimized coding gene 04147 of a half polysaccharide lyase shown in SEQ ID NO. 3 to an expression vector pET28a with a6×His tag, and transforming the gene into escherichia coli to obtain recombinant expression bacteria; and performing IPTG induction on the recombinant expression bacteria to express recombinant protein, and purifying by nickel affinity chromatography to obtain the recombinant peach gum polysaccharide hydrolase.

Because the full length of the polysaccharide lyase coding gene 04147 is relatively large, subsequent recombinant expression and purification are difficult, the invention performs truncated expression on the basis of retaining the key structural domain, and the retained amino acids 330 to 1110 of SEQ ID NO. 2, namely the optimized sequence shown as SEQ ID NO. 3, are used for constructing an expression vector to perform subsequent recombinant protein expression.

Preferably, the E.coli is E.coli BL21 (DE 3).

Preferably, A600 is used when recombinant expression bacterium IPTG induces expression starting point _nm 0.5 to 0.8.

Preferably, the IPTG induction temperature is 17-37 ℃, the IPTG concentration is 0.1-0.9 mM, and the induction time is 4-20 h.

Further preferably, the IPTG induction temperature is 37℃and the IPTG concentration is 0.3mM and the induction time is 6h.

The invention also claims the recombinant peach gum polysaccharide hydrolase prepared by any one of the methods.

The recombinant peach gum polysaccharide hydrolase prepared by the method can act on galactose glycosidic bond and arabinoside bond, and has peach gum splitting activity. Therefore, the invention also provides application of the recombinant peach gum polysaccharide hydrolase in splitting peach gum.

Preferably, the enzymolysis temperature is 30-50 ℃, and the pH value of the enzymolysis system is 6-10.

Further preferably, the enzymolysis temperature is 45 ℃, and the pH value of the enzymolysis system is 6.

Preferably, 10mM K is also added into the enzymolysis system ⁺ Or Na (or) ⁺ 。

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

the invention provides application of a polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase. The optimized polysaccharide lyase gene shown in SEQ ID NO. 3 is connected into an expression vector pET28a with a6 XHis tag and is transformed into escherichia coli to obtain recombinant expression bacteria; and then performing IPTG induction expression on the recombinant expression bacteria, purifying by nickel affinity chromatography to obtain recombinant peach gum polysaccharide hydrolase, and obtaining single enzyme, wherein the recombinant peach gum polysaccharide hydrolase acts on a galactoside bond and an arabinoside bond, has good peach gum cleavage activity, can be used for preparing commercial peach gum hydrolase, and has a large application prospect.

Drawings

FIG. 1 shows a double enzyme electrophoresis pattern of 04147 plasmid. 1: pET28a-04147-1;2: pET28a-04147-2;3: pET28a-04147-3;4: pET28a-04147-4;5: pET28a-04147-5;6: pET28a-04147-6.

FIG. 2 shows the result of SDS-PAGE analysis of 04147 recombinant protein. Lane M (Marker): protein molecular weight standard. Lane 1: wild type E.coli BL21. Lane 2: e.coli empty vector blank induction control. Lane 3, supernatant from centrifugation of recombinant protein 0447 culture. Lane 4, whole-cell lysate of 0447 protein recombinant. Lane 5, centrifugal supernatant of recombinant protein 0447 lysate. Lane 6: centrifugal precipitation of 04147 protein recombinant bacteria lysate.

FIG. 3 shows the result of Western-blotting analysis of 04147 recombinant proteins. Lane 1: BL21 whole bacteria lysate; lane 2: BL21-pET28a lysate; lane 3: recombinant bacterium culture solution supernatant; lane 4: recombinant bacteria whole bacterial lysate; lane 5: recombinant bacteria whole bacteria lysate supernatant; lane 6 whole bacterial lysate of recombinant bacteria precipitated.

FIG. 4 shows the effect of induction temperature on 04147 protein expression. Lane M: protein molecular weight standard; lane M: protein molecular weight standard; 17. 00578 recombinant bacterium lysate at 22, 27, 32 and 37 ℃ at different induced expression temperatures

FIG. 5 shows the effect of induction time on 04147 protein expression. Lane M: protein molecular weight standard; 0. and 2,4,6,8 and 10 hours are 00578 recombinant bacteria lysate under different induction expression time respectively.

FIG. 6 shows the effect of inducer concentration on 04147 protein expression. Lane M: protein molecular weight standard; and 0.1,0.2,0.3,0.4 and 0.5mM are respectively 00578 recombinant bacteria lysate under different inducer concentrations.

FIG. 7 shows the results of SDS-PAGE analysis of the nickel affinity chromatography purification of the recombinant proteins. Lane 1: bacterial lysate supernatant. Lane 2: purifying the column penetrating liquid. Lane 3: purifying the column washing liquid. Lane 4: purifying the column eluate.

FIG. 8 shows SDS-PAGE results of urea denatured recombinant protein by nickel affinity chromatography. Lane 1: bacterial lysate supernatant. Lane 2: purifying the column penetrating liquid. Lane 3: purifying the column washing liquid. Lane 4: purifying the column eluate.

FIG. 9 shows the results of mass spectrometry of protein peptide fragments.

FIG. 10 is a graph showing the comparison of peach gum degradation ability of the recombinant and host bacterial lysates of peach gum polysaccharide degrading enzymes.

FIG. 11 is a TLC profile of peach gum polysaccharide hydrolase 04147 for various glycosidic bond hydrolyses. pnpgl: p-nitrophenyl-galactoside; pnprara: p-nitrophenyl-arabinoside; pnpgu: p-nitrophenyl-glucoside; gal: galactose; ara: arabinose; glu: glucose.

FIG. 12 is a graph showing the effect of temperature on peach gum polysaccharide hydrolase activity.

FIG. 13 is a graph showing the effect of temperature on peach gum polysaccharide hydrolase stability.

FIG. 14 is a graph showing the effect of pH on peach gum polysaccharide hydrolase activity.

FIG. 15 is a graph showing the effect of pH on peach gum polysaccharide hydrolase stability.

FIG. 16 is a graph showing the effect of metal ions and their chelators on peach gum polysaccharide hydrolase activity.

FIG. 17 is a double reciprocal plot of the Miq equation for peach gum polysaccharide hydrolase 04147.

FIG. 18 shows the ability of peach gum polysaccharide lyase 04147 to hydrolyze peach gum to release glycogen with reduced end sugars.

FIG. 19 is a TLC profile of peach gum polysaccharide lyase 04147 on peach gum hydrolysis. 1, glucose, 2 galactose, 3, arabinogalactan+ 04174,4,

peach gum+

04147,5, pectin+ 04147. And B, 1, glucose, 2, galactose, 3, arabinogalactan, 4, peach gum and 5, pectin.

Detailed Description

The invention is further illustrated in the following drawings and 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 inventors have previously completed obtaining their genomic DNA information by whole genome de novo sequencing of Microbacterium sp.China. The DNA size of the Microbacterium sp.China genome is 3,478,219bp, and there are 3409 open reading frames in total. And (3) screening out a gene orf04147 with a potential peach gum polysaccharide degradation function by comparing with a CAZy database and combining pfam database domain comparison with equivalent bioinformatics analysis, so as to carry out recombinant construction, cloning, purification expression and subsequent function research.

