A pink bacillus JZB09, alginate lyase rAly06925 and application of the coding gene of the enzyme

Abstract

The present application relates to a kind of persicobacter sp. JZB09 is preserved in China typical culture preservation center, address: Wuhan University, Wuhan City, Wuchang District, Bayi Road 299, Wuhan, Hubei Province, preservation date April 12, 2021, preservation number CCTCC M 2021354;And provide a kind of alginate lyase rAly06925 derived from persicobacter sp. JZB09, its amino acid sequence is as shown in SEQ ID NO.2, the coding gene aly06925 of the alginate lyase rAly06925, its nucleotide sequence is as shown in SEQ ID NO.1, the alginate lyase rAly06925 can degrade alginate or alginate oligosaccharide production non-reducing end contains ΔM series G-rich unsaturated oligosaccharide fragment.

Description

[0001] This invention is a divisional application of application number 202110476331.2, application date 2021.04.29, entitled "A type of Chlorella JZB09, alginate lyase rAly06925 and the encoding gene of the enzyme and its application". Technical Field

[0002] This invention relates to a Chlorella vulgaris JZB09, an alginate lyase rAly06925, and the encoding gene of the enzyme and its applications, belonging to the field of biotechnology. Background Technology

[0003] Brown algin is a linear anionic polysaccharide composed of two sugar units, β-D-mannuronate (M) and α-L-guluronate (G), randomly linked by β-1,4 glycosidic bonds, with M and G being epimers at the C5 position. [1] Algin is mainly produced in the cell walls and intercellular matrix of large brown algae such as kelp, Sargassum, Fucus vesiculosus, and giant kelp. [2] In addition, opportunistic pathogens such as *Pseudomonas aeruginosa* and certain soil microorganisms such as *Azotobacter purpureus* can also secrete alginate. [3,4] The difference lies in the fact that alginate derived from microorganisms has acetylation modification. [5] Alginate is non-toxic and harmless, and can be used in the food industry to remove heavy metal ions from the body. [6] And increase the viscosity of ice cream [7] It can also be used in the pharmaceutical industry for hemostatic gauze, hemostatic agents, and bandages. [8] and drug embedding materials [9] Alginate, along with agar and carrageenan, is one of the three most abundant, economically valuable, and widely used marine polysaccharides. For a long time, the research on the high-value utilization of alginate has been a key focus in the field of marine biotechnology. Existing studies have shown that alginate oligosaccharides alter the nuclear morphology of U973 leukemia cells, thereby leading to apoptosis.

[10] This study achieved an inhibitory effect on U973 leukemia cells. Furthermore, recent research indicates that "mannan oligosaccharide disodium (GV971)," prepared from poly(M-segmented) alginate oligosaccharides, can inhibit β-amyloid protein precipitation, cytotoxicity, and cell aggregation.

[11] It can be used to treat mild to moderate Alzheimer's disease (AD). Therefore, alginate oligosaccharides with specific M / G ratios and degrees of polymerization have important application and economic value, and achieving efficient preparation of such oligosaccharides is of great significance.

[0004] Alginate lyases are a class of polysaccharide lyases (PL) that catalyze the cleavage of glycosidic bonds via β-elimination reactions, forming a C4=C5 unsaturated double bond at the non-reducing end of the oligosaccharide product. This bond forms a conjugated structure with the C=O (carboxyl carbonyl group) at the C5- position, resulting in an oligosaccharide product containing an unsaturated terminus (Δ) and exhibiting characteristic absorption around 232 nm.

[12] Alginate lyase has a wide range of sources, including soil microorganisms, marine algae, marine mollusks, echinoderms, and various symbiotic marine microorganisms on or within their bodies. Bacteria are the main source of alginate lyase. [13,14] However, due to the limited yield of natural alginate lyases and the fact that most transgenic alginate lyases, while having higher yields, exhibit poor water solubility and activity, research on substrate selectivity, substrate degradation patterns, and oligosaccharide formation characteristics is currently scarce. Only the G-specific endonuclease Aly5 has been studied.

[15] M-specific endonucleases Pae-AlgL and Avi-AlgL

[16] G-prone endonuclease Aly1

[17] and Aly2

[18] Such reports, however, fail to provide sufficient information on enzymatic properties, resulting in a shortage of tool enzymes and hindering their precise application. Especially in industry, endo-alginate lyases are highly efficient tool enzymes for preparing a series of alginate oligosaccharide products.

[0005] The inventors' existing research has focused on analyzing endo-alginate lyases, elucidating their substrate selectivity, substrate degradation patterns, and oligosaccharide formation characteristics. Through summarization, they found that the substrate selectivity and substrate degradation patterns jointly determine the oligosaccharide formation characteristics of the endo-alginate enzymes. Alginate endo-alginate enzymes have been found to exhibit a succession pattern of ΔM to ΔG or ΔG to ΔM in the structural characteristics of their oligosaccharide end products. [15-18] A specialized exoglucan lyase that produces G-fragment-rich monosaccharides at the ΔG-terminus.

[19] And the PL8 family of M-specific broad-spectrum polysaccharide-degrading enzymes that produce G-fragments rich in ΔM-terminus and use alginate as the optimal substrate.

[20] However, there are currently no reports on the alginate lyases, characteristics, and application value derived from the Persicobacter strain, especially alginate endonucleases that specifically produce ΔM-terminal end products. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a strain of Chlorella JZB09, alginate lyase rAly06925, the encoding gene of the enzyme, and its applications.

[0007] In the following text, Δ represents an unsaturated monosaccharide, X refers to G or M; M is β-1,4-D-mannuronic acid, and G is α-1,4-L-guluronic acid.

[0008] The alginate lyase rAly06925 is abbreviated as "rAly06925".

[0009] This enzyme specifically degrades mannuronic acid oligosaccharide fragments from alginate, making it an M-specific alginate lyase. Therefore, it is hypothesized that during alginate degradation, it specifically degrades M-rich fragments, namely M-MMXn, G-MMXn, and Δ-MMXn (n≥1, and a natural number; X is G or M), and efficiently cleaves the glycosidic bonds indicated by the '-' symbol, thereby generating a series of unsaturated oligosaccharide final products with only ΔM as the non-reducing terminator. Therefore, rAly06925 is the first reported endoglucose lyase specifically producing only ΔM-terminators. Based on this, it can be used to completely degrade alginate polysaccharides or oligosaccharides to specifically prepare a series of G-rich unsaturated oligosaccharide fragments with ΔM as the non-reducing terminator. Furthermore, the effects of a series of mutants on enzyme activity were investigated, and the potential catalytic motif was identified as N. 238 -N 239 -H 240 -G 241 -T 242 -H 243 And Y 87 Q 170 H 240 Y 295 These are key catalytic site residues. Specifically, the additional peptide K of rAly06925 is truncated. 45 -N 60 (i.e., T45-60N, which is the removal of amino acids from position 45 to 60 in the amino acid sequence) or Y 244 Mutations to amino acids other than aromatic hydrocarbon amino acids all resulted in the recombinant protein losing its water solubility; therefore, the additional peptide K... 45 -N 60 and the Y contained therein 244 Residues play a crucial role in maintaining protein conformation or determining the hydrophilicity of protein surfaces.

[0010] This invention is achieved through the following technical solution:

[0011] A Persicobacter sp. JZB09 strain is deposited at the China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The deposit date is April 12, 2021, and the accession number is CCTCCM2021354.

[0012] "Persicobacter sp. JZB09" is abbreviated as "Persicobacter JZB09".

[0013] The above-mentioned method for culturing Chlorella JZB09 includes the following steps:

[0014] (1) Inoculate Chlorella JZB09 into liquid culture medium and culture for 16-24 h at a temperature of 25-30℃ and a rotation speed of 180-220 rpm to obtain an activated strain;

[0015] (2) The activated strain obtained in step (1) is inoculated into liquid culture medium at a volume percentage of 1-3% and cultured for 16-24 hours at a temperature of 25-30℃ and a rotation speed of 180-220 rpm to obtain seed liquid.

[0016] (3) The seed culture obtained in step (2) is inoculated into the liquid culture medium at a volume percentage of 1-5%, and cultured for 4-6 days at a temperature of 25-30℃ and a rotation speed of 180-220 rpm to obtain the Chlorella JZB09 bacterial culture.

[0017] According to a preferred embodiment of the present invention, the liquid culture medium in steps (1), (2), and (3) has the following components per liter:

[0018] Tryptone 4 g / L, yeast extract 2.5 g / L, potassium chloride 7.5 g / L, sodium chloride 20 g / L, calcium chloride 1.1 g / L, magnesium sulfate 7.2 g / L, ammonium chloride 1.5 g / L, balance water, pH 7.0–7.5.

[0019] The application of the above-mentioned Chlorella JZB09 in the preparation of alginate lyase rAly06925.

[0020] The application of Persicobacter sp. JZB09 in the degradation of alginate or alginate oligosaccharides.

[0021] An alginate lyase rAly06925 derived from Chlorella JZB09 has the amino acid sequence shown in SEQ ID NO.2.

[0022] The alginate lyase rAly06925 contains only one domain, namely Alginate_lyase.

