WO2020029379A1 - Monosaccharide exotype algin lyase aly-6 having m-tendency, coding gene thereof and use thereof - Google Patents

Abstract

A monosaccharide exotype algin lyase Aly-6 having M-tendency, and a coding gene thereof and use thereof. The amino acid sequence of the monosaccharide exotype algin lyase Aly-6 having M-tendency is shown as SEQ ID NO. 2. The nucleotide sequence encoding Aly-6 is shown as SEQ ID NO. 1. Aly-6 is obtained from the genome of bacterium MY04 of genus Flammeovirga, and can not only continuously degrade guluronic acid fragments of algin by means of external cleavage of monosaccharides, but also continuously degrade mannuronic acid fragments by means of external cleavage of monosaccharides, however, is more apt in degrading the mannuronic acid fragments.

Description

M-prone exo-type alginate lyase Aly-6 and its coding gene and application Technical field

The invention relates to an M-prone exotype alginate lyase Aly-6 and its coding gene and application, and belongs to the technical field of genetic engineering.

Background technique

Fucoidan is a linear polysaccharide composed of two sugar units, α-L-Guluronic acid (G) and β-D-Mannuronic acid (M). G segments and MG or GM mixed segments are arranged alternately ^[1] . Fucoidan is usually prepared from large seaweeds such as kelp, sargassum, and giant algae. The latest research shows that the new candidate class 1 drug "GV971" prepared with poly-M-stage alginate oligosaccharide can inhibit the aggregation and cytotoxicity of β-amyloid cells and can be used to treat mild to moderate Alzheimer's disease ^[2] . Through phase III clinical studies; saturated poly-G oligosaccharides can work with antibiotics to inhibit multi-drug resistant pathogens ^[3] . This indicates that the alginate oligosaccharide with important polymerization degree and M / G ratio has important application value and economic value, so it is of great significance to realize the efficient preparation of this type of oligosaccharide.

Alginate lyases are a class of polysaceharide lyases (PLs) that catalyze the cleavage of glycosidic bonds in alginate molecules through the β-elimination mechanism, and form a non-reducing end of C4 = C5 in the oligosaccharide product. A saturated double bond forms a conjugated structure with C = O (carboxycarbonyl) at the C5-position, resulting in an oligosaccharide product containing an unsaturated terminal (Δ) ^[4] . Endoenzymes usually produce a series of unsaturated oligosaccharide products after the alginate is completely degraded. Exoenzymes usually produce a single oligosaccharide main product, such as a delta unit. Alginate lyases are selective for substrates with different M / G ratios. Combining enzymes with different endo / exo degradation modes of substrates, enzymes can be comprehensively divided into at least six categories: G-specific internal Dicer, exonuclease, M-specific endonuclease, exonuclease, and bifunctional endonuclease, exonuclease. Compared with chemical or physical degradation methods, the enzymatic degradation of alginate has the advantages of mild conditions, easy control, and certain substrate selectivity, so it has the potential for popularization and application ^[5] . At present: (1) although there are a large number of patent applications ^[6-8] and scientific research literature on alginate lyase, it is mainly focused on primary research such as enzyme-producing strains and enzyme resource exploration; (2) specificity of enzymes The analysis mostly uses poly M and G segments with limited purity as test substrates, while the structural characteristics of oligosaccharide products of enzymes lack spectral data such as mass spectrometry and nuclear magnetic resonance of oligosaccharide final products, and oligosaccharide production characteristics That is, the explanation of the corresponding substrate degradation mechanism (such as the minimum substrate, the minimum product, the position of the minimum product in the substrate, and the substrate propensity of the enzyme, etc.) is relatively small; (3) Molecular enzymology research uses gene cloning It is mainly related to heterologous expression, purification and functional identification of recombinases. There is less research on molecular modification such as active site mutation and functional module truncation, and molecular enzymatic modification and enzyme alginate degradation mode or oligosaccharide production characteristics. There is less awareness of regular relationships. Therefore, compared with other polysaccharide degrading enzymes such as β-agarase and cellulase that have been commercially produced and widely used, there are few commercially available alginate lyases that are widely used. In summary, the traditional research of alginate lyase is weak, the number of tool enzymes is scarce, and the mechanism is unclear. These shortcomings not only limit the precise application of the existing alginate lyase, but also severely restrict the enzymatic preparation of the alginate oligosaccharide technology. development of.

In the past 5 years, the patent inventors and research team have systematically conducted resource screening, genome sequencing, and gene discovery research on high-efficiency polysaccharide-degrading bacteria in response to the important and key needs of the aforementioned industries. The focus is on alginate lyase, and systematic analysis of multiple biochemical characteristics of a recombinase and its mutants, substrate selectivity, and mode of substrate degradation oligosaccharides characteristic, different values and comparing the characteristics of their application for the preparation of oligosaccharides.

For example, the inventor's research on the G-specific endophytic alginate lyase Aly5 derived from Flammeovirga sp. MY04 showed that ^[9 , ^10] , the final main product of this enzyme after complete degradation of alginate is a degree of polymerization of 2-7 Series of unsaturated oligosaccharides, including: (1) oligosaccharide end products with a degree of polymerization of 5-7, containing only ΔM non-reducing ends, which cannot be used by the G-specific endonuclease Aly5 because they are rich in M; Deep degradation can be considered as unsaturated poly-M-segment series oligosaccharides; (2) unsaturated disaccharides contain only ΔG units, and oligosaccharide products with a degree of polymerization of 3-4 contain ΔM or ΔG non-reducing ends and are rich in G, but Constrained by Aly5 because the disaccharide is the smallest product of the endonuclease and the smallest substrate is pentasaccharide, so it has not been completely digested; (3) the non-catalytic region of Aly5 is truncated, although the base of the truncated protein is not changed Bioselectivity and incision mode, but the minimum substrate of the truncated body becomes larger, so that in the end product of the truncated body degrading the alginate, the content of the series of oligosaccharides with a degree of polymerization of 5-7 increased significantly, while the degree of polymerization was 2 -4 series of oligosaccharide content is relatively reduced. This study confirmed for the first time that the application of G-specific alginate endonucleases can not only specifically produce two types of alginate oligosaccharide products, such as unsaturated polyM segment and unsaturated polyG segment, and their molecular weight distributions The ranges are significantly different, and the characteristics of truncated bodies to produce larger oligosaccharide fragments can be enhanced by molecular enzymatic modification.

The inventors have also studied two M-specific endophytic alginate lysing enzymes AlgL ^[11] derived from Pseudomonas aeruginosa and Azotobacter brown, and found that: in their oligosaccharide end products after degrading alginate , Oligosaccharides with a large degree of polymerization (5-7) contain only ΔG non-reducing ends and are rich in G, and can be regarded as unsaturated poly G-segment series oligosaccharides; unsaturated disaccharide products mainly contain ΔM, unsaturated trisaccharides The sugar and tetrasaccharide products mainly contain ΔM or ΔG termini. That is, these two M-specific alginate endonucleases, AlgL, are oligosaccharide end products after degrading alginate, and the G-specific alginase end products of oligosaccharides have opposite structural characteristics to each other, but are accompanied by The degree of aggregation succession is similar to each other.

