Sodium alginate lyase, preparation method therefor and use thereof

Abstract

Provided are a novel sodium alginate lyase AlyB, a preparation method therefor and the use thereof. The main cracking product of the novel sodium alginate lyase AlyB is a monosaccharide, which has the effects of resisting oxidation, resisting viruses and inhibiting an angiotensin converting enzyme, and thus can be applied to the fields of cosmetics, medicines, healthcare foods and the like.

Description

Sodium alginate lyase, preparation method and use thereof Technical Field

[0001] The present invention relates to an alginate lyase AlyB, its preparation method, and its use. Specifically, it relates to an alginate lyase AlyB that can cleave alginic acid and / or sodium alginate, with its primary cleavage product being 4-deoxy-L-erythro-hexoseulose uronic acid (DEH). Background Art

[0002] Sodium alginate is a polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). It is primarily found in the cell walls of brown algae such as kelp and Sargassum. Certain bacteria, such as Pseudomonas aeruginosa, also have the ability to synthesize sodium alginate. The enzyme that decomposes sodium alginate is called alginate lyase, also known as alginase or alginate depolymerase. It cleaves the glycosidic bonds of sodium alginate via β-elimination and is a member of the polysaccharide lyase family (EC 4.2.2). Based on the degree of similarity in their amino acid sequences, alginate lyases are classified into the polysaccharide lyase families PL5, PL6, PL7, PL14, PL15, PL17, and PL18. Alginate lyases from different sources exhibit distinct properties.

[0003] Alginate lyases can be categorized as endo- and exo-type based on their cleavage mechanism. Endo-type lyases can be further divided into (1) α-L-guluronate lyases, which degrade polyguluronic acid (PG); (2) β-D-mannuronic acid lyases, which degrade polymannuronic acid (PM); and (3) bifunctional lyases, which degrade both PG and PM. Endo-type alginate lyases can cleave alginate into oligosaccharides with a degree of polymerization (DP) of 2 to 7. Exo-type alginate lyases, on the other hand, cleave alginate into unsaturated monosaccharides that are then converted to 4-deoxy-L-erythro-hexoseulose uronic acid (DEH).

[0004] Alginate lyase has applications in medicine, agriculture, industry, and health foods. In medicine, alginate lyase can degrade vinylated alginate biofilms secreted by the mucoid bacterium Pseudomonas aeruginosa, and can be used to treat lung diseases caused by Pseudomonas aeruginosa. In agriculture, alginate lyase can be used to treat brown algae waste, converting it into oligosaccharides or monosaccharides. This not only addresses environmental pollution but also maximizes the value of agricultural waste resource recycling. In industry, researchers have used metabolic engineering to transform Escherichia coli or Saccharomyces cerevisiae into alginate lyase-producing strains, and the resulting DEH is used to produce ethanol, showing potential for bioethanol production. In the field of health foods, oligosaccharides obtained by the decomposition of sodium alginate by alginate lyase have various biological activities such as anti-inflammatory, antioxidant, immune regulation, anti-cancer, neuroprotective, antibacterial, antihypertensive, anti-lipid, anti-glycemic, anti-obesity and prebiotic properties, and are considered to have the potential to be developed into health food ingredients.

[0005] Summary of the Invention

[0006] Based on the versatility of sodium alginate lyase, the present invention aims to provide a new sodium alginate lyase that can lyse alginic acid and / or sodium alginate and produce functional lysates.

[0007] To achieve the above object, the present invention provides an alginate lyase AlyB, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] Preferably, the nucleotide sequence of the alginate lyase AlyB is shown in SEQ ID NO: 2.

[0009] Preferably, the optimal action pH of the alginate lyase AlyB is 7.0 to 9.0.

[0010] Preferably, the optimal operating temperature of the alginate lyase AlyB is 33 to 37°C.

[0011] Preferably, the sodium alginate lyase AlyB cleaves alginic acid and sodium alginate.

[0012] Preferably, the cleavage mode of the alginate lyase AlyB is exo-type.

[0013] Preferably, the main product after degradation of the alginate lyase AlyB is 4-deoxy-L-erythro-hexuronic acid (DEH).

[0014] Preferably, the alginate lyase AlyB is isolated from Paenibacillus sp.

[0015] According to another object of the present invention, a recombinant vector is provided, which comprises a nucleic acid encoding the aforementioned sodium alginate lyase AlyB.

[0016] Preferably, the recombinant vector further comprises chaperone protein expression vectors pG-KJE8, pGro7, pKJE7 and / or pTf16.

[0017] Preferably, the recombinant vector further comprises the nucleotide sequence shown in SEQ ID NO:5.

[0018] According to another object of the present invention, a transformant is provided, comprising the aforementioned recombinant vector.

[0019] According to another object of the present invention, a method for preparing the aforementioned sodium alginate lyase AlyB is provided, which comprises: preparing the aforementioned recombinant vector; transforming the recombinant vector into a host cell and obtaining the aforementioned transformant therefrom; culturing the transformant and expressing the sodium alginate lyase AlyB; and isolating and purifying the expressed sodium alginate lyase AlyB.

[0020] Preferably, the host cell is Escherichia coli BL21 (DE3).

[0021] According to one object of the present invention, a use of the aforementioned sodium alginate lyase AlyB is provided, which is used for lysing alginic acid and / or sodium alginate.

[0022] According to one object of the present invention, there is provided a use of the aforementioned sodium alginate lyase AlyB for preparing a cosmetic, food additive or pharmaceutical composition for scavenging DPPH and ABTS free radicals.

[0023] According to one object of the present invention, there is provided a use of the aforementioned sodium alginate lyase AlyB for preparing antioxidant cosmetics, food additives or pharmaceutical compositions.

[0024] According to one object of the present invention, there is provided a use of the aforementioned sodium alginate lyase AlyB for preparing a food additive or pharmaceutical composition for resisting viral infection.

[0025] According to one object of the present invention, there is provided a use of the aforementioned sodium alginate lyase AlyB for preparing a food additive or a pharmaceutical composition for inhibiting angiotensin-converting enzyme.

[0026] With the above-mentioned features, the present invention provides a novel sodium alginate lyase, its preparation method, vector, and transformant. This sodium alginate lyase can cleave alginic acid and / or sodium alginate and produce functional lysates, thereby achieving the purpose of cleaving alginic acid and / or sodium alginate to prepare cosmetics, food additives, or pharmaceutical compositions with antioxidant, antiviral, or angiotensin-converting enzyme inhibitory properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the amino acid sequence alignment of Paenibacillus alginate lyase AlyB with other Paenibacillus alginate lyases. PEAly-M63 is from Paenibacillus elgii; PEAly-AC13 is from Paenibacillus elgii; and PAAly-T-3040 is from Paenibacillus agaridevorans.

[0028] Figure 2 shows the analysis of recombinant alginate lyase AlyB expressed in E. coli BL21(DE3) using protein electrophoresis (A) and immunoblotting (B). Lane M represents Page Ruler TM Prestained Protein Ladder; Lane C represents total cell lysate from E. coli BL21(DE3) [pET-29a(+)] cells induced at 37°C with 0.1 mM IPTG for 4 hours; Lane T represents total cell lysate from E. coli BL21(DE3) (pET-AlyB) cells induced at 37°C with 0.1 mM IPTG for 4 hours. The arrow indicates the expected molecular weight of recombinant alginate lyase AlyB.

[0029] Figure 3 shows the effect of different induction temperatures on the soluble expression of recombinant alginate lyase AlyB in E. coli BL21(DE3) using protein electrophoresis (A) and immunoblotting (B). Lane M represents Page Ruler TM Prestained Protein Ladder; Lane T represents the total cell lysate after induction of E. coli BL21(DE3)(pET-AlyB) at different temperatures and 0.1 mM IPTG; Lane S represents the soluble protein fraction after induction of E. coli BL21(DE3)(pET-AlyB) at different temperatures and 0.1 mM IPTG. The arrow indicates the predicted molecular weight of recombinant alginate lyase AlyB.

