Sodium alginate lyase, method of preparation thereof, and use thereof.

Abstract

This invention provides a novel sodium alginate lyase AlyB, a method for producing the same, and its use. The main degradation product of the novel sodium alginate lyase AlyB is a monosaccharide, which has antioxidant, antiviral, and angiotensin-converting enzyme inhibitory effects, making it applicable to fields such as cosmetics, pharmaceuticals, and health foods.

Description

[Technical Field]

[0001] The present invention relates to alginate lyase AlyB, a method for preparing the same, and its use. Specifically, the present invention relates to alginate lyase AlyB, which can cleave alginate and / or sodium alginate, and whose main degradation product is 4-deoxy-L-erythro-5-hexoseuronic acid (DEH). [Background technology]

[0002] Sodium alginate is a polysaccharide polymerized from β-D-mannuronic acid (M) and α-L-guluronic acid (G), and is mainly found in the cell walls of brown algae such as kelp and Sargassum. Some bacteria, such as Pseudomonas aeruginosa, also have the ability to synthesize sodium alginate. The enzyme that breaks down sodium alginate is called alginate lyase, also known as alginase or alginate depolymerase. It is an enzyme that cleaves the glycosidic bonds of sodium alginate through a β-elimination reaction and belongs to the polysaccharide lyase family (EC4.2.2). Based on the similarity of their amino acid sequences, alginate lyases are classified into the PL5, PL6, PL7, PL14, PL15, PL17, and PL18 families of polysaccharide lyases. Different sources of alginate lyase exhibit different properties.

[0003] Alginate lyases can be classified into endo-lyases and exo-lyases based on their cleavage mechanisms. Endo-cleavage lyases can be further classified into (i) α-L-glucuronate lyases that can degrade polyguluronic acid (PG), (ii) β-D-mannuronate lyases that can degrade polymannuronic acid (PM), and (iii) bifunctional lyases that can degrade both PG and PM, depending on the substrate they can degrade. The aforementioned endo-lyases can cleave sodium alginate into oligosaccharides having a degree of polymerization of 2 to 7. Exo-lyases can cleave sodium alginate into unsaturated monosaccharides, which then undergo non-enzymatic conversion to 4-deoxy-L-erythro-5-hexoceuroseuronic acid (DEH).

[0004] Alginate lyase has applications in the fields of medicine, agriculture, industry, and health foods. In the medical field, alginate lyase can degrade alginate biofilms secreted by mucoid Pseudomonas aeruginosa and can be used to treat lung diseases caused by Pseudomonas aeruginosa. In the agricultural field, alginate lyase can be used to treat brown algae waste, converting the waste into oligosaccharides or monosaccharides, which not only solves environmental pollution problems but also realizes the value of agricultural waste resource recycling. In the industrial field, researchers have used metabolic engineering to create alginate lyase-producing strains from Escherichia coli (E. coli) or Saccharomyces cerevisiae, and then used the resulting DEH to produce alcohol, demonstrating its potential application in biomass alcohol production. In the health food sector, oligosaccharides obtained from the decomposition of sodium alginate by alginate lyase are thought to have various biological activities, including anti-inflammatory, antioxidant, immunomodulatory, anticancer, neuroprotective, antibacterial, blood pressure lowering, blood lipid lowering, blood glucose lowering, obesity suppression, and prebiotic properties, and are considered to have the potential to be developed as health food ingredients. [Overview of the project] [Problems that the invention aims to solve]

[0005] Based on the versatility of alginate lyase, the object of the present invention is to provide a novel alginate lyase that can cleave alginate and / or sodium alginate and produce functional degradation products. [Means for solving the problem]

[0006] To achieve the above objective, the present invention provides alginate lyase AlyB comprising the amino acid sequence shown in Sequence ID No. 1. Preferably, the alginate lyase AlyB is encoded by the nucleotide sequence shown in SEQ ID NO: 2.

[0007] Preferably, alginate lyase AlyB has an optimal pH range of 7.0 to 9.0. Preferably, alginate lyase AlyB has an optimal operating temperature range of 33°C to 37°C.

[0008] Preferably, alginate lyase AlyB cleaves alginate and sodium alginate. Preferably, alginate lyase AlyB is an exo-type alginate lyase.

[0009] Preferably, the main degradation product after dissolution by alginate lyase AlyB includes 4-deoxy-L-erythro-5-hexoseuronic acid (DEH). Preferably, alginate lyase AlyB is isolated from the genus Paenibacillus.

[0010] A further object of the present invention is provided: a recombinant vector comprising the nucleic acid encoding the alginate lyase AlyB described above. Preferably, the recombinant vector further comprises a chaperone expression vector selected from pG-KJE8, pGro7, pKJE7, and / or pTf16.

[0011] Preferably, the recombinant vector further comprises the nucleotide sequence shown in SEQ ID NO: 5. According to another object of the present invention, a transformant containing the above recombinant vector is provided.

[0012] According to still another object of the present invention, a method for preparing the alginate lyase AlyB is provided, the method comprising preparing the recombinant vector, obtaining the transformant by transforming a host cell with the recombinant vector, culturing the transformant to produce the expressed alginate lyase AlyB, and isolating and purifying the expressed alginate lyase AlyB.

[0013] [[ID=·10]] Preferably, the host cell is Escherichia coli BL21(DE3). According to one object of the present invention, the use of the alginate lyase AlyB for use in cleaving alginic acid and / or sodium alginate is provided.

[0014] According to one object of the present invention, the use of the alginate lyase AlyB for preparing a cosmetic, food additive, or pharmaceutical composition for capturing DPPH free radicals and ABTS free radicals is provided.

[0015] According to one object of the present invention, the use of the alginate lyase AlyB for preparing a cosmetic, food additive, or pharmaceutical composition having antioxidant activity is provided. According to one object of the present invention, the use of the alginate lyase AlyB for preparing a food additive or pharmaceutical composition for inhibiting viral infection is provided.

[0016] According to one object of the present invention, the use of the alginate lyase AlyB for preparing a food additive or pharmaceutical composition for inhibiting angiotensin converting enzyme is provided. Based on the above characteristics, the present invention provides a novel alginate lyase, a method for preparing the same, a vector, and a transformant. The alginate lyase can produce functional degradation products by cleaving alginate and / or sodium alginate, thereby achieving the objective of cleaving alginate and / or sodium alginate to prepare cosmetic, food additive, or pharmaceutical compositions having antioxidant properties, antiviral properties, or angiotensin-converting enzyme inhibitory properties. [Brief explanation of the drawing]