In addition, the inventors also studied the G-biasing bifunctional endonucleases Aly1 ^{[12, 13]} and Aly2 ^[ 14, 115 ^] derived from Flammeovirga sp. MY04, and confirmed that they are more suitable for thorough degradation of algin and efficient production Low molecular weight oligosaccharide fragments such as unsaturated disaccharides (ΔG) and unsaturated trisaccharides.

In summary, similar to the domestic counterparts, the existing research focus of the inventors has been on the analysis of endophytic alginate lyase. But the difference is that the inventors and the research group also focused on explaining the substrate selectivity, substrate degradation mode, and oligosaccharide generation characteristics of a series of endonucleases. The substrate degradation mode together determines the oligosaccharide product generation characteristics of the endonuclease. Related molecular enzymatic engineering research has also explored effective ways to transform natural enzymes and enhance their oligosaccharide production characteristics.

Moreover, there are few reports on the biochemical properties, catalytic mechanism, enzymatic properties and molecular modification of exofucoid lyases at home and abroad. For example, the application of the exotype alginate lyase in the Chinese invention patent application is only found in the 2017 Chinese patent document CN107177612A (application number 201710367707X), which first disclosed the exotype alginate lyase AlgL17 from the marine bacterium Microalgae ALW1. Basic biochemical properties and enzymatic hydrolysis of alginate can produce products: 4-deoxy-L-erythro-5-hexoseuronic acid (DEH). The substrate degradation characteristics and oligosaccharides of exotype alginate lyase are not described in detail. Similar studies on glycogenic properties. Domestic patent claims for bifunctional alginate lyases are also only seen in the inventors' open applications for endonucleases Aly-1 and Aly-2 ^[12-15] .

However, there are no reports on the degradation mechanism of M-biasing bifunctional monosaccharide exonuclease and its related substrates and the resulting oligosaccharide product generation characteristics.

Summary of the invention

The present invention aims at the shortcomings of the prior art, and provides an M-prone exo-type alginate lyase Aly-6 and its coding gene and application.

The technical solution of the present invention is as follows:

An M-prone exotype alginate lyase Aly-6, the amino acid sequence of which is shown in SEQ ID NO.2.

The above-mentioned M-prone exo-type alginate lyase Aly-6 can both degrade the guluronic acid oligosaccharide fragment from alginate and reduce the mannuronic acid oligosaccharide fragment, so it is facultative alginate Lyase.

The aforementioned M-prone exo-type alginate lyase Aly-6 contains two domains, one of which is classified as AlgLyase superfamily and the other as Heper_II_III superfamily.

In the process of degrading the alginate polysaccharide, the enzyme gradually degrades the intermediate product (unsaturated sugar chain) in the manner of monosaccharide excision, so the main product is the unsaturated monosaccharide unit (Δ) and the remaining sugar chain. When the enzyme does not completely degrade algin, the series of unsaturated oligosaccharide products produced have the common feature that the non-reducing end only contains the ΔG terminal structure.

After the enzyme has completely degraded the alginate polysaccharide, the final remaining oligosaccharide products are mainly unsaturated trisaccharide fragments, containing traces of unsaturated tetrasaccharides, and no unsaturated disaccharides are seen.

The smallest unsaturated oligosaccharide substrate of this enzyme is the disaccharide ΔM, and 2 molecules of Δ are produced after degradation of ΔM. This enzyme can degrade one molecule of unsaturated disaccharide ΔG in trace amounts, producing 2 molecules of Δ. This enzyme cannot degrade saturated disaccharides MM or GG.

In summary, the minimum oligosaccharide products of this enzyme include unsaturated monosaccharides Δ, saturated monosaccharides M and G.

An M-prone exotype alginate lyase encoding gene aly-6, the nucleotide sequence is shown in SEQ ID NO.1.

The encoding gene aly-6 of the aforementioned M-prone exo-type alginate lyase Aly-6 has a total length of 2238 bp, the encoded protein contains 745 amino acids, and the molecular weight is about 84.7 kD.

A recombinant expression vector in which the above-mentioned M-prone monosaccharide exo-type alginate lyase Aly-6 encoding gene aly-6 is inserted into the expression vector.

According to a preferred embodiment of the present invention, the expression vector is selected from the group consisting of: an E. coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, and an insect Expression vector or mammalian cell expression vector.

A recombinant strain in which a recombinant expression vector of the above-mentioned M-prone monosaccharide exophytic algal lyase Aly-6 is transferred into a host cell, or the above-mentioned M-prone monosaccharide exo-type alginate lyase is expressed in a host cell Aly-6.

According to a preferred embodiment of the present invention, the host cell is selected from the group consisting of: an E. coli host cell, a yeast host cell, a subtilis host cell, a lactic acid host cell, an actinomycete host cell, a filamentous fungal host cell, an insect cell, or a mammalian cell.

The encoding gene aly-6, recombinant expression vector, and recombinant bacteria of the above-mentioned M-prone monosaccharide exo-fucoid lyase Aly-6 are used in the preparation of the M-prone monosaccharide exo-fucoid lyase, rAly-6. application.

Application of the above-mentioned M-prone exo-type alginate lyase Aly-6 in thoroughly degrading alginate or degrading alginate oligosaccharides to produce unsaturated trisaccharides and unsaturated tetrasaccharides.

The above-mentioned M-prone exo-type alginate lyase Aly-6 does not completely degrade alginate or degrade alginate oligosaccharides to produce a large series of unsaturated oligosaccharide fragments and unsaturated monomers containing ΔG ends at non-reducing ends. Application of sugar delta.

Beneficial effect

1. The present invention discloses for the first time that an M-prone monosaccharide exophytic alginate lyase Aly-6 is obtained from the genome of Flammeovirga yaeyamensis MY04. The enzyme is a monosaccharide exophyte Glycolytic enzyme with obvious M tendency. The enzyme Aly-6 can both continuously degrade the guluronic acid fragments from alginate in a monosaccharide excision manner, and can also continuously degrade mannose in a monosaccharide excision manner. The uronic acid fragment, but it is easier to degrade the mannuronic acid fragment; the properties of this enzyme are significantly different from the existing known alginate lyase, the physical and chemical properties are stable, the activity is high, and it has the potential for industrial application.

