CN106811454B

CN106811454B - Truncated recombinant alginate lyase rAly1-T185N and coding gene and application thereof

Info

Publication number: CN106811454B
Application number: CN201710110627.6A
Authority: CN
Inventors: 韩文君; 程媛媛; 王丹丹; 刘会会; 古静燕; 李福川; 李新; 卫洁
Original assignee: Tengzhou Wutong Aroma Chemicals Co ltd
Current assignee: Tengzhou Wutong Aroma Chemicals Co ltd
Priority date: 2017-02-28
Filing date: 2017-02-28
Publication date: 2021-03-23
Anticipated expiration: 2037-02-28
Also published as: CN106811454A

Abstract

The invention relates to a truncated recombinant alginate lyase rAly1-T185N, a coding gene and application thereof, wherein the amino acid sequence of the truncated recombinant alginate lyase rT185N is shown as SEQ ID NO. 2; the nucleotide sequence of the coding gene of the truncated recombinant alginate lyase rT185N is shown in SEQ ID NO. 1. 185 amino acid residues are cut off from the N end of the full-length alginate lyase Aly-1 by utilizing a PCR technology, so that T185N truncated enzyme is obtained; the expression level of rT185N can reach 1259 +/-2.2 mg/L fermentation liquor, the specific enzyme activity can reach 2895 +/-10.3U/mg, and the expression level is improved by nearly 2.6 times compared with the expression level of the full-length protein rAly-1, so that the purposes of increasing yield, saving energy and improving economic benefit are achieved, and the one-step purification of the fusion protein can be realized by separating rT185N through a nickel column.

Description

Truncated recombinant alginate lyase rAly1-T185N and coding gene and application thereof

Technical Field

The invention relates to a truncated recombinant alginate lyase rAly1-T185N and a coding gene and application thereof, belonging to the technical field of genetic engineering and protein expression.

Background

Algin is a linear acidic polysaccharide formed by linking mannuronic acid (M) and guluronic acid (Guluronic acid, G) which is a C-5 differential isomer of mannuronic acid through glycosidic bonds, and uniform poly-M sections, poly-G sections and hybrid MG or GM sections exist in molecules. Algin is widely present in cell walls and intercellular matrix of large marine brown algae for both medicine and food, such as herba Zosterae Marinae and Sargassum. At present, the main body applied in the fields of daily chemical industry, food and medicine is algin from seaweed. Intensive research on glycobiology finds that the alginate oligosaccharide has important biological activity. For example, alginate oligosaccharides have important functions of dynamically regulating anti-inflammatory/pro-inflammatory and antioxidant/pro-oxidative, inhibiting alpha-glucosidase activity, and the like. The poly-M-segment algin oligosaccharide has the potential of becoming the medicine for resisting Alzheimer syndrome. In conclusion, the algin oligosaccharide with specific M/G ratio and polymerization degree has important application value and economic value, and has important significance for realizing the high-efficiency preparation of the oligosaccharide.

The alginate lyase catalyzes the breakage of glycosidic bonds through a beta-elimination mechanism to generate a series of oligosaccharide products with non-reducing ends containing unsaturated conjugated structures and characteristic absorption near 235 nm. Compared with chemical or physical degradation methods, the enzymatic degradation of the algin has the advantages of mild conditions, easy control, small environmental pollution and the like, thereby having potential of popularization and application. Generally, the endonuclease can degrade polysaccharide randomly and efficiently to generate series of unsaturated oligosaccharides with different polymerization degrees; the exonuclease can degrade the substrate orderly to generate basic saccharide units specifically for organism to absorb. Since algin is composed of M, G saccharide units, algin lyase is generally classified into M-specific endonuclease, M-specific exonuclease, G-specific endonuclease, G-specific exonuclease, M/G-facultative endonuclease, and the like, according to the selectivity of the enzyme to the substrate and the substrate degradation mode of the enzyme.

The algin lyase is mainly from digestive system of conch, seaweed degrading bacteria or virus, etc. At present, although a large number of patent applications and scientific research documents about alginate lyase are available, the research mainly focuses on primary researches such as enzyme-producing strains and enzyme resource discovery; most of the enzyme specificity analysis takes a poly-M segment and a poly-G segment with limited purity as test substrates, while the analysis of the structural characteristics of the oligosaccharide products of the enzyme lacks the nuclear magnetic resonance spectrum data of the oligosaccharide final products, and the analysis of the oligosaccharide generation characteristics, namely the corresponding degradation mechanisms of the substrates (such as minimum substrates, minimum products, the generation positions of the minimum products in the substrates, substrate tendencies of the enzyme and the like) is relatively less explained; the molecular enzymology research mainly takes gene cloning and heterologous expression, recombinase purification and function as identification, and the research on molecular modification such as active site mutation, functional module truncation and the like is less. There is less knowledge about the regularity of the relationship between molecular enzymatic engineering and the degradation pattern of algin. In conclusion, the conventional research of the alginate lyase has weak foundation and insufficient depth, so that compared with similar products such as beta-agarase, cellulase and the like, the wide application of commercial alginate lyase is rare, and the development of the oligosaccharide enzyme preparation technology is severely restricted.

In general, alginate lyase consists of a catalytic module domain and one or more non-catalytic regions. The catalytic structure domain determines the recognition, combination and degradation of the enzyme to the algin substrate, thereby determining the biochemical characteristics of the enzyme; although the non-catalytic region cannot degrade algin, the non-catalytic region can have various influences on the catalytic performance of the enzyme. Recently, studies on endo-alginate lyase Aly2 (from Agarivorans sp.strain L11, facultative) and Aly5 (i.e. AlgL-5, from Flammeovirga sp.strain MY04, G-specific, patent document CN104293754A, application No. 201410469861.4) have shown that the presence of a non-catalytic region can help the catalytic domain to bind and degrade small molecular weight oligosaccharide substrates more efficiently. The difference, however, is that truncating the facultative Aly2 is effective in relatively increasing the propensity of the enzyme to substrate poly-M fragments; however, no significant change in substrate orientation was seen following the same truncation of G-specific Aly 5. Then why is the non-catalytic domain only able to exert a substrate-biasing effect on this facultative alginate lyase, but not substantially on the specific alginate lyase? How does the non-catalytic domain influence the catalytic domain and thus change the substrate orientation for the ampholytic alginate lyase? At present, only reports of relevant experimental phenomena are found, and detailed mechanism discussion is not found.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a truncated recombinant alginate lyase rAly1-T185N and a coding gene and application thereof. The truncated alginate lyase Aly1-T185N (T185N) is subjected to molecular modification on original Aly-1 (application No. 201610838337.9), and related intrinsic mechanisms are analyzed according to the influence and characteristics of a non-catalytic region in a protein molecule on the function of a catalytic region.

The technical scheme of the invention is as follows:

a truncated recombinant alginate lyase rT185N is prepared by cutting 185 non-catalytic amino acid residues from the N-terminus of Aly-1 full-length recombinant alginate lyase to obtain a T185N truncated enzyme.

The amino acid sequence of the truncated recombinant alginate lyase rT185N is shown in SEQ ID NO. 2.