[0030] FIG4 shows the results of the activity of the recombinant alginate lyase AlyB in E. coli BL21(DE3)(pET-AlyB) after induction at different temperatures and 0.1 mM IPTG.

[0031] Figure 5 shows the effect of co-expressed partner proteins on the solubility of recombinant AlyB analyzed by protein electrophoresis (A) and immunoblotting (B). Lane M represents Page Ruler TM Prestained Protein Ladder; lane T represents the total cell lysate; lane S represents the soluble protein fraction. The arrow indicates the expected molecular weight position of recombinant alginate lyase AlyB.

[0032] Figure 6 shows the expression of recombinant alginate lyase AlyB by various E. coli transformants. (A) Comparison of the solubility of recombinant alginate lyase AlyB; (B) Comparison of the yield of soluble AlyB.

[0033] FIG. 7 shows the effect of co-expression of chaperone proteins on the activity of recombinant AlyB in E. coli BL21(DE3) (pET-AlyB).

[0034] Figure 8 shows the protein electrophoresis analysis of the recombinant alginate lyase AlyB purified from the intracellular soluble fraction of E. coli BL21 (DE3) / pET-AlyB / pGro7. Lane M represents Page Ruler TM Prestained Protein Ladder; lane F represents the flow-through; lanes 1 to 7 represent the eluate. Arrows indicate the expected molecular weight position of recombinant alginate lyase AlyB.

[0035] Figure 9 shows the analysis of purified recombinant alginate lyase AlyB by protein electrophoresis (A) and immunoblotting (B). Lane M represents Page Ruler TM Prestained Protein Ladder; lane 1 represents purified recombinant alginate lyase AlyB. The arrow indicates the expected molecular weight position of recombinant alginate lyase AlyB.

[0036] Figure 10 shows the effects of different pH values ​​and temperatures on the activity of recombinant alginate lyase AlyB. (A) Effect of different pH values ​​on the activity of recombinant alginate lyase AlyB. The buffer solutions used were 50 mM citrate buffer (pH 3 to 6, ●), phosphate buffer (pH 6 to 7, △), HEPES-NaOH buffer (pH 7 to 8, ■), bicine-NaOH buffer (pH 8 to 9, ○), and glycine-NaOH buffer (pH 9-10, ▲). (B) Effect of different temperatures on the activity of recombinant alginate lyase AlyB. The maximum enzyme activity was defined as 100%. All experiments were performed in triplicate, and the results are presented as mean ± SD.

[0037] Figure 11 shows the effects of different pH values ​​(A) and temperatures (B) on the stability of recombinant alginate lyase AlyB. The pH stability of recombinant alginate lyase AlyB was determined by placing the enzyme in buffer solutions of different pH values ​​and measuring the enzyme activity after storage at 4°C for 4 hours. The temperature stability of recombinant alginate lyase AlyB was determined by placing the enzyme in a pH 8.0 buffer solution and storing it at different temperatures for 4 hours, and then measuring the enzyme activity. The maximum enzyme activity was defined as 100%. All experiments were performed in triplicate, and the results are presented as mean ± standard deviation.

[0038] Figure 12 shows the enzyme kinetics analysis of recombinant alginate lyase AlyB. (A) Substrate reaction rate. (B) Double reciprocal plot. All experiments were performed in triplicate, and results are presented as mean ± standard deviation.

[0039] Figure 13 shows thin-layer chromatography analysis of alginate cleavage products by recombinant alginate lyase AlyB. Lane 1 represents sodium D-mannuronate (M); lane 2 represents D-mannuronate disaccharide (DP2); lane 3 represents D-mannuronate triose (DP3); lane 4 represents D-mannuronate tetraose (DP4); and lane 5 represents the alginate cleavage products by recombinant alginate lyase AlyB.

[0040] Figure 14 shows the ability of the products of alginate cleavage by recombinant alginate lyase AlyB to scavenge DPPH free radicals (A) and total antioxidant capacity (B). All experiments were performed in triplicate, and the results are presented as mean ± standard deviation.

[0041] FIG. 15 shows the effect of DEH on inhibiting the infection of feline macrophage cell line Fcwf-4 by serotype II feline infectious peritonitis virus (FIPV) after treatment with DEH at different concentrations mixed with FIPV for 1 hour.

[0042] FIG16 shows the effect of FIPV infection on Fcwf-4 cells and subsequent treatment with different concentrations of DEH.

[0043] FIG17 shows the effect of DEH on inhibiting the infection of African green monkey embryonic kidney cell line Marc-145 by porcine reproductive and respiratory syndrome virus (PRRSV) after being mixed with DEH at different concentrations for 1 hour.

[0044] FIG. 18 shows the effect of different concentrations of DEH treatment on PRRSV infection in Marc-145 cells.

[0045] Figure 19 shows the ability of the product of alginate cleavage by recombinant alginate lyase AlyB to inhibit angiotensin-converting enzyme (ACE). All experiments were performed in triplicate, and the results are presented as mean ± standard deviation. DETAILED DESCRIPTION

[0046] The following embodiments are described in detail with reference to the relevant drawings. However, these embodiments can be implemented in different forms, but this is not the only form of implementing or using the specific embodiments of the invention claimed in this case, and should not be understood as limiting the above embodiments. The features of multiple specific embodiments and the method steps and their sequence for constructing and operating these specific embodiments are covered in the implementation method. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences. On the contrary, these embodiments are provided so that this specification can be thoroughly and completely disclosed to fully express the spirit of the invention to those skilled in the art. Similar component symbols in the drawings refer to similar components. In the following description, conventional functions or structures will not be described in detail so as not to repeat unnecessary details in the embodiments.

[0047] Unless otherwise specified, any singular noun used in this specification includes the plural form of that noun, and any plural noun used in this specification also includes the singular form of that noun. Furthermore, in this specification and the claims, the expressions "at least one" and "one or more" have the same meaning, both representing one, two, three, or more.

[0048] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the numerical values ​​in the specific examples are presented herein as precisely as possible. However, any numerical value inherently and inevitably contains standard deviations due to individual testing methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard error of the mean, as determined by one skilled in the art. Except in the examples, or unless otherwise expressly indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe material amounts, time periods, temperatures, operating conditions, quantitative ratios, and the like) are to be understood as modified by the word "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and claims are approximate and may be modified as needed. At a minimum, these numerical parameters should be understood to include the number of significant digits indicated and to apply normal rounding. Herein, numerical ranges are expressed from one endpoint to another endpoint or between two endpoints; unless otherwise stated, the numerical ranges described herein include the endpoints.

[0049] The details of various embodiments of the present invention are as follows. Other features of the present invention will become apparent through the detailed description and scope of protection of each embodiment below. Without further elaboration, it is believed that those skilled in the art can utilize the present invention to the broadest extent based on the above description. Therefore, it should be understood that the following description is for illustrative purposes only and is not intended to limit the remainder of the disclosure in any way. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

[0050] When referring to concentration herein, "percentage", "%", etc., as used herein may refer to mass concentration or volume percentage concentration; mass concentration is defined as the mass of the solute divided by the volume of the corresponding mixture (including the volume of the solute), and may be expressed herein by "percentage", "%", etc. followed by "(w / v)"; volume percentage concentration is defined as the volume of the solute as a percentage of the total volume of the solution, and may be expressed herein by "percentage", "%", etc. followed by "(v / v)".