EXAMPLE 1 cloning of peach gum polysaccharide degradation related enzyme encoding gene orf-04147 and construction of recombinant plasmid

1. Method of

1. Design of PCR primers

Based on the ORF-04147 gene sequence obtained by sequencing the genome of Microbacterium, PCR primers were designed using software Primer premier 5.0, and the Primer sequences are shown in Table 1 below. The primer is delivered to Shanghai biological engineering company for synthesis.

TABLE 1

2. PCR reaction

(1) Adding each component by taking the genome DNA of the microbacterium as a PCR template according to the following table 2, and uniformly mixing;

TABLE 2

(2) The following procedure was set up for PCR reactions

PCR reaction conditions: pre-denaturation at 98℃for 10min; denaturation at 98℃for 45sec, annealing at 55℃for 40sec, elongation at 72℃for 1min for 35 cycles; extending at 72℃for 8min.

(3) The PCR reaction products were subjected to agarose gel electrophoresis.

4. The PCR product gel recovery was performed using a DNA gel recovery kit.

5. Double enzyme cutting

(1) Preparing a double enzyme digestion reaction system for the recovered PCR gel product according to Table 3

TABLE 3 Table 3

(2) Vector plasmid double enzyme cutting system

TABLE 4 Table 4

(3) After the components of the system are added and mixed uniformly, the mixture is placed in a constant-temperature water bath at 37 ℃ for incubation, and enzyme digestion is carried out for 2 hours.

(4) After enzyme cutting is finished, the liquid on the pipe wall and the pipe cover is thrown down by short centrifugation. Purification was performed using a DNA product purification kit.

6. Enzyme digestion product purification Using PCR product recovery kit

7. Connection

(1) The ligation system of the PCR products and the vector is shown in Table 5 below:

TABLE 5

(2) The components are added into a PCR tube and mixed evenly, and then put into a 25 ℃ metal bath connector for connection for 2 hours.

8. Coli DH 5. Alpha. Competent cells were prepared.

9. And (3) transformation.

10. The monoclonal was picked.

11. Plasmid extraction was performed according to the smallclothes root plasmid miniprep kit.

12. Double enzyme cutting identification

(1) The recombinant plasmid was subjected to double digestion and the components were added according to the following Table 6:

TABLE 6

(2) Adding the components into a PCR tube, uniformly mixing, and performing enzyme digestion for 1h in a water bath kettle at 37 ℃.

(3) The digested product was subjected to agarose gel electrophoresis at 180V for 20min with 1% agarose gel.

(4) After electrophoresis, the gel is placed in a gel imager for observation, photographing and recording. Plasmid samples meeting the expected molecular weight size were sent for biological sequencing.

(5) Sequencing results were aligned by NCBI sequences to confirm sequencing results.

13. The plasmid of interest was transformed into E.coli BL21 (DE 3).

2. Results

The fast extraction kit of bacterial genome is used to extract the genome DNA of Microbacterium sp.China, the target gene orf-04147 is amplified by PCR, and the orf-04147PCR product is separated by agarose electrophoresis, and the result is consistent with the expected result. Synthesizing 04147 truncated gene, constructing pET-28a-04147 expression plasmid, sequencing to match the theoretical gene sequence, and double enzyme digestion to identify the synthesized plasmid to match the theory, the result being shown in figure 1. pET-28a-04147 was transformed into BL21 (DE 3) for expression. The nucleotide sequence of orf-04147 is shown in SEQ ID NO:1, the coded amino acid sequence is shown as SEQ ID NO:2, the 04147 truncated gene sequence is shown in SEQ ID NO: 3.

EXAMPLE 2 analysis of expression of recombinant plasmid encoding orf-04147 gene of enzyme involved in degradation of peach gum polysaccharide

1. The growth curve of the recombinant escherichia coli BL21 is measured, the logarithmic growth period of the escherichia coli BL21 (DE 3) is measured to be 2-2.5 hours in feeding time, and the corresponding A600nm is 0.5-0.8, so that the time period is selected as the starting point of subsequent IPTG induction.

2. Inducible expression of recombinant proteins of interest

(1) Coli BL 21-expressing bacteria and plasmid empty bacteria were inoculated into liquid LB medium containing the corresponding antibiotics (Amp) in an inoculum size of 1%, and shake-cultured in a constant temperature shaker at 37℃and 220rpm for activation overnight.

(2) The following day, the transfer bacteria liquid is transferred into fresh LB culture medium according to 1 percent, and is cultured until the spectrophotometer detects the OD of the escherichia coli ₆₀₀ When the value is 0.5-0.6, adding proper amount of IPTG to make it become final concentrationThe degree was 0.3mM, and the culture was induced in a constant temperature incubator at 37℃and 220rpm for 6 hours.

(3) After the induction culture, two 1.5mL centrifuge tubes are taken for each culture solution, 1mL bacterial solution is taken for each culture solution, and the culture solution is centrifuged at 10000rpm for 1min at room temperature, so as to collect bacterial cells.

(4) Adding 1mLPBS buffer solution into the centrifuge tube, centrifuging at 10000rpm for 1min after re-suspending the thalli, removing the supernatant, and cleaning the thalli. After repeating three times, 500ml of LPBS was added to resuspend the cells.

(5) One of the tubes was directly added with 125mL of 5 XSDS-PAGE loading buffer, and after being blown uniformly, heated in a metal bath at 99℃for 10min to completely denature the protein. The refrigerator is kept at the temperature of minus 20 ℃ for standby.

(6) The other centrifuge tube was placed on ice and broken by ultrasound using an ultrasonic cytobreaker. The ultrasonic bacteria breaking condition is 400w, the working time is 3s, the gap is 3s, and the bacteria breaking is stopped until the bacterial liquid becomes clear and transparent. After the bacteria breaking is finished, the mixture is placed into a precooling centrifuge for centrifugation at 10000rpm for 10min at 4 ℃. After centrifugation, the supernatant was transferred to another clean Ep tube and the pellet was resuspended in 500. Mu.L PBS buffer. To both separation tubes, 125. Mu.L of 5 XSDS-PAGE loading buffer was added. After being blown uniformly by a pipette, the protein is heated in a metal bath at 99 ℃ for 10min to be completely denatured and subjected to SDS-PAGE electrophoresis.

Results: inoculating recombinant escherichia coli into a fresh culture medium containing ampicillin with one thousandth concentration, setting a control group at the same time, adding a proper amount of IPTG into the culture medium to enable the final concentration to reach 0.3mM when the A600nm value of the escherichia coli reaches the range of 0.5-0.8, and then respectively inducing and culturing the escherichia coli in a constant-temperature shaking incubator at 37 ℃ at a rotating speed of 220rpm for 6 hours. After the culture was completed, the cells were collected by centrifugation at 10000rpm, the supernatant was collected after ultrasonic disruption or high-pressure homogenization, and the samples were separated by SDS-PAAGE and the result of electrophoresis was visualized by Coomassie blue staining. As shown in fig. 2, the protein of interest 04147 can be successfully expressed at 37 ℃ and detected in lysate supernatant samples.

3. Western-binding identification of recombinant proteins

After the SDS-PAGE analysis 04147 recombinant protein is successfully expressed, the recombinant protein is identified by utilizing His-tag of the recombinant protein through Western-Blotting, and the result is shown in figure 3, wherein the recombinant protein 04147 is detected in the whole bacterial lysate of the recombinant bacterium, the supernatant and the sediment of the lysate, the amount of the protein in the supernatant is more, and the control group is not detected, so that the expression of the recombinant protein 04147 is proved to be successful according to the SDS-PAGE result.