Compared with the full-length protein rAly-1, the truncated recombinant alginate lyase rT185N has the advantages that through heterologous expression, the yield of the enzyme is increased, the enzyme activity is enhanced, and the purposes of increasing yield, saving energy and improving economic benefits are achieved. In contrast, the optimum temperature, pH and the influence of metal ions, chemical agents and other basic enzymological basic properties of the truncated rT185N are not significantly different. However, the degradation rate of the oligosaccharide substrate degraded by the truncation rT185N, the optimal substrate M/G ratio, the substrate cleavage pattern and other enzymatic properties are changed remarkably. Compared with the full-length protein rAly-1, (1) the truncated recombinase rT185N can degrade both guluronic acid oligosaccharide fragments and mannuronic acid oligosaccharide fragments from algin, and is still a facultative algin lyase; (2) after the recombinant enzyme rT185N degrades algin, a series of oligosaccharide products with unsaturated disaccharide and unsaturated trisaccharide as main products are still finally generated, the molar ratio of the products is still close to 1:1, wherein almost all the unsaturated disaccharide final product is delta G, the unsaturated trisaccharide product contains delta G, delta M and other two non-reducing ends, and the molar ratio of the two non-reducing ends is close to 5:8, which indicates that the content of the trisaccharide product containing the delta M end is increased by about 17%; (3) when recombinase rT185N degrades series of unsaturated oligosaccharides larger than trisaccharide, series of M oligosaccharides larger than tetrasaccharide or G oligosaccharides, a classical endonuclease mode is adopted, and the minimum product is a disaccharide fragment; (4) upon degradation of the M tetrasaccharide or the G tetrasaccharide, an unsaturated trisaccharide product is formed, with an accompanying monosaccharide product. (5) At degradation of saturated M pentasaccharide or G pentasaccharide, rT185N can generate disaccharide products from either the reducing end or the non-reducing end, respectively, and the final major product is all unsaturated trisaccharide products. The two are obviously different in that the degradation rate of the truncated rT185N on unsaturated tetrasaccharide fragments, saturated M tetrasaccharides, saturated M pentasaccharides, and fluorescently-labeled saturated poly M pentasaccharides (2AB-M5) and saturated poly G tetrasaccharides (2AB-G4) is obviously improved, but the significant changes on the saturated G tetrasaccharides, the saturated G pentasaccharides and larger oligosaccharide substrates are not seen. It is particularly noteworthy that excess of the full-length enzyme rAly-1 did not change before and after reaction with the fluorescently labeled G tetrasaccharide (2AB-G4), but rT185N completely degraded the fluorescently labeled G tetrasaccharide (2AB-G4) to an equimolar amount of 2AB-UG3, with the concomitant formation of saturated monosaccharides.

The coding gene of a truncated recombinant alginate lyase rT185N has a nucleotide sequence shown in SEQ ID NO. 1.

The total length of the coding gene of the truncated recombinant alginate lyase rT185N is 780bp, and the coded protein contains 259 amino acids and has the molecular weight of about 30.0 kD.

A recombinant vector, in the vector inserted the truncated recombinant alginate lyase rT185N coding gene.

The vector is selected from Escherichia coli expression vector, yeast expression vector, Bacillus subtilis expression vector, lactobacillus expression vector, Streptomyces expression vector, phage vector, filamentous fungi expression vector, plant expression vector, insect expression vector, or mammalian cell expression vector.

A recombinant bacterium or transgenic cell line, wherein the recombinant vector is transferred into a host cell or cell line.

The host cell or cell line is selected from Escherichia coli host cell, yeast host cell, Bacillus subtilis host cell, lactobacillus host cell, actinomycete host cell, filamentous fungi host cell, insect cell or mammal cell.

Recombinant bacteria or transgenic cell lines for recombinant expression of the truncated alginate lyase rT185N may be E.coli host cells (e.g.Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH 5. alpha. etc.), yeast host cells (e.g.Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces Iacta etc.), Bacillus subtilis host cells (e.g.Bacillus subtilis R25, Bacillus subtilis 9920 etc.), Lactic acid bacteria host cells (e.g.Lactic acid bacterium C0CC101 etc.), actinomycete host cells (e.g.Streptomyces spp. etc.), filamentous fungal host cells (e.g.Trichoderma viriti, Trichoderma reesei, Aspergillus niger etc.), insect cells (e.g.Bomori, kidney cells, baby hamster ovary cells, CHO etc.), insect cells (e.g.baby hamster ovary cells, baby hamster ovary cells, BHK, etc.).

The coding gene, recombinant vector, recombinant strain or transgenic cell line of the truncated recombinant alginate lyase rT185N is applied to the preparation of the truncated recombinant alginate lyase rT 185N.

The truncated recombinant alginate lyase rT185N can be used for degrading algin, alginate oligosaccharide or preparing unsaturated oligosaccharide.

Advantageous effects

1. 185 amino acid residues are cut off from the N end of the full-length alginate lyase Aly-1 by utilizing a PCR technology, so that rT185N truncated enzyme is obtained; the expression level of rT185N can reach 1259 +/-2.2 mg/L fermentation liquor, the specific enzyme activity can reach 2895 +/-10.3U/mg, and the expression level is improved by nearly 2.6 times compared with the expression level of the full-length protein rAly-1, thereby achieving the purposes of increasing yield, saving energy and improving economic benefit. And rT185N can be separated by a nickel column to achieve one-step purification of the fusion protein.

2. The truncated recombinant alginate lyase rT185N can degrade guluronic acid fragments from algin, can also reduce mannuronic acid fragments, and is still a facultative alginate lyase; the main product after rT185N completely degrading algin is unsaturated disaccharide and unsaturated trisaccharide with equal molar amounts, wherein the unsaturated disaccharide product is delta G, and the unsaturated trisaccharide product contains two non-reducing ends delta G, delta M and the like, and the molar ratio of the two ends is close to 5:8, which shows that the content of the trisaccharide product containing the delta M end is improved by about 17%; the degradation rate of rT185N on unsaturated tetrasaccharide fragments, saturated M tetrasaccharides, saturated M pentasaccharides, and fluorescence-labeled saturated poly M pentasaccharides (2AB-M5) and saturated poly G tetrasaccharides (2AB-G4) is remarkably improved;

drawings

FIG. 1 shows the BLASTp analysis results of the functional modules of truncated recombinant alginate lyase rT 185N;

FIG. 2 is a polyacrylamide gel electrophoresis (SDS-PAGE) of the expression and purification of truncated recombinant alginate lyase rT 185N;

in the figure: m, protein molecular weight standard, wherein the size of a strip from top to bottom is as follows: 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4 kD; lane 1, control strain before cell wall breaking, loading 5 μ L; lane 2, the recombinant bacteria before wall breaking, and the loading amount is 5 μ L; lane 3, supernatant after wall breaking of the recombinant bacteria, and sample loading amount of 5 μ L; lane 4, rT185N purified by nickel column, loading 5 μ L;

FIG. 3, graph of the effect of temperature on the activity of truncated recombinant alginate lyase rT 185N;

FIG. 4, graph of the effect of pH on the activity of truncated recombinant alginate lyase rT 185N;

FIG. 5, graph of the effect of temperature on the stability of truncated recombinant alginate lyase rT 185N;

FIG. 6, graph of the effect of pH on the stability of truncated recombinant alginate lyase rT 185N;

FIG. 7, bar graph of the effect of metal ions and chemicals on the activity of truncated recombinant alginate lyase rT 185N;

FIG. 8, molecular gel chromatography-HPLC analysis comparison of oligosaccharide products after complete degradation of alginate with recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT 185N;

in the figure: (1) UDP2, an unsaturated disaccharide; (2) UDP3, unsaturated trisaccharide; (3) UDP4, unsaturated tetrasaccharide;

FIG. 9 is a HPLC analysis chart of unsaturated oligosaccharide fragments UDP2 and UDP3 prepared after completely degrading algin with truncated recombinant alginate lyase rT 185N;