[0023] The nucleotide sequence of the gene encoding the alginate lyase rAly06925 is shown in SEQ ID NO.1.

[0024] The gene aly06925 is 1197 bp in length and encodes a protein containing 399 amino acids with a molecular weight of approximately 45.6 kDa.

[0025] A recombinant expression vector I contains the encoding gene aly06925 of the above-mentioned alginate lyase rAly06925.

[0026] A recombinant bacterium I containing the encoding gene aly06925 of the aforementioned alginate lyase rAly06925.

[0027] The application of the encoding gene aly06925 of the above-mentioned alginate lyase rAly06925, recombinant expression vector I, and recombinant bacteria I in the preparation of alginate lyase rAly06925.

[0028] Application of the above-mentioned alginate lyase rAly06925 in the degradation of alginate or alginate oligosaccharides.

[0029] According to a preferred embodiment of the present invention, the above-mentioned alginate lyase rAly06925 is used in the degradation of alginate or alginate oligosaccharides to produce a series of G-rich oligosaccharide fragments containing ΔM at non-reducing ends.

[0030] According to a preferred embodiment of the present invention, the above-mentioned alginate lyase rAly06925 is used in the degradation of alginate or alginate oligosaccharides to produce unsaturated disaccharides.

[0031] A mutant alginate lyase rAly06925, with the amino acid mutation site being that the 48th amino acid in the amino acid sequence SEQ ID NO.2 is changed from tyrosine to alanine;

[0032] Preferably, the encoding gene of the alginate lyase rAly06925 mutant enzyme is mutated at a specific site on the encoding gene SEQ ID NO.1 according to the mutation site of the amino acid;

[0033] Preferably, a recombinant expression vector II contains the gene encoding the mutant enzyme;

[0034] Preferably, a recombinant bacterium II contains the gene encoding the mutant enzyme.

[0035] The application of the encoding gene of the above-mentioned alginate lyase rAly06925 mutant enzyme, recombinant expression vector II, and recombinant strain II in the preparation of alginate lyase rAly06925 mutant enzyme.

[0036] Application of the above-mentioned alginate lyase rAly06925 mutant enzyme in the degradation of alginate or alginate oligosaccharides.

[0037] According to a preferred embodiment of the present invention, the above-mentioned alginate lyase rAly06925 mutant enzyme is used in the degradation of alginate or alginate oligosaccharides to produce a series of G-rich oligosaccharide fragments containing ΔM at non-reducing ends.

[0038] A mutant alginate lyase rAly06925, wherein the amino acid mutation sites are: amino acid position 53 of amino acid sequence SEQ ID NO.2 is changed from isoleucine to alanine, amino acid position 52 is changed from phenylalanine to alanine, amino acid position 115 is changed from histidine to alanine, amino acid position 118 is changed from phenylalanine to alanine, amino acid position 119 is changed from asparagine to alanine, or amino acid position 243 is changed from histidine to alanine.

[0039] Preferably, the encoding gene of the alginate lyase rAly06925 mutant enzyme is mutated at a specific site on the encoding gene SEQ ID NO.1 according to the mutation site of the amino acid;

[0040] Preferably, a recombinant expression vector III contains the gene encoding the mutant enzyme;

[0041] Preferably, a recombinant bacterium III contains the gene encoding the mutant enzyme.

[0042] Application of the encoding gene of the above-mentioned alginate lyase rAly06925 mutant enzyme, recombinant expression vector III, and recombinant strain III in the preparation of alginate lyase rAly06925 mutant enzyme.

[0043] Application of the above-mentioned alginate lyase rAly06925 mutant enzyme in the degradation of alginate or alginate oligosaccharides.

[0044] According to a preferred embodiment of the present invention, the above-mentioned alginate lyase rAly06925 mutant enzyme is used in the degradation of alginate or alginate oligosaccharides to produce a series of G-rich oligosaccharide fragments containing ΔM at non-reducing ends.

[0045] A mutant alginate lyase rAly06925, wherein the amino acid mutation site is the deletion of amino acids 1 to 65 in the amino acid sequence SEQ ID NO.2;

[0046] Preferably, the encoding gene of the alginate lyase rAly06925 mutant enzyme is mutated at a specific site on the encoding gene SEQ ID NO.1 according to the mutation site of the amino acid;

[0047] Preferably, a recombinant expression vector IV contains the gene encoding the mutant enzyme;

[0048] Preferably, a recombinant bacterium IV contains the gene encoding the mutant enzyme.

[0049] The application of the encoding gene of the above-mentioned alginate lyase rAly06925 mutant enzyme, recombinant expression vector IV, and recombinant strain IV in the preparation of alginate lyase rAly06925 mutant enzyme.

[0050] Application of the above-mentioned alginate lyase rAly06925 mutant enzyme in the degradation of alginate or alginate oligosaccharides.

[0051] A mutant alginate lyase rAly06925, with the amino acid mutation site being the deletion of amino acids 45 to 60 in the amino acid sequence SEQ ID NO.2;

[0052] Preferably, the encoding gene of the alginate lyase rAly06925 mutant enzyme is mutated at a specific site on the encoding gene SEQ ID NO.1 according to the mutation site of the amino acid;

[0053] Preferably, a recombinant expression vector V contains the gene encoding the mutant enzyme;

[0054] Preferably, a recombinant bacterium V contains the gene encoding the mutant enzyme.

[0055] The application of the encoding gene of the above-mentioned alginate lyase rAly06925 mutant enzyme, recombinant expression vector V, and recombinant strain V in the preparation of alginate lyase rAly06925 mutant enzyme.

[0056] Application of the above-mentioned alginate lyase rAly06925 mutant enzyme in the degradation of alginate or alginate oligosaccharides.

[0057] A mutant alginate lyase rAly06925, with the amino acid mutation site being the 244th amino acid in the amino acid sequence SEQ ID NO.2, which is mutated to an amino acid other than aromatic hydrocarbon amino acids;

[0058] Preferably, the encoding gene of the alginate lyase rAly06925 mutant enzyme is mutated at a specific site on the encoding gene SEQ ID NO.1 according to the mutation site of the amino acid;

[0059] Preferably, a recombinant expression vector VI contains the gene encoding the mutant enzyme;

[0060] Preferably, a recombinant bacterium VI contains the gene encoding the mutant enzyme.

[0061] The application of the encoding gene of the above-mentioned alginate lyase rAly06925 mutant enzyme, recombinant expression vector VI, and recombinant strain VI in the preparation of alginate lyase rAly06925 mutant enzyme.

[0062] Application of the above-mentioned alginate lyase rAly06925 mutant enzyme in the degradation of alginate or alginate oligosaccharides.

[0063] The amino acid sequence SEQ ID NO.2 contains amino acids 1 through 65, which are Met. 1 ~Ala 65 .

[0064] The amino acid sequence SEQ ID NO.2 contains amino acids K from position 45 to position 60. 45 -N 60 Lys 45 ~Asn 60 .

[0065] The non-catalytic domain T65N (T65N is the amino acid sequence of Met) of the above-mentioned alginate lyase rAly06925 1 ~Ala 65 The non-catalytic domain plays an indispensable role in maintaining protein conformation, determining the hydrophilicity of the protein surface, and exerting catalytic function.

[0066] The catalytic motif of the aforementioned alginate lyase rAly06925 is N. 238 -N 239 -H 240 -G 241 -T 242 -H 243 And Y 87 Q 170 H 240 Y 295 These are key catalytic site residues. Specifically, the additional peptide K of rAly06925 is truncated. 45 -N 60 Or Y 244 Mutations to amino acids other than aromatic hydrocarbon amino acids all resulted in the recombinant protein losing its water solubility; therefore, the additional peptide K... 45 -N 60 and the Y contained therein 244 Residues play a crucial role in maintaining protein conformation or determining the hydrophilicity of protein surfaces.

[0067] Beneficial effects

[0068] 1. This invention discloses for the first time an alginate lyase rAly06925 obtained from the genome of Persicobacter sp. JZB09. It is the first report on the characteristics and application value of an alginate lyase from the genus Persicobacter. It is also the first report on a monoculture endoglucose lyase containing only ΔM terminus. This enzyme is significantly different from existing known alginate lyases. It has stable physicochemical properties, high activity, and has the potential for industrial application.

[0069] 2. The alginate lyase rAly06925 prepared in this invention has an alginate degradation activity of 135 U / mg, and is suitable for the production of a series of unsaturated oligosaccharides.

[0070] 3. The alginate lyase rAly06925 prepared in this invention, when degrading alginate polysaccharides composed of a random mixture of M and G, can only specifically recognize M-MMXn, G-MMXn, Δ-MMXn (n≥1, and a natural number; X, M, or G) motifs rich in M, and efficiently cleaves the glycosidic bond positions indicated by –, thereby generating a series of unsaturated oligosaccharide final products with non-reducing ends containing only ΔM. Therefore, it can be used to degrade alginate or alginate oligosaccharides to produce a series of G-rich unsaturated oligosaccharide fragments with non-reducing ends containing ΔM and unsaturated monosaccharides Δ.