[0017] [Figure 1-1] This shows the amino acid sequence alignment of Paenibacillus alginate lyase AlyB with other Paenibacillus alginate lyases. PEAly-M63 is derived from Paenibacillus elgii, PEAly-AC13 is derived from Paenibacillus elgii, and PEAly-T-3040 is derived from Paenibacillus agaridevorans. [Figure 1-2] Same as above. [Figure 2] This diagram shows the analysis of recombinant alginate lyase AlyB expression in E. coli BL21(DE3) using protein electrophoresis (A) and Western blotting (B). Lane M represents the PageRuler-preserved protein ladder; lane C represents the total cell lysate of E. coli BL21(DE3)[pET-29a(+)] induced with 0.1 mM IPTG at 37°C for 4 hours; and lane T represents the total cell lysate of E. coli BL21(DE3)(pET-AlyB) induced with 0.1 mM IPTG at 37°C for 4 hours. The arrows indicate the expected molecular weight position of recombinant alginate lyase AlyB. [Figure 3]This diagram shows the effect of various induction temperatures on the soluble expression of recombinant alginate lyase AlyB in E. coli BL21(DE3) using protein electrophoresis (A) and Western blotting (B). Lane M represents the PageRuler-preserved protein ladder; lane T represents the total cell lysates of E. coli BL21(DE3)(pET-AlyB) induced at various temperatures and under 0.1 mM IPTG conditions; and lane S represents the soluble protein fraction of E. coli BL21(DE3)(pET-AlyB) induced at various temperatures and under 0.1 mM IPTG conditions. Arrows indicate the expected molecular weight position of recombinant alginate lyase AlyB. [Figure 4] The results of intracellular recombinant alginate lyase AlyB activity in *E. coli* BL21(DE3)(pET-AlyB) induced under various temperatures and 0.1 mM IPTG conditions are shown. [Figure 5] The effect of co-expressed chaperone proteins on the solubility of recombinant AlyB is shown using protein electrophoresis (A) and Western blotting (B). Lane M represents the PageRuler-preserved protein ladder; lane T represents the total cell lysate; and lane S represents the soluble protein fraction. Arrows indicate the expected molecular weight position of recombinant alginate lyase AlyB. [Figure 6] This shows the expression of recombinant alginate lyase AlyB in various E. coli transformants. (A) Comparison of the solubility of recombinant alginate lyase AlyB; (B) Comparison of the yield of soluble AlyB. [Figure 7] This study demonstrates the effect of co-expressed chaperone proteins on intracellular recombinant AlyB activity in Escherichia coli BL21(DE3)(pET-AlyB). [Figure 8] This shows the analysis of recombinant alginate lyase AlyB purified from the intracellular soluble fraction of E. coli BL21(DE3) / pET-AlyB / pGro7 using protein electrophoresis. Lane M represents the Page Ruler-preserved protein ladder; lane F represents the eluate; lanes 1-7 represent the eluate. Arrows indicate the expected molecular weight position of recombinant alginate lyase AlyB. [Figure 9] The analysis of purified recombinant alginate lyase AlyB using protein electrophoresis (A) and Western blotting (B) is shown. Lane M represents the PageRuler-preserved protein ladder; lane 1 represents purified recombinant alginate lyase AlyB. The arrows indicate the expected molecular weight position of recombinant alginate lyase AlyB. [Figure 10] This study demonstrates the effects of various pH values ​​and temperatures on the activity of recombinant alginate lyase AlyB. (A) Effects of various pH values ​​on the activity of recombinant alginate lyase AlyB. The buffers used were 50 mM citrate buffer (pH 3-6, ●), phosphate buffer (pH 6-7, △), HEPES-NaOH buffer (pH 7-8, ■), bicine-NaOH buffer (pH 8-9, ○), and glycine-NaOH buffer (pH 9-10, ▲). (B) Effects of various temperatures on the activity of recombinant alginate lyase AlyB. The highest enzyme activity was defined as 100%. All experiments were performed in triplicates, and the results are expressed as mean ± standard deviation. [Figure 11] This study demonstrates the effects of various 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 buffers of various pH values, storing them at 4°C for 4 hours, and then measuring the enzyme activity. The temperature stability of recombinant alginate lyase AlyB was determined by placing the enzyme in a pH 8.0 buffer, storing it at various temperatures for 4 hours, and then measuring the enzyme activity. The highest enzyme activity was defined as 100%. All experiments were performed in triplicates, and the results are expressed as mean ± standard deviation. [Figure 12] The enzymatic reaction rate analysis of recombinant alginate lyase AlyB is shown. (A) Michaelis-Menten plot. (B) Lineweaver-Burk plot. All experiments were performed in triplicates, and the results are expressed as mean ± standard deviation. [Figure 13]This shows the analysis of sodium alginate degradation products by recombinant alginate lyase AlyB using thin-layer chromatography. Lane 1 represents sodium D-mannuronate (M); Lane 2 represents D-mannuronobiose (DP2); Lane 3 represents D-mannuronotriose (DP3); Lane 4 represents D-mannuronotetraose (DP4); Lane 5 represents the sodium alginate degradation products by recombinant alginate lyase AlyB. [Figure 14] This paper shows the DPPH free radical scavenging ability (A) and total antioxidant capacity (B) of the product obtained from the degradation of sodium alginate by recombinant alginate lyase AlyB. All experiments were performed in triplicates, and the results are shown as mean ± standard deviation. [Figure 15] This study demonstrates the effect of DEH on inhibiting FIPV infection in the feline macrophage cell line Fcwf-4 after treatment with various concentrations of DEH mixed with serotype II feline infectious peritonitis virus (FIPV) for 1 hour. [Figure 16] This study demonstrates the effects of post-infection treatment with various concentrations of DEH on FIPV infection in Fcwf-4 cells. [Figure 17] This study demonstrates the effect of DEH on inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection in the African green monkey embryonic kidney cell line Marc-145 one hour after treatment with various concentrations of DEH in combination with PRRSV. [Figure 18] This study demonstrates the effects of post-infection treatment with various concentrations of DEH on PRRSV infection in Marc-145 cells. [Figure 19] This study demonstrates the inhibitory activity of the product formed by cleaving sodium alginate with recombinant alginate lyase AlyB against angiotensin-converting enzyme. All experiments were performed in triplicates, and the results are presented as mean ± standard deviation. [Modes for carrying out the invention]

[0018] Embodiments will be described in detail below with reference to the attached drawings. However, these embodiments may be carried out in different forms and are not the only forms for carrying out or utilizing a particular embodiment of the claimed invention. Therefore, they should not be construed as limitations on the embodiments described above. The embodiments encompass the features of several particular embodiments, as well as method steps and their sequence for constructing and operating these particular embodiments. However, other particular embodiments may also be used to achieve the same or equivalent functions and sequence of steps. Rather, these embodiments are provided so that this specification may fully and completely disclose the spirit of the invention to those skilled in the art. Similar element symbols in the drawings refer to similar elements. In the following description, prior art functions or structures will not be described in detail in order to avoid unnecessary details in the embodiments.

[0019] Where the context does not contradict the use of singular nouns herein, singular nouns encompass their plural forms, and vice versa. Furthermore, in this specification and in the claims, expressions such as “at least one” and “one or more” have the same meaning and both indicate one, two, three or more.

[0020] The numerical ranges and parameters used to define the broader scope of the present invention are approximate, but the relevant values ​​in specific embodiments are presented as accurately as possible. However, due to the individual test methods, all numerical values ​​inevitably include a standard deviation. Where used herein, “approximately” generally means that the actual value is within ±10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the word “approximately” may mean that the actual value falls within the acceptable standard error of the mean, as determined by those skilled in the art. Unless otherwise explicitly stated, or unless otherwise provided in embodiments, all ranges, quantities, values, and percentages used herein (e.g., to describe material usage, duration, temperature, operating conditions, quantity ratios, etc.) are modified with “approximately.” Thus, the numerical parameters disclosed herein and in the claims are approximations unless otherwise specified and are subject to modification as necessary. These numerical parameters should be understood as numerical values ​​obtained by applying the indicated significant figures and general carry methods. Here, a numerical range is expressed as from one endpoint to the other, or between two endpoints, and unless otherwise specified, all numerical ranges described herein include the endpoints.

[0021] A detailed description of various specific embodiments of the present invention is given below. Other features of the present invention will become apparent from the detailed description of the specific embodiments below and from the claims. Without further explanation, it is assumed that those skilled in the art can make the most use of the present invention based on the foregoing description. Therefore, it is understood that the following description is merely illustrative and is not intended to limit the remaining disclosure in any way. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0022] In this specification, when referring to concentration, the terms “percentage” and “%” may refer to mass concentration or volume percentage concentration. Mass concentration is defined as the percentage obtained by dividing the mass of the solute by the volume of the corresponding mixture (including the volume of the solute), and is expressed herein by “(w / v)” following “percentage” or “%”. Volume percentage concentration is defined as the percentage of the volume of the solute relative to the total volume of the solution, and is expressed herein by “(v / v)” following “percentage” or “%”.

