β-Agarase, its preparation methods, applications, and preparation methods of neo-agarbiose.

Abstract

This invention belongs to the field of biotechnology and discloses β-agarase, its preparation method, applications, and a method for preparing neo-agarbiose. The β-agarase provided by this invention specifically comprises an amino acid fragment with the sequence shown in SEQ ID NO:1. It can efficiently and specifically degrade agar oligosaccharides under mild conditions, yielding a single product, neo-agarbiose. It possesses advantages such as high enzyme activity, mild reaction temperature, high stability, and homogeneous enzymatic hydrolysis products, showing excellent application prospects in the large-scale industrial production of neo-agarbiose.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to a β-agarase, a method for preparing β-agarase, its application, and a method for preparing neo-agarbiose. Background Technology

[0002] Agar, also known as agar, has a complex structure. Its main chain is composed of alternating 1,3-β-D-galactopyranose and 1,4-3,6-endoether-α-L-galactopyranose residues. It is a major component of the cell walls of red algae such as Gracilaria and is one of the most abundant seaweed polysaccharides in the ocean.

[0003] Agarases are a class of glycosidic hydrolases that specifically degrade agar to produce agar oligosaccharides. Based on their sites of action and products, they can be specifically divided into α-agarases and β-agarases. α-Agarases hydrolyze the α-1,3-glycosidic bonds in agarose to produce agar oligosaccharides; while β-agarases act on β-1,4-glycosidic bonds, primarily producing neo-agar oligosaccharides with a degree of polymerization typically between 2 and 6. The neo-agarbioses produced by β-agarase degradation possess various biological activities, such as promoting the growth of human intestinal flora, inhibiting melanin production for a whitening effect, antioxidant effects, anti-inflammatory effects, and antibacterial effects, showing excellent prospects for practical applications.

[0004] However, the currently reported β-agarases usually generate mixed products of various oligosaccharide components, including neoagarbiose, tetrasaccharides, and hexasaccharides, during the enzymatic hydrolysis process. They also have problems such as long reaction time, harsh reaction conditions, poor enzymatic hydrolysis ability, and complex enzymatic hydrolysis products, which have significant limitations. Summary of the Invention

[0005] The primary objective of this invention is to address the problems of existing β-agarases, such as long reaction time, harsh reaction conditions, poor enzymatic hydrolysis ability, and complex enzymatic hydrolysis products, and to provide a β-agarase.

[0006] A second objective of this invention is to provide a nucleic acid molecule.

[0007] A third objective of this invention is to provide an expression strain.

[0008] A fourth objective of this invention is to provide a method for preparing the aforementioned β-agarase.

[0009] The fifth objective of this invention is to provide the application of the above-mentioned β-agarase, nucleic acid molecules or expression strains in the preparation of new agarobiose.

[0010] The sixth objective of this invention is to provide a method for preparing a novel agarobionic sugar.

[0011] Specifically, the β-agarase provided by the present invention comprises the amino acid fragment shown in SEQ ID NO:1.

[0012] The nucleic acid molecule provided by this invention includes a nucleotide fragment encoding the above-mentioned β-agarase.

[0013] Furthermore, the nucleic acid molecule includes the nucleotide fragment shown in SEQ ID NO:2.

[0014] The expression strain provided by this invention expresses the above-mentioned β-agarase.

[0015] The method for preparing the above-mentioned β-agarase provided by the present invention includes: introducing the encoding gene of the β-agarase into chassis cells to obtain an expression strain; and inducing the expression strain to obtain the β-agarase.

[0016] Further, the preparation method includes one or more of the following technical features: (1) the chassis cells are selected from one or more of Escherichia coli, Bacillus, and Saccharomyces cerevisiae; (2) the encoding gene of β-agarase is carried on a plasmid vector, the plasmid vector being selected from one or more of pET28a plasmid, pET29a plasmid, PHT43 plasmid, pPIC9K plasmid, pPIC3.5K plasmid, pGAPZαA plasmid, pGAPZαB plasmid, and pGAPZαC plasmid; (3) the method of introducing the encoding gene of β-agarase into the chassis cells includes one or more of CaCl2 chemical transformation, electroconversion, conjugation transfer, and phage transduction.