[0071] 4. The series of mutants of the alginate lyase rAly06925 prepared in this invention revealed that the catalytic motif of this enzyme is N. 238 -N 239 -H 240 -G 241 -T 242 -H 243 And Y 87 Q 170 H 240 Y 295 These are key catalytic site residues. Specifically, the additional peptide K of rAly06925 is truncated. 45 -N 60 Or Y 244 Mutations to amino acids other than aromatic hydrocarbon amino acids all resulted in the recombinant protein losing its water solubility; therefore, the additional peptide K... 45 -N 60 and the Y contained therein 244 Residues play a crucial role in maintaining protein conformation or determining the hydrophilicity of the protein surface. The conserved motif N of the alginate lyase rAly06925 has been preliminarily identified. 238 -N 239 -H 240 -G 241 -T 242 -H 243 and key active site residue Y 87 Q 170 H 240 Y 295 Y 244 This discovery reveals that it differs from all previously identified alginate lyase families, suggesting a novel catalytic mechanism that requires further detailed explanation through structural biology research. This effectively supplements our understanding of the novel alginate lyase family and its conserved motifs, providing theoretical insights for related enzymological research. Attached Figure Description

[0072] Figure 1 Figure (A) shows the BLASTp analysis results of the functional modules of alginate lyase rAly06925 and the phylogenetic analysis figure (B) shows that the functional characteristics of alginate lyases with high consistency with some identified and unidentified functional characteristics are highly consistent.

[0073] In the figure: Aly06925 is the alginate lyase rAly06925;

[0074] Aly06925-T65N is a truncated form rAly06925-T65N.

[0075] Figure 2 , Multiple sequence alignment analysis of alginate lyase rAly06925 with alginate lyases with currently unknown functions;

[0076] In the figure: Aly06925 is the alginate lyase rAly06925.

[0077] Figure 3 Polyacrylamide gel electrophoresis (SDS-PAGE) images of the expression and purification of alginate lyase rAly06925 and its truncated form rAly06925-T65N.

[0078] In the figure: A represents alginate lyase rAly06925

[0079] M. Protein molecular weight standards, with band sizes from top to bottom as follows: 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD; Lane 1: Whole bacterial suspension of control strain after cell wall disruption, 4μL loading; Lane 2: Whole bacterial suspension of recombinant strain after cell wall disruption, 4μL loading; Lane 3: Supernatant of recombinant strain after cell wall disruption, 4μL loading; Lane 4: rAly06925 purified by nickel column, 2μL loading.

[0080] B is a truncated form rAly06925-T65N

[0081] M. Protein molecular weight standards, with band sizes from top to bottom as follows: 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD; Lane 1: Whole bacterial suspension of control strain after cell wall disruption, 4μL loading; Lane 2: Whole bacterial suspension of recombinant strain after cell wall disruption, 4μL loading; Lane 3: Supernatant of recombinant strain after cell wall disruption, 4μL loading; Lane 4: Truncated form rAly06925-T65N purified by nickel column, 2μL loading.

[0082] Figure 4HPLC analysis chromatogram of alginate lyase rAly06925 and truncated rAly06925-T65N enzyme activities;

[0083] In the figure: UDP2, unsaturated disaccharide; UDP3, unsaturated trisaccharide; UDP4, unsaturated tetrasaccharide; UDP5, unsaturated pentasaccharide; UDP6, unsaturated hexasaccharide; E(-) is the negative control without enzyme addition.

[0084] Figure 5 The effect of temperature on the activity of alginate lyase rAly06925 is shown in the graph.

[0085] Figure 6 The effect of temperature on the stability of alginate lyase rAly06925 is shown in the graph.

[0086] Figure 7 The effect of pH on the activity of alginate lyase rAly06925 is shown in the graph.

[0087] Figure 8 Effect curves of pH on the stability of alginate lyase rAly06925.

[0088] Figure 9 Bar graph showing the effects of metal ions and chemical reagents on the activity of alginate lyase rAly06925.

[0089] Figure 10 Effect curves of NaCl concentration on the activity of alginate lyase rAly06925.

[0090] Figure 11 Molecular gel chromatography-HPLC analysis of oligosaccharide products during the degradation of alginate by alginate lyase rAly06925.

[0091] Figure 12 HPLC analysis chromatogram (A) of unsaturated oligosaccharide fragments UDP2, UDP3, UDP4, UDP5, and UDP6 prepared after complete degradation of alginate by alginate lyase rAly06925. 1 H-NMR analysis diagram (B).

[0092] Figure 13 HPLC analysis of the series of saturated oligosaccharides M3-M7(A) and G5(B) completely degraded by alginate lyase rAly06925.

[0093] Figure 14 HPLC analysis of the complete degradation of 2-AB-labeled saturated oligosaccharides M3-M6 by alginate lyase rAly06925;

[0094] In the figure: (-) is the negative control without enzyme.

[0095] Figure 15 HPLC detection chromatogram of characteristic unsaturated oligosaccharides UDP3-UDP6 degraded by alginate lyase rAly06925.

[0096] Figure 16 Three-dimensional simulated structure of alginate lyase rAly06925 (A) and comparison diagram (B);

[0097] In Figure B: red represents rAly06925, green represents Pae-AlgL, the structure of which has been identified in PL5, and yellow represents the additional peptide segment (T45-60N) of rAly06925.

[0098] Figure 17 Bar graph showing the relative enzyme activity analysis of the truncated form of alginate lyase rAly06925, T45-60N, and its series of mutants.

[0099] Figure 18 HPLC(A) analysis of the degradation end products of the alginate lyase rAly06925 mutant I53A and 1 H-NMR (B) analysis diagram. Detailed Implementation

[0100] The following embodiments are provided to fully disclose some common techniques for implementing the present invention, and not to limit the scope of application of the invention. The inventors have made every effort to ensure the accuracy of the parameters (e.g., quantities, temperatures, etc.) in the embodiments; however, some experimental errors and deviations should also be taken into account. Unless otherwise stated, molecular weight in the present invention refers to weight-average molecular weight, and temperature is in degrees Celsius.

[0101] Biological material sources

[0102] Persicobacter sp. JZB09 is deposited at the China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The deposit date is April 12, 2021, and the accession number is CCTCC M2021354.

[0103] The alginate used in this invention was purchased from Sigma, imidazole from Amresco, o-aminobenzamide from Sigma-aldrich, and the alginate series of saturated oligosaccharides from Qingdao Bozhi Huili Biotechnology Co., Ltd.

[0104] Example 1

[0105] Extraction of genomic DNA from Persicobacter sp. JZB09 strain

[0106] In October 2009, the inventors collected marine mud samples from the tidal flats near Hongdao Island in Jiaozhou Bay, Qingdao, and screened a polysaccharide-degrading bacterium, JZB09, using a single carbon source method. This bacterium was molecularly identified as *Persicobacter sp.* JZB09. The *Persicobacter sp.* JZB09 strain was inoculated into M10 liquid medium and cultured at 28°C and 200 rpm with shaking until an absorbance value of 600 nm (OD) was reached. 600 The value was 1.18; 20 mL of the culture solution was taken and centrifuged at 28℃ and 12,000 × g (g, the gravitational constant of the Earth) for 20 min, and the bacterial precipitate was collected.

[0107] The above-mentioned M10 liquid culture medium is composed of: 4 g / L tryptone, 2.5 g / L yeast extract, 7.5 g / L potassium chloride, 20 g / L sodium chloride, 1.1 g / L calcium chloride, 7.2 g / L magnesium sulfate, 1.5 g / L ammonium chloride, and the balance being water, with a pH of 7.2.

[0108] Add 12.0 mL of lysozyme buffer to the above bacterial precipitate to obtain approximately 14.0 mL of bacterial solution. Add 560 μL of lysozyme at a concentration of 20 mg / mL, resulting in a final concentration of approximately 800 μg / mL. Incubate in an ice-water bath for 1.0 h, then transfer to a 37°C water bath and incubate for 2 h until the reaction system becomes viscous. Add 0.82 mL of sodium hexadecyl sulfonate solution (100 mg / mL) and 60 μL of proteinase K solution (100 mg / mL), and incubate at 52°C for 1.0 h. Add 15 mL of Tris-equilibrated phenol / chloroform / isoamyl (volume ratio 25:24:1), and gently invert to mix until fully emulsified. Centrifuge at 10,000 × g and 4°C for 10 min, collect the supernatant, and add 2.0 mL of NaAc-HAc (pH 10). Mix 5.2M and 3.0M buffer with 17.0 mL of anhydrous ethanol (stored at -20℃) thoroughly; pick up the filamentous DNA with a pipette tip and transfer it to a 1.5 mL centrifuge tube, wash twice with 70% ethanol, centrifuge briefly and discard the supernatant; centrifuge at 10,000×g at 4℃ for 2 min and discard the supernatant completely; air dry the DNA precipitate in a sterile workbench, and then dissolve the DNA sample in sterile deionized water overnight at 4℃ to obtain genomic DNA.