[0051] As used herein, the term "produced from" is synonymous with "comprising." As used herein, the terms "includes," "including," "comprises," "comprising," "has," "having," "contains," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that contains a plurality of listed elements is not necessarily limited to only those listed elements but may include other elements not expressly listed but inherent to the composition, process, method, article, or apparatus. The term "comprising" is generally used in an inclusive sense, that is, allowing for the presence of one or more additional features or components.

[0052] The terms "host" and "host cell" are used interchangeably herein and may refer to prokaryotic cells. One or more vectors described herein may be introduced into the host cell, or a vector containing a gene to be expressed may be inserted through genetic engineering methods. As those skilled in the art will appreciate, these terms refer not only to specific cells but also to the progeny or potential progeny of such cells. Subsequent generations may exhibit changes due to genetic mutations or environmental influences, so the progeny of such cells may not be substantially identical to the parent cell; however, they are still encompassed within the scope of the terms as used herein.

[0053] The main purpose of the present invention is to clone a new alginate lyase gene alyB from Paenibacillus sp. and explore the effectiveness of producing the recombinant enzyme using an Escherichia coli expression system. Subsequently, the recombinant alginate lyase AlyB with a His tag at the C-terminus was purified using immobilized metal ion affinity chromatography, and the enzyme properties were investigated using the purified alginate lyase AlyB. Subsequently, the recombinant alginate lyase AlyB was used to cleave sodium alginate, and the properties of the cleavage products were analyzed. This provides a new alginate lyase AlyB that can cleave alginic acid and / or sodium alginate and produce a functional lysate.

[0054] According to one embodiment of the present invention, a sodium alginate lyase AlyB is provided, whose amino acid sequence is shown in SEQ ID NO: 1. According to another embodiment of the present invention, the nucleotide sequence of the sodium alginate lyase AlyB is shown in SEQ ID NO: 2. According to one embodiment of the present invention, the optimal action pH of the sodium alginate lyase AlyB is 7.0 to 9.0, preferably 8. In addition, its optimal action temperature is 33 to 37° C., preferably 35° C. The sodium alginate lyase AlyB according to an embodiment of the present invention cleaves alginic acid and sodium alginate in an exolytic manner, and the main product after degradation is 4-deoxy-L-erythro-hexuronic acid (DEH). The sodium alginate lyase AlyB is obtained from Paenibacillus sp., specifically Paenibacillus chondroitinus, and was purchased from the Bioresource Collection and Research Center of the Food Industry Development Research Institute, with the strain number BCRC 15913.

[0055] According to one embodiment of the present invention, a recombinant vector is provided, comprising a nucleic acid encoding alginate lyase AlyB. According to another embodiment of the present invention, the recombinant vector further comprises a chaperone protein expression vector pG-KJE8, pGro7, pKJE7, and / or pTf16 to enhance the activity of alginate lyase AlyB.

[0056] According to one embodiment of the present invention, a method for producing alginate lyase AlyB is provided, comprising: preparing a recombinant vector; transforming the recombinant vector into a host cell to obtain a transformant; culturing the transformant to express the alginate lyase AlyB; and isolating and purifying the expressed alginate lyase AlyB. The host cell may be Escherichia coli BL21(DE3) or other suitable host cells.

[0057] The sodium alginate lyase AlyB prepared according to the embodiment of the present invention can be used to cleave alginic acid and / or sodium alginate, and the cleavage products thereof can be used to prepare cosmetics, food additives or pharmaceutical compositions that are antioxidant or inhibit vasoconstriction.

[0058] The following describes the alginate lyase of the present invention, its preparation method, vector and transformant, and its use with reference to specific embodiments and accompanying drawings.

[0059] Materials and Methods

[0060] Use strains

[0061] Escherichia coli BL21(DE3)

[0062] Escherichia coli BL21 (DE3) was purchased from New England Biolabs (NEB), USA; its genotype was F - ompT gal dcm lon hsdSB(r B - m B - )λ(DE3). This host cell contains T7 RNA polymerase and is used as a host cell for recombinant protein expression.

[0063] Paenibacillus sp.

[0064] Paenibacillus chondroitinus was purchased from the Bioresource Collection and Research Center of the Food Industry Development Research Institute, with the strain number BCRC 15913. This strain was used as the source of the alginate lyase gene.

[0065] carrier

[0066] pJET1.2

[0067] This vector was purchased from Thermo Fisher Scientific, USA. It is a blunt-end cloning vector with a size of 2,974 bp and carries the ampicillin resistance gene.

[0068] pET29a(+)

[0069] This vector was purchased from Merck Millipore Corporation in the United States and is used for recombinant protein expression. It is 5,371 bp in size and carries a kanamycin resistance gene.

[0070] pG-KJE8

[0071] This vector was purchased from TaKaRa Co., Ltd. in Japan. The vector carries the chaperone gene dnaK-dnaJ-grpE-groES-groEL and is a chaperone protein expression vector. Its size is 11.1 kb and it carries the chloramphenicol resistance gene.

[0072] pGro7

[0073] This vector was purchased from TaKaRa Co., Ltd. in Japan. The vector carries the groES-groEL chaperone protein gene and is a chaperone protein expression vector with a size of 5.4 kb and a chloramphenicol resistance gene.

[0074] pKJE7

[0075] This vector was purchased from TaKaRa Co., Ltd. in Japan. The vector carries the dnaK-groES-groEL chaperone protein gene and is a chaperone protein expression vector with a size of 7.2 kb and a chloramphenicol resistance gene.

[0076] pTf16

[0077] This vector was purchased from TaKaRa Co., Ltd. of Japan. It carries the tlg chaperone protein gene and is a chaperone protein expression vector. It is 8.3 kb in size and carries the chloramphenicol resistance gene.

[0078] Cloning of alginate lyase gene alyB

[0079] According to the present invention, using the results of whole-genome sequencing and analysis, specific primers PCALYBF (5'-GATATAAGATCTATGACAACTCTGTCGCCATTATACG-3', SEQ ID NO: 3) and PCALYBR (5'-CAATATGTCGACTTAGTGGTGGTGGTGGTGGTG GTAAGCCT TAGGCACCTCAACACG-3', SEQ ID NO: 4) were designed for the alginate lyase gene aly. Using P. chondroitinus genome DNA as a template, PCR was used to amplify the target gene fragment. After completion of the PCR reaction, the PCR product was recovered using the Plus Gel Eluted Kit. AlyB was then cloned using the CloneJET PCR Cloning Kit. Transformants were screened by colony polymerase chain reaction. After confirming that the recombinant plasmid in the transformant contained the inserted DNA, the plasmid was extracted from the transformant using the Plasmid Miniprep Purification Kit II and DNA sequencing was performed. The plasmid with the correct DNA sequence was named pJET-AlyB.

[0080] Construction of expression vector for alginate lyase

[0081] According to an embodiment of the present invention, pJET-AlyB was cut with BglII and SalI, and then the alyB gene was inserted into pET29a(+) cut with BamHI and SalI using T4 DNA ligase. The ligated product was transformed into Escherichia coli ECOS TM 9-5. Transformants were screened using colony polymerase chain reaction. After confirming that the recombinant plasmid contained the inserted DNA, the plasmid was isolated and sequenced. The plasmid with the correct DNA sequence was named pET-AlyB.

[0082] Transformation and induced expression of alginate lyase expression vector

[0083] According to an embodiment of the present invention, the alginate lyase expression vector pET-AlyB was transformed into E. coli BL21(DE3) or E. coli BL21(DE3) containing a chaperone protein expression vector. The resulting transformants were designated E. coli BL21(DE3) / pET-AlyB, E. coli BL21(DE3) / pET-AlyB / pG-KJE8, E. coli BL21(DE3) / pET-AlyB / pGro7, E. coli BL21(DE3) / pET-AlyB / pKJE7, E. coli BL21(DE3) / pET-AlyB / pG-Tf2, and E. coli BL21(DE3) / pET-AlyB / pTf16.