2. The M-prone monosaccharide exo-type alginate lyase Aly-6 involved in the invention does not completely degrade alginate polysaccharides, and mainly produces unsaturated sugar units Δ through exo-cutting, while the remaining larger series do not The common structural feature of saturated oligosaccharide product fragments is that the non-reducing end contains only the ΔG-terminus. Therefore, Aly-6 can be used for the incomplete degradation of algin, so as to specifically prepare a series of unsaturated oligosaccharide fragments of only ΔG terminus;

3. When the M-prone exo-type alginic alginate lyase Aly-6 used in the present invention is used to completely degrade the alginate polysaccharide, the final remaining oligosaccharide product is an unsaturated trisaccharide containing a small amount of Saturated tetrasaccharides, unsaturated disaccharides were not seen, and the main product-unsaturated monosaccharide Δ-has been deeply converted into other products. Therefore, Aly-6 can completely digest alginate and provide an unsaturated monosaccharide carbon source for the growth of microorganisms for microbial growth and utilization;

4. The M-prone monosaccharide exotype alginate lyase Aly-6 according to the present invention contains two domains (AlgLyase, superfamily and Heper_II_III, superfamily), which were respectively truncated and expressed. As a result, neither of the two truncated proteins Alginate lyase activity, which indicates that two putative functional modules generate exophytic alginate lyase activity through molecular interactions, which is completely different from the existing known alginate lyase structure. To further study the alginate cleavage Enzymes lay the foundation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. BLASTp analysis results of functional modules of recombinant alginate lyase rAly-6;

Figure 2. Expression and purification of polyacrylamide gel electrophoresis (SDS-PAGE);

Among them, A. Recombinant alginate lyase rAly-6 expression and purified polyacrylamide gel electrophoresis (SDS-PAGE); In the figure: M, protein molecular weight standard, band size from top to bottom is 116kD, 66.2kD , 45kD, 35kD, 25kD, 18.4kD, 14.4kD; Lane 1, control strain before breaking the bacterial cells, load 10 μL, lane 2, recombinant bacteria before breaking the wall, 10 μL, lane 3, recombinant After the wall was broken, the supernatant was loaded with 10 μL, lane 4 and rAly-6 purified by nickel column, and the load was 10 μL;

B. Polyacrylamide gel electrophoresis (SDS-PAGE) expression and purification of truncated rAly-6Lm; in the figure: M, protein molecular weight standard, band size from top to bottom: 116kD, 66.2kD, 45kD, 35kD , 25kD, 18.4kD, 14.4kD; Lane 1, control strain before breaking the wall, 10 μL of sample load, lane 2, recombinant bacteria before breaking the wall, 10 μL of sample load, lane 3, after the recombinant bacteria break the wall Clear, load 10 μL, lane 4, purified rAly-6 by nickel column, load 10 μL;

C, rAly-6Hpm expression and purification of polyacrylamide gel electrophoresis (SDS-PAGE); in the picture: M, protein molecular weight standard, the size of the band from top to bottom is 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD; lane 1, control strain before breaking the wall, 10 μL of sample load, lane 2, recombinant bacteria before breaking the wall, 10 μL, lane 3, recombinant bacteria after breaking the wall, supernatant The sample volume is 10 μL, lane 4 and rAly-6 purified by nickel column, and the sample volume is 10 μL;

Figure 3, the effect of temperature on the activity of recombinant alginate lyase rAly-6;

Figure 4: Effect of temperature on stability of recombinant alginate lyase rAly-6;

Figure 5: Effect of pH on the activity and stability of recombinant alginate lyase rAly-6;

Figure 6. Histogram of the effects of metal ions and chemical reagents on the activity of recombinant alginate lyase rAly-6;

Figure 7. Molecular gel chromatography-HPLC analysis of oligosaccharide products during the degradation of algin by recombinant alginate lyase rAly-6;

In the picture: (1) UDP3, unsaturated trisaccharide; (2) UDP4, unsaturated tetrasaccharide; (3) UDP5, unsaturated pentasaccharide; (4) UDP6, unsaturated hexasaccharide

Figure 8: HPLC analysis of unsaturated oligosaccharide fragments UDP3, UDP4, UDP5, and UDP6 prepared by recombinant alginate lyase rAly-6 that does not completely degrade alginate;

In the picture: (1) UDP3, unsaturated trisaccharide; (2) UDP4, unsaturated tetrasaccharide; (3) UDP5, unsaturated pentasaccharide; (4) UDP6, unsaturated hexasaccharide

FIG. 9, ¹ H-NMR chart of unsaturated oligosaccharide tablets UDP3, UDP4, UDP5, and UDP6 prepared by recombinant alginate lyase rAly-6 incompletely degrading alginate;

FIG. 10 is an HPLC analysis diagram of UDP2 and UDP3 isolated from a product that is completely degraded by the recombinant alginate lyase rAly-1;

FIG. 11 is an HPLC analysis diagram of UDP2 isolated from the product of completely degraded alginate by the recombinant alginate lyase Pae-rEAlgL;

Figure 12: HPLC analysis of oligosaccharide UDP3-UDP6 prepared by rAlgL-6 and UDP3 prepared by rAly-1 using recombinant alginate lyase rAly-6;

Figure 13: HPLC analysis of the reaction product of excess recombinant alginate lyase rAly-6 with four disaccharides (ΔM, ΔG, MM, GG) containing different components;

FIG. 14 is an HPLC analysis chart (ultraviolet) of a reaction product of an excessive amount of recombinant alginate lyase rAly-6 and saturated polyM pentaose (M5);

Figure 15. HPLC analysis chart (ultraviolet) of the reaction product of excess alginate lyase rAly-6 with saturated polyGpentaose (G5);

Figure 16. HPLC analysis (fluorescence) of degradation of reducing poly-M-pentaose (M5) labeled with 2-AB at the reducing end by an excess of recombinant alginate lyase rAly-6.

Figure 17. HPLC analysis (fluorescence) of degradation of reducing poly-G-pentaose (G5) at the reducing end by 2-AB with a recombinant alginate lyase rAly-6;

Figure 18. HPLC analysis (fluorescence) of the degradation of the unsaturated pentasaccharide (UDP5) from rAly-6 by reducing the reducing end with 2-AB using a recombinant alginate lyase rAly-6.

detailed description

The following embodiments are described in order to comprehensively disclose some common techniques of how the present invention is implemented, and not to limit the scope of application of the present invention. The inventors have done their best to ensure the accuracy of the parameters in the embodiments (such as amount, temperature, etc.), but some experimental errors and deviations should also be considered. Unless otherwise stated, molecular weight in the present invention refers to weight average molecular weight, and temperature is in degrees Celsius.

Source of biological material

Flammeovirga yaeyamensis strain MY04 is derived from the General Microbiology Center of the China Microbial Species Collection Management Committee, Address: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, China, date of deposit: November 27, 2008 , Deposit number CGMCC NO. 2777.

Example 1. Extraction of genomic DNA from Flammeovirga yaeyamensis strain MY04

Flammeovirga yaeyamensis MY04 was inoculated into liquid medium YT04, and cultured at 28 ° C and 200 rpm with shaking to a 600 nm absorbance (OD ₆₀₀ ) of 1.2; 10 mL of the culture broth was taken at 12,000 × g ( g, earth's gravitational constant) centrifugation for 15min to collect bacterial cell pellets; suspend bacterial cells with 10mL of lysozyme buffer (10mM Tris-HCl, pH 8.0), and centrifuge at 12,000rmp for 15min to collect bacterial cell pellets.