[0023] As used herein, the terms “produced from” and “comprising” are synonymous. As used herein, the terms “includes, including,” “comprises, comprising,” “has, having,” “contains, containing,” or any other variation thereof, are intended to include nonexclusivity. For example, a composition, process, method, article, or device containing any of the listed elements is not necessarily limited to those listed and may include other elements that are not expressly listed but are associated with that composition, process, method, article, or device. The term “comprising” is used generally to mean including, i.e., allowing the presence of one or more other features or components.

[0024] As used herein, the terms “host” and “host cell” are interchangeable and may refer to a prokaryotic cell. One or more vectors described in the present invention can be introduced into a host cell, or a vector containing a gene to be expressed can be inserted into a host cell using genetic engineering methods. As those skilled in the art will understand, the above terms refer not only to specific cells but also to the offspring or potential offspring of such cells. Changes may occur in subsequent generations due to genetic mutations or environmental influences, and therefore the offspring of these cells may not be identical to the parent cells, but they are still included within the scope of the terms used herein.

[0025] The main objective of this invention is to clone a novel alginate lyase gene, AlyB, from the genus Paenibacillus and to explore the effectiveness of producing recombinant enzymes using an E. coli expression system. Subsequently, recombinant alginate lyase AlyB having a His tag at the C-terminus is purified by immobilized metal ion affinity chromatography, and the enzyme properties of the purified alginate lyase AlyB are investigated. Next, sodium alginate is cleaved using recombinant alginate lyase AlyB, and the properties of the degradation products are analyzed. This will lead to the development of a novel alginate lyase AlyB capable of cleaving alginate and / or sodium alginate to produce functional degradation products.

[0026] According to one embodiment of the present invention, alginate lyase AlyB having the amino acid sequence shown in SEQ ID NO: 1 is provided. According to another embodiment of the present invention, alginate lyase AlyB has the nucleotide sequence shown in SEQ ID NO: 2. According to one embodiment of the present invention, alginate lyase AlyB has an optimal operating pH range of 7.0 to 9.0, preferably 8. Furthermore, its optimal operating temperature is 33°C to 37°C, preferably 35°C. Alginate lyase AlyB according to some embodiments of the present invention cleaves alginate and sodium alginate via an exo-type mechanism, and its main degradation product is 4-deoxy-L-erythro-5-hexoceuroseuronic acid (DEH). Alginate lyase AlyB is derived from the genus Paenibacillus, particularly Paenibacillus chondroitinus, and was purchased from the Bioresource Collection and Research Center of the Food Industry Development Foundation. Its strain number is BCRC 15913.

[0027] According to one embodiment of the present invention, a recombinant vector comprising a nucleic acid encoding alginate lyase AlyB is provided. According to another embodiment of the present invention, the recombinant vector may further comprise a chaperone expression vector selected from the group consisting of pG-KJE8, pGro7, pKJE7, and pTf16 in order to improve enzyme activity and solubility.

[0028] One embodiment of the present invention provides a method for preparing alginate lyase AlyB, comprising: preparing a recombinant vector; transforming host cells with the recombinant vector to obtain transformants; culturing the transformants to produce expressed alginate lyase AlyB; and isolating and purifying the expressed alginate lyase AlyB. The host cells may be Escherichia coli BL21(DE3) or other suitable host cells.

[0029] Alginate lyase AlyB, prepared according to embodiments of the present invention, is applicable to the decomposition of alginate and / or sodium alginate, and the resulting decomposition products are suitable for use in the preparation of cosmetic, food additive, or pharmaceutical compositions having antioxidant properties or inhibiting vasoconstriction.

[0030] The following specific embodiments, along with the accompanying drawings, illustrate the alginate lyase of the present invention, its preparation method, vector, transformant, and its use. Materials and methods Bacterial strains used: E. coli BL21(DE3) We purchased E. coli BL21(DE3) from New England Biolabs (NEB), USA; its genotype is F-ompT gal dcm lon hsdSB(r B - m B - )λ(DE3). This host cell contains T7RNA polymerase and was used as a host cell for recombinant protein expression.

[0031] Paenibacillus genus Paenibacillus chondroitinus was purchased from the Biological Resource Conservation and Research Center of the Food Industry Development Research Foundation, and its strain number is BCRC15913. This strain was used as a source of alginate lyase gene.

[0032] vector pJET1.2 This vector, purchased from Thermo Fisher Scientific in the United States, is a blunt-ended cloning vector. Its size is 2,974 bp, and it contains an ampicillin resistance gene.

[0033] pET29a(+) This vector, purchased from Merck Millipore in the United States, is a vector for recombinant protein expression. Its size is 5,371 bp, and it contains a kanamycin resistance gene.

[0034] pG-KJE8 This vector was purchased from TaKaRa Co., Ltd. in Japan. This vector contains the dnaK-dnaJ-grpE-groES-groEL chaperone genes, is an 11.1kb chaperone expression vector, and contains the chloramphenicol resistance gene.

[0035] pGro7 This vector was purchased from TaKaRa Co., Ltd. in Japan. This vector contains the groES-groEL chaperone gene, is a 5.4kb chaperone expression vector, and has a chloramphenicol resistance gene.

[0036] pKJE7 This vector was purchased from TaKaRa Co., Ltd. in Japan. This vector contains the dnaK-groES-groEL chaperone gene, is a 7.2kb chaperone expression vector, and contains the chloramphenicol resistance gene.

[0037] pTf16 This vector was purchased from TaKaRa Co., Ltd. in Japan. This vector contains the tlg chaperone gene, is an 8.3kb chaperone expression vector, and has a chloramphenicol resistance gene.

[0038] Cloning of the alginate lyase gene alyB According to embodiments of the present invention, the specific primers PCALYBF (5'-GATATAAGATCTATGACAACTCTGTCGCCATTATACG-3', SEQ ID NO: 3) and PCALYBR (5'-CAATATGTCGACTTAGTGGTGGTGGTGGTGGTGGTAAGCCT TAGGCACCTCAACACG-3', SEQ ID NO: 4) are designed to target the alginate lyase gene alyB using whole-genome sequencing and analysis results. The target gene fragment is amplified by PCR using P. chondroitinus genomic DNA as a template. After the PCR reaction is complete, the PCR product is recovered using the Plus Gel Eluted Kit. Next, the alyB gene is cloned using the CloneJET PCR Cloning Kit. Transformants are screened by colony polymerase chain reaction (PCR). After confirming that the inserted DNA is supported on the recombinant plasmid of the transformant, the plasmid is extracted from the transformant using the Plasmid Miniprep Purification Kit II and DNA sequencing is performed. The plasmid with the correct DNA sequence will be named pJET-AlyB.

[0039] Construction of an alginate lyase expression vector According to one embodiment of the present invention, pJET-AlyB is cleaved with BglII and SalI, and the alyB gene is inserted into pET29a(+) cleaved with BamHI and SalI using T4 DNA ligase. Then, the ligation product is used to form E. coli ECOS TMTransform cells 9-5. Screen the transformants by colony polymerase chain reaction (PCR). After confirming that the inserted DNA is supported on the recombinant plasmid of the transformant, extract the plasmid from the transformant and perform DNA sequencing. Name the plasmid with the correct DNA sequence pET-AlyB.