[0084] 1. Induced expression of E. coli BL21(DE3) / pET-AlyB

[0085] A single colony was selected and inoculated into 12 mL of LB medium containing kanamycin (final concentration of 30 μg / mL) and cultured at 37°C and 180 rpm. After overnight culture, 10 mL of the pre-culture solution was added to 1 L of LB medium containing kanamycin (final concentration of 30 μg / mL) and cultured with shaking (37°C, 180 rpm) until the OD 600 The OD value was about 0.4 to 0.6. 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and the recombinant protein was induced at 18°C, 25°C, 30°C and 37°C. After 4 hours of induction at 25°C, 30°C and 37°C or 20 hours of induction at 18°C, the OD value per ml was collected. 600= 2. Centrifuge at 20,630 × g and 4°C for 10 minutes, then discard the supernatant. Separate the centrifuged cells for soluble and insoluble protein.

[0086] 2. Induced expression of E. coli BL21(DE3) / pET-AlyB / pG-KJE8

[0087] A single colony was selected and inoculated into 12 mL of LB medium containing kanamycin (final concentration of 30 μg / mL) and chloramphenicol (final concentration of 20 μg / mL) and cultured at 37°C and 180 rpm. After overnight culture, 10 mL of the pre-culture was added to 1 L of LB medium containing kanamycin (final concentration of 30 μg / mL), chloramphenicol (final concentration of 20 μg / mL), tetracycline stock solution (final concentration of 5 ng / mL), and arabinose (final concentration of 0.5 mg / mL). The culture was shaken (37°C, 180 rpm) until the OD reached 0. 600 The OD value was about 0.4 to 0.6. 0.1 mM IPTG was added and the recombinant protein was induced at 25°C. After 4 hours of induction, the OD value per ml was collected. 600 = 2. Centrifuge at 20,630 × g and 4°C for 10 minutes, then discard the supernatant. Separate the centrifuged cells for soluble and insoluble protein.

[0088] 3. Induction Culture of E. coli BL21(DE3) / pET-AlyB / pGro7

[0089] A single colony was selected and inoculated into 12 mL of LB medium containing kanamycin (final concentration of 30 μg / mL) and chloramphenicol (final concentration of 20 μg / mL) and cultured at 37°C and 180 rpm. After overnight culture, 10 mL of the pre-culture was added to 1 L of LB medium containing kanamycin (final concentration of 30 μg / mL), chloramphenicol (final concentration of 20 μg / mL), and arabinose (final concentration of 0.5 mg / mL) and cultured with shaking (37°C, 180 rpm) until the OD reached 0. 600 The OD value was about 0.4 to 0.6. 0.1 mM IPTG was added and the recombinant protein was induced at 25°C. After 4 hours of induction, the OD value per ml was collected.600 = 2. Centrifuge at 20,630 × g and 4°C for 10 minutes, then discard the supernatant. Separate the centrifuged cells for soluble and insoluble protein.

[0090] IV. Induction Culture of E. coli BL21(DE3) / pET-AlyB / pKJE7

[0091] A single colony was selected and inoculated into 12 mL of LB medium containing kanamycin (final concentration of 30 μg / mL) and chloramphenicol (final concentration of 20 μg / mL) and cultured at 37°C and 180 rpm. After overnight culture, 10 mL of the pre-culture was added to 1 L of LB medium containing kanamycin (final concentration of 30 μg / mL), chloramphenicol (final concentration of 20 μg / mL), and arabinose (final concentration of 0.5 mg / mL) and cultured with shaking (37°C, 180 rpm) until the OD reached 0. 600 The OD value was about 0.4 to 0.6. 0.1 mM IPTG was added and the recombinant protein was induced at 25°C. After 4 hours of induction, the OD value per ml was collected. 600 = 2. Centrifuge at 20,630 × g and 4°C for 10 minutes, then discard the supernatant. Separate the centrifuged cells for soluble and insoluble protein.

[0092] 5. Induction culture of E. coli BL21(DE3) / pET-AlyB / pG-Tf2

[0093] A single colony was selected and inoculated into 12 mL of LB medium containing kanamycin (final concentration of 30 μg / mL) and chloramphenicol (final concentration of 20 μg / mL) and cultured at 37°C and 180 rpm. After overnight culture, 10 mL of the pre-culture was added to 1 L of LB medium containing kanamycin (final concentration of 30 μg / mL), chloramphenicol (final concentration of 20 μg / mL), and tetracycline stock solution (final concentration of 5 ng / mL) and cultured with shaking (37°C, 180 rpm) until the OD reached 0. 600 The OD value was about 0.4 to 0.6. 0.1 mM IPTG was added and the recombinant protein was induced at 25°C. After 4 hours of induction, the OD value per ml was collected. 600= 2. Centrifuge at 20,630 × g and 4°C for 10 minutes, then discard the supernatant. Separate the centrifuged cells for soluble and insoluble protein.

[0094] VI. Induction Culture of E. coli BL21(DE3) / pET-AlyB / pTf16

[0095] A single colony was selected and inoculated into 12 mL of LB medium containing kanamycin (final concentration of 30 μg / mL) and chloramphenicol (final concentration of 20 μg / mL) and cultured at 37°C and 180 rpm. After overnight culture, 10 mL of the pre-culture solution was added to 1 L of LB medium containing kanamycin (final concentration of 30 μg / mL), chloramphenicol (final concentration of 20 μg / mL), and arabinose (final concentration of 0.5 mg / mL) and cultured with shaking (37°C, 180 rpm) until the OD reached 0. 600 The OD value was about 0.4 to 0.6. 0.1 mM IPTG was added and the recombinant protein was induced at 25°C. After 4 hours of induction, the OD value per ml was collected. 600 = 2. Centrifuge at 20,630 × g and 4°C for 10 minutes, then discard the supernatant. Separate the centrifuged cells for soluble and insoluble protein.

[0096] Classification of soluble and insoluble proteins

[0097] When recombinant proteins are expressed in E. coli, the expressed proteins may form inclusion bodies. Inclusion bodies are denatured and insoluble proteins. To restore the protein's conformation and biological activity, refolding is required. However, the protein refolding process is cumbersome and time-consuming, and the folding efficiency varies depending on the protein's characteristics. To prevent the formation of inclusion bodies in cells, the solubility of recombinant proteins can be improved by co-expressing chaperone proteins. Chaperone proteins are a class of proteins that assist in the assembly of molecules and protein folding within the cell, such as GroES, GroEL, DnaK, DnaJ, and GrpE.

[0098] According to the embodiment of the present invention, the centrifuged bacteria were suspended in a lysis buffer solution and stirred evenly. The final concentration of the broken bacteria was OD 600=4. Bacteria were disrupted using an ultrasonic disruptor (Sonics VCX-750, USA). The conditions for disruption were 25% amplitude, intermittent treatment (9 seconds on, 9 seconds off), and 10,800 joules per treatment. The disrupted bacterial solution was centrifuged (15,900 × g, 30 minutes), and the supernatant was collected. Protein electrophoresis and immunoblotting were used to investigate the induced expression of recombinant alginate lyase AlyB.

[0099] Recombinant alginate lyase AlyB

[0100] According to an embodiment of the present invention, E. coli BL21(DE3) / pET-AlyB / pGro7 was cultured, and the recombinant alginate lyase AlyB was obtained by purification, and the protein concentration and activity were determined.