The above liquid medium YT04 has the following components per liter:

10 g tryptone, 5.0 g yeast extract, 30 g sodium chloride, dissolved in water and made up to 1 L, pH 7.2.

To the above bacterial cell pellet, 6.0 mL of lysozyme buffer solution (purchased from Shanghai Biotech Engineering Co., Ltd.) was added to each tube to obtain about 7.0 mL of bacterial solution, and 280 μL each of lysozyme solution at a concentration of 20 mg / mL was added. The final lysozyme concentration was 800 μg / mL; placed in an ice-water bath for 1.0 h, then transferred to a 37 ° C water bath and warmed for 2 h until the reaction system was viscous; 100 mg / mL sodium cetylsulfonate solution was added 30 μL of 0.41mL, 100mg / mL proteinase K solution, incubate at 52 ℃ for 1.0h; add 7.5mL of Tris-equilibrated phenol / chloroform / isoamyl alcohol (volume ratio 25: 24: 1) solution, mix gently by inversion ; Centrifuge at 10,000 × g and 4 ° C for 10min, collect the supernatant, add 1.0mL of NaAc-HAc (pH5.2, 3.0M) buffer, and 8.5mL of absolute ethanol, mix thoroughly; use a gun Pick out the filamentous DNA, transfer to a 1.5mL centrifuge tube, wash twice with 70% ethanol (stored at -20 ℃), discard the supernatant after microcentrifugation; centrifuge at 10,000 × g, 4 ℃ 2min, completely discard the supernatant; the DNA precipitate was blown to dryness in a sterile workbench, and then the DNA sample was dissolved at 4 ° C overnight with sterile deionized water to prepare a base. Group DNA.

Example 2. Scanning and sequencing of the genome of Flammeovirga yaeyamensis MY04 strain.

The genomic DNA prepared in Example 1 was scanned and sequenced by the genome using pyrosequencing technology, which was completed by Shanghai Meiji Biological Company. The online sequencing software of NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/) was used to analyze the DNA sequencing results. The analysis software of the NCBI website used is Open Reading Frame Finder (ORF Finder, http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and Basic Local Alignment Search Tool (BLAST, http: /// blast.ncbi.nlm.nih.gov/Blast.cgi).

The results of analysis by the above biological software showed that the genomic DNA of Flammeovirga yaeyamensis MY04 strain carried a gene encoding algin-6 of alginate lyase, the coding region of gene aly-6 was 2238bp, and the nucleotide sequence was SEQ ID NO.1. The recombinant alginate lyase rAly-6 encoded by the gene aly-6 contains 745 amino acids in total, and its amino acid sequence is shown in SEQ ID NO.2.

Using BLASTp software for online analysis, the results showed that the NCBI database with amino acid sequence similarity to the recombinant alginate lyase rAly-6 greater than 30% is a protein with unidentified functions. As shown in Figure 1, BLASTp analysis also speculated that the N-terminus of Aly-6 protein contains a putative catalytic domain AlgLyase superfamily, which is conserved in the alginate lyase superfamily, named Aly-6Lm, and the C-terminus contains the heparinase. The conserved putative catalytic domain Heper_II_III superfamily in the II_III superfamily, named Aly-6Hpm. Analysis using the biological software BioEdit 7.0.5.3 showed that the theoretical molecular weight of the protein Aly-6 was approximately 84.7 kD. The signal peptide online prediction software SignalP4.1Server (http://www.cbs.dtu.dk/services/SignalP/) was used for online analysis. The N-terminal amino acids 1-23 of the protein were secreted signal peptides.

Example 3 Recombinant expression of genes aly-6 and truncated aly-6Lm, aly-6Hpm in E. coli BL21 (DE3) strain

The genomic DNA prepared in Example 1 was used as a template for PCR amplification. The primer sequences are as follows:

aly-6 amplified forward primer Aly6-F: 5'-gg CATATG CAGAACGGAAATTTAATTACTTCCG-3 '(NdeI);

aly-6 amplified reverse primer Aly6-R: 5'-gc TCTAGA AATTTCTTCCAATAGGTAAACCCC-3 '(Xba I);

aly-6Lm amplified forward primer Aly6Lm-F: 5'-gg CATATG CAGAACGGAAATTTAATTACTTCCGAG-3 '(Nde I);

aly-6Lm amplified reverse primer Aly6Lm-R: 5'-gc TCTAGA CAAACTTCTTTTTTGGTAAGTCGTTGC-3 '(Xba I);

aly-6Hpm amplified forward primer Aly6Hpm-F: 5'-gg CATATG TACTATTCAAGAGAATTGAAACTAGCC-3 '(Nde I);

aly-6Hpm amplified reverse primer Aly6Hpm-R: 5'-gc TCTAGA AATTTCTTCCAATAGGTAAACCCC-3 '(Xba I);

The forward primer underlined the restriction enzyme Nde I site, and the reverse primer underlined the restriction enzyme Xba I site. The high-fidelity DNA polymerase PrimeSTAR, HS, and DNA Polymerase used were purchased from China's Dalian Bao Biological Company, and the PCR reaction reagents used were operated according to the product instructions provided by the company.

PCR reaction conditions: pre-denaturation at 95 ° C for 4min; denaturation at 94 ° C for 40s, annealing at 60 ° C for 30s, extension at 72 ° C for 135s, 35 cycles; extension at 72 ° C for 10min; stabilization at 4 ° C for 10min.

The PCR products were double-digested with restriction enzymes Nde I and Xba I, and the digested PCR products were recovered by agarose gel electrophoresis. The product pCold TF plasmid DNA purchased from Dalian Bio-Bio Co., Ltd. was double-digested with Nde I and Xba I, agarose gel electrophoresis was performed, and the digested product fragment was recovered. Restriction enzymes Nde I and Xba I were purchased from China's Dalian Bao Biological Company. The system, temperature and time for the reaction between the enzyme and the substrate used for the enzyme digestion were in accordance with the product instructions provided by the company.

The PCR products digested with Nde I and Xba I were double-digested with the pCold TF plasmid vector also digested with DNA ligase. The ligation products were transformed into E. coli DH5a strain and coated with 100 μg / mL ampicillin Luria-Bertani medium solid plate, after incubating at 37 ° C for 16h, select the monoclonal; the monoclonal was inserted into liquid Luria-Bertani medium containing 100 μg / mL ampicillin and cultured to extract the plasmid; The plasmid was verified by PCR with amplification primers. The result was an amplification product with a size of 2.3 kb, which initially proved that the constructed recombinant plasmid was correct. The sequencing of the recombinant plasmid showed that the Nde I and Xba enzymes in pColdTF Genes aly-6, aly-6Lm, aly-6Hpm were inserted between the cleavage sites, and the insertion direction was correct, so it was further proved that the constructed recombinant plasmid was correct. The recombinant plasmids were named pCTF-Aly6, pCTF-Aly6Lm, and pCTF, respectively. -Aly6Hpm.