FIG. 10-1, UDP2 of unsaturated oligosaccharide fragment prepared by completely degrading algin with truncated recombinant alginate lyase rT185N¹H-NMR chart;

FIG. 10-2, preparation of unsaturated oligo by completely degrading algin with truncated recombinant alginate lyase rT185NOf sugar fragments UDP3¹H-NMR chart;

FIG. 11 is a HPLC analysis chart of UDP2-UDP6 separated from the product of non-completely degrading algin by recombinant algin lyase rAlgL-5;

FIG. 12, a comparative graph of HPLC analysis of UDP2-UDP6 oligosaccharides prepared with rAlgL-5 for complete degradation using recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT 185N;

FIG. 13, comparative HPLC analysis chart (UV) of complete degradation of saturated poly-M series oligosaccharides (M3-5) with recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT 185N;

FIG. 14, comparative HPLC analysis chart (UV) of complete degradation of saturated poly G-series oligosaccharides (G3-5) with recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT 185N;

FIG. 15, comparative HPLC analysis chart (fluorescence) of complete degradation of saturated polyM-series oligosaccharides (M3, M4, M5) with the reducing end labeled with 2-AB using recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT 185N;

FIG. 16, comparative HPLC analysis chart (fluorescence) of complete degradation of saturated poly G-series oligosaccharides (G3, G4, G5) whose reducing ends are labeled with 2-AB using recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT 185N;

FIG. 17, HPLC analysis (fluorescence) of the degradation process of saturated poly G tetrasaccharide (G4) with a reducing end labeled with 2-AB using truncated recombinant alginate lyase rT 185N;

FIG. 18, HPLC analysis (fluorescence) of the process of degradation of saturated poly G pentasaccharide (G5) with a reducing end labeled with 2-AB using truncated recombinant alginate lyase rT 185N;

Detailed Description

The following examples are set forth so as to provide a thorough disclosure of some of the commonly used techniques of how the present invention may be practiced, and are not intended to limit the scope of the invention. The inventors have made the best effort to ensure accuracy with respect to various parameters (e.g., amounts, temperature, etc.) in the examples, but some experimental errors and deviations should be accounted for. Unless otherwise indicated, molecular weight in the present invention refers to weight average molecular weight and temperature is in degrees Celsius.

In the following examples, unless otherwise specified, all methods are conventional.

Source of biological material

Flammeovirga yaeyamensis MY04 was purchased from China general microbiological culture Collection center, address: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, with a preservation date of 2008, 11 months and 27 days, and a preservation number of CGMCC NO. 2777.

Example 1 extraction of genomic DNA of Flammeovirga yaeyamensis MY04 Strain

Inoculating Flammeovirga yaeyamensis MY04 into liquid culture medium YT04, culturing at 28 deg.C and 200rpm under shaking to 600nm absorbance (OD)₆₀₀) Is 1.2; taking 10mL of culture solution, centrifuging for 15min under the condition of 12,000 Xg (g, gravity constant of the earth), and collecting thalli sediment; the cells were suspended in 10mL of lysozyme buffer (10mM Tris-HCl, pH 8.0), centrifuged at 12,000rmp for 15min, and the pellet was collected.

The liquid culture medium YT04 comprises the following components per liter:

10g of tryptone, 5.0g of yeast extract and 30g of sodium chloride, and dissolving the components in water to obtain a constant volume of 1L and pH of 7.2.

Adding 6.0mL of lysozyme buffer solution (purchased from Shanghai Biotechnology engineering Co., Ltd.) into each tube to obtain about 7.0mL of bacterial liquid, and respectively adding 280 μ L of 20mg/mL lysozyme solution to make the final concentration of lysozyme be 800 μ g/mL; placing in ice water bath for 1.0h, transferring to 37 deg.C water bath, and warm-bathing for 2h until the reaction system is viscous; adding 0.41mL of hexadecyl sodium sulfonate solution with the concentration of 100mg/mL and 30 mu L of proteinase K solution with the concentration of 100mg/mL, and bathing for 1.0h at the temperature of 52 ℃; adding 7.5mL of Tris-balanced phenol/chloroform/isoamyl alcohol (volume ratio is 25:24:1), and mixing by gentle inversion; centrifuging at 4 deg.C for 10min at 10,000 Xg, collecting supernatant, adding 1.0mL NaAc-HAc (pH 5.2, 3.0M) buffer solution and 8.5mL anhydrous ethanol, and mixing well; picking out filamentous DNA with a gun head, transferring into a centrifugal tube of 1.5mL, washing for 2 times with 70% ethanol (stored at-20 ℃), and discarding supernatant after microcentrifugation; centrifuging at 10,000 Xg and 4 deg.C for 2min, and completely discarding supernatant; the DNA precipitate was air-dried in a sterile bench, and then the DNA sample was dissolved with sterile deionized water overnight at 4 ℃ to prepare genomic DNA.

Example 2 scanning of genome of Flammeovirga yaeyamensis MY04 strain and sequence analysis thereof.

Scanning sequencing of the genomic DNA prepared in example 1 was carried out by pyrosequencing technology, and was carried out by Meiji Biochemical, Shanghai. The DNA sequencing results were analyzed with the online software of the NCBI (National Center for Biotechnology Information, http:// www.ncbi.nlm.nih.gov /) website. The analytical software used for the NCBI website is Open Reading Frame Finder (ORF Finder, http:// www.ncbi.nlm.nih.gov/gorf. html.) and Basic Local Alignment Search Tool (BLAST, http:// BLAST. NCBI. nlm. nih. gov/BLAST. cgi).

The results of the analysis by the above biological software show that the genomic DNA of strain MY04 of Flammeovirga yaeyamensis carries a gene aly-1 encoding alginate lyase, and the results of the online analysis by the BLASTp software show that the protein C-terminal of Aly-1 is presumed to contain a putative catalytic domain conserved in the super family 2 of alginate lyase, and the putative catalytic domain contained in the C-terminal of Aly-1 protein is truncated by the molecular PCR technique and is named rT185N (as shown in FIG. 1). The nucleotide sequence is shown in SEQ ID NO. 1. The amino acid sequence is shown in SEQ ID NO. 2. Analysis with the biological software BioEdit 7.0.5.3 showed that the theoretical molecular weight of the protein rT185N was about 30.0 kD. The protein has no secretory signal peptide by using signal peptide online prediction software SignalP 4.1Server (http:// www.cbs.dtu.dk/services/SignalP /).

Example 3 recombinant expression of Gene aly-1 in E.coli BL21(DE3) Strain

PCR amplification was performed using the genomic DNA prepared in example 1 as a template. The primer sequences are as follows:

forward primer Aly 1-F: 5' -cgcGGATCCAACAATAAAGTAGAGGACGAG-3’(BamH I)；

Reverse primer Aly 1-R: 5' -ccgCTCGAGTATAAGTTTCTTTTAATTCTATAG-3’(Xho I)；

The restriction enzyme BamH I site is underlined in the forward primer Aly1-F, and the restriction enzyme Xho I site is underlined in the reverse primer Aly 1-R. The high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase used was purchased from Dalibao, China, and the PCR reagents used were operated according to the product instructions provided by this company.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 4 min; denaturation at 94 ℃ for 40s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 75s, and 35 cycles; extending for 10min at 72 ℃; stabilizing at 4 deg.C for 10 min.

The PCR product is subjected to double digestion by restriction enzymes BamH I and Xho I, and the digested PCR product is recovered by agarose gel electrophoresis. A product pET-30a (+) plasmid DNA purchased from Invitrogen, USA, was double-digested with BamH I and Xho I, subjected to agarose gel electrophoresis, and the product fragment after the digestion was recovered. Restriction enzymes BamH I and Xho I were purchased from Dalibao, China, and the system, temperature and time of reaction between enzyme and substrate used in enzyme digestion were operated according to the product specifications provided by the company.