[0109] Example 2

[0110] Genome scanning and sequence analysis of Persicobacter sp. JZB09 strain

[0111] The genomic DNA obtained in Example 1 was sequenced using pyrosequencing technology by Shanghai Meiji Biotechnology Co., Ltd. The DNA sequencing results were analyzed using online software from the NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ) website. The NCBI analysis software used was 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).

[0112] Analysis using the aforementioned biological software revealed that the genomic DNA of *Persicobacter sp.* JZB09 carries a candidate alginate lyase gene, aly06925, encoding an alginate lyase rAly06925. The full-length alginate lyase rAly06925 is 1197 bp, and its nucleotide sequence is shown in SEQ ID NO.1, containing 399 amino acids. The N-terminal amino acids 1-18 form a type I signal peptide, and its amino acid sequence is shown in SEQ ID NO.2. Online BLAST analysis showed that the alginate lyase rAly06925 contains a putative domain, namely the Alginate_Lyase superfamily (e.g., ...). Figure 1 -A shows that it has the highest sequence identity (9%) with AlgL from the PL-5 family of alginate lyases (derived from Azotobacter vinelandii). Phylogenetic analysis shows that rAly06925 is most closely related to the PL-5 and PL-17 families (e.g., as shown in Figure A). Figure 1 (as shown in -B), but it does not cluster with any of the identified PL-5 or PL-17 family members. Instead, it clusters with unidentified alginate lyases and is located within a branch. Therefore, it should be classified into a new, unreported family of polysaccharide lyases. Multiple sequence comparison with currently unidentified alginate lyases revealed that rAly06925 contains two potential catalytic motifs (such as...). Figure 2 As shown), that is, N respectively. 238 -N 239 -H 240 -G 241 -T 242 -H 243 N 113 -N114 -H 115 -T 116 -D 117 -F 118 .

[0113] Example 3

[0114] Recombinant expression of gene aly06925 and its truncated form aly06925-T65N in Escherichia coli BL21(DE3) strain

[0115] Using the genomic DNA obtained in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0116] Forward primer rAly06925-F: 5'-g CATATG CAAAAAACCATTTCATTAACGG-3'(Nde I), SEQ IDNO.3;

[0117] Reverse primer rAly06925-R: 5'-g CTCGAG TTGGAAAAACGCTTCCACGAAATTCC-3'(Xho I), SEQ ID NO.4;

[0118] Forward primer rAly06925-T65N-F: 5'-GTTACGCATAAAACGGGTGTTCCACC-3', SEQ ID NO.5;

[0119] Reverse primer rAly06925-T65N-R: 5'-CATATGTATATCTCCTTCTTAAAGTTAAAC-3', SEQ ID NO.6.

[0120] The underlined site in the forward primer rAly06925-F is the restriction endonuclease Nde I site, and the underlined site in the reverse primer rAly06925-R is the restriction endonuclease Xho I site. The high-fidelity DNA polymerase PrimeSTARHS was purchased from Takara Bio Inc., Dalian, China, and the PCR reagents were used according to the company's product instructions.

[0121] PCR reaction conditions: 94℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s; 65℃ annealing for 30 s; 72℃ extension for 170 s; 35 cycles; 72℃ extension for 10 min; 5℃ stabilization for 10 min.

[0122] The PCR product was ligated into the pEASY-Blunt simple vector and transformed into *E. coli* Trans1-T1 strain. The transformed product was plated on Luria-Bertani agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 16 h. Single colonies were then picked and inoculated into liquid Luria-Bertani agar containing 50 μg / mL kanamycin. Plasmids were extracted and verified by PCR using amplification primers. The verified recombinant plasmid was then analyzed using Nde I and Xho... The plasmid was double-digested with enzyme I and ligated with the pET30a plasmid vector, which had also been double-digested, under the catalysis of DNA ligase. The ligation product was transformed into Escherichia coli DH5α strain, plated on Luria-Bertani agar plates containing 100 μg / mL kanamycin, and incubated at 37°C for 16 h. Single clones were then picked and inoculated into liquid Luria-Bertani agar containing 100 μg / mL kanamycin. The plasmid was then extracted. The plasmid was double-digested to verify the amplification product, and the results showed that the size and orientation of the amplified product were correct, which initially proved that the constructed recombinant plasmid was correct. The recombinant plasmid was then sequenced, and the results showed that the gene aly06925 shown in SEQ ID NO.1 was inserted between the Nde I and Xho I restriction sites of the pET30a plasmid, and the insertion orientation was correct. Therefore, the construction of the recombinant plasmid was further confirmed to be correct, and the recombinant plasmid was named pET30a-Aly06925.

[0123] To verify the function of the non-catalytic domain of this alginate lyase rAly06925, the following was performed: Figure 1 The shown alginate lyase rAly06925 functional module performs T65N (removing Met from the amino acid sequence) 1 ~Ala 65The non-catalytic domain portion was truncated. To obtain the truncated recombinant plasmid rAly06925-T65N, PCR amplification was performed using recombinant plasmid pET30a-Aly06925 as a template and primers rAly06925-T65N-F and rAly06925-T65N-R. After gel recovery of the PCR product, terminal phosphorylation and circularization ligation were performed. The ligation product was transformed into E. coli DH5α, plated on LB agar plates containing 50 μg / mL Kana, and incubated at 37°C for 14 h. Single colonies were picked and inoculated into agar plates containing 50 μg / mL Kana. Kana was cultured in liquid LB medium at 37°C with shaking for 12 hours. PCR verification was performed using amplification primers, which initially confirmed the correctness of the constructed recombinant plasmid. Plasmid extraction was then performed using a plasmid miniprep kit (Tiangen Biotech Co., Ltd.). The recombinant plasmid was then sequenced to verify the insertion of the correct size and orientation of the target fragment. The successfully constructed recombinant plasmid was named pET30a-Aly06925-T65N. However, after induction, the expression resulted in inclusion bodies. Therefore, the recombinant plasmid pET30a-Aly06925-T65N was double-digested with restriction endonucleases Nde I and Xho I, and the digested fragments were recovered by agarose gel electrophoresis. Similarly, the pCold TF plasmid was digested with restriction enzymes Nde I and Xho I, and the digested fragments were also recovered by agarose gel electrophoresis. Then, the two fragments were ligated using DNA ligase. The ligation product was transformed into *E. coli* DH5α, plated on LB agar plates containing 50 μg / mL Amp, and incubated upside down at 37°C for 14 h. Single colonies were picked and inoculated into a medium containing 50 μg / mL Amp. The plasmid was cultured in liquid LB medium at 37°C with shaking for 12 hours. PCR verification was performed using amplification primers, which initially confirmed the correctness of the constructed recombinant plasmid. Plasmid extraction was then performed using a plasmid miniprep kit (Tiangen Biotech Co., Ltd.). The recombinant plasmid was then sequenced to verify the size and orientation of the inserted gene sequence. The successfully constructed recombinant plasmid was named pColdTF-Aly06925-T65N.

[0124] Recombinant plasmids pET30a-Aly06925, pET30a, and pCold TF-Aly06925-T65N were transformed into *E. coli* strain BL21(DE3) (purchased from Invitrogen, USA). Following the company's instructions, the expression of alginate lyase rAly06925 and the truncated form rAly06925-T65N was induced using isopropyl thiogalactoside (IPTG). The cells were collected by centrifugation at 8,000 × g, 4 °C for 15 min, resuspended in buffer A, and sonicated in an ice-water bath. The cells were further centrifuged at 15,000 × g, 4 °C for 30 min, and the water-soluble fraction was collected. The alginate lyase rAly06925 and rAly06925-T65N were purified using Ni-agarose gel electrophoresis. Gradient elution was performed using buffer A (50 mM Tris, 150 mM NaCl, pH 8.0) containing imidazole at concentrations of 10, 50, 250, and 500 mM, following the purification conditions specified in the gel's product manual. The purification status of the recombinase was assessed using polyacrylamide gel electrophoresis. Results are shown below. Figure 3 As shown in -A: After IPTG induction, the recombinant plasmid pET30a-Aly06925 was expressed in E. coli BL21(DE3) strain. The water-soluble product accounted for 95%. The alginate lyase rAly06925 purified by nickel column affinity chromatography showed a single band on electrophoresis, and the position matched the predicted molecular weight. The purified alginate lyase rAly06925 and rAly06925-T65N samples were placed in dialysis bags with a minimum molecular weight cutoff of 10 kDa and dialyzed at 4°C to prepare alginate lyase rAly06925 enzyme solution with a concentration of 3 μg / μL and a truncated rAly06925-T65N enzyme solution with a concentration of 2 μg / μL. These enzyme solutions were used for subsequent experiments.

[0125] Example 4

[0126] Activity verification of alginate lyase rAly06925 and its truncated form rAly06925-T65N

[0127] A 12 g / L alginate substrate solution prepared with deionized water was mixed with the alginate lyase rAly06925 enzyme solution or the truncated rAly06925-T65N enzyme solution prepared in Example 3, and 150 mM HAc-NaAc (pH 6.0) buffer at a ratio of 1:1:1 (v / v) and reacted at 30°C for 4 h. The reaction product was heated in a boiling water bath for 10 min to inactivate the enzyme, transferred to an ice-water bath for 5 min, and centrifuged at 12,000 × g at 4°C for 15 min. The supernatant was collected and analyzed by HPLC. A Superdex Peptide 10 / 300GL (GE) molecular gel chromatography column was equilibrated with 0.20 M NH4HCO3 solution at a flow rate of 0.40 mL / min for at least two column beds. A portion of the supernatant obtained above was loaded with 20 μg / sample using an autosampler, and the other conditions remained unchanged. Detection was performed at 232 nm.