4. Condition optimization of inducible expression recombinant proteins

Judging whether the recombinant protein is expressed in the form of inclusion bodies according to SDS-PAGE results: if the target band is displayed on the bacterial sediment sample in the SDS-PAGE electrophoresis result, judging that the protein is expressed as inclusion body; if the target band appears in the bacterial supernatant sample, the protein is considered to be soluble expressed.

(1) Optimization of induction temperature: according to the experimental method of induced expression recombinant protein, adding appropriate amount of IPTG to reach final concentration of 0.3mM, and performing induced culture in a constant temperature incubator at 17, 22, 27, 32 and 37 ℃ for 6h, and setting negative control without adding IPTG. SDS-PAGE was used to detect the relative protein content after harvesting the protein samples, and the optimal induction temperature for recombinant protein expression was determined by comparison of the individual conditions.

Results: 04147 recombinant strains are respectively induced and cultured for 6 hours in an IPTG with the final concentration of 0.3mM at a constant temperature of 17 ℃,22 ℃,27 ℃,32 ℃ and 37 ℃ in a shaking incubator, and the expression quantity of the recombinant proteins is detected by SDS-PAGE. As a result, as shown in FIG. 4, the most soluble samples were detected in the supernatant samples at 37℃and the optimum induction temperature was determined at 37 ℃.

(2) Optimization of induction time: according to the experimental method of inducing and expressing recombinant protein, adding appropriate amount of IPTG to make the final concentration 0.3mM, and respectively inducing and culturing at optimal induction temperature for different time (if the optimal induction temperature is 37 ℃, the induction and culturing time is respectively set to 0h,2h,4h,6h,8h and 10h, and if the optimal temperature is below 30 ℃, the induction and culturing time is respectively set to 0h,4h,8h,12h,16h and 20 h). After harvesting the protein samples, the relative amounts of protein were detected using SDS-PAGE, and the optimal induction time for recombinant protein expression was determined by comparing the amounts of protein for each condition.

Results: the 04147 recombinant strain was induced and cultured in a constant temperature shaking incubator at 37℃for 2,4,6,8 and 10 hours with IPTG at a final concentration of 0.3mM, respectively, and the recombinant protein expression level was detected by SDS-PAGE. As a result, as shown in FIG. 5, the soluble sample detected in the supernatant sample at 6 hours was large and the subsequent expression level was not changed much, and it was confirmed that 6 hours was the optimal induction time.

(3) Optimization of inducer concentration: according to the experimental method operation of inducing and expressing recombinant protein, adding appropriate amount of IPTG to reach final concentration of 0mM,0.1mM,0.2mM,0.3mM,0.4mM and 0.5mM, inducing and culturing at optimal induction temperature for optimal induction time, detecting relative content of protein by SDS-PAGE after harvesting protein sample, and determining optimal inducer concentration of recombinant protein expression by comparing protein amount of each condition.

Results: the 04147 recombinant strain is induced and cultured for 6 hours in a constant temperature shaking incubator at 37 ℃, and the final concentration of inducer IPTG is respectively set to be 0.1,0.2,0.3,0.4 and 0.5mM. SDS-PAGE detects recombinant protein expression. As a result, as shown in FIG. 6, the supernatant sample was added at a final concentration of 0.3mM to obtain a more soluble sample, and the concentration of the inducer was determined to be 0.3 mM.

(4) Expansion culture of recombinant strains: according to the determined optimal expression conditions, the recombinant escherichia coli expression bacteria are induced and cultured, and the final volume of the expanded culture of the culture solution is 500mL. After the cultivation is finished, the mixture is split into 50mL centrifuge tubes, put into a centrifuge for centrifugation at 8000rpm for 5min at room temperature, taken out, and the supernatant is poured out. And (3) adding a proper amount of PBS buffer solution into the residual bacterial precipitate for resuspension, and putting the bacterial precipitate into a centrifugal machine for centrifugation at 8000rpm at room temperature for 5min. After centrifugation, the supernatant was decanted. The washing step is repeated three times to ensure that the excess culture medium is removed, and the bacterial precipitate can be subjected to subsequent experiments or stored at-20 ℃ for later use.

(5) High pressure homogenized bacterial disruption: about 70mL of a bacterial lysate containing 10mM imidazole was added to the washed cells, and the cells were sufficiently resuspended, so that the bulk cells could not remain. And (3) starting a low-temperature cooling liquid circulating pump to pre-cool to-10 ℃, assembling a sample injection cup, opening a high-pressure homogenizer, and setting the circulating power to be 50hz. Ethanol, ultrapure water and bacterial lysate are added one by one in sequence, so that the aim of cleaning and infiltrating channels is fulfilled. After the components flow out, pouring bacterial suspension into the sample adding cup, placing the sample outlet pipe into the sample adding cup to enable the sample outlet pipe to circularly flow, and discharging all air in the channel. The manometer was turned on, the pressurization valve was slowly and carefully twisted, the pressure was slowly increased to 800-900bar, and the mixture was homogenized for several minutes until the liquid in the cup was clear and transparent, and the sample inlet was observed. The crushed bacterial liquid is collected and filtered by a 0.45 mu m filter membrane, and the bacterial liquid is stored at 4 ℃ or directly subjected to subsequent experiments.

5. Nickel affinity chromatography purification condition research of recombinant protein

(1) The Ni-NTA Superflow was resuspended by stirring with a glass rod, slowly introduced into a clean, empty chromatography column, allowed to settle slowly to form a stationary phase column, and continuously added with 30% ethanol to prevent drying of the column.

(2) Balance: and balancing the preloaded nickel affinity chromatographic column by using a constant flow pump at a flow rate of 3mL/min, wherein the dosage of the balancing solution is about 2-3 column volumes.

(3) Loading: and (3) loading the sample at a flow rate of 1mL/min by using a constant flow pump, and continuing to balance after the sample is loaded, and starting to collect the penetrating fluid until the baseline is stable when the protein detector detects the protein.

(4) Washing: washing the nickel affinity chromatographic column with a constant flow pump at a flow rate of 3mL/min, and collecting the washing liquid until the baseline is stable.

(5) Eluting: the eluent is washed by a constant flow pump at a flow rate of 3mL/min for the nickel affinity chromatography column, and the eluent is collected until the baseline is stable.

(6) Cleaning: and cleaning the nickel affinity chromatographic column with a constant flow pump at a flow rate of 3mL/min, and cleaning the volume of 2-3 columns.

(7) And (3) preserving: and (3) cleaning the nickel affinity chromatography column with 30% ethanol at a flow rate of 3mL/min by using a constant flow pump, cleaning the volume of 2-3 columns, closing the inlet and outlet of the column, and storing in a refrigerator at 4 ℃.

(8) And (3) taking a part of each of the sample before purification, the penetrating fluid, the washing fluid and the eluent, adding a proper amount of 5X SDS loading buffer solution for SDS-PAGE, and detecting the protein content of each component to judge the purification effect.

(9) Because the purification effect of electrophoresis verification is not ideal, 5M urea is added into all purification systems (including bacterial lysate, balance solution, washing solution and eluent) to denature proteins, his-tag and Ni-NTA are exposed to combine, so that good purification effect is achieved, and the eluent is detected by SDS-PAGE after dialysis.