Carrying out double enzyme digestion on the PCR product subjected to the BamH I and Xho I and a pET-30a (+) plasmid vector subjected to the double enzyme digestion in the same way, and carrying out grafting under the catalysis of DNA ligase; the ligation product is transformed into an Escherichia coli DH5 alpha strain, the strain is spread on a Luria-Bertani culture medium solid plate containing 50 mu g/mL kanamycin, after culture for 16h at 37 ℃, a single clone is picked; inoculating the single clone into a liquid Luria-Bertani culture medium containing 50 mu g/mL kanamycin for culture, and extracting plasmids; carrying out PCR verification on the plasmid by using an amplification primer, obtaining an amplification product with the size of 1.3kb as a result, and preliminarily proving that the constructed recombinant plasmid is correct; the recombinant plasmid was then sequenced, and the result showed that aly-1 gene shown in SEQ ID NO.1 was inserted between the BamH I and Xho I sites of pET-30a (+) in the correct direction, thus confirming that the constructed recombinant plasmid was correct and was named pE30a-Aly 1.

The recombinant plasmid pE30a-Aly1 was transformed into E.coli strain BL21(DE3) (purchased from Invitrogen, USA), namely BL21(DE3)/pE30a-Aly1, to obtain an engineered bacterium expressing pE30a-Aly 1.

The plasmid pET-30a (+) is transformed into an Escherichia coli strain BL21(DE3) to obtain an engineering bacterium of an empty vector control BL21(DE3)/pET-30a (+).

The following operations will be performed in parallel on the above two engineering bacteria.

Inducible expression of the engineered bacteria was performed using isopropyl thiogalactoside (IPTG) at a final concentration of 0.05mM according to the protocol provided by Invitrogen. Centrifuging at 8,000 Xg and 4 deg.C for 15min, collecting thallus, resuspending thallus with buffer solution A, and ultrasonicating in ice water bath. Further centrifugation was carried out at 15,000 Xg at 4 ℃ for 30min, and water-soluble fractions were collected and purified by Ni-Sepharose gel to obtain the alginate lyase rAly-1. Gradient elution was performed with buffer A containing imidazole at concentrations of 10, 50, 100, 250, 500mM, and the purification conditions were as per the gel's product manual. And (3) detecting the purification condition of the recombinant alginate lyase rAly-1 by polyacrylamide gel electrophoresis. And (3) filling the purified recombinant alginate lyase rAly-1 sample into a dialysis bag with the minimum molecular cut-off of 10kD, and dialyzing the buffer solution A at the temperature of 4 ℃. The buffer solution A comprises 50mM Tris and 150mM NaCl, and has pH of 7.9, and the recombinant alginate lyase rAly-1 enzyme solution is prepared.

Example 4 recombinant expression of Gene aly1-T185N 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:

forward primer Aly 1-T185N-F: 5' -cgcGGATCCTACAAAGAAGACGAAGTGCCAG-3’(BamH I)；

Reverse primer Aly 1-T185N-R: 5' -ccgCTCGAGTATAAGTTTCTTTTAATTCTATAG-3’(Xho I)；

The forward primer Aly1-T185N-F is underlined the restriction enzyme BamH I site, and the reverse primer Aly1-T185N-R is underlined the restriction enzyme Xho I site. The high fidelity DNA Polymerase PrimeSTAR HS DNA Polymerase (available from Dalibao, China) was used, and the PCR reagents used were manipulated according to the product instructions.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 40s, annealing at 59 ℃ for 40s, extension at 72 ℃ for 60s, and 35 cycles; extending for 10min at 72 ℃; stabilizing at 4 deg.C for 10 min.

Connecting the PCR product subjected to double enzyme digestion of BamH I and Xho I with a pET-30a (+) plasmid vector subjected to double enzyme digestion in the same way under the catalysis of DNA ligase; the ligation product is transformed into an Escherichia coli DH5 alpha strain, the strain is spread on a Luria-Bertani culture medium solid plate containing 50 mu g/mL kanamycin, after culture for 16h at 37 ℃, a single clone is picked; inoculating the single clone into a liquid Luria-Bertani culture medium containing 50 mu g/mL kanamycin for culture, and extracting plasmids; carrying out PCR verification on the plasmid by using an amplification primer, obtaining an amplification product with the size of 0.78kb as a result, and preliminarily proving that the constructed recombinant plasmid is correct; the recombinant plasmid was then sequenced, and it was confirmed that aly1-T185N gene represented by SEQ ID No.1 was inserted between BamH I and Xho I sites of pET-30a (+) in the correct direction, thus confirming the correctness of the constructed recombinant plasmid, which was named pE30a-Aly 1-T185N.

The recombinant plasmid pE30a-Aly1-T185N is transformed into an Escherichia coli strain BL21(DE3) (purchased from Invitrogen corporation, USA), namely BL21(DE3)/pE30a-Aly1-T185N, so as to obtain an engineering bacterium for expressing pE30a-Aly 1-T185N.

Inducible expression of the engineered bacteria was performed using isopropyl thiogalactoside (IPTG) at a final concentration of 0.05mM according to the protocol provided by Invitrogen. Centrifuging at 8,000 Xg and 4 deg.C for 15min, collecting thallus, resuspending thallus with buffer solution A, and ultrasonicating in ice water bath. Further centrifugation was carried out at 15,000 Xg at 4 ℃ for 30min, and water-soluble fractions were collected and purified from recombinant alginate lyase rT185N using Ni-Sepharose. Gradient elution was performed with buffer A containing imidazole at concentrations of 10, 50, 250, 500mM, and the purification conditions were as per the gel's product manual. The purification condition of the recombinant alginate lyase rT185N is detected by polyacrylamide gel electrophoresis.

The results are shown in FIG. 2: in BL21(DE3)/pE30a-Aly1-T185N thalli after IPTG induction expression, the yield of recombinant alginate lyase rT185N is not lower than 1259 +/-2.2 mg/L of thalli culture, and the empty vector control thalli does not have the expression of the band; the purified recombinant alginate lyase rT185N is in a single band on the electrophoresis gel, and the position of the single band is matched with the predicted molecular weight; the purified recombinant alginate lyase rT185N sample is filled into a dialysis bag with a minimum molecular cut-off of 3kD, and the buffer A is dialyzed in an environment of 4 ℃. The buffer solution A comprises the following components: 50mM Tris, 150mM NaCl, pH 7.9, to prepare the recombinant alginate lyase rT185N enzyme solution.

Example 5 determination of enzyme Activity of recombinant alginate lyase rT185N by DNS-reducing sugar method

Mixing 0.1-1.2% of algin prepared by deionized water, 10 mu g/mL of recombinant algin lyase rT185N enzyme solution, 150mmol/L of HAc-NaAc (pH 6.0) buffer solution and water according to the ratio of 1:1:1 (volume ratio) and reacting for 4 hours at 40 ℃. Heating the reaction product in boiling water bath for 10min to inactivate enzyme, transferring into ice water bath for 5min, centrifuging at 12,000 Xg and 4 deg.C for 15min, and collecting supernatant; mixing a certain volume of supernatant with DNS (3, 5-p-nitroxylene) reaction solution with the same volume, heating in boiling water bath for 10min, cooling to room temperature, and measuring the absorption value at 540 nm. Analytically pure glucuronolactone as standard, and the same method for operation, the molar concentration and OD of glucuronolactone were plotted₅₄₀The dose-effect relationship curve between. Determination of weight with protein quantitative kit purchased from Shanghai Biotechnology engineering Co., LtdProtein content in the enzyme solution of the group alginate lyase rT 185N. The units of enzyme activity were calculated according to the international standard definition, i.e. the amount of enzyme required to produce 1. mu. mol of product per minute under standard conditions was 1 IU. The results show that: the recombinant rT185N can take algin as a substrate, carry out enzymolysis to generate a reducing sugar product, and have the enzyme activity of 2895 +/-10.3U/mg.