[0017] Further, the induction culture includes: inoculating the expression strain into a fermentation medium and culturing for 8h~24h, and adding feed medium when the DO value of the solution is greater than 60% until the DO value is less than 35%, and adjusting the pH value of the solution to 6.9~7.1 by using ammonia and phosphoric acid solution; wherein, the induction culture includes at least one of the following technical features: (1) the initial OD of the solution in the induction culture. 600The value is 25~35; (2) The fermentation medium includes 6g / L~10g / L glucose, 0.5g / L~2g / L magnesium sulfate, 10g / L~16g / L potassium dihydrogen phosphate, 1g / L~4g / L sodium sulfate, 2g / L~6g / L diammonium hydrogen phosphate, 1g / L~3g / L sodium citrate, 0.05%(v / v)~0.2%(v / v) antifoaming agent, and 30mg / L ~60 mg / L sodium citrate dihydrate, 1 mg / L~3 mg / L cobalt chloride hexahydrate, 0.8 mg / L~1.5 mg / L sodium molybdate dihydrate, 0.6 mg / L~1 mg / L copper sulfate pentahydrate, 0.5 mg / L~1 mg / L manganese chloride tetrahydrate, 0.5 mg / L~1 mg / L zinc chloride and 0.3 mg / L~1 mg / L boric acid; (3) the supplementary culture medium package This includes glucose monohydrate (400 g / L~700 g / L), magnesium sulfate heptahydrate (5 g / L~10 g / L), sodium citrate dihydrate (5 mg / L~15 mg / L), ferric chloride hexahydrate (1 mg / L~10 mg / L), cobalt chloride hexahydrate (0.1 mg / L~0.5 mg / L), sodium molybdate dihydrate (0.1 mg / L~0.5 mg / L), and sulfuric acid pentahydrate (0.1 mg / L~1 mg / L). Copper, 1 mg / L~3 mg / L manganese chloride tetrahydrate, 1 mg / L~3 mg / L zinc chloride, 0.5 mg / L~1 mg / L calcium chloride dihydrate and 1 mg / L~3 mg / L boric acid; (4) the concentration of the ammonia water is 25% (w / v)~30% (w / v) and the concentration of the phosphoric acid solution is 15% (w / v)~25% (w / v); (5) the induction culture temperature is 20℃~25℃, the rotation speed is 200 rpm~220 rpm, and the time is 8h~24h.

[0018] This invention provides the application of the above-mentioned β-agarase, nucleic acid molecules, or expression strains in the preparation of new agarobiose.

[0019] The method for preparing the new agaroblocene provided by the present invention includes: taking the above-mentioned β-agarase and the new agarobloc oligosaccharide for enzymatic hydrolysis to obtain the new agaroblocene.

[0020] Further, the preparation method includes one or more of the following technical features: (1) the degree of polymerization of the new agarose oligosaccharide is 4 to 40; (2) the concentration of the β-agarase is 0.1% (w / v) to 5% (w / v), and the concentration of the new agarose oligosaccharide is 1% (w / v) to 20% (w / v); (3) the temperature of the enzymatic hydrolysis reaction is 30℃ to 40℃, the pH value is 6.5 to 7.5, and the time is 0.1h to 10h.

[0021] Beneficial effects: The β-agarase provided by this invention specifically includes an amino acid fragment with the sequence shown in SEQ ID NO:1. It can efficiently and specifically degrade agar oligosaccharides under mild conditions, and the degradation product is a single product, neo-agarbiose. It has the advantages of high enzyme activity, mild reaction, high stability, and homogenization of enzymatic hydrolysis products. It has excellent application prospects in realizing the large-scale industrial production of neo-agarbiose. Attached Figure Description