The recombinant plasmids pCTF-Aly6, pCTF-Aly6Lm, and pCTF-Aly6Hpm were transformed into E. coli strain BL21 (DE3) (purchased from Invitrogen, USA), and then according to the procedures provided by the company, using isopropylthiogalactoside (IPTG) ) For inducing expression of recombinant alginate lyases rAly-6, rAly-6Lm, rAly-6Hpm. Centrifuge at 8,000 × g and 4 ° C for 15min, collect the bacterial cells, resuspend the cells with buffer A, and sonicate in an ice-water bath environment. After centrifugation at 15,000 × g and 4 ° C for 30 minutes, the water-soluble components were collected, and the recombinant alginate lyases rAly-6, rAly-6Lm, and rAly-6Hpm were purified by Ni-sepharose. Gradient elution was performed with buffer A containing imidazole concentrations of 10, 50, 100, 250, and 500 mM. Purification conditions were in accordance with the gel's product manual. Polyacrylamide gel denaturing gel electrophoresis was used to detect the purification of recombinant alginate lyases rAly-6, rAly-6Lm, and rAly-6Hpm. The results are shown in FIG. 2A: In BL21 (DE3) cells induced by IPTG, the yield of recombinant alginate lyase rAly-6 is not less than 200 mg / L of the cell culture; the purified recombinant alginate lyase rAly-6 showed a single band on the electrophoresis gel, and its position coincided with the predicted molecular weight, and rAly-6 showed a single band on the electrophoresis gel, and its position matched the predicted molecular weight. The results are shown in Figures 2B and 2C. Glyases rAly-6Lm and rAly-6Hpm also exhibit water-soluble expression. Purified recombinant alginate lyase The purified alginate lyase rAly-6 showed a single band on the electrophoresis gel, and its location was the same as the predicted molecular weight. Coincide. The purified recombinant alginate lyases rAly-6, rAly-6Lm, and rAly-6Hpm were loaded into a dialysis bag with a minimum molecular retention of 10 kD, and the buffer A was dialyzed at 4 ° C. The components of the buffer solution A are 50 mM Tris, 150 mM NaCl, and pH 7.9, and a pure alginate lyase rAly-6, rAly-6Lm, and rAly-6Hpm pure enzyme solution are prepared.

Example 4 Determination of Optimal Temperature of Recombinases rAly-6, rAly-6Lm, rAly-6Hpm

Alginate with a mass-volume concentration of 0.1-1.2% is prepared with deionized water. After heating and dissolving, it is placed in a water bath environment at 0 ° C, 10 ° C, 20 ° C, 30 ° C, 35 ° C, 40 ° C, 45 ° C, and 50 ° C to cool down. 1h. To each 900 μL of the substrate solution, 100 μL of the dilute solution of the recombinant alginate rAly-6 prepared in Example 3 was added. The concentration of the dilute solution of the recombinant alginate rAly-6 was 10 μg / mL, and the reaction was continued after mixing. , Sampling at intervals. Three parallel samples at each temperature were used as a control group with a boiling water bath-inactivated recombinant enzyme preparation. rAly-6Lm and rAly-6Hpm were also measured for their optimal temperatures by referring to the above method.

The DNS-reducing sugar method was used to determine the concentration of newly formed reducing sugars (OD ₅₄₀ ) in each reaction system, and the average value was calculated for deviation analysis. The reaction temperature corresponding to the maximum absorption value is the optimal temperature of the recombinase, and the relative enzyme activity (RA) is defined as the percentage of each absorption value to the maximum absorption value. The results are shown in Figure 3: When the enzyme activity was measured using the alginate as described above, the recombinant alginate lyase rAly-6 reached its maximum activity at 40 ° C. This indicates that the recombinant alginate lyase rAly-6 The optimal reaction temperature is 40 ° C.

However, when the activities of rAly-6Lm and rAly-6Hpm were measured at the above-mentioned different temperatures, neither of them was found to be active. This shows that the two hypothesized catalytic domains of Aly6 are inactive alone, and the activity of Aly6 requires the cooperation of the two domains to display. It further shows that truncated expression can easily inactivate exofucolytic enzyme Aly-6.

Example 5 Determination of Optimal pH of Recombinant Alginate Lyase rAly-6

Use 50mM NaAc-HAC buffer, 50mM NaH ₂ PO ₄ -Na ₂ HPO ₄ buffer, and 50mM Tris-HCl buffer to prepare algin with mass volume concentration (g / mL) of 0.1 ～ The 1.2% alginate substrate has corresponding pH values of 5, 6, 6, 7, 8, 7, 8, 9, and 10, and each pH value is adjusted at the optimum temperature. After dissolving the substrate, incubate at the optimal temperature for 1 hour, and then add 100 μL of the diluted solution of the recombinant alginate lyase rAly-6 prepared in Example 3 to each 900 μL of the substrate, and start the reaction after mixing. sampling. Three parallel samples were taken at each pH, and a reconstituted enzyme preparation inactivated by a boiling water bath was used as a control group. The DNS-reducing sugar method was used to determine the concentration of newly generated reducing sugars (OD ₅₄₀ ) in each reaction system, and the average and deviation were calculated. Relative enzyme (RA) activity was defined as the percentage of the average absorption value to the maximum absorption value of each group. The pH corresponding to the maximum absorption value is the optimum pH of the recombinase. The results are shown in Figure 5: The optimal reaction pH of the recombinant alginate lyase rAly-6 was 6.0.

Example 6: Temperature stability analysis of recombinant alginate lyase rAly-6

The recombinant alginate lyase rAly-6 enzyme solution prepared in Example 3 after being heat-treated at 0 ° C, 20 ° C, 30 ° C, 40 ° C, 50 ° C, and 60 ° C for different times was separately configured with the quality of distilled water Fucoidan with a volume concentration (g / mL) of 0.1 to 1.2% is mixed at a ratio of 1: 9 (volume ratio), and then the residual enzyme activity is measured at the optimal temperature. The enzyme activity of the enzyme solution without heat treatment is defined as 100% relative vitality. The results are shown in Fig. 4: After the recombinant alginate lyase rAly-6 was pretreated at 40 ° C for 1 hour, it still had a residual activity of> 50%, indicating that the enzyme has certain thermal stability.

Example 7 pH stability analysis of recombinant alginate lyase rAly-6

The enzyme solution of the recombinant alginate lyase rAly-6 prepared in Example 3 was pre-incubated at the optimal temperature (40 ° C) and different pH (pH 5-10) for 2 hours, and then the mass and volume concentration (g Alginate substrate solution with a concentration of 0.1 to 1.2% was mixed at a ratio of 1: 9 (volume ratio), and then the residual enzyme activity was measured at the optimum temperature. The enzyme activity without the pretreatment was defined as 100. % Relative vitality. The results are shown in Fig. 4. After pre-treating for 2 h in the range of pH 6-7, rAly-6 enzyme activity remained above 90%.