[0128] The results are as follows Figure 4 As shown, the alginate lyase rAly06925 can degrade alginate well, but the truncated form rAly06925-T65N cannot degrade alginate. This indicates that the non-catalytic domain of rAly06925 plays an indispensable role in maintaining protein conformation, determining the hydrophilicity of the protein surface, and exercising catalytic function. Furthermore, the enzyme activity of rAly06925, determined by the DNS reducing sugar method, is approximately 135 U / mg.

[0129] Example 5

[0130] Determination of the optimal temperature for alginate lyase rAly06925

[0131] Alginate substrate with a mass concentration of 12 g / L was prepared using deionized water, sterilized, and cooled to room temperature. It was then incubated in water baths at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C for 30 min each. 30 μL of the alginate lyase rAly06925 prepared in Example 3, 100 μL of 150 mM HAc-NaAc (pH 6.0) buffer, and 70 μL of deionized water were added to each 100 μL substrate solution. After mixing, the reaction was continued for 4 h. Three parallel samples were prepared at each temperature condition, with a recombinant enzyme preparation inactivated by boiling water bath serving as a control. The concentration (OD) of newly generated reducing sugar in each reaction system was determined using the DNS-reducing sugar method. 540 The average value was calculated, and a deviation analysis was performed. The reaction temperature corresponding to the maximum absorbance value is the optimum temperature of the recombinant enzyme. The relative enzyme activity (RA) is defined as the percentage of each absorbance value to the maximum absorbance value.

[0132] The results are as follows Figure 5As shown, when using alginate as a substrate to determine enzyme activity, alginate lyase rAly06925 reached its maximum activity at 35℃, indicating that the optimal reaction temperature for alginate lyase rAly06925 is 35℃. Compared to other temperatures, alginate lyase rAly06925 only exhibits a relative enzyme activity greater than 80% at 30℃-35℃, suggesting that alginate lyase rAly06925 is an alginate lyase adapted to the marine environment.

[0133] Example 6

[0134] Temperature stability analysis of alginate lyase rAly06925

[0135] The alginate lyase rAly06925 prepared in Example 3 was treated at different temperatures (0–60°C) for 0.5 h, 1 h, 2 h, 4 h, 12 h, and 24 h, respectively. Each mixture was then mixed with alginate substrate prepared in distilled water at a mass-to-volume concentration of 12 g / L at a volume ratio of 1:9. The mixtures were then reacted at the optimal temperature for 4 h, and residual enzyme activity was measured. Three parallel samples were prepared at each temperature, with the alginate lyase preparation inactivated by boiling water bath serving as the control group. The enzyme activity of the untreated enzyme solution was defined as 100% relative activity.

[0136] The results are as follows Figure 6 As shown, after pretreatment at temperatures below 20°C for 12 hours or at 30°C for 2 hours, the alginate lyase rAly06925 still retained more than 50% of its residual enzyme activity. However, after pretreatment at temperatures above 40°C for 2 hours, the residual enzyme activity of alginate lyase rAly06925 decreased sharply, and the rate of decrease accelerated at higher temperatures. This indicates that alginate lyase rAly06925 possesses a certain degree of thermostability.

[0137] Example 7

[0138] Determination of the optimal pH and stability of alginate lyase rAly06925

[0139] Alginate substrates with a mass concentration of 12 g / L were prepared by reacting alginate with 50 mM NaAc-HAc buffer, NaH2PO4-Na2HPO4 buffer, and Tris-HCl buffer, respectively, corresponding to three pH ranges: 5, 6; 6, 7, 8; and 8, 9, 10. Each pH value was adjusted at the optimum temperature. After dissolving the substrate, the mixture was incubated at the optimum temperature for 30 min. Then, 30 μL of the alginate lyase rAly06925 prepared in Example 3, 100 μL of each of the above buffer solutions, and 70 μL of deionized water were added to every 100 μL of substrate solution. The mixture was then stirred and the reaction was continued for 4 h. Three parallel samples were prepared for each pH condition, with alginate lyase preparation inactivated by boiling water bath serving as a control. The concentration of newly generated reducing sugar (OD) in each reaction system was determined using the DNS-reducing sugar method. 540 The average value was calculated, and a deviation analysis was performed. The reaction pH corresponding to the maximum absorbance value is the optimal pH for alginate lyase. The relative enzyme activity (RA) is defined as the percentage of each absorbance value relative to the maximum absorbance value.

[0140] Simultaneously, 30 μL of the alginate lyase rAly06925 prepared in Example 3 was mixed with 100 μL of each of the above buffer solutions, and incubated at 35°C for 2 h. Then, it was mixed with 100 μL of alginate substrate with a mass concentration of 12 g / L, and reacted at the optimal temperature for 4 h, with residual enzyme activity measured. Three parallel samples were prepared for each pH condition, with the alginate lyase preparation inactivated by boiling water bath serving as a control. The concentration of newly generated reducing sugar (OD) in each reaction system was determined using the DNS-reducing sugar method. 540 The enzyme activity was calculated and averaged, and a deviation analysis was performed. The enzyme activity of the untreated enzyme solution was defined as 100% relative activity.

[0141] The results are as follows Figure 7 As shown, the optimal reaction pH for alginate lyase rAly06925 is 6.0. In slightly acidic or alkaline environments, the activity of rAly06925 decreases sharply. These results indicate that the enzyme is adapted to neutral to slightly acidic buffer conditions but not to harsh environments such as strong acids or bases. After pretreatment with different buffers for 2 hours, the activity of alginate lyase rAly06925 remained above 50% within the pH range of 6.0-8.0; however, at pH 5.0, pH 9.0, and pH 10.0, the residual enzyme activity of alginate lyase rAly06925 decreased sharply, falling below 20%. This indicates that alginate lyase rAly06925 has a certain pH tolerance and is relatively adapted to neutral environments. Figure 8 As shown.

[0142] Example 8

[0143] Effects of metal ions and chemical reagents on the activity of alginate lyase rAly06925

[0144] A reaction system was prepared by mixing a 12 g / L alginate substrate (prepared with deionized water), the alginate lyase rAly06925 prepared in Example 3, and water in a volume ratio of 5:1:4. Different metal ions or chemical reagents were then added to the reaction system until the final concentration reached 1 mM or 10 mM. The reaction was then carried out at 35°C for 4 hours, with three replicates for each sample. Enzyme activity was determined using the DNS-reducing sugar method described above. The control group, representing the activity of rAly06925 without the addition of any metal ions or chemical reagents, was set at 100%.

[0145] The results are as follows Figure 9 As shown, 1mM Ca 2+ 1 mM DTT (dithiothreitol) significantly promoted the activity of alginate lyase rAly06925, increasing the relative activity by approximately 25%; K + Li + Na + Mg 2+ The chemical reagent Glyerol had no significant effect on the enzyme activity of alginate lyase rAly06925; however, at concentrations of 1 mM or 10 mM, Ag... + Cu 2+ Hg 2+ Cr 3+ Mn 2+ Ni 2+ Zn 2+ Fe 2+ Fe 3+ Heavy metal ions and chemical reagents such as SDS, EDTA, and imidazole significantly inhibited the activity of alginate lyase rAly06925; in addition, 1 mM Co 2+ 10mM Pb 2+ It also has a significant inhibitory effect on the enzyme activity of alginate lyase rAly06925.

[0146] Example 9

[0147] Effect of NaCl on the activity of alginate lyase rAly06925

[0148] Prepare a 5M NaCl stock solution using deionized water. Add 30μL of the alginate lyase rAly06925 prepared in Example 3, 100μL of 150mM HAc-NaAc (pH 6.0) buffer, and 70μL of the NaCl stock solution diluted with an appropriate amount of deionized water to each 100μL substrate solution, resulting in final NaCl concentrations of 0M, 0.2M, 0.4M, 0.6M, 0.8M, and 1M. React at the optimal temperature for 4 hours at each concentration. Three parallel groups were set up for each concentration, with the enzyme preparation inactivated by boiling water bath serving as the control group. Enzyme activity was determined using the aforementioned DNS-reducing sugar method. The control group represented the activity of rAly06925 without NaCl, set at 100%.

[0149] The results are as follows Figure 10 As shown, when the NaCl concentration is 0-0.2M, the activity of alginate lyase rAly06925 increases with increasing NaCl concentration; however, when the NaCl concentration is 0.2-1.0M, the activity of alginate lyase rAly06925 decreases with increasing NaCl concentration. Among these, 0.2M NaCl has the highest promoting effect on alginate lyase rAly06925, increasing its relative activity by about 60%. In the range of 0-0.6M, the activity of alginate lyase rAly06925 remains above 50%. This indicates that alginate lyase rAly06925 has a certain degree of NaCl tolerance.