[0040] Transformation and inducible expression of alginate lyase expression vector According to an example of the present invention, Escherichia coli BL21(DE3) or Escherichia coli BL21(DE3) containing a chaperone expression vector is transformed with the alginate lyase expression vector pET-AlyB. These transformants are named Escherichia coli BL21(DE3) / pET-AlyB, Escherichia coli BL21(DE3) / pET-AlyB / pG-KJE8, Escherichia coli BL21(DE3) / pET-AlyB / pGro7, Escherichia coli BL21(DE3) / pET-AlyB / pKJE7, Escherichia coli BL21(DE3) / pET-AlyB / pG-Tf2, and Escherichia coli BL21(DE3) / pET-AlyB / pTf16, respectively.

[0041] I. Inducible expression of Escherichia coli BL21(DE3) / pET-AlyB Select a single colony and inoculate it into 12 mL of LB medium containing kanamycin (final concentration 30 μg / mL), then culture at 37°C and 180 rpm. After overnight culture, add 10 mL of the pre-culture to 1 L of LB medium containing kanamycin (final concentration 30 μg / mL), and perform OD (Oral Dissociation). 600 Incubate with shaking (37°C, 180 rpm) until the OD is approximately 0.4-0.6. Add 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and induce recombinant protein expression at 18°C, 25°C, 30°C, and 37°C. After induction for 4 hours at 25°C, 30°C, and 37°C, or after induction for 20 hours at 18°C, add 2 OD per mL. 600 The bacterial culture containing [the specified substance] was collected. After centrifugation at 20,630 × g and 4°C for 10 minutes, the supernatant was discarded. The resulting cell pellet was subjected to protein fractionation to separate soluble and insoluble proteins.

[0042] II. Inducible Expression of Escherichia coli BL21(DE3) / pET-AlyB / pG-KJE8 Select a single colony and inoculate it into 12 mL of LB medium containing kanamycin (final concentration 30 μg / mL) and chloramphenicol (final concentration 20 μg / mL), and culture it at 37°C and 180 rpm. After overnight culture, add 10 mL of the preculture to 1 L of LB medium containing kanamycin (final concentration 30 μg / mL), chloramphenicol (final concentration 20 μg / mL), tetracycline stock solution (final concentration 5 ng / mL), and arabinose (final concentration 0.5 mg / mL), and shake culture (37°C, 180 rpm) until the OD 600 reaches approximately 0.4 - 0.6. Then, add 0.1 mM IPTG and induce recombinant protein expression at 25°C. After 4 hours of induction, collect the bacterial culture with an OD 600 of 2 per mL. After centrifugation at 20,630×g and 4°C for 10 minutes, discard the supernatant. Subject the obtained cell pellet to protein fractionation to separate soluble protein and insoluble protein.

[0043] III. Inductive Culture of Escherichia coli BL21(DE3) / pET-AlyB / pGro7 Select a single colony and inoculate it into 12 mL of LB medium containing kanamycin (final concentration 30 μg / mL) and chloramphenicol (final concentration 20 μg / mL), and culture it at 37°C and 180 rpm. After overnight culture, add 10 mL of the preculture to 1 L of LB medium containing kanamycin (final concentration 30 μg / mL), chloramphenicol (final concentration 20 μg / mL), and arabinose (final concentration 0.5 mg / mL), and shake culture (37°C, 180 rpm) until the OD 600 reaches approximately 0.4 - 0.6. Then, add 0.1 mM IPTG and induce recombinant protein expression at 25°C. After 4 hours of induction, collect the bacterial culture with an OD 600 of 2 per mL. After centrifugation at 20,630×g and 4°C for 10 minutes, discard the supernatant. Subject the obtained cell pellet to protein fractionation to separate soluble protein and insoluble protein.

[0044] IV. Inducible culture of Escherichia coli BL21(DE3) / pET-AlyB / pKJE7 Select a single colony and inoculate it into 12 mL of LB medium containing kanamycin (final concentration 30 μg / mL) and chloramphenicol (final concentration 20 μg / mL), then culture at 37°C and 180 rpm. After overnight culture, add 10 mL of the preculture to 1 L of LB medium containing kanamycin (final concentration 30 μg / mL), chloramphenicol (final concentration 20 μg / mL), and arabinose (final concentration 0.5 mg / mL), then perform OD (Oral Dissociation). 600 The culture is performed with shaking (37°C, 180 rpm) until the volume is approximately 0.4-0.6. Then, 0.1 mM IPTG is added and recombinant protein expression is induced at 25°C. After 4 hours of induction, 2 OD per 1 mL is obtained. 600 The bacterial culture containing [the specified substance] was collected. After centrifugation at 20,630 × g and 4°C for 10 minutes, the supernatant was discarded. The resulting cell pellet was subjected to protein fractionation to separate soluble and insoluble proteins.

[0045] V. Inducible culture of Escherichia coli BL21(DE3) / pET-AlyB / pG-Tf2 Select a single colony and inoculate it into 12 mL of LB medium containing kanamycin (final concentration 30 μg / mL) and chloramphenicol (final concentration 20 μg / mL), then culture at 37°C and 180 rpm. After overnight culture, add 10 mL of the pre-culture to 1 L of LB medium containing kanamycin (final concentration 30 μg / mL), chloramphenicol (final concentration 20 μg / mL), and tetracycline stock solution (final concentration 5 ng / mL), then perform OD (Oral Dissociation). 600 The culture is performed with shaking (37°C, 180 rpm) until the volume is approximately 0.4-0.6. Then, 0.1 mM IPTG is added and recombinant protein expression is induced at 25°C. After 4 hours of induction, 2 OD per 1 mL is obtained. 600 The bacterial culture containing [the specified substance] was collected. After centrifugation at 20,630 × g and 4°C for 10 minutes, the supernatant was discarded. The resulting cell pellet was subjected to protein fractionation to separate soluble and insoluble proteins.

[0046] VI. Inducible culture of Escherichia coli BL21(DE3) / pET-AlyB / pTf16 Select a single colony and inoculate it into 12 mL of LB medium containing kanamycin (final concentration 30 μg / mL) and chloramphenicol (final concentration 20 μg / mL), then culture at 37°C and 180 rpm. After overnight culture, add 10 mL of the preculture to 1 L of LB medium containing kanamycin (final concentration 30 μg / mL), chloramphenicol (final concentration 20 μg / mL), and arabinose (final concentration 0.5 mg / mL), then perform OD (Oral Dissociation). 600 The culture is performed with shaking (37°C, 180 rpm) until the volume is approximately 0.4-0.6. Then, 0.1 mM IPTG is added and recombinant protein expression is induced at 25°C. After 4 hours of induction, 2 OD per 1 mL is obtained. 600 The bacterial culture containing [the specified substance] was collected. After centrifugation at 20,630 × g and 4°C for 10 minutes, the supernatant was discarded. The resulting cell pellet was subjected to protein fractionation to separate soluble and insoluble proteins.

[0047] Fractionation of soluble and insoluble proteins When recombinant proteins are expressed using E. coli, the expressed proteins may form inclusion bodies. Inclusion bodies are denatured, insoluble proteins. Refolding is necessary to restore the protein's three-dimensional structure and biological activity, but the refolding process is complex and time-consuming, and the folding efficiency varies depending on the protein's properties. To avoid the formation of inclusion bodies in cells, the solubility of recombinant proteins can be improved by co-expressing chaperones. Chaperones are a class of proteins that assist in intracellular molecular assembly and protein folding, such as GroES, GroEL, DnaK, DnaJ, and GrpE.