Example 8. Effects of metal ions and chemical reagents on the activity of recombinant alginate lyase rAly-6

Alginate substrate prepared with deionized water at a mass concentration of 0.1 to 1.2%, the recombinant alginate lyase rAly-6 enzyme solution prepared in Example 3, and water at a ratio of 5: 1: 4 (volume ratio) After mixing, different metal ions are then added to the reaction system, the final ion concentration is 1 mM or 10 mM, and then the reaction is carried out at 40 ° C. for 4 h, and the enzyme activity is measured according to the aforementioned DNS-reducing sugar method. The control group was rAly-6 activity (set to 100%) without any metal ions. The results are shown in Fig. 6. At the concentration of 1 mM or 10 mM: (1) three monovalent metal reagents of K ⁺ , Li ⁺ , Na ⁺ showed a promoting effect on rAly-6 activity at 10 mM, but Ag ⁺ had significant effects. Inhibition; (2) Co ²⁺ , Mg ²⁺ , Mn ²⁺ and other divalent metal reagents can promote enzyme activity at 10 mM, while the remaining divalent and trivalent metal ions can inhibit enzyme activity; (3) ) Glycerol, imidazole, and DTT can promote the activity of rAly-6 at 1 mM and 10 mM, and the remaining reagents can inhibit the enzyme activity.

Example 9: Determination of Enzymatic Activity of Recombinant Alginate Lyase rAly-6 by DNS-reducing Sugar Method

Alginate with a mass concentration of 0.1-1.2% prepared with deionized water, a recombinant alginate lyase rAly-6 enzyme solution with a concentration of 10 μg / mL, 150 mmol / L of HAc-NaAc (pH 6.0) buffer solution, and water After mixing at a ratio of 2: 1: 3: 4 (volume ratio), the reaction was performed at 40 ° C for 4 hours. The reaction product was heated in a boiling water bath for 10 min, transferred to an ice water bath for 5 min, and centrifuged at 12,000 × g, 4 ° C for 15 min, and the supernatant was collected; a certain volume of the supernatant and an equal volume of DNS (3, 5-pair Nitroxylene)-The reaction solution was mixed well, heated in a boiling water bath for 10 min, lowered to room temperature, and the absorption value was measured at 540 nm. Using the analysis of pure sodium glucurolactone as a standard, the same method was used to draw the dose-effect relationship curve between the molar concentration of glucuronide and OD ₅₄₀ . The protein content in the recombinant alginate lyase rAly-6 enzyme solution was determined using a protein quantification kit purchased from Shanghai Shenggong Biological Engineering Co., Ltd. Calculate the unit of enzyme activity according to the international standard definition, that is, under standard conditions, the amount of enzyme required to produce 1 μmol of product per minute is 1 IU. The results showed that the recombinant rAly-6 could use alginate as a substrate to hydrolyze and produce reducing sugar products. The enzyme activity was 726 ± 2.2U / mg.

Example 10: High Performance Liquid Chromatography (HPLC) Analysis of Recombinant Alginate Lyase Enzyme rAly-6

A fucoidan substrate having a mass-volume concentration (g / mL) of 0.1 to 1.2% was prepared with deionized water, heated and dissolved, and then cooled in a 40 ° C water bath environment for 1 h. 10-100 μL of the diluted solution of the recombinase rAly-6 prepared in Example 3 was added to every 100 μL of the substrate. When the volume was less than 200 μL, it was made up with sterile deionized water; the reaction was continued after mixing, and samples were taken at intervals. The reaction product was heated in a boiling water bath for 10 minutes and transferred to an ice-water bath for 5 minutes. Centrifuge at 12,000 × g and 4 ° C for 15min, and collect the supernatant.

Superdex Peptide 10 / 300GL (GE) molecular gel chromatography column was equilibrated with a NH ₄ HCO ₃ solution with a concentration of 0.20 mol / L, with a flow rate of 0.40 mL / min, and at least 2 column beds. The samples of the above alginate with different enzymolysis time were loaded with 100 μg / sample by an autosampler, other conditions remained unchanged, and detection was performed at 235 nm. The HPLC operating software was used to analyze the integrated area of each oligosaccharide component and calculate the relative molar concentration.

As shown in Figure 7, under the above conditions, after the alginate substrate is degraded, as the enzymatic hydrolysis reaction time increases, the degree of polymerization of the oligosaccharide product with a characteristic absorption of 235 nm decreases with time, and eventually changes. An unsaturated trisaccharide with a peak time of 40.5 '. This preliminary indicates that the recombinant enzyme rAly-6 is an alginate lyase.

Example 11. Molecular weight identification of the main product of the oligosaccharide of the alginate incomplete degradation by the recombinant alginate lyase rAly-6

As described in Example 10, a total of 200 mg of alginate was incompletely digested with the recombinant alginate lyase rAly-6. Samples were loaded in batches and passed through a Superdex Peptide 10 / 300GL molecular gel chromatography column (GE), and Peak out time Four oligosaccharide samples with peak times of 34.8 ', 36.0', 38.2 'and 40.5' were collected. After collecting the four oligosaccharide samples collected multiple times, the samples were repeatedly freeze-dried to desalinate. The obtained oligosaccharide samples were dissolved with sterile deionized water and analyzed by first-order mass spectrometry (MS) to determine the relative molecular weight of each oligosaccharide. The obtained oligosaccharide sample was dissolved in heavy water (D ₂ O), and repeatedly freeze-dried to complete the hydrogen-deuterium replacement, and ¹ H-NMR analysis was performed to finally determine the chemical structure and characteristics of each oligosaccharide.

As shown in Figure 8, HPLC analysis of unsaturated oligosaccharide fragments UDP6, UDP5, UDP4, and UDP3 prepared after the alginate was not completely degraded by the recombinant alginate lyase rAly-6 showed that the product had a characteristic absorption at 235 nm. The peak times were 34.8 ', 36.0', 38.2 ', and 40.5', respectively, and the purity was greater than 99%, which met the requirements of further experiments.

The structural characteristics of the two oligosaccharides were further analyzed by ¹ H-NMR data. As shown in FIG. 9, the characteristic chemical shift value at 5.65 ppm showed that the recombinant alginate lyase rAly-6 did not completely degrade the alginate. The non-reducing ends of this product are all ΔG units.

Example 12 Preparation of Recombinant Fucoidan Lyase Enzyme rAly-1 for Degradation of Fucoidan Products UDP2 and UDP3

According to Example 11 in the patent application (Application No. 201610838337.9, a facultative endogenous recombinant alginate lyase rAly-1 and its coding gene and application), a total of 200 mg of alginate was completely treated with the recombinase rAly-1 Enzymolysis, the product was loaded in batches, passed through a molecular gel column Superdex Peptide 10 / 300GL (GE), and two oligosaccharide samples with peak times of 40.5 ′ and 43.2 ′ were collected according to the peak time. The two oligosaccharide samples were collected multiple times and collected separately, and then repeatedly freeze-dried to desalinate.