[0150] Example 10

[0151] High-performance liquid chromatography (HPLC) analysis of products from the degradation of alginate by the alginate lyase rAly06925.

[0152] Prepare 100 μL each of 12 g / L alginate and 150 mM NaAc-HAc (pH 6.0) buffer using deionized water. Add 70 μL of deionized water, mix well, and incubate at 35°C for 1 h. Add 30 μL of the alginate lyase rAly06925 prepared in Example 3, mix well, and continue the reaction, taking samples at intervals. Heat the reaction product in a boiling water bath for 10 min, then transfer it to an ice-water bath for 5 min. Centrifuge at 12,000 × g and 4°C for 15 min, and collect the supernatant.

[0153] A Superdex Peptide 10 / 300GL (GE) molecular gel chromatography column was equilibrated with 0.20 M NH4HCO3 solution at a flow rate of 0.40 mL / min for at least two column beds. The alginate-digested sample was loaded at 20 μg / sample using an autosampler, with other conditions unchanged, and detected at 232 nm. The integral area of ​​each oligosaccharide component was analyzed using HPLC software, and the relative molar concentration was calculated. Figure 11 As shown, under the above conditions, alginate lyase rAly06925 initially produced oligosaccharides with relatively large molecular weights when degrading alginate substrates. With increasing reaction time, the content of oligosaccharide products with a characteristic absorption peak at 235 nm gradually increased and eventually stabilized. The final main products of the reaction were five oligosaccharide products with elution times of 41.8', 39.1', 36.5', 34.7', and 33.4' as detected by HPLC. Referring to the elution times of molecular weight standards, each oligosaccharide product was identified as UDP2, UDP3, UDP4, UDP5, and UDP6, with a molar ratio of approximately 6:4:3:2:1. This indicates that alginate lyase rAly06925 is an endo-type alginate lyase.

[0154] Example 11

[0155] The alginate lyase rAly06925 degrades alginate end products. 1 H-NMR spectral analysis

[0156] Following the optimal reaction system, 36 mg of alginate was completely degraded using the alginate lyase rAly06925. After the reaction was complete, the sample underwent a series of treatments, and the supernatant was obtained. The sample was then analyzed using a Superdex molecular gel chromatography column. TM Peptide 10 / 300GL (GE) was used to separate and purify the final product of alginate completely degraded by the alginate lyase rAly06925. The purity was determined by HPLC after combining the precipitates, with chromatographic conditions as described in Example 10. Figure 12 As shown in -A, the purity of the final product is above 99%. After freeze-drying and desalting, followed by hydrogen-deuterium replacement with heavy water, it is then subjected to... 1 The structural characteristics of the above oligosaccharide products were analyzed by ¹H-NMR. The results are as follows: Figure 12 As shown in Figure -B, all the series of unsaturated oligosaccharide products exhibited a characteristic absorption peak at 5.57 ppm, with only a ΔM signal peak observed and no ΔG signal peak detected. This indicates that the non-reducing ends of the final oligosaccharide products obtained from the degradation of M / G segmental alginate by rAly06925 are all ΔM structures.

[0157] Example 12

[0158] The degradation characteristics of alginate lyase rAly06925 on alginate-saturated oligosaccharide substrates.

[0159] To further elucidate the oligosaccharide generation characteristics of rAly06925, the substrate selectivity and oligosaccharide substrate degradation mode of this enzyme were systematically analyzed. Equal molar amounts of saturated oligosaccharides, including poly-M segment oligosaccharides (M3-M7) and poly-G segment oligosaccharides (G3-G7), were reacted with alginate lyase rAly06925 under optimal conditions for 24 h. After processing the reaction solution, HPLC analysis was performed. The degree of polymerization of each oligosaccharide product was determined by referring to molecular weight standards. The integral area of ​​each oligosaccharide component was analyzed, and the relative molar concentration was calculated. The chromatographic conditions were the same as in Example 10. The results are as follows: Figure 13 As shown in -A, significant degradation of M-series saturated oligosaccharides was observed, but M3 was not degraded. Degradation of M7, M6, and M5 primarily produced UM2 and UM3, with some UM4. Degradation of M4 primarily produced UM3 and some UM2. No degradation of G-series saturated oligosaccharides was observed (only the detection results of alginate lyase rAly06925 on G5 degradation are shown here), as... Figure 13 -B is shown.

[0160] The above results indicate that alginate lyase rAly06925 is an M-specific alginate endonuclease, M4 is the smallest saturated oligosaccharide substrate of alginate lyase rAly06925, and M is the smallest saturated oligosaccharide product of rAly06925.

[0161] Example 13

[0162] Degradation characteristics of alginate lyase rAly06925 on fluorescently labeled alginate saturated oligosaccharide substrates

[0163] Approximately 10 μg of saturated poly(M) oligosaccharides was added to a dimethyl sulfoxide (DMSO) solution containing excess o-aminobenzamide (2-AB) and sodium borohydride. After mixing, the mixture was incubated in a 60°C water bath for 2 h. The mixture was then evaporated to dryness by rotation. 500 μL of deionized water was added to dissolve the sample. The sample was then shaken with 200 μL of chloroform, centrifuged, and the supernatant was collected. The sample was repeatedly extracted with chloroform at least 7 times to obtain a reaction solution with fluorescently labeled reducing ends. Saturated poly(M) trisaccharides (2AB-M3), tetrasaccharides (2AB-M4), pentasaccharides (2AB-M5), and hexasaccharides (2AB-M6) with fluorescently labeled reducing ends were used as substrates. The substrates were reacted with alginate lyase rAly06925 under optimal conditions for 24 h. After processing, the reactants were analyzed by HPLC. The relative molecular weights of each oligosaccharide product were determined by referring to molecular weight standards. The integral area of ​​each oligosaccharide component was analyzed, and the relative molar concentration was calculated. HPLC detection conditions: Column: Superdex TM Peptide 10 / 300GL; Detector: Fluorescence detector, excitation wavelength Ex 330nm, detection wavelength Em 420nm; Mobile phase: 0.2M NH4HCO3.

[0164] like Figure 14 As shown, when the alginate lyase rAly06925 degrades 2AB-M6, the main products are 2AB-UM3 and 2AB-UM4 in a molar ratio of approximately 1.4:1, with small amounts of 2AB-UM5 and 2AB-UM2 also produced. When degrading 2AB-M5, the main products are 2AB-UM3 and 2AB-UM4 in a molar ratio of approximately 2:1, with a small amount of 2AB-UM2 also produced. Furthermore, when 2AB-M4 is incompletely degraded, the main product is 2AB-UM3, but 2AB-M3 is not degraded.

[0165] The above results indicate that 2AB-M4 is the smallest artificially synthesized M-series saturated oligosaccharide substrate of rAly06925, with M being the smallest saturated oligosaccharide product exhibiting a variable substrate degradation mode. The 2-AB labeling at the reducing end has no significant effect on the enzyme activity, indicating that the enzyme degrades the alginate substrate from the non-reducing end.

[0166] Example 14

[0167] The degradation characteristics of alginate lyase rAly06925 on unsaturated oligosaccharides in alginate substrates.

[0168] To further investigate the enzymatic properties of this enzyme, based on the existing experimental foundation of our research group, we used the M-prone alginate exonuclease Aly6.

[19] Incomplete degradation of alginate was achieved using a Superdex molecular gel chromatography column. TM The degradation products were separated and purified using Peptide 10 / 300GL (GE) to prepare a series of characteristic oligosaccharide products with only ΔG as the non-reducing terminator. These oligosaccharide fragments were then reacted with alginate lyase rAly06925 under optimal conditions for 24 h. After processing the reaction solution, HPLC analysis was performed, with chromatographic conditions as described in Example 10.

[0169] like Figure 15 As shown, the alginate lyase rAly06925 partially degrades UDP6 to produce UDP4, UDP3, and UDP2, and minutely degrades UDP5 to produce UDP4 and unsaturated monosaccharides (Δ), without degrading UDP4 and UDP3. These results indicate that the alginate lyase rAly06925 degrades unsaturated oligosaccharides via an endoglucosamine process. The smallest unsaturated oligosaccharide substrate is UDP5, but the degradation is very weak. This is presumably because the aforementioned series of unsaturated oligosaccharides are obtained from the incomplete degradation of alginate by Aly6. Their non-reducing ends were identified as ΔG units, which is consistent with the finding that rAly06925 is an M-specific alginate lyase.

[0170] Furthermore, combined Figure 11 , Figure 12 , Figure 13 and Figure 15It is speculated that the oligosaccharide generation characteristics of alginate lyase rAly06925 during alginate degradation are determined by both substrate selectivity and substrate degradation mode: when degrading alginate polysaccharide substrates, rAly06925 specifically degrades M-rich regions, namely M-MMXn, G-MMXn, Δ-MMXXn (n≥1 and a natural number; X, G or M), and efficiently cleaves the glycosidic bond positions shown in the figure, thereby generating a series of unsaturated oligosaccharide final products with only ΔM as the non-reducing terminus.