[0048] According to one embodiment of the present invention, centrifuged bacterial cells are OD 600The cells are suspended in lysis buffer to a cell density equivalent to 4, and the suspension is stirred until homogeneous before being subjected to cell lysis. The suspended bacterial cells are then treated with an ultrasonic disruptor (Sonics VCX-750, USA). The disruption conditions are 25% amplitude, intermittent treatment (treatment for 9 seconds, then pause for 9 seconds), and one treatment at 10,800 joules. The disrupted bacterial suspension is centrifuged (15,900 × g, 30 minutes), and the supernatant is collected. Induced expression of recombinant alginate lyase AlyB is confirmed using protein electrophoresis and Western blotting.

[0049] Purification of alginate lyase AlyB According to one embodiment of the present invention, after culturing Escherichia coli BL21(DE3) / pET-AlyB / pGro7, recombinant alginate lyase AlyB is obtained by purification, and then its protein concentration is determined and its activity is analyzed.

[0050] Protein concentration determination According to embodiments of the present invention, protein quantitative analysis is performed using the Protein Quantification Assay Kit (MACHEREY-NAGEL, Germany) or the Bio-Rad Protein Assay (Bio-Rad, USA). The procedure for using the Protein Quantification Assay Kit is as follows: Dilute the protein sample 20-fold with buffer (20 mM sodium phosphate, pH 7.0). Thoroughly mix 20 μL of the diluted sample, 40 μL of protein lysis buffer, and 40 μL of the quantitative reagent, and incubate at room temperature for 30 minutes. Measure the absorbance at 570 nm using an Infinite® 200 Pro microdisc analyzer (Tecan, USA). Create a standard protein concentration curve using 2 mg / mL bovine serum albumin (BSA) as a standard. The concentration of the protein sample can be calculated by comparing the absorbance of the sample with the standard curve. The procedure for using the Bio-Rad Protein Assay is as follows: Thoroughly mix 10 μL of protein sample with 200 μL of diluted dry reagent and incubate at room temperature for 5 minutes. Measure the absorbance at 595 nm using an Infinite® 200 Pro microdisc analyzer (Tecan, USA). Create a standard protein concentration curve using 2 mg / mL bovine serum albumin (BSA) as a standard. Calculate the protein sample concentration by comparing the sample absorbance with the standard curve.

[0051] Activity analysis of recombinant alginate lyase AlyB According to one embodiment of the present invention, the activity analysis of alginate lyase is performed using the thiobarbituric acid (TBA) colorimetric method, as described below. A substrate solution containing 0.22% (w / v) sodium alginate is prepared using phosphate buffer (20 mM sodium phosphate, pH 7.4). 900 μL of this substrate solution is thoroughly 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 thoroughly mixed with 200 μL of reagent A [25 mM periodic acid (H5IO6) / 0.125N H2SO4] and allowed to stand at room temperature for 20 minutes to oxidize the enzymatic degradation product and formylpyruvic acid. Next, 500 μL of reagent B [2% sodium arsenite (NaAsO2) / 0.5N HCl] is added to the above reaction solution to terminate the enzymatic reaction. After shaking and mixing, the solution is allowed to stand at room temperature for 2 minutes. Next, 2 mL of 0.3% thiobarbituric acid solution is added, and the reaction is carried out at 100°C for 10 minutes to react methyl pyruvate with thiobarbituric acid and produce a colorimetric result. After cooling, 100 μL of the reaction solution is transferred to a 96-well plate, and the absorbance is measured at 548 nm using an Infinite® 200 Pro microdisc analyzer (Tecan, USA). A standard curve of alginate lyase activity corresponding to the absorbance value is established by performing a TBA colorimetric reaction using commercially available alginate lyase (Sigma, USA) solutions with different active units. The activity (active units, U) of purified alginate lyase AlyB is calculated using the standard curve.

[0052] Characterization of recombinant alginate lyase AlyB According to one embodiment of the present invention, purified alginate lyase AlyB is used for enzyme characterization. The analytical project and operating steps are as follows:

[0053] Optimal pH Substrate solutions with various pH values ​​are prepared by uniformly mixing 800 μL of 0.25% sodium alginate solution with 100 μL each of 0.5 M citrate buffer (pH 3.0-6.0), 0.5 M phosphate buffer (pH 6.0-7.0), 0.5 M 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid [HEPES] buffer (pH 7.0-8.0), 0.5 M N-dihydroxyethylglycine-sodium hydroxide (bicine-NaOH) buffer (pH 8.0-9.0), and 0.5 M glycine-sodium hydroxide (glycine-NaOH) buffer (pH 9.0-10.0). After incubating the substrate solutions at 37°C for 5 minutes, 100 μL of 0.4 U / mL purified enzyme solution is added, and the mixture is reacted at 37°C for 30 minutes. The TBA colorimetric method and the method for calculating enzyme activity are as described above. The highest enzyme activity measured during the reaction is defined as 100%, representing the enzyme activity at the optimal pH value. The relative enzyme activity at other pH values ​​is then calculated.

[0054] Optimal operating temperature A substrate solution is prepared by uniformly mixing 800 μL of 0.25% sodium alginate solution with 100 μL of 0.5 M HEPES buffer (pH 8.0). After incubating the substrate solution at 20°C to 60°C for 5 minutes, 100 μL of 0.4 U / mL enzyme solution is added, and the reaction is carried out at various temperatures (20°C to 60°C) for 30 minutes. The method for calculating the color development of TBA and enzyme activity is as described above. The highest enzyme activity measured during the reaction is defined as 100%, and this is the enzyme activity at the optimal operating temperature. The relative enzyme activity at other temperatures is then calculated.

[0055] pH stability analysis The enzyme solution was diluted to 0.4 U / mL with buffers of various pH values ​​and incubated at 35°C for 2 hours and 4 hours, respectively. A substrate solution was prepared by uniformly mixing 800 μL of 0.25% sodium alginate solution with 100 μL of 0.5 M HEPES buffer (pH 8.0). After incubating the substrate solution at 35°C for 5 minutes, 100 μL of one of the enzyme solutions treated at various pH values ​​was added and the mixture was reacted at 35°C for 30 minutes. Subsequent TBA color development and enzyme activity calculations were performed as described above. The highest enzyme activity measured during the reaction was defined as 100%, and the relative enzyme activity at other pH values ​​was calculated.

[0056] Temperature stability analysis The enzyme solution is diluted to 0.4 U / mL with HEPES buffer (pH 8.0) and incubated at various temperatures for 2 hours and 4 hours, respectively. A substrate solution is prepared by uniformly mixing 800 μL of 0.25% sodium alginate solution with 100 μL of 0.5 M HEPES buffer (pH 8.0). After incubating the substrate solution at 35°C for 5 minutes, 100 μL of one of the enzyme solutions treated at various temperatures is added and the mixture is reacted at 35°C for 30 minutes. Subsequent TBA color development and enzyme activity calculations are performed as described above. The highest enzyme activity measured during the reaction is defined as 100%, and the relative enzyme activity at other temperatures is calculated.

[0057] Effects of various chemicals on the activity of recombinant alginate lyase AlyB According to embodiments of the present invention, the effects of various chemical substances, including metal salts, sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA), on the activity of recombinant alginate lyase AlyB are investigated. A substrate solution is prepared by uniformly mixing 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. After incubating the substrate solution at 35°C for 5 minutes, 50 μL of enzyme solution diluted to 0.8 U / mL is added, and the mixture is reacted at 35°C for 30 minutes. Subsequent TBA color development and enzyme activity calculations are performed as described above. The highest enzyme activity measured during the reaction is defined as 100%, and the relative enzyme activity under various chemical reactions is calculated. The experimental data are then analyzed using the Dunnett method in Minitab14 software to determine if there is a statistically significant difference.