Example 6 determination of the optimum temperature of recombinase rT185N

Preparing alginate substrate with mass volume concentration (g/mL) of 0.1-1.2% with deionized water, heating to dissolve, and cooling in water bath environment at 0 deg.C, 10 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 1 h. To 900. mu.L of the substrate solution was added 100. mu.L of the recombinant alginate lyase rT185N prepared in example 4, and the concentration of the recombinant alginate lyase rT185N was 10. mu.g/mL, and the mixture was mixed, followed by further reaction and sampling at intervals. 3 parallel samples at each temperature were used as controls with a boiling water bath inactivated recombinase preparation. The concentration (OD) of newly formed reducing sugar in each reaction system was measured by the DNS-reducing sugar method₅₄₀) And calculating the average value, and performing deviation analysis. The reaction temperature corresponding to the maximum absorbance is the optimal temperature of the recombinase, and the relative enzyme activity (RA) is defined as: percentage of each absorption value to the maximum absorption value.

The results are shown in FIG. 3: when the enzyme activity is measured by taking the algin as a substrate, the recombinant algin lyase rT185N achieves the maximum activity when reacting at 40 ℃, which shows that the optimal reaction temperature of the recombinant algin lyase rT185N is 40 ℃. Meanwhile, the recombinant alginate lyase rT185N shows 81% of relative enzyme activity at 0 ℃, which indicates that the recombinant alginate lyase rT185N has cold-adapted enzymological properties; the recombinant alginate lyase rT185N has certain thermophilic enzymology property, and also shows more than 69.2% of relative enzyme activity at 80 ℃.

Example 7 determination of optimum pH for recombinant alginate lyase rT185N

Respectively using NaAc-HAC buffer solution with the concentration of 50mM and NaH with the concentration of 50mM₂PO₄-Na₂HPO₄Buffer solution, 50mM Tris-HCl buffer solution, and brown algae with the mass volume concentration (g/mL) of 0.1-1.2% respectively prepared by mixing with alginThe gum substrate has corresponding pH values of 5, 6, 7, 8, 9 and 10, and each pH value is set at the optimal temperature. After dissolving the substrate, placing the substrate in the optimum temperature and incubating for 1h, then adding 100. mu.L of the recombinant alginate lyase rT185N diluent prepared in example 4 into every 900. mu.L of the substrate, mixing uniformly, starting the reaction, and sampling at intervals. 3 replicates of each pH were treated with a boiling water bath inactivated recombinase preparation as a control. The concentration of newly formed reducing sugar (OD) in each reaction system was measured by the DNS-reducing sugar method₅₄₀) And the mean and deviation are calculated. Relative enzyme (RA) activity is defined as: percentage of the mean absorption value to the maximum absorption value for each group. The pH corresponding to the maximum absorbance is the optimum pH for the recombinase.

The results are shown in FIG. 4: the optimal reaction pH of the recombinant alginate lyase rT185N is 6.0. Within the pH range of 5-10, the relative enzyme activity of the recombinant alginate lyase rT185N is above 65.6%, and the enzyme has wide pH reaction activity.

Example 8 analysis of temperature stability of recombinant alginate lyase rT185N

The recombinant alginate lyase rT185N prepared in example 4 is subjected to heat treatment at different temperatures (0-70 ℃) for 0.5h, 1h and 2h, and then is mixed with alginate with the mass volume concentration (g/mL) of 0.1-1.2% prepared by distilled water according to the ratio of 1:9 (volume ratio), and then the residual enzyme activity is determined at the optimal temperature, wherein the enzyme activity of the enzyme solution without heat treatment is defined as 100% relative activity. The results are shown in FIG. 5: the recombinant alginate lyase rT185N still has the residual activity of > 87% after being pretreated for 2 hours at the temperature of lower than 50 ℃, which indicates that the enzyme has certain thermal stability.

Example 9 analysis of pH stability of recombinant alginate lyase rT185N

The recombinant alginate lyase rT185N prepared in example 4 is preincubated for 2 hours in the environment with the optimal temperature (40 ℃) and different pH (pH 5-10) respectively, then mixed with the alginate substrate solution with the mass volume concentration (g/mL) of 0.1-1.2% according to the proportion 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 pretreatment is defined as 100% relative activity, the result is shown in FIG. 6, the enzyme activity of rT185N is pretreated for 2 hours in the range of pH 5-10, and the enzyme activity of rT185N is still maintained above 40%, which shows that the tolerance range of the recombinant alginate lyase rT185N to the pH value is wider.

Example 10 Effect of Metal ions and chemical reagents on the Activity of recombinant alginate lyase rT185N

Preparing an alginate substrate with mass concentration (g/mL) of 0.1-1.2% by using deionized water, a recombinant alginate lyase rT185N enzyme solution prepared in example 4, and water according to the weight ratio of 5: 1:4 (volume ratio), then adding different metal ions to the reaction system to a final concentration of 1mM or 10mM, and reacting at 40 ℃ for 4 hours, and measuring the activity of the enzyme by the DNS-reducing sugar method as described above. The control group is the activity of rT185N without any metal ions (set at 100%). Results are shown in FIG. 7, at 1mM or 10mM concentrations: (1) na (Na)⁺、K⁺、Li⁺The activity of the three monovalent metal reagents on rT185N shows weak inhibition effect, but Ag⁺Has obvious inhibiting effect; (2)1mM Mg²⁺10mM Fe²⁺、Mn²⁺The divalent metal examples have the promoting effect on enzyme activity, and the rest divalent and trivalent metal ions have the inhibiting effect on the enzyme activity; (3) glycerol, beta-mercaptoethanol and DTT had significant activity-promoting activity against rT185N at 10 mM.

Example 11 High Performance Liquid Chromatography (HPLC) comparative analysis of the product of the recombinant alginate lyase rAly-1 and the truncated recombinant alginate lyase rT185N for complete degradation of algin

Preparing an alginate substrate with the mass volume concentration (g/mL) of 0.1-1.2% by using deionized water, heating and dissolving, and then placing in a water bath environment at 40 ℃ for cooling for 1 h. Adding 10-100. mu.L of a dilution of recombinase rAly-1 prepared in example 3 and recombinase rT185N prepared in example 4 to 100. mu.L of a substrate, respectively, and supplementing with sterile deionized water when the volume is less than 200. mu.L; after mixing, the reaction is continued for 24 h. Heating the reaction product in boiling water bath for 10min, and transferring into ice water bath for 5 min. The mixture was centrifuged at 12,000 Xg at 4 ℃ for 15min, and the supernatant was collected.

With NH at a concentration of 0.20mol/L₄HCO₃Solution, equilibration of Superdex Peptide10/300GL (GE corporation) molecular gel chromatography column with flow rate of 0.40mL/min, at least 2 beds. Loading 100 mu g/sample of the sample subjected to algin enzymolysis by using an automatic sample injector, and detecting the sample at 235nm under the unchanged other conditions. The integrated area of each oligosaccharide component was analyzed using HPLC operating software to calculate the relative molarity.