[0101] Protein concentration determination

[0102] According to embodiments of the present invention, protein quantification analysis is performed using a Protein Quantification Assay Kit (MACHEREY-NAGEL, Germany) or a Bio-Rad Protein Assay (Bio-Rad, USA). The Protein Quantification Assay Kit is operated as follows. A protein sample is diluted 20-fold with a buffer solution (20 mM sodium phosphate, pH 7.0). 20 μL of the diluted sample, 40 μL of Protein Solving Buffer, and 40 μL of Quantification Reagent are mixed and incubated at room temperature for 30 minutes. The absorbance is measured at 570 nm using an Infinite 200Pro microplate reader (Tecan, USA). A protein concentration standard curve is generated using 2 mg / mL bovine serum albumin (BSA) as a standard. The sample absorbance is compared to the standard curve to calculate the protein sample concentration. The Bio-Rad Protein Assay is operated as follows. Mix 10 μL of protein sample with 200 μL of diluted Dry reagent and incubate at room temperature for 5 minutes. Measure absorbance at 595 nm using an Infinite 200Pro microplate reader (Tecan, USA). Prepare a protein concentration standard curve using 2 mg / mL bovine serum albumin (BSA) as a standard. Compare the sample absorbance to the standard curve to calculate the protein sample concentration.

[0103] Activity analysis of recombinant alginate lyase AlyB

[0104] According to an embodiment of the present invention, the activity of alginate lyase is analyzed using a thiobarbituric acid (TBA) colorimetric assay as described below. A substrate solution containing 0.22% (w / v) sodium alginate is prepared in phosphate buffer (20 mM sodium phosphate, pH 7.4). 900 μL of the substrate solution is mixed with 100 μL of appropriately diluted purified enzyme solution and incubated at 37°C for 30 minutes. After the reaction is complete, 200 μL of the reaction solution is mixed with 200 μL of Reagent A [25 mM periodic acid (H₅IO₆) / 0.125 N H₂SO₄] and allowed to stand at room temperature for 20 minutes to allow the cleavage product to oxidize to formyl pyruvic acid. Afterwards, 500 μL of reagent B (2% sodium arsenite (NaAsO2) / 0.5N HCl) was added to the reaction solution to terminate the enzyme reaction. After vortexing, the mixture was allowed to stand at room temperature for 2 minutes. Next, 2 mL of 0.3% thiobarbituric acid solution was added and the mixture was incubated at 100°C for 10 minutes to allow the formylpyruvate to react with thiobarbituric acid, resulting in color development. After cooling, 100 μL of the reaction solution was transferred to a 96-well plate and the absorbance was measured at 548 nm using an Infinite 200Pro microplate reader (Tecan, USA). A TBA color reaction was performed using commercially available alginate lyase (Sigma, USA) solutions with varying activity units, and a standard curve was established comparing alginate lyase activity to absorbance. The activity (activity unit, U) of the purified alginate lyase AlyB was calculated using this standard curve.

[0105] Characterization of recombinant alginate lyase AlyB

[0106] According to an embodiment of the present invention, the purified sodium alginate lyase AlyB was used to perform enzyme property analysis, and the analysis items and operation steps are as follows.

[0107] Optimal pH

[0108] Substrate solutions of varying pH values ​​were prepared by mixing 800 μL of 0.25% sodium alginate solution with 100 μL of 0.5 M citric acid buffer (pH 3.0 to 6.0), 0.5 M phosphate buffer (pH 6.0 to 7.0), 0.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.0 to 8.0), 0.5 M bicine-NaOH buffer (pH 8.0 to 9.0), and 0.5 M glycine-NaOH buffer (pH 9.0 to 10.0). After incubating the substrate solutions at 37°C for 5 minutes, 100 μL of 0.4 U / mL purified enzyme solution was added and the reaction was continued at 37°C for 30 minutes. TBA color development and enzyme activity calculation were performed as described above. The highest enzyme activity measured in the reaction was defined as 100%, which was the enzyme activity at the optimal pH value, and the relative enzyme activity at other pH values ​​was calculated.

[0109] Optimum working temperature

[0110] 800 μL of 0.25% sodium alginate solution was evenly mixed with 100 μL of 0.5 M HEPES buffer (pH 8.0) to prepare a substrate solution. After the substrate solution was incubated at 20 to 60°C for 5 minutes, 100 μL of 0.4 U / mL enzyme solution was added and the reaction was incubated at different temperatures (20 to 60°C) for 30 minutes. TBA color development and enzyme activity calculation methods were as described above. The highest enzyme activity measured in the reaction was defined as 100%, which was the enzyme activity at the optimal reaction temperature. The relative enzyme activity at other temperatures was then calculated.

[0111] pH stability analysis

[0112] The enzyme solution was diluted to 0.4 U / mL with buffer solutions of different pH values ​​and placed at 35°C for 2 hours and 4 hours respectively. 800 μL of 0.25% sodium alginate solution was evenly mixed with 100 μL of 0.5 M HEPES buffer solution (pH 8.0) to prepare a substrate solution. After the substrate solution was placed at 35°C for 5 minutes, 100 μL of enzyme solution treated at different pH values ​​was added and reacted at 35°C for 30 minutes. The subsequent TBA color development and enzyme activity calculation methods were as described above. The highest enzyme activity measured in the reaction was defined as 100%, and the relative enzyme activity at other pH values ​​was calculated.

[0113] Temperature stability analysis

[0114] The enzyme solution was diluted to 0.4 U / mL with HEPES buffer (pH 8.0) and placed at different temperatures for 2 hours and 4 hours respectively. 800 μL of 0.25% sodium alginate solution was evenly mixed with 100 μL of 0.5 M HEPES buffer solution (pH 8.0) to prepare a substrate solution. After the substrate solution was placed at 35°C for 5 minutes, 100 μL of enzyme solution treated at different temperatures was added and reacted at 35°C for 30 minutes. The subsequent TBA color development and enzyme activity calculation method were as described above. The highest enzyme activity measured in the reaction was defined as 100%, and the relative enzyme activity at other temperatures was calculated.

[0115] Effects of different chemicals on the activity of recombinant alginate lyase AlyB

[0116] According to an embodiment of the present invention, the effects of different chemical substances, including metal salts, sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA), on the activity of recombinant sodium alginate lyase AlyB were investigated. 800 μL of 0.25% sodium alginate solution, 100 μL of 0.5 M HEPES buffer (pH 8.0), and 50 μL of 20 mM chemical solution were uniformly mixed to prepare a substrate solution. After the substrate solution was incubated at 35°C for 5 minutes, 50 μL of enzyme solution diluted to 0.8 U / mL was added and the reaction was continued at 35°C for 30 minutes. Subsequent TBA color development and enzyme activity calculation methods were as described above. The highest enzyme activity measured in the reaction was defined as 100%. The relative enzyme activities under the reactions with different chemical substances were calculated, and the experimental data were analyzed for significant differences using the Dunnett method in Mintabe 14 software.

[0117] Substrate Specificity Analysis of Recombinant Alginate Lyase AlyB

[0118] According to an embodiment of the present invention, substrates that can be cleaved by recombinant sodium alginate lyase AlyB are analyzed. The substrates measured include agarose, agar, sodium alginate, alginic acid, carrageenan, and carboxymethyl cellulose. 800 μL of a 0.25% substrate solution was evenly mixed with 100 μL of a 0.5 M HEPES buffer solution (pH 8.0) to prepare a substrate solution. After the substrate solution was placed at 35°C for 5 minutes, 100 μL of an enzyme solution diluted to 0.4 U / mL was added and reacted at 35°C for 30 minutes. The subsequent TBA color development and enzyme activity calculation method are as described above. The highest enzyme activity measured in the reaction was defined as 100%, and the relative enzyme activity under other different substrate reactions was calculated.