[0058] Substrate specificity analysis of recombinant alginate lyase AlyB According to one embodiment of the present invention, substrates that can be cleaved by recombinant alginate lyase AlyB are analyzed. The substrates to be measured include agarose, agar, sodium alginate, alginic acid, carrageenan, and carboxymethylcellulose. A substrate solution is prepared by uniformly mixing 800 μL of a 0.25% substrate solution with 100 μL of 0.5 M HEPES buffer (pH 8.0). After incubating the substrate solution at 35°C for 5 minutes, 100 μL of enzyme solution is added to obtain a final enzyme activity of 0.4 U / mL, and the reaction is carried out at 35°C for 30 minutes. The subsequent TBA colorimetric method and the method for calculating enzyme activity are as described above. The highest enzyme activity measured during the reaction is defined as 100%, and the relative enzyme activity in other different substrate reactions is calculated.

[0059] Analysis of products from the degradation of sodium alginate by recombinant alginate lyase AlyB Thin-layer chromatography (TLC) was used to analyze the products of sodium alginate degradation by recombinant alginate lyase AlyB. A 900 mL solution of 0.555% sodium alginate was prepared, the pH was adjusted to 8.0 with NaOH, and then 10,000 U of recombinant alginate lyase AlyB was added. Deionized water was then added to bring the volume to 1 L, and the solution was incubated at 35°C for 16-24 hours. After centrifugation at 38,400 × g at room temperature for 30 minutes, the solution was filtered through a 0.2 μm sterile filter (Thermo Fisher Scientific, USA). 4 μL of supernatant or 4 μL of 1 μg / μL sodium D-mannuronate (M), mannuronate oligosaccharides (DP2), mannuronate oligosaccharides (DP3), mannuronate oligosaccharides (DP4), and 4 μL of 10 mg / mL sample solution, placed on silica gel at 60°F. 254 Spot onto a TLC slide (Millipore, USA). After air-drying the sample, tilt the TLC slide and place it in a developing tank containing developing buffer (n-butanol:formic acid:water = 4:6:1). After development, remove the TLC slide and air-dry. 100 mL of 99% ethanol, 20 mg of Ce(SO4)2, 50 mg of (NH4)6Mo7O 24 A colorimetric solution is prepared by thoroughly mixing the sample with 5 mL of H2SO4. This solution is then sprayed onto a TLC slide, dried with hot air, and then fired at 180°C for 20 minutes using a stirrer (IKA® C-MAG HS hot plate stirrer) to develop the color. The Rf values ​​(retention coefficient values) of the sample and standard signal are calculated. The Rf values ​​are used to determine the type of degradation product.

[0060] DPPH radical scavenging ability of degradation products According to one embodiment of the present invention, the antioxidant capacity of the degradation products of alginate lyase is determined using a DPPH (α,α-diphenyl-β-picrylhydrazyl) scavenging ability test. The experimental procedure is modified according to the method of Shimamura et al. (2014). Solutions at concentrations of 40 mg / mL, 30 mg / mL, 20 mg / mL, 10 mg / mL, and 5 mg / mL are prepared by reconstituting lyophilized powder of sodium alginate degradation products by recombinant alginate lyase AlyB with deionized water. 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; dissolved in anhydrous ethanol) are thoroughly mixed and incubated at 25°C in the dark for 30 minutes. The mixture is then centrifuged at 20,640 × g for 5 minutes at room temperature. After centrifugation, the supernatant is collected and thoroughly stirred. Next, 200 μL of supernatant is collected, and the absorbance at 517 nm is measured using an Infinite® 200 Pro microdisc analyzer. A lower absorbance indicates a stronger DPPH capture ability of the sample. Capture effect % = [(AC - (AS - A sample blank)] / AC × 100; where AC = control group without sample; AS = sample; A sample blank = group without DPPH solution. The half-capture concentration of the sample is further calculated using the capture effect.

[0061] Analysis of the Trolox equivalent antioxidant capacity (TEAC) of degradation products According to one embodiment of the present invention, the equivalent antioxidant capacity of Trolox was analyzed, and the experimental procedure was as follows: Solutions at concentrations of 40 mg / mL, 30 mg / mL, 20 mg / mL, 10 mg / mL, and 5 mg / mL were prepared by reconstituting lyophilized oligosaccharide powder with deionized water. 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 thoroughly mixed and allowed to stand in the dark. After the formation of stable blue-green ABTS+ cationic free radicals, 0.25 mL of the sample was added and allowed to stand 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 at 734 nm was measured using an Infinite® 200 Pro microdisc analyzer. A lower absorbance indicates a stronger TEAC of the sample. Capture effect % = [1 - (Absorbance of the sample at 734 nm) / (Absorbance of the control group without the sample at 734 nm)] × 100. The half-capture concentration of the sample is further calculated using the capture effect.

[0062] Analysis of angiotensin I-converting enzyme inhibitory activity The angiotensin I-converting enzyme inhibitory activity of degradation products will be analyzed using the ACE Kit-WST kit (Dojindo, Japan). The experimental procedure is as follows: Solutions at concentrations of 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL are prepared by reconstituting the lyophilized lysate powder with deionized water. 20 μL of each solution is pipetted into a 96-well plate, and the reagent from the kit is added. The absorbance of the sample is measured at 450 nm. The inhibition rate is calculated using the following formula: Inhibition Rate (%) = [(OD 450_ブランク1 -OD 450_試料 ) / (OD 450_ブランク1 -OD 450_ブランク2 )] × 100; in the formula, Blank 1 represents the group without the inhibitor, and Blank 2 represents the group without the enzyme. Using the inhibition rate, the median inhibitory concentration (IC) of the sample is calculated. 50 ) is then calculated further.

[0063] Antiviral activity of degradation products Virus-susceptible Fcwf-4 and Marc-145 cells were treated with various concentrations of DEH before and after FIPV and PRRSV infection, respectively. The antiviral activity of 4-deoxy-L-erythro-5-hexoseuronic acid (DEH) was evaluated by examining viral titers after infection.

[0064] I. Evaluation of the antiviral activity of the feline macrophage cell line Fcwf-4 infected with serotype II feline infectious peritonitis virus (FIPV). cell culture The Fcwf-4 cell line is purchased from the American Type Culture Collection (ATCC). The culture medium used to cultivate the cells contains 90% Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS), while the culture medium used to infect the cells contains 98% DMEM and 2% FBS. The cell cryopreservation tubes are placed in a 37°C water bath. After complete thawing, the cell suspension is transferred to a container with an appropriate amount of culture medium and cultured in an incubator. Subculturing is performed when the cell density reaches 80% or higher. When subculturing, the original culture medium is removed, the container is washed with phosphate-buffered saline (PBS), and then trypsin is added to suspend the cells. After that, an appropriate amount of cells is transferred to a container with new culture medium and cultured further.

[0065] Viral titer measurement The viral titer assay is performed using two protocols: "DEH pretreatment" and "DEH post-infection treatment." In the "DEH pretreatment" protocol, Fcwf-4 cells are seeded at an appropriate density in a 96-well cell culture dish. The viral solution is then mixed with various concentrations of DEH to obtain DEH concentrations of 0, 0.0625, 0.5, 1, 2, 5, 10, and 20 mg / mL. After reacting for 1 hour, the viral solution containing the different DEH concentrations is added to the cells. After incubation for 2 days, the culture medium is collected for viral titer measurement. In the "DEH post-infection treatment" protocol, the viral solution is added to the cells and infected for 1 hour. After removing the viral solution, infection medium containing DEH at concentrations of 0, 0.05, 0.4, 1.6, 2, 4, and 8 mg / mL, respectively, is added to the cells. After incubation for 2 days, the culture medium is collected for viral titer assay.