Results: recombinant protein 04147 was purified by nickel ion affinity chromatography using a Ni-NTA column. In the purification, a denaturing agent is not added first, and the mixture is subjected to nickel ion affinity chromatography in a natural conformation. However, none of the native conformation of 04147 was bound to the nickel filler and the protein of interest was not isolated from the sample, see figure 7. Therefore, the purification method is optimized, and 5M urea is added into each reagent in the purification system to expose the His-tag of the recombinant protein, so that the His-tag is combined with the nickel filler on the chromatographic column. As a result, as shown in FIG. 8, the target protein 04147 can be isolated from the sample for purification purposes.

6. Mass spectrometric identification of proteins of interest

The obtained purified protein is separated by SDS-PAGE and then is shown by a coomassie blue staining method, a gel sample containing a target band is cut off by a knife according to a molecular weight standard, and the gel sample is sent to Shenzhen large gene technology Co-Ltd for protein gel point mass spectrum identification. The identification is that SDS-PAGE gel is treated by pancreatin, protein is extracted, the obtained peptide is separated by high performance liquid phase and then is detected by mass spectrum, the mass spectrum is obtained, and protein identification software is used for identifying protein.

The sequences and the identification results of the peptide fragments are shown in Table 7. 04147 can be matched to the corresponding protein of Microbacterium sp.str 'China' in a database. The recombinant protein obtained by purification is proved to be the target protein which is designed and expressed by us, which indicates that the recombinant protein is successfully expressed and purified. The mass spectrum of the corresponding peptide fragment of 04147 is shown in fig. 9.

Table 7 04147 LC-MS/MS identification results of proteins

EXAMPLE 3 04147 polysaccharide hydrolysis function verification of the protein of interest

1. Preparation of standard curve for measuring reducing sugar by DNS (Domain name System) chromogenic method

(1) Taking 6 EP pipes to number in sequence;

(2) 200 mu L of standard glucose solution with different concentrations is added respectively, and the concentrations are 0, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL and 0.5mg/mL respectively;

(3) Adding 100 mu L of 3, 5-dinitrosalicylic acid reagent and shaking uniformly;

(4) Heating in boiling water bath for 5min, immediately taking out and cooling after water bath is finished, and measuring the change of absorbance value of each system at 540 nm;

(5) And drawing a standard curve according to the absorbance value and the concentration of the reducing sugar solution.

2. Determination of enzyme Activity of galactosidase by DNS (Domain name System) chromogenic method

A control group was set while adding 40. Mu.L of a 6% peach gum solution and 40. Mu.L of a crude enzyme solution (i.e., recombinant bacterial lysate supernatant) to the PCR tube (blank control group added with equal amounts of peach gum solution and ddH) ₂ O), incubate for a certain period of time at 37 ℃ in a thermostated incubator. Then 40. Mu.L of 3, 5-dinitrosalicylic acid reagent was added and shaken well, heated in a boiling water bath for 5min, immediately cooled after completion of the water bath, and 100. Mu.L was taken and the absorbance measured at 550 nm. The absorbance of each group was subtracted from the absorbance of the blank group and substituted into the standard curve to calculate the yield of reducing sugar.

The results are shown in FIG. 10: the recombinant bacterium lysate of the over-expressed recombinant peach gum polysaccharide degrading enzyme 04147 can degrade peach gum to release reducing sugar or reducing oligosaccharide chains, which indicates that the constructed genetically engineered bacterium can over-express the recombinant enzyme with peach gum degrading activity.

EXAMPLE 4 enzymatic Property study of Gene recombinant peach gum polysaccharide hydrolase 04147

Because peach gum polysaccharide has complex structure and different polymerization degree, even though components and structures of the same batch of products may have larger difference, the research of enzymology property requires quantitative experiments using substrates with clear components and stable quality. Therefore, the synthetic glycoside analogue p-Nitrophenyl-beta-D-Galactopyranoside (pNPGal) is used for replacing peach gum polysaccharide, and the type of the recombinant enzymatic hydrolysis glycosidic bond is studied initially.

1. Genetic recombinant peach gum polysaccharide hydrolysis glycosidic bond specificity analysis

To the PCR tube, 90. Mu.L of 1M pNPGal (p-nitrophenylgalactoside), 1M pNPAra (p-nitrophenylalanoside) and 1M pNPGlu (p-nitrophenylglucoside) were added, respectively, and 10. Mu.L of enzyme was added to the PCR tube to add 10. Mu.L of deionized water as a control group, and after mixing the enzyme and the substrate, the reaction was carried out for 15 minutes under the optimal conditions of the enzyme. After the completion of the incubation, the reaction was terminated by adding 1.5 times the volume of 1M sodium carbonate solution, and then 2. Mu.L of the reaction solution was taken, and the substrate and the product were separated by TLC to determine the substrate specificity of the enzyme.

TLC method: n-butanol: acetic acid: water = 2:1:1 (volume ratio) is used as a developing agent, silica gel G254 on an aluminum foil plate is used as a stationary phase, and the gel is developed in a chromatographic cylinder after sample application. And after the development is finished, taking out the aluminum foil plate, spraying a color development liquid prepared from dihydroxynaphthalene sulfuric acid ethanol solution, and heating at 110 ℃ for 3-5 min to develop color.

Results: three glycoside compounds pNPGal, pNPAra and pNPGlu were selected as substrates for the enzymatic hydrolysis glycosidic bond specificity experiments to determine if 04147 was able to hydrolyze such artificial glycoside substrates. The product was isolated by thin layer chromatography hydrolysis experiments and as shown in figure 11, 04147 acts on galactosides and arabinosides, degrading pnpgl and pnpgara, releasing Gal and Ara, but not glucose on glucosides. 04147 shows galactosidase and arabinosidase activities. Since the peach gum polysaccharide backbone is mainly composed of galactans, subsequent experiments explored some of the enzymatic properties of 04147 using pnpgl as a substrate.

2. Influence of temperature on activity and stability of gene recombinant peach gum polysaccharide hydrolase

Determination of optimum temperature: to the PCR tube, 99. Mu.L of 1M pNPGal and 1. Mu.L of enzyme were added, and incubated at 15℃at 20℃at 25℃at 30℃at 35℃at 40℃at 45℃at 50℃at 55℃at 60℃for 15min, with three replicates per group. After the incubation was completed, the reaction was stopped by adding 1.5 volumes of 1M sodium carbonate solution, and then 100. Mu.L of the mixture was taken to measure A405nm, and the data was recorded and plotted. Comparing the enzyme activity of each temperature, or obtaining the optimal temperature, and taking the optimal temperature as the temperature condition of the subsequent experiment.

Temperature stability determination: the enzymes were pre-incubated at 15℃at 20℃at 25℃at 30℃at 35℃at 40℃at 45℃at 50℃at 55℃at 60℃for 2h,4h,6h,8h and 10h, respectively. Thereafter, 99. Mu.L of 1M pNPGal and 1. Mu.L of pre-incubated enzyme were added to the PCR tube, and incubated at the optimum temperature for 15min, and three replicates were set for each group. After the incubation was completed, 150. Mu.L of 1M sodium carbonate solution was added to terminate the reaction and develop the color, and then 100. Mu.L of the mixture was taken to measure A405nm, the data was recorded, the results were analyzed and a curve was drawn.