[0119] Analysis of products from the cleavage of sodium alginate by recombinant alginate lyase AlyB

[0120] The products of alginate cleavage by recombinant alginate lyase AlyB were analyzed by thin layer chromatography (TLC). 900 mL of a 0.555% sodium alginate solution was prepared and the pH was adjusted to 8.0 with NaOH. Then, 10,000 U of recombinant alginate lyase AlyB was added. Deionized water was added to bring the volume to 1 L and the mixture was incubated at 35°C for 16 to 24 hours. The solution was centrifuged at 38,400 × g for 30 minutes at room temperature and filtered using a 0.2 μm sterile filter cup (Thermo Fisher Scientific, USA). 4 μL of supernatant or 4 μL of 1 μg / μL mannuronic acid monosaccharide (D-mannuronic acid sodium, M), mannuronic acid disaccharide (mannuronic acid disaccharide, DP2), mannuronic acid trisaccharide (mannuronic acid oligosaccharides, DP3), mannuronic acid tetrasaccharide (mannuronic acid oligosaccharides, DP4) and 4 μL of 10 mg / mL sample solution were spotted on silica gel 60F 254 After the sample was air-dried, the TLC plate was tilted and placed in a developing tank containing a developing solution (n-butanol: formic acid: water = 4:6:1). After the development was complete, the TLC plate was removed and air-dried. 100 mL of 99% alcohol, 20 mg of Ce(SO4)2, 50 mg of (NH4)6Mo7O 24After mixing evenly with 5mL H2SO4, prepare the color solution and spray it onto the TLC sheet. Bake at 180°C for 20 minutes using C-MAG HS hotplate stirrers for color development. Calculate the Rf (retention factor) value of the sample and standard signals. Identify the type of pyrolysis product based on the Rf value.

[0121] DPPH radical scavenging ability of cleavage products

[0122] According to an embodiment of the present invention, the antioxidant capacity of alginate lyase cleavage products was determined using a DPPH (α,α-Dipenyl-β-picrylhydrazyl) scavenging assay. The experimental procedure was modified from the method of Shimamura et al. (2014). The freeze-dried powder of alginate cleavage products by recombinant alginate lyase AlyB was re-dissolved in deionized water to prepare solutions with concentrations of 40 mg / mL, 30 mg / mL, 20 mg / mL, 10 mg / mL, and 5 mg / mL. 100 μL of the test sample, 400 μL of 0.1 M Tris-HCl (pH 7.4), and 500 μL of DPPH (0.2 mM in absolute ethanol) were mixed and allowed to stand in the dark at 25°C for 30 minutes. The solution was then centrifuged at 20,640 × g for 5 minutes at room temperature. After centrifugation, remove the supernatant and shake well. Then, take 200 μL of the supernatant and measure the absorbance at 517 nm using an infinite 200Pro microplate reader. Lower absorbance values ​​indicate a greater DPPH-scavenging ability of the sample. Scavenging effect (%) = [(AC - (AS - A sample blank)] / AC × 100; where AC = control group without sample addition; AS = sample; A sample blank = group without DPPH solution addition. The scavenging effect (%) is further used to calculate the 50% scavenging concentration of the sample.

[0123] Analysis of the total antioxidant capacity (TEAC) of pyrolysis products

[0124] According to the examples of the present invention, total antioxidant capacity analysis was performed using the following experimental procedures. Freeze-dried oligosaccharide powder was re-dissolved in deionized water to prepare solutions with concentrations of 40 mg / mL, 30 mg / mL, 20 mg / mL, 10 mg / mL, and 5 mg / mL. 0.25 mL of peroxidase (44 U / mL), 0.25 mL of 1 mM ABTS solution, 1.5 mL of deionized water, and 0.25 mL of 500 μM H₂O₂ were mixed thoroughly and allowed to stand in the dark. After stable blue-green ABTS+ cation radicals were generated, 0.25 mL of sample was added and the reaction was allowed to proceed for 10 minutes. After centrifugation at 20,630 × g for 5 minutes at room temperature, 100 μL of the supernatant was transferred to a 96-well plate and the absorbance was measured at 734 nm using an Infinite 200Pro microplate reader. Lower absorbance values ​​indicate greater total antioxidant capacity. Scavenging effect % = [1 - (absorbance of sample at 734 nm) / (absorbance of control group without sample added at 734 nm)] × 100. The scavenging effect was used to calculate the half-scavenging concentration of the sample.

[0125] Analysis of angiotensin I-converting enzyme inhibitory activity

[0126] The ACE Kit-WST kit (Dojindo, Japan) was used to analyze the ability of the cleavage products to inhibit angiotensin-converting enzyme. The experimental steps are as follows. The freeze-dried cleavage product powder was dissolved back in deionized water to prepare solutions with concentrations of 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL and 1.25 mg / mL. 20 μL of the above solutions were respectively drawn into a 96-well plate, and the reagents in the kit were added. The absorbance of the samples was measured at 450 nm. The inhibition rate was calculated using the following formula. Inhibition rate (%) = [(OD 450_blank 1 -OD 450_sample ) / (OD 450_balnk 1 -OD 450_blank 2 )]×100; blank 1 represents the group without inhibitor; blank 2 represents the group without enzyme. The inhibition rate was used to further calculate the half maximal inhibitory concentration (IC 50 ).

[0127] Antiviral ability of lysis products

[0128] Virus-sensitive Fcwf-4 and Marc-145 cells were treated with different concentrations of DEH before and after infection with FIPV and PRRSV, respectively. Viral titers were assessed post-infection to assess the antiviral activity of 4-deoxy-L-erythro-hexoseulose uronic acid (DEH).

[0129] 1. Evaluation of antiviral activity using serotype II feline infectious peritonitis virus (FIPV) to infect the feline macrophage cell line Fcwf-4

[0130] Cell culture

[0131] The Fcwf-4 cell line was purchased from the American Type Culture Collection (ATCC). The culture medium used for culturing cells consists of 90% Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS). The culture medium used for infected cells consists of 98% DMEM and 2% FBS. Place the cryovial in a 37°C water bath. Once completely thawed, transfer the cell suspension to a container containing an appropriate amount of culture medium and culture in an incubator. Cells are passaged when the cell density reaches 80% or higher. To passage cells, remove the original culture medium, rinse the plate with phosphate-buffered saline (PBS), and then add trypsin to suspend the cells. Subsequently, transfer an appropriate amount of cells to a container containing fresh culture medium for culture.

[0132] Virus titer determination

[0133] Virus titer determination is divided into two implementation modes: "DEH pre-treatment" and "DEH post-infection treatment". In the "DEH pre-treatment" mode, Fcw-4 is first planted in a 96-well cell culture dish at an appropriate density. The virus solution is also mixed with different concentrations of DEH, and the final DEH concentration is 0, 0.0625, 0.5, 1, 2, 5, 10 and 20 mg / mL. After 1 hour of reaction, the virus solution containing different concentrations of DEH is added to the cells. After culturing for 2 days, the culture fluid is collected for virus titer determination. In the "DEH post-infection treatment" implementation mode, the virus solution is added to the cells and infected for 1 hour. After removing the virus solution, infection culture medium containing 0, 0.05, 0.4, 1.6, 2, 4 and 8 mg / mL DEH is added to the cells. After culturing for 2 days, the culture fluid is collected for virus titer determination.

[0134] 2. Evaluation of antiviral activity using porcine reproductive and respiratory syndrome virus (PRRSV) to infect African green monkey embryonic kidney cell line Marc-145

[0135] Cell culture

[0136] The Marc-145 cell line was obtained from the Swine Science Research Institute in Taiwan (Chang et al., 1993). The culture medium used to culture the cells consists of 98% DMEM and 2% FBS. Place the cryovial in a 37°C water bath. Once completely thawed, remove the cell suspension and add it to a container containing an appropriate amount of culture medium. Incubate the cells in an incubator. Once the cell density reaches 80% or higher, proceed to cell passage. To passage cells, remove the original culture medium, rinse the plate with PBS, and then add trypsin to suspend the cells. Next, transfer an appropriate amount of cells to a container containing fresh culture medium for incubation.