[0171] Example 15

[0172] Homology modeling of alginate lyase rAly06925

[0173] Bioinformatics analysis revealed that the sequence of alginate lyase rAly06925 is novel, with an amino acid sequence share of only 9% with the identified alginate lyases (AlgL from the PL5 family derived from Azotobacter vinelandii). Furthermore, rAly06925 exhibits a unique evolutionary position; although closely related to the PL5 and PL17 families, it does not cluster with any identified PL5 or PL17 family members, instead forming its own distinct branch. Multiple sequence alignment analysis identified two sets of candidate motifs, NNH (N... 113 -N 114 -H 115 and N 238 -N 239 -H 240 This sequence (NNHSYW) is not entirely identical to the reported conserved sequence of PL5, making it impossible to identify convincing key catalytic residues through bioinformatics analysis, nor can it be directly classified into a family, and no similar studies have been reported. Furthermore, we speculate that rAly06925 may contain other key amino acid residues besides NNH. Therefore, we used SWISS-MODEL online to simulate its three-dimensional structure, using Pae-AlgL and AI-Ⅲ, which have been identified in PL5, as templates for the three-dimensional simulation. Figure 16 As shown in Figure -A, rAly06925 consists of numerous α-helical structures and possesses an α / α-column structure, similar in structure to the AlgL alginate lyases identified in the PL5 family derived from *Pseudomonas aeruginosa* and *Azotobacter vinelandii*. It also contains a tunnel-like catalytic cavity, presumably the basis for its alginate lysin activity. Comparison using PyMOL software revealed that rAly06925 has an additional α-helix (K... 45 -N 60 ) and some assumed key amino acid residues (Y 48 F52 I 53 Y 87 H 115 F 118 N 119 Q 170 H 240 H 243 Y 244 Y 295 ),like Figure 16 -B is shown.

[0174] Example 16

[0175] Molecular modification and catalytic mechanism study of alginate lyase rAly06925

[0176] To obtain the recombinant plasmid T45-60N, a truncated form of the alginate lyase rAly06925, PCR amplification was performed using the recombinant plasmid pET30a-Aly06925 as a template and Aly06925-T45-60N-F and Aly06925-T45-60N-R as primers. After gel extraction, the PCR product was phosphorylated at the 5' end and ligated using T4 DNA ligase. The ligation product was transformed into *E. coli* DH5α, plated on LB agar plates containing 50 μg / mL Kana, and incubated at 37°C for 14 h. Single colonies were picked and inoculated into agar plates containing 50 μg / mL Kana. Kana was cultured in liquid LB medium at 37°C with shaking for 12 hours. PCR verification was performed using amplification primers, which initially confirmed the correctness of the constructed recombinant plasmid. Plasmid extraction was then performed using a plasmid miniprep kit (Tiangen Biotech Co., Ltd.). The recombinant plasmid was then sequenced to verify the insertion of the target fragment of the correct size and orientation. The successfully constructed recombinant plasmid was named pET30a-Aly06925-T45-60N. However, after induction, the expression occurred in the form of inclusion bodies. The recombinant plasmid pET30a-Aly06925-T45-60N was also double-digested with restriction endonucleases Nde I and Xho I, and the digested fragments were recovered by agarose gel electrophoresis. Similarly, the pCold TF plasmid was digested with restriction enzymes Nde I and Xho I, and the digested fragments were also recovered by agarose gel electrophoresis. The two fragments were then ligated using DNA ligase. The ligation products were transformed into *E. coli* DH5α, plated on LB agar plates containing 50 μg / mL Amp, and incubated upside down at 37°C for 14 h. Single colonies were picked and inoculated into a medium containing 50 μg / mL Amp. The plasmid was cultured in liquid LB medium at 37°C with shaking for 12 hours. PCR verification was performed using amplification primers, which initially confirmed the correctness of the constructed recombinant plasmid. Plasmid extraction was then performed using a plasmid miniprep kit (Tiangen Biotech Co., Ltd.). The recombinant plasmid was then sequenced to verify the size and orientation of the inserted gene sequence. The successfully constructed recombinant plasmid was named pCold TF-Aly06925-T45-60N.

[0177] To obtain recombinant plasmids of the alginate lyase rAly06925 series mutants, the recombinant plasmid pET30a-Aly06925 was used as a template. PCR amplification was performed using the corresponding mutant primers (Table 1) and the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase to obtain the amplified fragments. The methylated template plasmid in the amplified product was removed using the enzyme DpnI. Homologous recombination was then performed at the 5' and 3' ends of the amplified product under the catalysis of the enzyme Exnase II, completing the circularization of the amplified product. After the reaction was complete, the circularized amplified product was transformed into *E. coli* DH5α, plated on LB agar plates containing 50 μg / mL Kana, and incubated at 37°C for 14 h. Single clones were picked and inoculated into agar plates containing 50 μg / mL Kana. Kana was cultured in liquid LB medium at 37°C with shaking for 12 hours. PCR verification using mutant primers initially confirmed the correctness of the constructed recombinant plasmid. Plasmid extraction was performed using a plasmid miniprep kit (Tiangen Biotech Co., Ltd.). The recombinant plasmid was then sequenced to verify the correct size and orientation of the inserted target fragment. The successfully constructed recombinant plasmids were named pET30a-Aly06925-Y48A, pET30a-Aly06925-F52A, pET30a-Aly06925-I53A, and p... ET30a-Aly06925-Y87A, pET30a-Aly06925-H115A, pET30a-Aly06925-F118A, pET30a-Aly06925-N119A, pET30a-Aly06 925-Q170A, pET30a-Aly06925-H240A, pET30a-Aly06925-H243A, pET30a-Aly06925-Y244A, pET30a-Aly06925-Y295A. And to Y 244 Saturation mutations were performed at the site; primer sequences are shown in Table 1. The induction expression and purification conditions for the series of mutant proteins are as described in Example 3, and the activity detection conditions are as described in Example 4.

[0178] The DNS-reducing sugar method was used to detect the activity of the truncated form of alginate lyase rAly06925, T45-60N, and a series of mutants. Figure 17 As shown: Y in alginate lyase rAly06925 87 Q 170 H 240 Y 295 All mutations resulted in inactivation, suggesting that the residues were at key sites, and confirming that N... 238 -N 239 -H 240 It is a conserved motif. Furthermore, Y was found... 244The site mutation resulted in inclusion body expression of all amino acids except aromatic amino acids, suggesting that Y... 244 The aromatic groups at the site have a high electron cloud density, which plays a crucial role in maintaining the protein's conformation or determining the hydrophilicity or hydrophobicity of the protein surface. Similarly, the extra α-helix (K... 45 -N 60 It also plays a crucial role in maintaining protein conformation and exerting catalytic activity. In summary, the conserved motif N of the novel alginate lyase rAly06925 has been preliminarily identified. 238 -N 239 -H 240 -G 241 -T 242 -H 243 and key active site residue Y 87 Q 170 H 240 Y 295 Y 244 This discovery reveals that it differs from all previously identified alginate lyase families, suggesting a novel catalytic mechanism that requires further detailed explanation through structural biology research. This effectively supplements our understanding of the novel alginate lyase family and its conserved motifs, providing theoretical insights for related enzymological research.

[0179] Using Superdex molecular gel chromatography column TM Peptide 10 / 300GL (GE) was used to separate and purify the final product obtained from the degradation of alginate by the recombinant enzyme rAly06925 mutant I53A. The purity was then determined by HPLC after combining the purified products. Figure 18 As shown in -A, the purity is all above 99%; after freeze-drying desalination and hydrogen-deuterium replacement with heavy water, the following processes are carried out: 1 ¹H-NMR detection and analysis using relevant NMR software revealed that all the unsaturated oligosaccharide products exhibited a characteristic absorption peak at 5.57 ppm. Only the ΔM signal peak was detected, while the ΔG signal peak was not observed. Figure 18 -B shows that the non-reducing ends of the final oligosaccharide products obtained by the alginate lyase rAly06925 mutant I53A from the degradation of M / G polyglucose are all ΔM units, and changes in amino acid residues at this site do not affect the structural characteristics of the final product.

[0180] Example 17

[0181] Cellulose, microcrystalline cellulose, carboxymethyl cellulose, starch, pectin, agar, mannan, alginate, carrageenan, κ-carrageenan, λ-carrageenan, τ-carrageenan, hyaluronic acid (HA), chondroitin sulfate A (CSA), chondroitin sulfate C (CSC), chondroitin sulfate E (CSE), dermatan sulfate (HS / DS), chitin, xanthan gum (Xanthan), alginate lyase rAly06925 prepared in Example 3, and 150 mM HAc-NaAc (pH 6.0) buffer were mixed at a volume ratio of 1:1:1 and reacted at 50°C for 72 h. The reaction product was heated in a boiling water bath for 10 min to inactivate the enzyme, transferred to an ice water bath for 5 min, and centrifuged at 12,000 × g at 4°C for 15 min. The supernatant was collected and subjected to DNS analysis. A certain volume of supernatant was mixed with an equal volume of DNS (3,5-p-nitroxylene) reaction solution, heated in a boiling water bath for 10 min, cooled to room temperature, and the absorbance was measured at 540 nm. The results showed that the alginate lyase rAly06925 had a significant degradation effect on alginate, but no degradation effect on other polysaccharides.