As shown in FIG. 10, the prepared oligosaccharide fragments UDP2 and UDP3 were analyzed by HPLC, and the results showed that the product had characteristic absorption at 235 nm, and the purity was more than 99%, which met the requirements of further experiments. In the above patent application (application number 201610838337.9, a facultative endogenous recombinant alginate lyase rAly-1 and its coding gene and application), Example 10 proves that almost all UDP2 prepared by rAly-1 degradation of alginate is ΔG Composition: There are two types of non-reducing end of UDP3: ΔG and ΔM, and the molar ratio is about 5: 4.

Example 13 Preparation of Recombinant Alginate Lyase Pae-rEAlgL for Degradation of Alginate Product UDP2

According to Example 11 in the patent application (application number 2017106518145, application of alginate lyase in the preparation of a series of oligosaccharide products), a total of 200 mg of alginate was thoroughly digested with the recombinant enzyme Pae-rEAlgL, and the products were loaded in batches 1. Supermolecular gel column Superdex Peptide 10 / 300GL (GE), and collect individual oligosaccharide fragments according to the peak time. These oligosaccharide samples were collected multiple times and collected separately, and then repeatedly freeze-dried to desalinate.

As shown in Figure 11, the prepared oligosaccharide fragment UDP2 was analyzed by HPLC, and the results showed that the product had characteristic absorption at 235nm, and the purity was greater than 99%, which met the requirements of further experiments. In the above patent application (application number 2017106518145, application of alginate lyase in preparing a series of oligosaccharide products), Example 12 proves that almost all UDP2 prepared by degradation of Pae-rEAlgL is a ΔM component.

Example 14: Analysis of Degradation of Oligosaccharides by Recombinant Alginate Lyase rAly-6

The oligosaccharide fragments prepared by using deionized water and Examples 11, 12 and 13 include: unsaturated pentasaccharide (UDP5), unsaturated tetrasaccharide (UDP4), unsaturated trisaccharide (UDP3: containing only ΔG terminal substrate), unsaturated trisaccharides (UDP3: ΔM and ΔG terminal mixed substrate), unsaturated disaccharides (UDP2: ΔG), unsaturated disaccharides (UDP2: ΔM), and purchased saturated oligosaccharide fragments ( Rauronic acid disaccharide (GG), mannuronic acid disaccharide (MM)). These oligosaccharide samples were respectively prepared with a substrate solution, a recombinant alginate lyase rAly-6 enzyme solution with a concentration of 10 μg / mL, a 150 mmol / L HAc-NaAc (pH 6.0) buffer solution, and water at a ratio of 2: 1: After mixing at a ratio of 3: 4 (volume ratio), the reaction was carried out at 40 ° C for 24 hours. HPLC analysis was performed according to the chromatographic operating conditions described in Example 10.

The results are shown in Figures 12 and 13, the recombinant alginate lyase rAly-6 described in this application:

(1) After degrading unsaturated pentasaccharide (UDP5), an equivalent amount of unsaturated trisaccharide and a small amount of unsaturated disaccharide are produced;

(2) After degrading unsaturated tetrasaccharide (UDP4), an equivalent amount of unsaturated trisaccharide and a small amount of unsaturated disaccharide are produced;

(3) The same amount of unsaturated disaccharide is produced after slight degradation of unsaturated trisaccharide (UDP3: containing only ΔG terminal substrate), and the degradation rate is about 5%;

(4) Degradation of unsaturated trisaccharides (UDP3: mixed substrate of ΔM and ΔG termini) yields an equivalent amount of unsaturated disaccharides with a degradation rate of about 60%;

(5) Minimal degradation of unsaturated disaccharide (UDP2: ΔG) produces unsaturated monosaccharide Δ, with a degradation rate of about 5%;

(6) Degradation of unsaturated disaccharide (UDP2: ΔM) produces unsaturated monosaccharide Δ, with a degradation rate of about 90%;

(7) No significant change before and after reaction with saturated disaccharides (MM and GG).

These results indicate that rAly-6 can effectively degrade trisaccharides and above oligosaccharide fragments; the enzyme cannot degrade saturated disaccharides GG or MM; unsaturated disaccharide fragments (ΔM or ΔG) are recombinant alginate lyase rAly-6 The smallest oligosaccharide substrate, ΔM is more easily degraded than ΔG, but neither can be completely degraded; saturated monosaccharides M, G or unsaturated monosaccharides Δ are the smallest oligosaccharides of recombinant alginate lyase rAly-6 Sugar product type. In summary, rAly-6, a recombinant alginate lyase, is an M-prone alginate lyase, which easily degrades sugar chains that are larger than trisaccharides or contain ΔM terminus, but not easy to degrade trisaccharides and disaccharides. The sugar size is in particular a sugar chain substrate containing ΔG, GG or MM termini.

Example 15 High Performance Liquid Chromatography (HPLC) Analysis of Recombinant Alginate Lyase rAly-6

Take a solution containing 30 μg of saturated polyM pentasaccharide (M5) and polyG pentasaccharide (G5), 150 mmol / L of HAc-NaAc (pH 6.0) buffer, and the recombinant alginate lyase rAly-6 prepared in Example 3. The diluted solution and water were mixed in a volume ratio of 2: 1: 3: 4 and placed in a water bath at 40 ° C for 12 hours. The reaction system was placed in a boiling water bath for 10 minutes, transferred to an ice water bath for 5 minutes, and centrifuged at 12,000 × g and 4 ° C for at least 15 minutes. The supernatant was collected and used as the oligosaccharide degradation product of the recombinant alginate lyase rAly-6. Recombinant alginate lyase rAly-6 enzyme solution inactivated in a boiling water bath in advance was used as a negative control reaction.

According to the chromatographic conditions described in Example 10, the recombinant alginate lyase rAly-6 was used to hydrolyze a sample of saturated polypentasaccharide (M5) and polypentasaccharide (G5), and an autosampler was loaded with 20 μg / sample. Other conditions remain unchanged, detection at 235nm. The HPLC operating software was used to analyze the integrated area of each oligosaccharide component and calculate the relative molar concentration. With reference to molecular weight standards, the relative molecular weight of each oligosaccharide was determined.

As shown in Figure 14, the recombinant alginate lyase rAly-6 degrades the saturated polyM pentasaccharide (M5) to produce unsaturated trisaccharide UM3 and unsaturated disaccharide UM2. As shown in FIG. 15, the recombinant alginate lyase rAly-6 degrades saturated polyG pentasaccharide (G5) to produce unsaturated trisaccharide UG3 and unsaturated disaccharide UG2. This disaccharide residue is consistent with the result that the enzyme cannot completely degrade the disaccharide ΔM or ΔG shown in FIG. 13. However, the series of oligosaccharide products produced by rAly-6 degradation of M5 has an absorption value intensity (area integral) at 235 nm of 8-10 times that of the degradation G5 product. This fully shows that the recombinant enzyme rAly-6 can degrade both M-rich oligosaccharide fragments and G-rich oligosaccharide fragments, but has M-proneness as a whole.