Results: the experimental results of the effect of temperature on 04147 enzyme activity are shown in FIG. 12, and the activity of both enzymes on pNPGal can be maintained to be more than 50% of the optimal activity in the temperature range of 30-50 ℃, the optimal reaction temperature of 04147 is 40 ℃, and the subsequent experiments are carried out at the optimal temperature. The 04147 enzyme activity was maintained at 60% or more of the highest activity within 10 hours at a temperature ranging from 30 to 50 ℃, but the enzyme was rapidly deactivated at a temperature higher than 60 ℃.

3. Influence of pH value on activity and stability of gene recombinant peach gum polysaccharide hydrolase

Determination of optimum pH: to the PCR tube, 99. Mu.L of 1M pNPGal and 1. Mu.L of enzyme were added, and incubated at

pH

4,5,6,7,8,9, 10 for 15min, respectively, and three replicates were set for each group. After the incubation, the reaction was stopped and the color was developed by adding 150. Mu.L of 1M sodium carbonate solution, and then 100. Mu.L of the mixture was taken for measurement of A405nm, the data was recorded and the curve was drawn. The obtained optimum pH was measured as the temperature condition for the subsequent experiments.

Determination of pH stability: the enzyme was pre-incubated in buffer at

pH

4,5,6,7,8,9, 10 for 2h,4h,6h,8h,10h at 4 ℃. Then 99. Mu.L of 1M pNPGal and 1. Mu.L of pre-incubated enzyme were added to the PCR tube and incubated at the optimum temperature and pH for 15min, and three replicates were set for each group. After the incubation was completed, the reaction was stopped by adding 1.5 volumes of 1M sodium carbonate solution, and then 100. Mu.L of the mixture was taken to measure A405nm, and the data was recorded and plotted.

As a result, as shown in FIG. 14, the pH range of the activity of 04147 enzyme on pNPGal was 6 to 10, and the optimal pH for 04147 enzyme activity was 7;04147 maintains over 70% of the enzyme activity at pH 6-10, and this is deactivated at pH below 6 (FIG. 15), so that acidic conditions should be avoided during storage or subsequent experiments to prevent deactivation of the enzyme.

4. Influence of Metal ions and chelators thereof on recombinant peach gum polysaccharide hydrolase Activity

mu.L of 1M pNPGal was added to the PCR tube, and various metal ions or metal ion chelators (Na ⁺ ，K ⁺ ，Ca ²⁺ ，Mg ²⁺ ，Zn ²⁺ ，Fe ³⁺ ，Ni ²⁺ And EDTA) was 10mmol/L, and three replicates were set per group incubated for 15min under optimum conditions. After the incubation was completed, the reaction was stopped by adding 1.5 volumes of 1M sodium carbonate solution, and then 100. Mu.L of the mixture was taken to measure A405nm, and the data was recorded and plotted.

Results: part of the glycoside hydrolase requires metal ions as enzyme activators to increase the enzyme activity, and several common metal ions are selected for determining the effect on 04147 enzyme activity. As a result, as shown in FIG. 16, the metal ion Zn ²⁺ ，Fe ³ ⁺ ，Ni ²⁺ ，Ca ²⁺ ，Mg ²⁺ And EDTA inhibits the viability of 04147pNPGal, wherein Zn ²⁺ ，Fe ³⁺ ，Ni ²⁺ And EDTA has obvious inhibiting effect (only 7-21 percent is reserved), however K ⁺ And Na (Na) ⁺ The enzyme activity of 04147 can be slightly improved. These results demonstrate that no matter 04147, it should be avoided to react with Zn ²⁺ ，Fe ³⁺ ，Ni ²⁺ Used with EDTA, but potassium and sodium ions are contemplated for use in maintaining physiological ion concentrations in the preservation or application of both enzymes.

5. Determination of enzymatic kinetic parameters of recombinant peach gum polysaccharide hydrolase

A gradient of pNPGal solution (0.6,1.2,1.8,2.4,3) was prepared, 90. Mu.L of pNPGal was added to each PCR tube, 10. Mu.L of enzyme was added thereto, and the reaction was carried out for 15 minutes under the optimal conditions of the enzyme, and three replicates were set for each set. After the incubation was completed, the reaction was terminated by adding 150. Mu.L of 1M sodium carbonate solution, and then measuring A405nm by taking 100. Mu.L of the mixture. The amount of enzyme required to degrade peach gum per ml of enzyme solution per unit time and to produce 1. Mu. Mol of reducing sugar was defined as one enzyme activity unit (U), and finally the kinetic parameters of the enzyme were calculated by the double reciprocal mapping method (Lineweaver-Burk method).

Results: the kinetic parameters of the enzyme reaction include various factors that influence the reaction rate of the enzyme catalysis. We define the amount of enzyme required to degrade peach gum per ml of enzyme solution per unit time and to produce 1. Mu. Mol of reducing sugar as one enzyme activity unit (U), and finally calculate the kinetic parameters of the enzyme by the double reciprocal mapping method (Lineweaver-Burk method). The results of experiments calculated by studying the effect of the concentration of pNPGal on the enzyme activity are shown in FIG. 17 and Table 8, FIG. 17 is a double reciprocal plot of the Mimi equation of 04147, and Table 8 is the kinetic parameters of the enzyme. 04147 the maximum reaction rate Vmax for pNPGal is 4.878 (. Mu.mol min) ^-1 ml ^-1 ) The Michaelis constant Km was 0.642 (mM), and the ratio Kcat/Km of the reaction constant to the Michaelis constant was 8.24(s) ^-1 mM ^-1 )。

Enzymatic kinetic parameters of Table 8 04147

Example 5 degradation Effect of Gene recombinant peach gum polysaccharide hydrolase 04147 on peach gum substrate

04147 (3.2 mg/ml) and 6% peach gum are uniformly mixed in equal volume, incubated at a constant temperature of 45 ℃, the reducing sugar production amount is detected by a DNS method every other hour, 40 mu L of 6% peach gum solution and 40 mu L of enzyme solution (equal amounts of peach gum solution and ddH2O are added in a blank control group) are added into a PCR tube, and the mixture is incubated for a certain time in a constant temperature incubator or a water bath. Then 40. Mu.L of 3, 5-dinitrosalicylic acid reagent was added and shaken well, heated in a boiling water bath for 5min, immediately cooled after completion of the water bath, and 100. Mu.L was taken and the absorbance measured at 550 nm. The absorbance of each group was subtracted from the absorbance of the blank group and substituted into the standard curve to calculate the yield of reducing sugar. The amount of reducing sugar produced represents the amount of polysaccharide fragment with reducing end sugar released by the degradation of peach gum by the recombinase. Definition of enzyme activity unit (U): the amount of enzyme required for degrading peach gum to produce 1 μg of reducing sugar per milliliter of enzyme solution per unit time is one enzyme activity unit (U), and the specific activity of enzyme refers to the activity of enzyme contained in every milligram of protein.

04147 (3.2 mg/ml) was mixed with 6% peach gum, arabinogalactan, pectin, etc. in volume, incubated at 45℃for 8 hours, and TLC plate chromatography was performed with glucose and galactose, PBS+peach gum, PBS+pectin, PBS+arabinogalactan as controls, with the following conditions: ethanol: water = 3:2:5

As a result, as shown in FIG. 18, recombinase 04147 can degrade peach gum to produce oligosaccharides and reduced terminal glycosyl fragments. 04147 recombinase and 6% peach gum are evenly mixed in equal volume, incubated for 12 hours at a constant temperature of 30 or 45 ℃, the enzyme activity of 04147 is detected to be 15.09U/mg by a DNS reducing sugar method, the specific activity of the original strain lysate to the degradation of the peach gum is only 1.01U/mg, and the specific activity of the enzyme is obviously improved. The amount of reducing sugar increases as the time of action of the enzyme with peach gum increases. The degradation of peach gum by recombinant 04147 enzyme was detected by TLC to yield oligosaccharide products, as shown in fig. 19.