[0182] This invention discloses for the first time an alginate lyase rAly06925 obtained from the genome of Persicobacter sp. JZB09. The encoding gene of this enzyme is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention also reports for the first time an alginate lyase from the genus Persicobacter, its characteristics, and application value. Furthermore, it is the first report of a specialized endoglucose lyase containing only ΔM-termini. This enzyme differs significantly from existing known alginate lyases, exhibiting stable physicochemical properties, high activity, and potential for industrial application. The alginate lyase rAly06925 prepared in this invention has an enzyme activity of 135 U / mg in degrading alginate, making it suitable for the production of a series of unsaturated oligosaccharides.

[0183] The alginate lyase rAly06925 mutant enzyme has the following changes: amino acid position 48 is changed from tyrosine to alanine (Y48A); amino acid position 53 is changed from isoleucine to alanine (I53A); amino acid position 52 is changed from phenylalanine to alanine (F52A); amino acid position 115 is changed from histidine to alanine (H115A); amino acid position 118 is changed from phenylalanine to alanine (F118A); amino acid position 119 is changed from asparagine to alanine (N119A); amino acid position 243 is changed from histidine to alanine (H243A). All of these mutant enzymes can effectively degrade alginate. (See [link to relevant documentation]). Figure 17 .

[0184] The alginate lyase rAly06925 mutant enzyme has the following amino acid mutation sites: the amino acid mutation sites are the deletion of amino acids 1 to 65 in the amino acid sequence SEQ ID NO.2, i.e., rAly06925-T65N; the amino acid mutation sites are the deletion of amino acids 45 to 60 in the amino acid sequence SEQ ID NO.2, i.e., Aly06925-T45-60N; or the amino acid mutation site is the mutation of amino acid 244 in the amino acid sequence SEQ ID NO.2 to an amino acid other than aromatic hydrocarbon amino acids. After induced expression, the mutants form inclusion bodies, which facilitates the extraction and purification of the mutant enzyme. The enzyme in the inclusion bodies has enzymatic activity for degrading alginate.

[0185] Table 1

[0186]

[0187]

[0188]

[0189] References

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[0191] [2]Kloareg B, Quatrano R S.Structure of the cell walls of marine algaeand ecophysiological functions of the matrix polysaccharides[J].OCEANOGRAPHYAND MARINE BIOLOGY:AN ANNUAL REVIEW,1988,26:259-315.

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[0194] [5]Linker,A,Jones R S.A polysaccharide resembling alginic acid from aPseudomonas microorganism[J].Nature,1964,204(4954):187-188.

[0195] [6]Papageorgiou S K,Katsaros F K,Kouvelos E P,et al.Heavy metalsorption by calcium alginate beads from Laminaria digitata[J].Journal ofHazardous Materials,2006,137(3):1765-1772.

[0196] [7]Ensor S A,Sofos J N,Schmidt G R.Optimization of algin / calciumbinder in restructured beef[J].Journal of Muscle Foods,1990,1(3):197-206.

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[10] Iwamoto Y,Xu X,Tamura T,et al.Enzymatically depolymerizedalginate oligomers that cause sytotoxic cytokine production in humanmononuclear cells[J].Bioscience,biotechnology,and biochemistry,2003,67(2):258-263.

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[13] Wong TY, Preston LA, Schiller N L.Alginate lyase: review of majorsources and enzyme characteristics, structure-function analysis, biological roles, and applications[J]. Annual Reviews in Microbiology, 2000, 54(1): 289-340.

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[14] Li Liyan, Guan Huashi, Jiang Xiaolu, et al. Research progress on seaweed tool enzyme—algin lyase [J]. Chinese Journal of Biotechnology, 2011, 27(6):838-845.

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[15] Han W, Gu J, Cheng Y, et al. Novel alginate lyase(Aly5) from apolysaccharode-degrading marine bacterium, Flammeovirga sp.MY04: effects of module truncation on biochemical characteristics, alginate degradation patterns, and oligosaccharide-yielding properties[J].Appl.Environ.Microbiol.,2016,82(1):364-374.

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[16] Han Wenjun, Cheng Yuanyuan, Wang Dandan, et al. Application of alginate lyase in the preparation of a series of oligosaccharide products. China, 2017, 1065-1814 [P]. 2018.01.12.

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[17] Cheng Y, Wang D, Gu J, et al. Biochemical characteristics and variable alginate-degrading modes of a novel bifunctional endolytic alginatelyase[J]. Appl. Environ. Microbiol., 2017, 83(23):e01608-17.

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Claims

1. A type derived from Chlorella vulgaris ( Persicobacter sp.) JZB09's alginate lyase rAly06925, characterized in that, The amino acid sequence is shown as SEQ ID NO.

2.

2. A gene encoding the alginate lyase rAly06925 according to claim 1 aly06925 characterized in that, The nucleotide sequence is shown as SEQ ID NO.

1.

3. A recombinant expression vector I, characterized in that, A gene encoding the alginate lyase rAly06925 according to claim 1 aly0692 5.

4. A recombinant bacterium I, characterized in that, A gene encoding the alginate lyase rAly06925 according to claim 1 aly06925 .

5. A gene encoding the alginate lyase rAly06925 according to claim 1. aly06925 The use of the recombinant expression vector I according to claim 3 or the recombinant bacteria I according to claim 4 for the production of the alginate lyase rAly06925.

6. Use of the alginate lyase rAly06925 of claim 1 in degrading alginate or alginate oligosaccharides that do not comprise G-series saturated oligosaccharides.

7. Use according to claim 6, wherein The brown algin lyase rAly06925 according to claim 1 in the degradation of algin or algin oligosaccharides to produce non-reducing end containing Use of the series of G-rich unsaturated oligosaccharide fragments of M.

8. The use according to claim 6, wherein Use of the alginate lyase rAly06925 of claim 1 in degrading alginate or alginate oligosaccharides to produce unsaturated disaccharides.

9. A mutant alginate lyase rAly06925 enzyme, characterized in that, The amino acid mutation site is the 48th amino acid of the amino acid sequence SEQ ID NO. 2, which is changed from tyrosine to alanine.

10. The gene encoding the alginate lyase rAly06925 mutant enzyme according to claim 9, characterized in that, Site-directed mutagenesis is performed on the coding gene SEQ ID NO. 1 according to the amino acid mutation site.

11. A recombinant expression vector II, characterized in that, The coding gene comprising the mutant enzyme of claim 9.

12. A recombinant bacterium II, characterized in that, The coding gene comprising the mutant enzyme of claim 9.

13. Use of the coding gene of the alginate lyase rAly06925 mutant enzyme of claim 9, the recombinant expression vector II of claim 11 or the recombinant bacteria II of claim 12 in preparing the alginate lyase rAly06925 mutant enzyme.

14. Use of the alginate lyase rAly06925 mutant enzyme of claim 9 in degrading alginate or alginate oligosaccharides that do not comprise G-series saturated oligosaccharides.

15. The use according to claim 14, wherein the compound is ###00010### 15 The alginate lyase rAly06925 mutant enzyme of claim 9, wherein the alginate lyase rAly06925 mutant enzyme degrades alginate or alginate oligosaccharides to produce non-reducing terminal containing Use of the series of G-rich unsaturated oligosaccharide fragments of M.

16. A mutant alginate lyase rAly06925 enzyme, characterized in that, The amino acid mutation site is the 53rd amino acid of the amino acid sequence SEQ ID NO. 2, which is changed from isoleucine to alanine, the 52nd amino acid is changed from phenylalanine to alanine, the 115th amino acid is changed from histidine to alanine, the 118th amino acid is changed from phenylalanine to alanine, the 119th amino acid is changed from asparagine to alanine, or the 243rd amino acid is changed from histidine to alanine.

17. A gene encoding the alginate lyase rAly06925 mutant enzyme according to claim 16, characterized in that, Site-directed mutagenesis is performed on the coding gene SEQ ID NO. 1 according to the amino acid mutation site.

18. A recombinant expression vector III, characterized in that, The coding gene comprising the mutant enzyme of claim 16.

19. A recombinant bacterial strain III, wherein, The coding gene comprising the mutant enzyme of claim 16.

20. Use of the coding gene of the alginate lyase rAly06925 mutant enzyme of claim 16, the recombinant expression vector III of claim 18 or the recombinant bacteria III of claim 19 in preparing the alginate lyase rAly06925 mutant enzyme.

21. Use of the alginate lyase rAly06925 mutant enzyme of claim 16 in degrading alginate or alginate oligosaccharides that do not comprise G-series saturated oligosaccharides.

22. The use of claim 21, wherein, The alginate lyase rAly06925 mutant enzyme of claim 16, wherein the alginate lyase rAly06925 mutant enzyme degrades alginate or alginate oligosaccharides to produce non-reducing terminal containing Use of the series of G-rich unsaturated oligosaccharide fragments of M.

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