As shown in FIG. 8, under the above conditions, the oligosaccharide product after complete degradation of the alginate substrate has a characteristic absorption of 235 nm. This preliminary suggests that rAly1 and rT185N are alginate lyases. Meanwhile, when the algin is used as a substrate, the rT185N is basically consistent with the rAly-1 product, and the content is slightly different. Wherein the final main products of the reaction for digesting the algin by the truncated recombinant algin lyase rT185N are two oligosaccharide products with the peak-out time of 40.5 'and 43.2' in HPLC analysis, and the area ratio is stable and is 51:49, which shows that the molar ratio of the two products is about 51: 49; while the area ratio of the two oligosaccharide products of the final main products 40.5 'and 43.2' of the rAly-1 enzymolysis reaction is stabilized to 48:52, which shows that the molar ratio of the two products is about 48: 52.

Example 12 molecular weight identification of major oligosaccharide products from the complete degradation of algin by recombinant alginate lyase rT185N

A total of 200mg of alginate was digested thoroughly with recombinant alginate lyase rT185N as described in example 11, the samples were loaded in batches, passed through Superdex Peptide 10/300GL molecular gel chromatography column (GE Co.), and two oligosaccharide samples with peak times of 40.5 'and 43.2' were collected according to peak times, respectively. The two oligosaccharide samples collected many times were collected separately and then freeze-dried repeatedly for desalting. The oligosaccharide samples were dissolved in sterile deionized water and subjected to primary Mass Spectrometry (MS) analysis to determine the relative molecular weight of each oligosaccharide. Heavy water (D) is also used₂O) dissolving the resulting oligosaccharide sample, freeze-drying repeatedly to complete the replacement with deuterium and hydrogen, and carrying out¹H-NMR analysis is carried out, and the chemical structure and the characteristics of each oligosaccharide are finally determined.

As shown in FIG. 9, HPLC analysis of the unsaturated oligosaccharide fragments UDP2 and UDP3 prepared after completely degrading algin with recombinant alginate lyase rT185N showed that the product had characteristic absorption at 235nm, peak-off times of 40.5 'and 43.2', respectively, and purity was greater than 99%.

The results of the primary mass spectrometry performed in the anionic mode showed that the molecular weights of these two oligosaccharide primary products were 352, 528, respectively. This indicates that the two oligosaccharide main products are unsaturated disaccharide (UDP2) and unsaturated trisaccharide (UDP3) generated after the recombinant alginate lyase rT185N thoroughly degrades the algin.

Combining the results of fig. 8 and 9, the unsaturated disaccharide is the main oligosaccharide product with the minimum polymerization degree after the recombinant alginate lyase rT185N degrades the alginate.

Further through¹H-NMR data analyze the structural characteristics of the two oligosaccharides:

(1) as shown in FIG. 10-1, the characteristic chemical shift value at 5.75ppm indicates that almost all of the unsaturated disaccharide UDP2 after the recombinant alginate lyase rT185N degrades alginate is Δ G.

(2) The chemical shift values at 5.70 and 5.60ppm shown in FIG. 10-2 indicate that the non-reducing end of the main product unsaturated trisaccharide UDP3 has two types: Δ G and Δ M; and the ratio of the area integrals of the two peaks is 100:161, which indicates that the molar ratio of the two types of unsaturated trisaccharides is about 5: 8. Compared with the molar ratio of two types of unsaturated trisaccharides of the main product for degrading the algin by rAly-1, which is about 5:4, the content of the trisaccharide product containing the delta M terminal is improved by about 17 percent.

Example 13 preparation of recombinant alginate lyase rAlgL-5 UDP2-UDP6 product not completely degrading algin

According to the patent application (application No. 201410469861.4, an incision type alginate lyase and its coding gene and application) described in example 10, a total of 200mg of alginate was subjected to incomplete enzymolysis using recombinase rAlgL-5, the product was loaded in batches, passed through molecular gel column Superdex Peptide 10/300GL (GE Co.), and single oligosaccharide fragments were collected according to the peak-out time. These oligosaccharide samples were collected several times, pooled separately, and freeze-dried repeatedly for desalting.

As shown in FIG. 11, the prepared oligosaccharide fragment UDP2-UDP6 was analyzed by HPLC detection, and the result showed that the product had characteristic absorption at 235nm and had purity of more than 99%.

Example 14 comparative analysis of recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT185N minimal unsaturated oligosaccharide substrates

Using deionized water and the oligosaccharide fragment prepared in example 13, comprising: unsaturated hexasaccharide (UDP6), unsaturated pentasaccharide (UDP5), unsaturated tetrasaccharide (UDP4), unsaturated trisaccharide (UDP3) and unsaturated disaccharide (UDP 2). The oligosaccharide samples are respectively prepared into substrate solutions, recombinant alginate lyase rAly1 and rT185N enzyme solutions with the concentration of 10 mug/mL, HAc-NaAc (pH 6.0) buffer solution with the concentration of 150mmol/L and water according to the ratio of 1:1:1 (volume ratio) and reacting at 40 ℃ for 24 hours. HPLC analysis was carried out according to the chromatographic conditions described in example 11. The results are shown in fig. 12, the truncated recombinant alginate lyase rT185N described in the present application:

(1) degradation of unsaturated hexasaccharides (UDP6) to yield unsaturated tetrasaccharides, unsaturated trisaccharides and unsaturated disaccharides;

(2) degradation of unsaturated pentasaccharide (UDP5) to yield equivalent amounts of unsaturated trisaccharide and unsaturated disaccharide;

(3) degradation of the unsaturated tetrasaccharide (UDP4) results in twice as much unsaturated disaccharide;

(4) there was no significant change before and after the reaction with the unsaturated trisaccharide or unsaturated disaccharide.

This is a good indication, unsaturated four sugar fragments are recombinant alginate lyase rT185N minimum unsaturated oligosaccharide substrate, and recombinant alginate lyase rT185N endonucleases degradation unsaturated oligosaccharide substrate.

Compared with rAly-1, the obvious difference is that the degradation rate of rT185N to unsaturated tetrasaccharide (UDP4) is obviously higher than that of rAly-1, the conversion efficiency can reach more than 95%, and the conversion efficiency of rAly-1 is only about 50%.

Example 15 comparative analysis by High Performance Liquid Chromatography (HPLC) of cleavage patterns of recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT185N

A solution containing about 30. mu.g of saturated poly-M sugar (M3, M4, M5) and poly-G sugar (G3, G4, G5), 150mmol/L HAc-NaAc (pH 6.0) buffer, and a diluted solution of recombinase rAly-1 prepared in example 3 and recombinant alginate lyase rT185N prepared in example 4 were mixed in a volume ratio of 1:1:1, and reacted in a water bath at 40 ℃ for 24 hours. Placing the reaction system in boiling water bath for 10min, transferring to ice water bath for 5min, and centrifuging at 4 deg.C under 12,000 Xg for at least 15 min. The supernatant was collected as the oligosaccharide degradation products of recombinant alginate lyase rAly-1 and rT 185N. The negative control reaction was performed with recombinant alginate lyase rAly-1 and rT185N enzyme solutions inactivated in a boiling water bath.

According to the example 11 of the chromatographic conditions, recombinant alginate lyase rAly-1 and rT185N enzymatic hydrolysis of saturated poly M sugar (M3, M4, M5), poly G sugar (G3, G4, G5) samples, with automatic sample feeder loading 20 u G/sample, other conditions are unchanged, 235nm detection. The integrated area of each oligosaccharide component was analyzed using HPLC operating software to calculate the relative molarity. The relative molecular weights of the oligosaccharides are determined with reference to molecular weight standards.