[0066] II. Evaluation of the antiviral activity of the African green monkey embryonic kidney cell line Marc-145 infected with porcine reproductive and respiratory syndrome virus (PRRSV). cell culture The Marc-145 cell line was obtained from Swine Research Institute in Taiwan (Chang et al., 1993). The culture medium used for cell culture contained 98% DMEM and 2% FBS. The cell cryopreservation tubes were placed in a 37°C water bath. After complete thawing, the cell suspension was transferred to a container with an appropriate amount of culture medium and cultured in an incubator. Subculturing was performed when the cell density reached 80% or higher. When subculturing, the original culture medium was removed, the container was washed with PBS, and trypsin was added to suspend the cells. Then, an appropriate amount of cells was transferred to a container with fresh culture medium and cultured further.

[0067] Viral titer measurement The viral titer assay is performed using two protocols: "DEH pretreatment" and "DEH post-infection treatment." In the "DEH pretreatment" protocol, Marc-145 cells are seeded at an appropriate density in a 96-well cell culture dish. The viral solution is then mixed with DEH to obtain final DEH concentrations of 0, 0.3125, 0.625, 2.5, 5, and 10 mg / mL. After 1 hour of incubation, viral solutions containing different concentrations of DEH are added to the cells. After incubation for 4 days, the culture medium is collected for viral titer measurement. In the "DEH post-infection treatment" protocol, Marc-145 cells are seeded at an appropriate density in a 96-well cell culture dish, and the viral solution is added to the cells for 1 hour of infection. After removing the viral solution, infection medium containing 0, 0.06, 0.13, 0.25, 0.5, and 1 mg / mL of DEH, respectively, is added to the cells. After incubation for 4 days, the culture medium is collected for the viral titer assay.

[0068] Example 1 Cloning of the alginate lyase alyB gene from the genus Paenibacillus and construction of an expression vector.

[0069] Whole-genome sequencing revealed that the genus Paenibacillus can possess two alginate lyase genes, named AlyA and AlyB. Bioinformatics analysis showed that the AlyA gene sequence is 1,128 bp long, with ATG as the start codon and TGA as the stop codon. The AlyB gene sequence is 2,304 bp long, with ATG as the start codon and TGA as the stop codon, encoding 767 amino acids. Analysis of the amino acid sequences of AlyA and AlyB using the SignalP 5.0 Server (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) predicted that these proteins lack a signal peptide and are intracellular proteins. The inventors previously performed gene cloning, recombinant protein expression, recombinant protein purification, and enzyme activity analysis of AlyA. As a result, it was shown that AlyA does not possess alginate lyase activity. In this invention, alyB was further investigated. Amino acid sequence alignment of AlyB using the NCBI BLASTP program showed that AlyB contains an unknown functional DUF4962 block whose amino acid sequence is similar to that of Flavobacterium heparinum heparinase II and heparinase III, and is also predicted to contain the Hepar_II_III functional block. A comparison of the amino acid sequences of the three predicted enzymes—Paenibacillus ergyi M63 alginate lyase (GenBank accession number KZE76488.1), Paenibacillus ergyi AC13 alginate lyase (GenBank accession number PUA38957.1), and Paenibacillus agarideborans T-3040 alginate lyase (GenBank accession number GBG06091.1)—showed similarities of 75.98%, 75.85%, and 65.32%, respectively (Figure 1).

[0070] To confirm that the protein encoded by AlyB actually possesses alginate lyase activity, further gene cloning, recombinant protein expression, recombinant protein purification, and enzyme characterization were performed. In gene cloning, specific primers targeting the alyB gene sequence were designed, and the target gene fragment was amplified using polymerase chain reaction (PCR). Next, the alyB gene was cloned using the CloneJET PCR Cloning Kit. The plasmid containing the alyB gene was named pJET-AlyB. Subsequently, pJET-AlyB was cleaved with BglII and SalI, and the alyB gene was inserted into pET29a(+) cleaved with BamHI and SalI using T4 DNA ligase to construct the alginate lyase expression vector pET-AlyB.

[0071] The pET-AlyB recombinant vector contains the nucleotide sequence shown in Sequence ID No. 5, specifically the combination shown in Table 1 below.

[0072] [Table 1]

[0073] Example 2 Expression of recombinant alginate lyase AlyB in Escherichia coli E. coli BL21(DE3) was transformed with the alginate lyase expression vector pET-AlyB to induce protein expression. SDS-PAGE and Western blotting results showed that E. coli BL21(DE3) transformants with the pET-AlyB expression vector were able to express recombinant alginate lyase with a C-terminal His tag, and the molecular weight of the band was close to the expected 89 kDa, compared to E. coli BL21(DE3) / pET-29a(+) (Figure 2).

[0074] Example 3 Effects of induction temperature and co-expression of chaperone proteins on the solubility of recombinant alginate lyase AlyB Induction temperature assays showed that lowering the induction temperature increased the solubility of recombinant alginate lyase AlyB. When recombinant alginate lyase AlyB was induced and expressed at temperatures of 37°C, 30°C, 25°C, and 18°C, the solubility of recombinant alginate lyase AlyB was 14.5%, 35.8%, 66.5%, and 89.5%, respectively (Figure 3). Intracellular enzyme activity analysis also showed that the intracellular enzyme activity induced at 18°C ​​(0.82 U / mL) was 20.5 times higher than that induced at 37°C (0.04 U / mL) (Figure 4). Subsequently, the effect of co-expression of different chaperone proteins on the solubility of recombinant alginate lyase AlyB was investigated by transforming E. coli BL21(DE3) / pET-AlyB with expression vectors containing different chaperone protein genes, including pG-KJE8, pGro7, pKJE7, pG-Tf2, and pTf16. SDS-PAGE and Western blotting results showed that co-expression of chaperone proteins, combined with an induction temperature of 25°C, improved the solubility and expression level of recombinant alginate lyase AlyB (Figure 5). Using ImageQuant software, the volume of soluble target protein and the volume of target protein in total cell lysates were analyzed 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 times and the protein expression level by 2.5 times (Figure 6). Intracellular enzyme activity analysis also showed that co-expression of GroES-GroEL significantly increased enzyme activity (Figure 7). Compared to 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 27.49 times.

[0075] Example 4 Purification and characterization of recombinant alginate lyase AlyB Recombinant alginate lyase AlyB was expressed using E. coli BL21(DE3) / pET-AlyB / pGro7, and intracellularly soluble recombinant alginate lyase AlyB was purified by immobilized metal ion affinity chromatography. SDS-PAGE results showed that affinity chromatography can purify recombinant alginate lyase AlyB with a C-terminal His tag (Figure 8). After mixing tubes 1-7 of the eluate, protein electrophoresis and Western blotting were performed again (Figure 9). Western blotting results confirmed that the purified protein was indeed recombinant alginate lyase AlyB. The purity of the purified enzyme reached 94.15%, and the specific activity was 142.85 U / mg. Approximately 5.43 mg of recombinant alginate lyase AlyB could be purified from 1 L of cultured cells.

[0076] Analysis of the optimal pH and temperature for enzyme activity showed that recombinant alginate lyase AlyB had an optimal pH of 8.0. Enzyme activity significantly decreased at pH values ​​below 7.0 or above 9.0. The optimal temperature for recombinant alginate lyase AlyB was 35°C. Enzyme activity decreased sharply at 40°C (Figure 10). Analysis of the enzyme's pH and temperature stability showed that recombinant alginate lyase AlyB maintained over 80% of its activity after 4 hours of incubation at pH 5.0–10.0 and temperatures of 4°C–35°C (Figure 11).