Sequence listing

<110> and university of south China

<120> application of polysaccharide lyase coding gene 04147 in preparation of recombinant peach gum polysaccharide hydrolase

<141> 2021-03-15

<160> 3

<170> SIPOSequenceListing 1.0

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gacgacgcgg cctccggcac gatcgacgac cccctcgcga cgctggaagg cgcccgcgac 180

cggatccgcg cgctgaaggc ggactccgcg ctcccggacg acggcctcac ggtgtacctc 240

cgggagggca cgtacccccg ctcggcgtcg ttcgagatcg gcgcgcagga ctcgggcact 300

gccgacgccc ccatcaccta ccgctcgtac ccgggggaga ccgccaccct caccggcggt 360

cgcgagctgc cgcgcgacca gttcgccgcg gtcgacgacg cggcggtgac cgagcgcatc 420

atcgacccgg ctgcccgcga ccgtgtcgtc gggatcgacc tcgccgacct cggcatcacc 480

gactacggcc agctcagccg ccacggctac tggaaggcca acgacgtcag caccaccccg 540

cccatggagc tgttcatcga cggccagggc atgaccctcg cgcgctggcc caacgccgac 600

gcggccaccc cgaccgtgca gatgggcgac atcatcgacg ccgggccgga ccgcaacgac 660

gccgacctgc aggagcgcgg cggcacgttc agctacggct acgaccgccc gaagcactgg 720

acccaggccg aggacgtctg gctggacggc atcttcggct acagctggga gtggtcgtac 780

aacaagatcg agagcatcga caccgaggcg aagaccatca ccctccgcta cggcgagatg 840

tccgggatca tgaagagctg gttccccgac ttccacttcg cgcagaacct gctggaggag 900

ctcgacgccc ccggcgagta ctacatcgac cgcgacgccg ggaagctgta cctcatcccg 960

aacgcggcct tcacgtccgg ccgcggcgcc gtgacggtca cgacgctcga cgagccgatg 1020

ctgcgcgccg acggggcctc ctacgtgaac ttcgacgacc tcgtgatgga gtacggccgc 1080

gcgacggccg cggtgatcct cggcggctcg cacgtgacga tctcgcacag cgacatccgc 1140

aacttcaccg acggcggcgt gctcatcaac tcgccggggc gctacacgta cgacggcatt 1200

ccggtgaacc gcggcggccg cgaccacgcc gtgaccgaca gccggctcac tcacgtcggc 1260

ggcgtcggcg tggtgctcca gggcggcgac aagacgacgc tcgaacccgg ccgcaaccgg 1320

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ttcgtcgacg cctacgcccc gtacgacaac tacgagcagt ggatgggcga ccaggagggc 1800

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ttcgtcggca cgccgtatct gacgaagtac cccgagctcg cgcacttctt cgaggacgac 1920

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gacccgaacc gctccatcca ggtggatgcg tcgggcgaca aggtgtaccg catcctcaag 2580

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gccccggcca cgacccccgc caacgcggga ctcatcgtct acgaccgcaa cggcgagggc 2700

ggcggctaca tccgcttccg ccaggccgcg gcggggccga cctggacgat cttcgacgac 2760

gcctggaagg tcgtcgccga gaaggtcgtg ccggcggcgc agggcctgac ccccggggag 2820

acctcgcacg tccgcatcgc tgtgcaggac gggcagatcc ggatctccgt gaacggggcg 2880

atcgcgttgg agggcgccga ccccggcccc ggcaaggccg gtcgggtcgg gttctacgtg 2940

gagaactacg cctcgctcga cttcgacgac atcgggttct cgctctccgg ggtgccggtg 3000

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gatgccgagt acccgaccac ccgcctggac ggccagctga aggacggggc gaactggagc 3300

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ccgccgcccg cgacggtgac cggcatctgc tgggcgcccg aagccgaccc gaagacccgc 3840

tacgtgcgcg gcgaggagct cgacgtgacc ggcatgctgc tgggcgtgga ctggagcgac 3900

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gtgcgcccgc accacacgct caccgtgacg tacgctggcg cgagcgtcga gctcccgatc 4020

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<212> PRT

<213> Microbacterium A5 (Microbacterium sp. China)

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Met Lys Arg Val Leu Ala Ser Leu Thr Leu Ala Ala Val Phe Gly Ser

1 5 10 15

Ala Leu Ala Ala Ala Val Pro Ser Ala Ala Ala Ala Ala Asp Asp Asp

20 25 30

Ala Leu Tyr Leu Ala Pro Gly Gly Asp Asp Ala Ala Ser Gly Thr Ile

35 40 45

Asp Asp Pro Leu Ala Thr Leu Glu Gly Ala Arg Asp Arg Ile Arg Ala

50 55 60

Leu Lys Ala Asp Ser Ala Leu Pro Asp Asp Gly Leu Thr Val Tyr Leu

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Arg Glu Gly Thr Tyr Pro Arg Ser Ala Ser Phe Glu Ile Gly Ala Gln

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Asp Ser Gly Thr Ala Asp Ala Pro Ile Thr Tyr Arg Ser Tyr Pro Gly

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Glu Thr Ala Thr Leu Thr Gly Gly Arg Glu Leu Pro Arg Asp Gln Phe

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Ala Ala Val Asp Asp Ala Ala Val Thr Glu Arg Ile Ile Asp Pro Ala

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Ala Arg Asp Arg Val Val Gly Ile Asp Leu Ala Asp Leu Gly Ile Thr

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Asp Tyr Gly Gln Leu Ser Arg His Gly Tyr Trp Lys Ala Asn Asp Val

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Ser Thr Thr Pro Pro Met Glu Leu Phe Ile Asp Gly Gln Gly Met Thr

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Leu Ala Arg Trp Pro Asn Ala Asp Ala Ala Thr Pro Thr Val Gln Met

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Gly Asp Ile Ile Asp Ala Gly Pro Asp Arg Asn Asp Ala Asp Leu Gln

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Glu Arg Gly Gly Thr Phe Ser Tyr Gly Tyr Asp Arg Pro Lys His Trp

225 230 235 240

Thr Gln Ala Glu Asp Val Trp Leu Asp Gly Ile Phe Gly Tyr Ser Trp

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Glu Trp Ser Tyr Asn Lys Ile Glu Ser Ile Asp Thr Glu Ala Lys Thr

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Ile Thr Leu Arg Tyr Gly Glu Met Ser Gly Ile Met Lys Ser Trp Phe

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Pro Asp Phe His Phe Ala Gln Asn Leu Leu Glu Glu Leu Asp Ala Pro

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Gly Glu Tyr Tyr Ile Asp Arg Asp Ala Gly Lys Leu Tyr Leu Ile Pro

305 310 315 320

Asn Ala Ala Phe Thr Ser Gly Arg Gly Ala Val Thr Val Thr Thr Leu

325 330 335

Asp Glu Pro Met Leu Arg Ala Asp Gly Ala Ser Tyr Val Asn Phe Asp

340 345 350

Asp Leu Val Met Glu Tyr Gly Arg Ala Thr Ala Ala Val Ile Leu Gly

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Gly Ser His Val Thr Ile Ser His Ser Asp Ile Arg Asn Phe Thr Asp