[0137] Virus titer determination

[0138] Virus titer determination is divided into two implementation modes: "pre-DEH treatment" and "DEH treatment after infection". In the "pre-DEH treatment" mode, Marc-145 is first planted at an appropriate density in a 96-well cell culture dish. The virus solution is mixed with DEH, and the final DEH concentration is 0, 0.3125, 0.625, 2.5, 5 and 10 mg / mL. After one hour of reaction, virus solution containing different concentrations of DEH is added to the cells. After culturing for 4 days, the culture fluid is collected for virus titer determination. In the "DEH treatment after infection" implementation mode, Marc-145 is planted at an appropriate density in a 96-well cell culture dish, and the virus solution is added to the cells for infection for 1 hour. After removing the virus solution, culture medium containing 0, 0.06, 0.13, 0.25, 0.5 and 1 mg / mL DEH is added to the cells. After culturing for 4 days, the culture fluid is collected for virus titer determination.

[0139] Example 1

[0140] Cloning and Expression Vector Construction of Paenibacillus alginate lyase AlyB Gene

[0141] Whole-genome sequence analysis revealed that Paenibacillus may harbor two alginate lyase genes, designated alyA and alyB. Bioinformatics analysis revealed that the alyA gene sequence is 1,128 bp long, encoding 375 amino acids, with a start codon and a terminator sequence of ATG and TGA, respectively. The alyB gene sequence is 2,304 bp long, with a start codon and a terminator sequence of ATG and TAG, respectively, encoding 767 amino acids. Analysis of the amino acid sequences of AlyA and AlyB using SignalP 5.0 Server (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) predicted that these proteins lack signal peptides and are intracellular. The inventors previously conducted gene cloning, recombinant protein expression, purification, and enzyme activity analysis for AlyA. The results showed that AlyA lacks alginate lyase activity. This present invention further investigates alyB. Comparison of the AlyB amino acid sequence using the NCBI BLASTP program revealed that AlyB is predicted to contain the DUF4962 functional block of unknown function and the Hepar_II_III functional block, which is similar to the amino acid sequences of Flavobacterium heparinase II and III. The amino acid sequences of three predicted enzymes, including Paenibacillus elgii M63 alginate lyase (GenBank accession No. KZE76488.1), Paenibacillus elgii AC13 alginate lyase (GenBank accession No. PUA38957.1), and Paenibacillus agaridevorans T-3040 alginate lyase (GenBank accession No. GBG06091.1), showed 75.98%, 75.85%, and 65.32% identity, respectively (Figure 1).

[0142] To verify that the protein encoded by alyB indeed possesses alginate lyase activity, further steps were taken to clone the gene, express the recombinant protein, purify the recombinant protein, and analyze its enzyme properties. For gene cloning, specific primers were designed targeting the alyB gene sequence, and the target gene fragment was amplified using polymerase chain reaction. Next, the alyB gene was cloned using the CloneJET PCR Cloning Kit. The plasmid containing the alyB gene was named pJET-AlyB. After pJET-AlyB was cleaved with BglII and SalI, the alyB gene was ligated into pET29a(+) cleaved with BamHI and SalI using T4 DNA ligase to construct the alginate lyase expression vector pET-AlyB.

[0143] The pET-AlyB recombinant vector comprises the nucleotide sequence shown in SEQ ID NO: 5, and specifically comprises the combination shown in Table 1 below:

[0144] Table 1

[0145] Example 2

[0146] Expression of recombinant alginate lyase AlyB in Escherichia coli

[0147] The alginate lyase expression vector pET-AlyB was transformed into E. coli BL21(DE3) for induced protein expression. SDS-PAGE and immunoblotting revealed that, compared to E. coli BL21(DE3) / pET-29a(+), the E. coli BL21(DE3) transformant harboring the pET-AlyB expression vector expressed recombinant alginate lyase with a His-tag at the C-terminus. The molecular weight of the band was close to the expected molecular weight of 89 kDa (Figure 2).

[0148] Example 3

[0149] Effects of induction temperature and co-expressed chaperone protein on the solubility of recombinant alginate lyase AlyB

[0150] Induction temperature tests showed that lowering the induction temperature increased the solubility of recombinant alginate lyase AlyB. When inducing expression of recombinant alginate lyase AlyB at 37°C, 30°C, 25°C, and 18°C, the solubility levels of recombinant alginate lyase AlyB were 14.5%, 35.8%, 66.5%, and 89.5%, respectively (Figure 3). Intracellular enzyme activity analysis also revealed that compared to the intracellular enzyme activity (0.04 U / mL) induced at 37°C, the intracellular enzyme activity induced at 18°C ​​(0.82 U / mL) was increased 20.5-fold (Figure 4). Subsequently, expression vectors containing different chaperone protein genes, including pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTf16, were transformed into E. coli BL21(DE3) / pET-AlyB to investigate the effect of co-expression of different chaperone proteins on the solubility of recombinant alginate lyase AlyB. SDS-PAGE and immunoblotting showed that co-expression of the chaperone proteins combined with an induction temperature of 25°C enhanced the solubility and expression level of recombinant alginate lyase AlyB (Figure 5). ImageQuant software was used to analyze the volume of soluble target protein and the volume of target protein in total cell lysate for each expression group, and the solubility of the target protein was calculated. The results showed that co-expression of GroES-GroEL increased the solubility of recombinant alginate lyase AlyB by 1.2-fold and the protein expression level by 2.5-fold (Figure 6). Intracellular enzyme activity analysis also demonstrated that co-expression of GroES-GroEL significantly enhanced enzyme activity (Figure 7). Compared with the intracellular enzyme activity of E. coli BL21 (DE3) / pET-AlyB (0.051 U / mL), the intracellular enzyme activity of E. coli BL21 (DE3) / pET-AlyB / pGro7 co-expressing GroES-GroEL (1.402 U / mL) increased by 27.49 times.

[0151] Example 4

[0152] Purification and Characterization of Recombinant Alginate Lyase AlyB

[0153] Recombinant alginate lyase AlyB was expressed using E. coli BL21(DE3) / pET-AlyB / pGro7, and intracellular soluble recombinant alginate lyase AlyB was purified by immobilized metal ion affinity chromatography. SDS-PAGE results showed that recombinant alginate lyase AlyB with a His-tag at the C-terminus could be purified by affinity chromatography (Figure 8). The eluates from tubes 1 to 7 were combined and analyzed again by protein electrophoresis and immunoblotting (Figure 9). The immunoblotting results confirmed that the purified protein was indeed recombinant alginate lyase AlyB. The purity of the purified enzyme reached 94.15%, with a specific activity of 142.85 U / mg. Approximately 5.43 mg of recombinant alginate lyase AlyB was purified from 1 L of bacterial culture.

[0154] Analysis of the enzyme's optimal pH and temperature showed that the recombinant alginate lyase AlyB has an optimal pH of 8.0. Enzyme activity significantly decreased at pH levels below 7.0 or above 9.0. The optimal temperature for recombinant alginate lyase AlyB was 35°C, with activity dropping sharply at 40°C (Figure 10). Analysis of the enzyme's pH and temperature stability showed that recombinant alginate lyase AlyB maintained over 80% activity after 4 hours at pH levels of 5.0 to 10.0 and 4 to 35°C (Figure 11).

[0155] Example 5

[0156] Effects of chemicals on the activity of recombinant alginate lyase AlyB.