[0077] Example 5 Effects of chemical substances on the enzymatic activity of recombinant alginate lyase AlyB In the analysis of the effects of various chemicals on the activity of recombinant alginate lyase AlyB, Co 2+ Cu 2+ Ca 2+ Zn 2+ Fe 2+It was shown that SDS significantly inhibited enzyme activity (Table 2). Substrate-specific analysis showed that recombinant alginate lyase AlyB showed the best resolution for alginate, followed by sodium alginate, with a relative activity of 61.85%, but was unable to cleave agarose, agar, carrageenan, and carboxymethylcellulose (Table 3).

[0078] [Table 2]

[0079] [Table 3]

[0080] Example 6 Reaction kinetics analysis of recombinant alginate lyase AlyB The reaction kinetics of recombinant alginate lyase AlyB were analyzed using sodium alginate as a substrate. As a result, the Vmax of recombinant alginate lyase AlyB, determined using the Lineweaver-Burk plot double reciprocal method, was 220.30 U / mg and the Km was 3.60 mg / mL (Figure 12). TLC analysis revealed that the main product of sodium alginate degradation by recombinant alginate lyase AlyB is 4-deoxy-L-erythro-5-hexoseuroseuronic acid (DEH) (Figure 13), indicating that recombinant alginate lyase AlyB is an exo-type alginate lyase.

[0081] Exo-type alginate lyases currently being studied include A1-IV from Sphingomonas sp. A1 (Miyake et al., 2003), Atu3025 from Agrobacterium tumefaciens C58 (Ochiai et al., 2006), Alg17C from Saccharophagus degradans 2-40 (Kim et al., 2012), AlyFRB from Falsirhodobacter sp. alg1 (Mori et al., 2016), AlgL17 from Microbulbifer sp. ALW1 (Jiang et al., 2019), and Vibrio xyamenensis. VxAly7D of xiamenensis QY104 (Tang et al., 2020), Aly1281 of Pseudoalteromonas carrageenovora (Zhang et al., 2020), Alteromonas portus HB161718 T Examples include Alg2951 (Huang et al., 2021) from *Paradendryphiella salina*, PsMan8A (Pilgaad et al., 2021) from *Microbarbifer* SH-1 (Yang et al., 2021) from *Tamlana sp.* s12 (Yin et al., 2021) and VsAly7D (Zhang et al., 2021) from *Vibrio* QY108. The AlyB of the present invention is a novel exo-type alginate lyase that differs from the enzymes described in the above literature in both amino acid sequence and properties.

[0082] Example 7 Antioxidant activity analysis of alginate degradation products This invention also measures the DPPH (α,α-diphenyl-2-picrylhydrazyl) free radical scavenging ability and Trolox equivalent antioxidant capacity of products formed from the degradation of sodium alginate by recombinant alginate lyase AlyB, in order to understand the antioxidant activity of the degradation products. The results are shown in Figure 14. The products formed from the degradation of sodium alginate by recombinant alginate lyase AlyB possess antioxidant activity, and the antioxidant effect increases in a dose-dependent manner. The median inhibitory concentrations for DPPH and ABTS scavenging by alginate degradation products, obtained from regression analysis of data detected by SigmaPlot software, were 31.5 mg / mL and 8.05 mg / mL, respectively. Studies have shown that free radicals in the human body and the surrounding environment cause oxidative stress in the human body, leading to many health problems such as inflammation, cardiovascular disease, diabetes, and cancer. The recombinant alginate lyase AlyB product according to the present invention has the ability to scavenge DPPH free radicals, possesses antioxidant activity, and can be used to prepare antioxidant food additives, antioxidant pharmaceutical compositions, or antioxidant cosmetic additives.

[0083] Example 8 Analysis of the antiviral activity of alginate degradation products This invention also investigates the antiviral activity of alginate degradation products. The results showed that pretreatment with DEH resulted in an EC of 2.3583 mg / mL. 50 This effectively reduced FIPV infection in Fcwf-4 cells (Figure 15). Post-infection DEH treatment also effectively reduced 1.584 mg / mL EC2. 50 This effectively reduced persistent FIPV infection (Figure 16). Furthermore, pretreatment with DEH involved 2.9174 mg / mL of EC2. 50 This effectively reduced PRRSV infection in Marc-145 cells (Figure 17). Post-infection DEH treatment also effectively reduced EC2 at 0.1141 mg / mL. 50 This effectively reduced persistent PRRSV infection (Figure 18). These results demonstrate that DEH can effectively reduce FIPV and PRRSV infection in Fcwf-4 and Marc-145 cells.

[0084] These experiments clearly demonstrate that alginate degradation products have the ability to inhibit viral infection and can be used to prepare food additives or pharmaceutical compositions that inhibit viral infection.

[0085] Example 9 Analysis of the angiotensin I-converting enzyme (ACE) inhibitory activity of alginate degradation products The present invention also investigates the inhibitory activity of alginate degradation products against angiotensin I-converting enzyme (ACE). The results showed that the product formed by the cleavage of sodium alginate by recombinant alginate lyase AlyB possessed ACE inhibitory activity, and this inhibitory activity increased in a dose-dependent manner (Figure 19). The median inhibitory concentration of the alginate degradation product against ACE was 7.41 mg / mL. Since the recombinant alginate degradation product has the ability to inhibit ACE, it can be used to prepare food additives or pharmaceutical compositions that inhibit ACE.

Claims

1. Alginate lyase AllyB containing the amino acid sequence shown in Sequence ID No.

1.

2. Alginate lyase AllyB according to claim 1, encoded by the nucleotide sequence shown in Sequence ID No.

2.

3. Alginate lyase AllyB according to claim 1, having an optimal operating pH range of 7.0 to 9.

0.

4. Alginate lyase AllyB according to claim 1, having an optimal operating temperature range of 33°C to 37°C.

5. Alginate lyase AllyB according to claim 1, which cleaves alginic acid and sodium alginate.

6. Alginate lyase AllyB according to claim 1, which is an exo-type alginate lyase.

7. Alginate lyase AllyB according to claim 6, wherein the main degradation product after dissolution is 4-deoxy-L-erythro-5-hexoseuroseuronic acid (DEH).

8. Alginate lyase AllyB according to any one of claims 1 to 7, isolated from the genus Paenibacillus.

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

10. The recombinant vector according to claim 9, further comprising at least one chaperone expression vector selected from pG-KJE8, pGr7, pKJE7, and pTf16.

11. The recombinant vector according to claim 9, further comprising the nucleotide sequence shown in Sequence ID No.

5.

12. A transformant comprising the recombinant vector according to any one of claims 9 to 11.

13. A method for preparing alginate lyase AllyB according to any one of claims 1 to 8, To prepare a recombinant vector according to any one of claims 9 to 11, By transforming host cells with the recombinant vector, the transformant described in claim 12 is obtained. By culturing the aforementioned transformant, the expressed alginate lyase AllyB is produced, and The expressed alginate lyase AllyB is isolated and purified. A method that includes this.

14. The method according to claim 13, wherein the host cell is Escherichia coli BL21 (DE3).

15. Use of alginate lyase AllyB according to any one of claims 1 to 8 for cleaving alginate and / or sodium alginate.

16. Use of alginate lyase AllyB according to any one of claims 1 to 8 for preparing a cosmetic, food additive, or pharmaceutical composition for capturing DPPH free radicals and ABTS free radicals.

17. Use of alginate lyase AllyB according to any one of claims 1 to 8 for preparing cosmetics, food additives, or pharmaceutical compositions having antioxidant activity.

18. Use of alginate lyase AllyB according to any one of claims 1 to 8 for preparing a food additive or pharmaceutical composition that inhibits viral infection.

19. Use of alginate lyase AllyB according to any one of claims 1 to 8 for preparing a food additive or pharmaceutical composition that inhibits angiotensin-converting enzyme.

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