As shown in FIG. 13, rT185N compares to rAly-1, and similarly: (1) degradation of the saturated poly-M pentasaccharide (M5) results in an unsaturated trisaccharide (UM3) and an unsaturated disaccharide (UM 2); (2) degradation of the saturated poly-M tetrasaccharide (M4) results in an unsaturated trisaccharide (UM3) and an unsaturated disaccharide (UM 2); (3) both were not significantly changed before and after the reaction with saturated poly-M trisaccharide (M3).

As shown in FIG. 14, rT185N compares to rAly-1, and similarly: (1) degradation of the saturated poly-G pentasaccharide (G5) followed by the production of unsaturated trisaccharide (UG3) and trace unsaturated disaccharide (UG 2); (2) degradation of the saturated poly-G tetrasaccharide (G4) followed by the production of unsaturated trisaccharide (UG3) and trace unsaturated disaccharide (UG 2); (3) both were not significantly changed before and after the reaction with the saturated poly-G trisaccharide (G3).

Comparing FIGS. 13 and 14, it is shown that truncated rT185N remains a facultative alginate lyase; rT185N and rAly-1 can respectively generate disaccharide products from a reducing end or a non-reducing end when degrading saturated poly M pentasaccharide (M5) and saturated poly G pentasaccharide (G5), and finally, the main products are unsaturated trisaccharide products; both of them, when degrading the saturated poly-M tetrasaccharide (M4) and the saturated poly-G tetrasaccharide (G4), showed that it was possible to cleave the saturated tetrasaccharide both exoenzymatically to yield one molecule of saturated monosaccharide and one molecule of unsaturated trisaccharide and endoenzymatically to yield one molecule of saturated disaccharide and one molecule of unsaturated disaccharide. The two are obviously different in that the degradation rate of the truncated rT185N to saturated M tetrasaccharide and saturated M pentasaccharide is obviously improved, and the obvious change of substrates of the saturated G tetrasaccharide, the saturated G pentasaccharide and larger oligosaccharides is not seen.

Example 16 fluorescence-High Performance Liquid Chromatography (HPLC) comparative analysis of cleavage patterns of recombinant alginate lyase rAly-1 and truncated recombinant alginate lyase rT185N

A solution containing about 10. mu.g of saturated poly-M saccharide (M3, M4, M5) and poly-G saccharide (G3, G4, G5) was rotary evaporated to dryness. Adding dimethyl sulfoxide (DMSO) solution containing excessive anthranilamide (2-AB) and sodium boronitrile, mixing, and incubating in 60 deg.C water bath for 2 h. Spin-dry to dryness, add 500. mu.L of deionized water to dissolve the sample, shake the sample with 200. mu.L of chloroform, centrifuge, and collect the supernatant. The extraction with chloroform was continued for not less than 7 times to obtain saturated polyM saccharides (2AB-M3, 2AB-M4, 2AB-M5) whose reducing ends were fluorescently labeled and saturated polyG saccharides (2AB-G3, 2AB-G4, 2AB-G5) which were fluorescently labeled. The molecular weight standards for saturated oligosaccharides were labeled in the same manner.

The above 2AB-M3, 2AB-M4, 2AB-M5, 2AB-G3, 2AB-G4 and 2AB-G5 samples, 150mmol/L HAc-NaAc (pH 6.0) buffer solution, and a diluent of recombinase rAly-1 prepared according to example 3 and recombinant alginate lyase rT185N prepared according to example 4 were mixed in a volume ratio of 1:1:1, and placed in a water bath at 40 ℃ for reaction for 24 hours. Placing the reaction system in boiling water bath for 10min, transferring to ice water bath for 5min, and centrifuging at 4 deg.C under 12,000 Xg for at least 15 min. The supernatant was collected as the oligosaccharide degradation products of recombinant alginate lyase rAly-1 and rT 185N. The negative control reaction was performed with recombinant alginate lyase rAly-1 and rT185N enzyme solutions inactivated in a boiling water bath.

According to the chromatographic conditions described in example 11, labeled samples of 2AB-M3, 2AB-M4, 2AB-M5, 2AB-G3, 2AB-G4, 2AB-G5 and their enzymatic products were loaded with 50-200 ng/sample using an autosampler, and other conditions were unchanged, 330nm excitation, and 420nm detection. The integrated area of each oligosaccharide component was analyzed using HPLC operating software to calculate the relative molarity. The relative molecular weights of the oligosaccharides are determined with reference to molecular weight standards.

As shown in FIGS. 15 and 16, rAly-1 was compared with rT 185N:

(1) after reaction with 2AB-M5 with an excess of recombinant alginate lyase rAly-1 and rT185N, only a small amount of degradation occurred and an equimolar amount of 2AB-UM3 was produced;

(2) the two have no obvious change before and after reaction with 2AB-M4 and 2 AB-M3;

(3) rAly-1 and rT185N completely degraded 2AB-G5 and produced equimolar amounts of 2AB-UG 3;

(4) there was no significant change before and after reaction of rAly-1 and rT185N with 2 AB-G3.

The significant difference between the two is that rT185N completely degraded 2AB-G4 and produced equimolar amounts of 2AB-UG3, whereas there was no significant change before and after reaction of rAly-1 with 2 AB-G4.

Examples 13 and 15, and examples 14 and 16, illustrate:

(1)2-AB marks the end of the saturated oligosaccharide, and inhibits the degradation activity of the enzyme rT185N on the oligosaccharide;

(2) compared with rAly-1, (a) the size of the oligosaccharide substrate which can be degraded by the truncated rT185N is obviously smaller than that of the rAly-1, and (b) the relative degradation activity of the truncated rT185N on polyM is obviously improved.

Example 17 fluorescent-High Performance Liquid Chromatography (HPLC) comparative analysis of the Process of cleaving Poly G oligosaccharides (G4, G5) with truncated recombinant alginate lyase rT185N

Samples of 2AB-G4 and 2AB-G5 prepared in example 16, 150mmol/L HAc-NaAc (pH 6.0) buffer solution, and a diluted solution of recombinant alginate lyase rT185N prepared in example 4 were mixed in a volume ratio of 1:1:1, reacted in a 40 ℃ water bath, and sampled at intervals. Placing the reaction system in boiling water bath for 10min, transferring to ice water bath for 5min, and centrifuging at 4 deg.C under 12,000 Xg for at least 15 min. The supernatant was collected as an oligosaccharide degradation product of rT 185N. A negative control reaction was performed with rT185N enzyme solution previously heated in a boiling water bath for 10 min.

Following the chromatographic conditions described in example 11, labeled samples of 2AB-G4, 2AB-G5 and their enzymatic products were loaded with 50-200 ng/sample using an autosampler, and other conditions were unchanged, 330nm excitation, and 420nm detection. The integrated area of each oligosaccharide component was analyzed using HPLC operating software to calculate the relative molarity. The relative molecular weights of the oligosaccharides are determined with reference to molecular weight standards.

As shown in FIG. 17, under the above conditions, after 2AB-G4 is degraded, the content of 2AB-UG3 oligosaccharide product gradually increases with the increase of the enzymolysis reaction time, and the relative content finally tends to be stable. This indicates that the truncated recombinant alginate lyase rT185N cleaves 2AB-G4 in an exonuclease manner, cleaving a molecule of monosaccharide from the non-reducing end to produce a molecule of 2AB-UG 3. As shown in FIG. 18, after 2AB-G5 is degraded, the content of 2AB-UG3 oligosaccharide product is gradually increased along with the increase of the enzymolysis reaction time, and the relative content is finally stable and can not catch the generation of 2AB-UG 4; the experiment was then repeated with the enzyme amount reduced to one percent of the original amount, and the production of 2AB-UG4 was still not captured. This indicates that the truncated recombinant alginate lyase rT185N cleaves 2AB-G5 in an endonuclease manner and cleaves one molecule of disaccharide from the non-reducing end to generate 2AB-UG 3.