[0157] The results of the analysis of the effects of different chemicals on the activity of recombinant sodium alginate lyase AlyB showed that Co 3+ 、Cu 2+ , Ca 2+ 、Zn 2+ 、Fe 2+ and SDS significantly inhibited enzyme activity (Table 2). Substrate specificity analysis showed that recombinant sodium alginate lyase AlyB had the best cleavage activity against alginate, followed by sodium alginate, with a relative activity of 61.85%. However, it was unable to cleave agarose, agar, carrageenan, or carboxymethyl cellulose (Table 3).

[0158] Table 2

[0159] Table 3

[0160] Example 6

[0161] Kinetic analysis of recombinant alginate lyase AlyB.

[0162] Kinetic analysis of recombinant alginate lyase AlyB was performed using sodium alginate as a substrate. The results showed that the Vmax of recombinant alginate lyase AlyB was 220.30 U / mg, and the Km was 3.60 mg / mL, as determined by the Lineweaver-Burk plot method (Figure 12). TLC analysis revealed that the primary product of alginate cleavage by recombinant alginate lyase AlyB was 4-deoxy-L-erythro-hexuronic acid (DEH) (Figure 13), indicating that recombinant alginate lyase AlyB is an exo-acting alginate lyase.

[0163] Exo-alginate lyases that have been studied include A1-IV of Sphingomonas sp.A1 (Miyake et al., 2003), Atu3025 of Agrobacterium tumefaciens C58 (Ochiai et al., 2006), Alg17C of Saccharophagus degradans 2-40 (Kim et al., 2012), AlyFRB of Falsirhodobacter sp.alg1 (Mori et al., 2016), AlgL17 of Microbulbifer sp.ALW1 (Jiang et al., 2019), VxAly7D of Vibrio xiamenensis QY104 (Tang et al., 2020), Aly1281 of Pseudoalteromonas carrageenovora (Zhang et al., 2020), and Alteromonas portus HB161718 (Zhang et al., 2016). TAlg2951 from (Huang et al., 2021), PsMan8A from Paradendryphiella salina (Pilgaard et al., 2021), AlgSH17 from Microbulbifer sp. SH-1 (Yang et al., 2021), Alys1 from Tamlana sp. s12 (Yin et al., 2021), and VsAly7D from Vibrio sp. QY108 (Zhang et al., 2021). AlyB of the present invention differs from the enzymes described in the aforementioned literature in both amino acid sequence and properties and is a novel exo-acting alginate lyase.

[0164] Example 7

[0165] Analysis of the antioxidant capacity of sodium alginate pyrolysis products

[0166] The present invention also measured the DPPH (α,α-diphenyl-2-picrylhydrazyl) free radical scavenging capacity and total antioxidant capacity (TAC) of the products formed by the cleavage of sodium alginate by recombinant alginate lyase AlyB to understand their antioxidant activity. As shown in Figure 14, the products formed by the cleavage of sodium alginate by recombinant alginate lyase AlyB exhibit antioxidant activity, with the antioxidant effect increasing with increasing concentration of the cleavage product. The half-inhibitory concentrations (CI50s) of the alginate cleavage products for DPPH and ABTS scavenging, as determined by regression analysis of the assay data using SigmaPlot software, were 31.5 mg / mL and 8.05 mg / mL, respectively. Research indicates that free radicals in the human body and the surrounding environment can cause oxidative stress, leading to numerous health problems such as inflammation, cardiovascular disease, diabetes, and cancer. The product of the recombinant sodium alginate lyase AlyB of the present invention has the ability to scavenge DPPH free radicals and has antioxidant capacity, and can be used to prepare antioxidant food additives, antioxidant pharmaceutical compositions or antioxidant cosmetic additives.

[0167] Example 8

[0168] Analysis of antiviral ability of sodium alginate lysis products

[0169] The present invention also studies the antiviral ability of sodium alginate lysate. The results show that pre-treatment with DEH can effectively reduce the infection of FIPV with Fcwf-4, EC 50Reaching 2.3583 mg / mL (Figure 15). Post-infection treatment with DEH can also effectively reduce the persistent infection of FIPV, EC 50 In addition, pre-treatment with DEH can also effectively reduce the infection of PRRSV in Marc-145 cells. 50 Reaching 2.9174 mg / mL (Figure 17). Post-infection treatment with DEH can also effectively reduce the persistent infection of PRRSV. 50 The above results show that DEH can effectively reduce the infection of FIPV and PRRSV in Fcwf-4 cells and Marc-145 cells.

[0170] The above experiments indicate that the sodium alginate lysis product has the ability to inhibit viral infection and can be used to prepare a food additive or pharmaceutical composition that inhibits viral infection.

[0171] Embodiment 9

[0172] Analysis of the ability of sodium alginate lysis products to inhibit angiotensin-converting enzyme

[0173] The present invention also investigated the ability of sodium alginate cleavage products to inhibit angiotensin-converting enzyme (ACE). Results showed that the product formed by cleaving sodium alginate using recombinant alginate lyase AlyB had the ability to inhibit ACE; this inhibition increased with increasing cleavage product concentration (Figure 19). The half-inhibition rate of the sodium alginate cleavage product on ACE was 7.41 mg / mL. Because the recombinant sodium alginate cleavage product has the ability to inhibit ACE, it can be used to prepare a food additive or pharmaceutical composition that inhibits ACE.

Claims

1. An alginate lyase AlyB, whose amino acid sequence is shown in SEQ ID NO:

1.

2. The alginate lyase AlyB according to claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO:

2.

3. The alginate lyase AlyB according to claim 1, wherein the optimal action pH thereof is 7.0 to 9.

0.

4. The alginate lyase AlyB according to claim 1, wherein the optimal action temperature is 33 to 37°C. The sodium alginate lyase AlyB according to claim 1 , which cleaves alginic acid and sodium alginate. The alginate lyase AlyB according to claim 1 , wherein the cleavage mode is exo-type.

7. The alginate lyase AlyB according to claim 6, wherein the main product after degradation is 4-deoxy-L-erythro-hexuronic acid (DEH). 8 . The alginate lyase AlyB according to claim 1 , which is isolated from Paenibacillus.

9. A recombinant vector comprising a nucleic acid encoding the alginate lyase AlyB according to any one of claims 1 to 8.

10. The recombinant vector according to claim 9, further comprising chaperone protein expression vectors pG-KJE8, pGro7, pKJE7 and / or pTf16. The recombinant vector according to claim 9 , further comprising the nucleotide sequence shown in SEQ ID NO:

5. 12 . A transformant comprising the recombinant vector according to claim 9 .

13. A method for preparing the sodium alginate lyase AlyB according to any one of claims 1 to 8, comprising: Prepare the recombinant vector according to any one of claims 9 to 11; Transforming the recombinant vector into a host cell and obtaining a transformant as claimed in claim 12 therefrom; The transformant is cultured and expresses alginate lyase AlyB; The expressed alginate lyase AlyB was isolated and purified. The method according to claim 13 , wherein the host cell is Escherichia coli BL21 (DE3).

15. Use of the sodium alginate lyase AlyB according to any one of claims 1 to 8, for lysing alginic acid and / or sodium alginate.

16. Use of the alginate lyase AlyB according to any one of claims 1 to 8 for preparing cosmetics, food additives or pharmaceutical compositions for scavenging DPPH and ABTS free radicals.

17. Use of the sodium alginate lyase AlyB according to any one of claims 1 to 8 for preparing antioxidant cosmetics, food additives or pharmaceutical compositions.

18. Use of the sodium alginate lyase AlyB according to any one of claims 1 to 8 for preparing a food additive or a pharmaceutical composition for inhibiting viral infection.

19. Use of the sodium alginate lyase AlyB according to any one of claims 1 to 8 for preparing a food additive or a pharmaceutical composition for inhibiting angiotensin converting enzyme.

Images