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Gly Gly Val Leu Ile Asn Ser Pro Gly Arg Tyr Thr Tyr Asp Gly Ile

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Pro Val Asn Arg Gly Gly Arg Asp His Ala Val Thr Asp Ser Arg Leu

405 410 415

Thr His Val Gly Gly Val Gly Val Val Leu Gln Gly Gly Asp Lys Thr

420 425 430

Thr Leu Glu Pro Gly Arg Asn Arg Val Glu Asn Ser Glu Ile Ala Asp

435 440 445

Phe Ala Tyr Tyr His Lys Ala Tyr Asn Pro Gly Val Met Phe Asp Gly

450 455 460

Val Gly Asn Ile Ala Arg Asp Asn Glu Ile His Asp Ala Pro His Pro

465 470 475 480

Gly Ile Ile Val His Gly Asn Asp His Leu Phe Glu Arg Asn Glu Val

485 490 495

Tyr Asp Val Cys Lys Gln Phe His Asp Leu Gly Ala Ile Tyr Met Asn

500 505 510

Ser Gly Lys Thr Pro Gln Gln Arg Gly His Val Phe Arg Glu Asn Tyr

515 520 525

Phe His Asp Ile Gly Val Gly Met Ala Gly Val Glu Gly Ile Tyr Ala

530 535 540

Asp Asn Phe Thr Trp Asp Leu Thr Ile Glu Lys Asn Val Phe Val Asn

545 550 555 560

Met Gly Asn Gly Ala Ile Lys Ser Gly Ser Ala Asp Tyr Ile Glu Ala

565 570 575

Arg Asn Asn Val Phe Val Asp Ala Tyr Ala Pro Tyr Asp Asn Tyr Glu

580 585 590

Gln Trp Met Gly Asp Gln Glu Gly Asn Val Val Asp Arg Asp Tyr Met

595 600 605

Pro Ala Trp Glu Lys Val Phe Ala Asp Asn Asn Asp Phe Val Gly Thr

610 615 620

Pro Tyr Leu Thr Lys Tyr Pro Glu Leu Ala His Phe Phe Glu Asp Asp

625 630 635 640

His Tyr Phe Pro Asn His Ser Thr Phe Ala Glu Asn Val Val Trp Asn

645 650 655

Pro Asn Arg Ala Arg Met Ala Gly Val Asn Glu His Gly Ala Lys Asp

660 665 670

Gly Lys Asn Leu Leu Asn Tyr Glu Asp Asn Trp Val Ala Asp Ala Asp

675 680 685

Pro Gly Phe Val Asp Ala Ala Asn Gly Asp Tyr Thr Leu Lys Ala Asp

690 695 700

Ala Ala Val Phe Asp Gln Ile Pro Gly Phe Glu Ala Ile Ala Phe Gly

705 710 715 720

Glu Ile Gly Val Asp Gly Ala Ile Gly Gln Thr Gln Gln Pro Gln Thr

725 730 735

Ile Pro Leu Glu Asp Ile Ala Phe Asp Ser Asp Thr Leu Thr Ile Asp

740 745 750

Ala Gly Asp Glu Val Arg Val Arg Ala Val Pro Leu Pro Trp Asn Ala

755 760 765

Asp Asp Ala Ala Val Thr Tyr Ala Ser Ala Asp Thr Ala Val Ala Ser

770 775 780

Val Asn Asp Lys Gly Val Val Leu Gly Met Gly Pro Gly Thr Thr Thr

785 790 795 800

Val Thr Ala Thr Ala Lys Ala Asp Ala Ala Lys Thr Ala Thr Ile Glu

805 810 815

Val Ile Val Glu Glu Gly Asp Gly Val Leu His Phe Thr Asp Phe Glu

820 825 830

Ser Gly Ala Asn Gly Trp Pro Thr Asp Pro Asn Arg Ser Ile Gln Val

835 840 845

Asp Ala Ser Gly Asp Lys Val Tyr Arg Ile Leu Lys Gly Ala Asn Ser

850 855 860

Ile Leu Pro Arg Asp Phe Thr Glu Phe Val Leu Asp Phe Asp Val Thr

865 870 875 880

Ala Pro Ala Thr Thr Pro Ala Asn Ala Gly Leu Ile Val Tyr Asp Arg

885 890 895

Asn Gly Glu Gly Gly Gly Tyr Ile Arg Phe Arg Gln Ala Ala Ala Gly

900 905 910

Pro Thr Trp Thr Ile Phe Asp Asp Ala Trp Lys Val Val Ala Glu Lys

915 920 925

Val Val Pro Ala Ala Gln Gly Leu Thr Pro Gly Glu Thr Ser His Val

930 935 940

Arg Ile Ala Val Gln Asp Gly Gln Ile Arg Ile Ser Val Asn Gly Ala

945 950 955 960

Ile Ala Leu Glu Gly Ala Asp Pro Gly Pro Gly Lys Ala Gly Arg Val

965 970 975

Gly Phe Tyr Val Glu Asn Tyr Ala Ser Leu Asp Phe Asp Asp Ile Gly

980 985 990

Phe Ser Leu Ser Gly Val Pro Val Thr Gly Val Ser Leu Asp Ala Asp

995 1000 1005

Ala Val Gly Leu Thr Val Gly Glu Arg Arg Ser Val Ala Ala Thr Val

1010 1015 1020

Ala Pro Glu Asp Ala Ser Asp Ala Arg Val Thr Trp Thr Thr Asp Ala

1025 1030 1035 1040

Pro Glu Val Ala Thr Val Ser Gly Gly Arg Ile Ala Gly Val Thr Ala

1045 1050 1055

Gly Thr Ala Thr Ile Thr Ala Thr Ser Val Ala Asp Pro Ser Leu Ser

1060 1065 1070

Asp Thr Val Thr Val Thr Val Asp Asp Ala Glu Tyr Pro Thr Thr Arg

1075 1080 1085

Leu Asp Gly Gln Leu Lys Asp Gly Ala Asn Trp Ser Gln Ser Asp Leu

1090 1095 1100

Ile Ala Val Asp Asp Thr Gly Val Val Ile Ser Gly Gln Gly Val His

1105 1110 1115 1120

Gly Tyr Glu Ala Glu Arg Phe Gly Asp Thr Leu Leu Gln Phe Glu Ala

1125 1130 1135

Glu Phe Gly Ala Phe Asp Gly Gly Trp Tyr Gly Phe Gln Ala Arg Ser

1140 1145 1150

Asp Gln Thr Gly Leu Pro Ala Trp Gln Asn Ser Asn Thr Gly Tyr Leu

1155 1160 1165

Ala Val Ile Lys Glu Asp Val Ile Glu Phe Gln Ser Trp Thr Pro Gly

1170 1175 1180

Gln Thr Thr Leu Asp Ser Ile Pro Asn Thr Val Ile Glu Pro Asn Ser

1185 1190 1195 1200

Thr His Arg Ile Glu Phe Gly Ala Val Ala Glu Asp Gly Gly Thr Arg

1205 1210 1215

Ile Val Leu Arg Val Asp Asp Val Thr Val Trp Asn Met Val Asp Ala

1220 1225 1230

Arg Glu Asn Leu Arg Ile Gly Ala Asp Gly Phe Phe Asn Val Tyr His

1235 1240 1245

Tyr Gly Lys Thr Asn Thr Leu Ala Val Arg Pro Thr Pro Pro Pro Ala

1250 1255 1260

Thr Val Thr Gly Ile Cys Trp Ala Pro Glu Ala Asp Pro Lys Thr Arg

1265 1270 1275 1280

Tyr Val Arg Gly Glu Glu Leu Asp Val Thr Gly Met Leu Leu Gly Val

1285 1290 1295

Asp Trp Ser Asp Gly Ser Arg Thr Thr Gln Gln Val Thr Ala Asp Met

1300 1305 1310

Val Ser Gly Phe Asp Ser Ser Lys Val Arg Pro His His Thr Leu Thr

1315 1320 1325

Val Thr Tyr Ala Gly Ala Ser Val Glu Leu Pro Ile Ser Val Arg Pro

1330 1335 1340

Lys Leu Lys Asn Asp Glu Gln Asp Val Pro Arg Cys Gly

1345 1350 1355

<210> 3

<211> 2343

<212> DNA

<213> Microbacterium A5 (Microbacterium sp. China)