References cited in the specification

1.Hu J,Geng M,Li J,Xin X,Wang J,Tang M,Zhang J,Zhang X,Ding J.2004.Acidic oligosaccharide sugar chain,a marine-derived acidic oligosaccharide,inhibits the cytotoxicity and aggregation of amyloid beta protein.Journal of Pharmacological Science 95:248-255

2.Xu X,Bi D,Li C,Fang W,Zhou R,Li S,Chi L,Wan M,Shen L.2015.Morphological and proteomic analyses reveal that unsaturated guluronate oligosaccharide modulates multiple functional pathways in murine macrophage RAW264.7cells.Marine Drugs 13:1798-1818

3.Zhu B,Yin H.2015.Alginate lyase:review of major resources and classification,properties,structure-function analysis and application.Bioengineered 6:125-131

4.MacDonald LC,Berger BW.2014.A polysaccharide lyase from Stenotrophomonas maltophilia with a unique,pH-regulated substrate specificity.The Journal of Biological Chemistry 289:312-325

5.Lundqvist LCE,Jam M,Barbeyron T,Czjzek M,Sandstrom C.2012.Substrate specificity of the recombinant alginate lyase from the marine bacteria Pseudomonas alginovora.Carbohydrate Reasearch 352:44-50

6.Thomas F,Lundqvist LCE,Jam M,Jeudy A,Barbeyron T,Sandstrom C,Michel G,Czjzek M.2013.Comparative characterization of two marine alginate lyases from Zobellia galactanivorans reveals distinct models of action and exquisite adaption to their natural substrate.The Journal of Biological Chemistry 288:23021-23037

7.Han W,Gu J,Cheng Y,Liu H,Li Y,Li F.2016.Novel alginate lyase(Aly5)from a polysaccharide-degrading marine bacterium,Flammeovirga sp.strain MY04:effects of module truncation on biochemical characteristics,alginate degradation patterns,and oligosaccharide-yielding properties.Applied and Environmental Microbiology 82:364-374

8.Li S,Yang X,Bao M,Wu Y,Yu W,Han F.2015.Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp.L11 enhances its catalytic efficiency and thermostability,and alters its substrate preference and product distribution.FEMS Microbiology Letters 362:1-7

9.Li S,Wang L,Hao J,Xing M,Sun J,Sun M.2017.Purification and Characterization of a New Alginate Lyase from Marine Bacterium Vibrio sp.SY08.Mar Drugs 15:1-11

SEQUENCE LISTING

<110> Wutong perfumery, Inc. of Tengzhou City

<120> truncated recombinant alginate lyase rAly1-T185N, and coding gene and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 780

<212> DNA

<213> Flammeovirga yaeyamensis

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gaatggtttc cttacttcca cgcaaaaaat aacgaagtat tatttaaagg acattgtgca 240

ggaacaacca ctagaggttc ttactaccca cgttgcgagt taagacaacg tgttggtggt 300

ggagacaatt attggagtgt tcaacagtat caatatttaa aaacggtatt aagagtaaca 360

cacttaccgg tggtcaagcc agaggtttcc atggtacaga tccacgggcc tcaggatgaa 420

ccactgagag tgcagtactc ggaaagtaca cgttcgtcga ccccttgggg attacacatc 480

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ggacaacgtt acaacaaaac gtatacttct gtagatcaaa caggatattt taaagtgggt 660

tgttatacac aatccacaaa attcctctct caagtaaaac cagaatatga tcaagacgag 720

ccaaacgatg cttatgcaga ggtggcagtt caatctatag aattaaaaga aacttattaa 780

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<211> 259

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<213> Flammeovirga yaeyamensis

<400> 2

Tyr Lys Glu Asp Glu Val Pro Asp Ala Asp Gln Lys Pro Thr Asp Val

1 5 10 15

Ile Pro Ser Leu Val Gln Trp Lys Val Thr Leu Pro Val Asp Ala Asp

20 25 30

Gly Asn Asp Asn Arg Tyr Val Thr Asp Pro Glu Leu Arg Asn Arg Asn

35 40 45

Pro Leu Glu Ile Lys Asp Gly Asp Leu Val Asp Tyr Glu Trp Phe Pro

50 55 60

Tyr Phe His Ala Lys Asn Asn Glu Val Leu Phe Lys Gly His Cys Ala

65 70 75 80

Gly Thr Thr Thr Arg Gly Ser Tyr Tyr Pro Arg Cys Glu Leu Arg Gln

85 90 95

Arg Val Gly Gly Gly Asp Asn Tyr Trp Ser Val Gln Gln Tyr Gln Tyr

100 105 110

Leu Lys Thr Val Leu Arg Val Thr His Leu Pro Val Val Lys Pro Glu

115 120 125

Val Ser Met Val Gln Ile His Gly Pro Gln Asp Glu Pro Leu Arg Val

130 135 140

Gln Tyr Ser Glu Ser Thr Arg Ser Ser Thr Pro Trp Gly Leu His Ile

145 150 155 160

Val Tyr Asn Glu Asn Phe Lys Glu Arg Thr Asn Ile Glu Tyr Lys Met

165 170 175

Gly Asp Arg Leu Glu Val Glu Val Ile Val Asp Lys Gly Asp Ile Thr

180 185 190

Val Asn Ile Arg Asn Leu Glu Asn Gly Gln Arg Tyr Asn Lys Thr Tyr

195 200 205

Thr Ser Val Asp Gln Thr Gly Tyr Phe Lys Val Gly Cys Tyr Thr Gln

210 215 220

Ser Thr Lys Phe Leu Ser Gln Val Lys Pro Glu Tyr Asp Gln Asp Glu

225 230 235 240

Pro Asn Asp Ala Tyr Ala Glu Val Ala Val Gln Ser Ile Glu Leu Lys

245 250 255

Glu Thr Tyr

Claims

1. A truncated recombinant alginate lyase rT185N is constructed by cutting 185 non-catalytic amino acid residues from the N-terminus of Aly-1 of full-length recombinant alginate lyase, and the amino acid sequence is shown in SEQ ID NO. 2.

2. The coding gene of a truncated recombinant alginate lyase rT185N has a nucleotide sequence shown in SEQ ID NO. 1.

3. A recombinant vector, in which the truncated recombinant alginate lyase rT185N gene coding for claim 2 is inserted.

4. The recombinant vector of claim 3, wherein the 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 fungi expression vector, a plant expression vector, an insect expression vector, and a mammalian cell expression vector.

5. A recombinant bacterium or transgenic cell line having the recombinant vector according to claim 3 transferred into a host cell or cell line.

6. The recombinant bacterium or transgenic cell line of claim 5, wherein the host cell or cell line is selected from an E.coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungal host cell, an insect cell, or a mammalian cell.

7. The use of the gene encoding the truncated recombinant alginate lyase rT185N of claim 2, the recombinant vector of claim 3, the recombinant strain or transgenic cell line of claim 5 for the preparation of the truncated recombinant alginate lyase rT 185N.

8. The use of the truncated recombinant alginate lyase rT185N of claim 1 for degrading alginate, degrading alginate oligosaccharides or preparing unsaturated oligosaccharides.