Example 16. Fluorescence-High Performance Liquid Chromatography (HPLC) Analysis of Recombinant Alginate Lyase rAly-6

A solution containing 10 μg of saturated polyM pentasaccharide (M5) and polyG pentasaccharide (G5), the unsaturated pentasaccharide (UDP5) prepared in Example 10 was taken, and evaporated to dryness by rotation. Add dimethyl sulfoxide (DMSO) solution containing excess anthranilamide (2-AB) and sodium boronitrile, mix well, and incubate in a 60 ° C water bath for 2 h. Rotate to dryness, add 500 μL of deionized water to dissolve the sample, shake the sample with 200 μL of chloroform, centrifuge, and collect the supernatant. Continue to extract with chloroform repeatedly, no less than 7 times, to obtain saturated poly M pentasaccharide (2AB-M5), fluorescently labeled saturated poly G pentasaccharide (2AB-G5), fluorescently labeled Unsaturated pentasaccharide (2AB-UDP5).

Take the above 2AB-M5, 2AB-G5, 2AB-UDP5 samples, the dilute solution of the recombinant alginate lyase rAly-6 prepared in Example 3, 150 mmol / L of HAc-NaAc (pH 6.0) buffer and water, according to The volume ratio was 2: 1: 3: 4, and the mixture was placed in a water bath at 40 ° C for 12 hours. The reaction system was placed in a boiling water bath for 10 minutes, transferred to an ice water bath for 5 minutes, and centrifuged at 12,000 × g and 4 ° C for at least 15 minutes. The supernatant was collected and used as the oligosaccharide degradation product of the recombinant alginate lyase rAly-6. Recombinant alginate lyase rAly-6 enzyme solution heated in a boiling water bath for 10 minutes was used as a negative control reaction.

According to the chromatographic conditions described in Example 10, the labeled samples of 2AB-M5, 2AB-G5, 2AB-UDP5 and their enzymatic hydrolysis products were loaded with 50-200ng / sample in an autosampler, and other conditions remained unchanged, and excited at 330nm. , 420nm detection. The HPLC operating software was used to analyze the integrated area of each oligosaccharide component and calculate the relative molar concentration. With reference to molecular weight standards, the relative molecular weight of each oligosaccharide was determined.

As shown in Figure 16, under the above conditions, after 2AB-M5 is degraded, as the enzymatic hydrolysis reaction time increases, 2AB-UM4, 2AB-UM3, and 2AB-UM2 are gradually formed, and the final oligosaccharide product is 2AB. -UM2, its relative content eventually stabilizes. This indicates that the recombinant alginate lyase rAly-6 cleaves 2AB-M5 by means of a monosaccharide exonuclease, and gradually cuts one molecule of saturated monosaccharide M and two molecules of unsaturated monosaccharide Δ from the non-reducing end until one molecule remains 2AB-UM2.

As shown in FIG. 17, after reacting with 2AB-G5 with an excess of recombinant alginate lyase rAly-6, about 45% of the substrate is degraded, and an equimolar amount of 2AB-UG2 is produced. After 2AB-G5 is degraded, With the increase of the enzymolysis reaction time, 2AB-UG4, 2AB-UG3, and 2AB-UG2 were gradually formed, and the final oligosaccharide final product was 2AB-UG2, and its relative content eventually stabilized. This indicates that the recombinant alginate lyase rAly-6 cleaves 2AB-G5 by means of a monosaccharide exonuclease, and gradually cuts one molecule of saturated monosaccharide G and two molecules of unsaturated monosaccharide Δ from the non-reducing end, until one molecule remains in the end. 2AB-UG2.

As shown in FIG. 18, after reacting with 2AB-UDP5 with an excessive amount of recombinant alginate lyase rAly-6, with the increase of the enzymatic hydrolysis reaction time, 2AB-UDP4, 2AB-UDP3, and 2AB-UDP2 were gradually formed, and finally formed The final oligosaccharide product is 2AB-UDP2, and its relative content eventually stabilizes. This indicates that the recombinant alginate lyase rAly-6 cleaves one molecule of 2AB-UDP5 by means of a monosaccharide exonuclease, and gradually cleaves three molecules of unsaturated monosaccharide Δ from the non-reducing end, until finally one molecule of 2AB-UDP2 remains.

Further, comparing the results of FIG. 14 and FIG. 16 and the results of FIG. 15 and FIG. 17, it is shown that: (1) 2-AB is labeled with a saturated pentasaccharide terminal, which inhibits the degradation activity of the enzyme rAly-6 on oligosaccharides, especially This makes the enzyme unable to degrade the 2-AB labeled disaccharide ΔM; (2) rAly-6 is a monosaccharide exo-type alginate lyase, which has a preference for polyM over polyG.

References involved in the description:

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Claims (9)

1. An M-prone exotype alginate lyase Aly-6, the amino acid sequence of which is shown in SEQ ID NO.2.
2. An M-prone exotype alginate lyase encoding gene aly-6, the nucleotide sequence is shown in SEQ ID NO.1.
3. A recombinant expression vector in which the gene aly-6 encoding the M-prone monosaccharide exotype alginate lyase Aly-6 according to claim 2 is inserted into the expression vector.
4. The recombinant expression vector of claim 3, wherein the expression vector is selected from the group consisting of: an E. coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a streptomyces expression vector, a phage vector, and a filamentous shape Fungal expression vector, plant expression vector, insect expression vector, or mammalian cell expression vector.
5. A recombinant strain in which a recombinant expression vector of the above-mentioned M-prone monosaccharide exophytic algal lyase Aly-6 is transferred into a host cell, or the above-mentioned M-prone monosaccharide exo-type alginate lyase is expressed in a host cell Aly-6.
6. The recombinant bacterium according to claim 5, wherein the host cell is selected from the group consisting of an E. coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid host cell, an actinomycete host cell, a filamentous fungal host cell, Insect or mammalian cells.
7. The M-prone monosaccharide exotype alginate lyase Aly-6 encoding gene aly-6 according to claim 2, the recombinant expression vector of claim 3, and the recombinant bacterium of claim 4 in the preparation of M-prone Application of exo-type alginate lyase rAly-6.
8. The use of the M-prone exo-type alginate lyase Aly-6 according to claim 1 in thoroughly degrading alginate or degrading alginate oligosaccharides to produce unsaturated trisaccharides and unsaturated tetrasaccharides.
9. The M-preferred monosaccharide exofucoid lyase Aly-6 of claim 1 produces a larger series of unsaturated oligosaccharide fragments containing a ΔG terminus at the non-reducing end by degrading the alginate or the alginate oligosaccharide And unsaturated monosaccharide Δ.

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