<400> 3

gccgtgacgg tcacgacgct cgacgagccg atgctgcgcg ccgacggggc ctcctacgtg 60

aacttcgacg acctcgtgat ggagtacggc cgcgcgacgg ccgcggtgat cctcggcggc 120

tcgcacgtga cgatctcgca cagcgacatc cgcaacttca ccgacggcgg cgtgctcatc 180

aactcgccgg ggcgctacac gtacgacggc attccggtga accgcggcgg ccgcgaccac 240

gccgtgaccg acagccggct cactcacgtc ggcggcgtcg gcgtggtgct ccagggcggc 300

gacaagacga cgctcgaacc cggccgcaac cgggtcgaga actccgagat cgccgacttc 360

gcgtactacc acaaggccta caatccgggc gtgatgttcg acggcgtcgg caacatcgcc 420

agggacaacg agatccacga cgccccgcat cccgggatca tcgtgcacgg caacgaccac 480

ctgttcgaac gcaacgaggt gtacgacgtc tgcaagcagt tccacgacct cggtgccatc 540

tacatgaact ccggcaagac cccgcagcag cgcggccacg tgttccggga gaactacttc 600

cacgacatcg gcgtcggcat ggcgggcgtc gagggcatct acgccgacaa cttcacgtgg 660

gacctcacga tcgagaagaa cgtgttcgtg aacatgggca acggcgcgat caagagcggc 720

tcggccgact acatcgaggc acgcaacaac gtcttcgtcg acgcctacgc cccgtacgac 780

aactacgagc agtggatggg cgaccaggag ggcaacgtcg tcgaccgcga ctacatgccg 840

gcctgggaga aggtgttcgc cgacaacaac gacttcgtcg gcacgccgta tctgacgaag 900

taccccgagc tcgcgcactt cttcgaggac gaccactact tcccgaacca cagcacgttc 960

gcggagaacg tcgtgtggaa cccgaaccgg gcccgcatgg ccggcgtcaa cgagcacggc 1020

gcgaaagacg ggaagaacct cctgaactac gaggacaact gggtggccga cgccgacccc 1080

ggcttcgtgg acgccgcgaa cggcgactac acgctgaagg cggacgcggc cgtgttcgac 1140

cagatcccgg gcttcgaggc catcgcgttc ggcgagatcg gcgtcgacgg cgcgatcggg 1200

cagacgcagc agccgcagac catcccgctc gaggacatcg cgttcgacag cgacacgctc 1260

acgatcgacg cgggcgacga ggtgcgcgtg cgcgccgttc cgctgccctg gaacgccgac 1320

gacgccgcgg tgacctacgc ctcggccgat accgccgtcg cctccgtcaa cgacaagggc 1380

gtggtgctcg gcatgggccc cggcacgacc acggtcacgg cgacggcgaa ggccgatgcc 1440

gccaagaccg cgaccatcga ggtgatcgtc gaggagggcg acggcgtgct gcacttcacc 1500

gacttcgagt cgggggcgaa cggctggccg accgacccga accgctccat ccaggtggat 1560

gcgtcgggcg acaaggtgta ccgcatcctc aagggagcca acagcatcct gccgcgggac 1620

ttcacggagt tcgtgctcga cttcgacgtc acggccccgg ccacgacccc cgccaacgcg 1680

ggactcatcg tctacgaccg caacggcgag ggcggcggct acatccgctt ccgccaggcc 1740

gcggcggggc cgacctggac gatcttcgac gacgcctgga aggtcgtcgc cgagaaggtc 1800

gtgccggcgg cgcagggcct gacccccggg gagacctcgc acgtccgcat cgctgtgcag 1860

gacgggcaga tccggatctc cgtgaacggg gcgatcgcgt tggagggcgc cgaccccggc 1920

cccggcaagg ccggtcgggt cgggttctac gtggagaact acgcctcgct cgacttcgac 1980

gacatcgggt tctcgctctc cggggtgccg gtgacgggcg tgagcctgga cgccgacgcc 2040

gtgggactga ccgtggggga gcggcggtcc gtcgcggcca cggtcgcccc ggaggacgcc 2100

agcgacgccc gggtcacctg gacgaccgac gcgccggagg tcgccacggt ctccggcgga 2160

cgcatcgccg gggtcacggc cggcacggcg acgatcaccg cgacctcggt cgccgatccg 2220

agcctcagcg acacggtcac cgtgaccgtg gacgatgccg agtacccgac cacccgcctg 2280

gacggccagc tgaaggacgg ggcgaactgg agccagtccg acctcatcgc ggtggacgac 2340

acg 2343

Claims

1. The optimized polysaccharide lyase coding gene 04147 shown in SEQ ID NO. 3 or the application of the coding protein thereof in degradation of peach gum.

2. The optimized polysaccharide lyase coding gene 04147 shown in SEQ ID NO. 3 is applied to the preparation of recombinant peach gum polysaccharide hydrolase.

3. A preparation method of recombinant peach gum polysaccharide hydrolase is characterized in that an optimized polysaccharide lyase coding gene 04147 shown in SEQ ID NO. 3 is connected to an expression vector pET28a with a6 XHis tag, and is transformed into escherichia coli to obtain recombinant expression bacteria; and performing IPTG induction on the recombinant expression bacteria to express recombinant protein, and purifying by nickel affinity chromatography to obtain the recombinant peach gum polysaccharide hydrolase.

4. A method according to claim 3, wherein the IPTG induction temperature is 17 ℃ to 37 ℃, IPTG concentration is 0.1 to 0.9mM, and induction time is 4 to 20 hours.

5. The method of claim 4, wherein the IPTG induction temperature is 37℃and the IPTG concentration is 0.3. 0.3mM and the induction time is 6h.

6. Recombinant peach gum polysaccharide hydrolase prepared by the method of any of claims 3-5.

7. The use of the recombinant peach gum polysaccharide hydrolase of claim 6 for splitting peach gum.

8. The use according to claim 7, wherein the enzymolysis temperature is 30-50 ℃ and the pH value of the enzymolysis system is 6-10.

9. The use according to claim 8, wherein the enzymatic hydrolysis temperature is 45 ℃ and the enzymatic hydrolysis system pH is 7.

10. The use according to claim 8, wherein the enzymatic hydrolysis systemAlso added with 10mM K ⁺ Or Na (or) ⁺ 。