[0022] Figure 1 The figure shows the experimental results of the optimal temperature test for β-agarase AgaSE05 provided in Example 2 of this invention; Figure 2 The figure shows the experimental results of the optimal pH test for β-agarase AgaSE05 provided in Example 2 of this invention; Figure 3 The figure shows the experimental results of the temperature stability test of β-agarase AgaSE05 provided in Example 2 of this invention; Figure 4 The figure shows the results of the stability test of β-agarase AgaSE05 provided in Example 2 of this invention under optimal conditions; Figure 5 This is a graph showing the experimental results of testing the enzymatic hydrolysis product of β-agarase AgaSE05 provided in Example 2 of this invention. Detailed Implementation

[0023] The inventors of this invention obtained a marine microbial strain capable of degrading agar by screening a large number of marine bacteria, based on a β-agarase that can catalyze the hydrolysis of highly polymerized agar oligosaccharides under mild conditions to generate a single degradation product, neoagarbiose. Marinimicrobium sp. Based on this, genomic analysis and screening were conducted to obtain a β-agarase with high enzyme activity, good temperature stability, and uniform enzymatic digestion products. Based on this, the technical solution of this invention was obtained.

[0024] The β-agarase provided by this invention specifically includes the amino acid fragment shown in SEQ ID NO:1.

[0025] The nucleic acid molecule provided by this invention specifically includes a nucleotide fragment encoding the aforementioned β-agarase. The nucleotide sequence of the nucleotide fragment encoding the aforementioned β-agarase is determined based on the structure of the β-agarase. Deriving the nucleotide sequence encoding the aforementioned β-agarase from the β-agarase is a conventional technique in the existing biotechnology field. Those skilled in the art can make adaptive designs based on the chassis cells intended for expression, and this invention does not impose any particular limitations on it.

[0026] In some specific embodiments, the nucleic acid molecule preferably includes the nucleotide fragment shown in SEQ ID NO:2. In this case, the nucleic acid molecule can achieve efficient expression of β-agarase in E. coli cells.

[0027] The expression strain provided by this invention expresses the above-mentioned β-agarase. More specifically, the expression strain specifically includes a nucleotide fragment encoding the above-mentioned β-agarase and a protein expression system for achieving β-agarase synthesis. Various existing options can be used, limited only by their ability to achieve β-agarase expression; this invention does not impose any particular limitation on them.

[0028] The method for preparing the above-mentioned β-agarase provided by the present invention specifically includes: introducing the encoding gene of the β-agarase into chassis cells to obtain an expression strain; and inducing the expression strain to obtain the β-agarase.

[0029] In this invention, the chassis cell refers to a host cell that has been screened or moderately modified in genetic engineering technology and has the ability to carry exogenous genes and express proteins. It can be any of the existing choices, and this invention does not particularly limit it. Specific examples include, but are not limited to, one or more of Escherichia coli, Bacillus, and Saccharomyces cerevisiae; preferably Escherichia coli.

[0030] In this invention, the β-agarase encoding gene may exist in the chassis cells in one or more of the following forms, but is not limited to: existing in the chassis cell cytoplasm as a free circular DNA molecule carried on a plasmid vector, existing in the chassis cell cytoplasm as a free mRNA molecule, or integrated into the chromosomal DNA of the chassis cells through homologous recombination.

[0031] In some specific embodiments, the β-agarase is preferably mounted on a plasmid vector, which can be of various existing types and is not particularly limited thereto. Specific examples include, but are not limited to, one or more of the following: pET28a plasmid, pET29a plasmid, pET48a plasmid, PHT43 plasmid, pPIC9K plasmid, pPIC3.5K plasmid, pGAPZαA plasmid, pGAPZαB plasmid, and pGAPZαC plasmid.

[0032] In this invention, the method of introducing the encoding gene of the β-agarase into the chassis cells can be any of the existing options, and this invention does not particularly limit it. Specific examples include, but are not limited to, one or more of the following: CaCl2 chemical conversion method, electroconversion method, conjugation transfer method, and phage transduction method.

[0033] In this invention, the induction culture specifically includes: inoculating the expression strain into a fermentation medium and culturing for 8-24 hours; when the DO value of the solution is greater than 60%, adding feed medium until the DO value is lower than 35%; and adjusting the pH of the solution to 6.9-7.1 using ammonia and phosphoric acid solutions. At this point, using ammonia as the nitrogen source and employing an inorganic high-density fermentation method to prepare β-agarase has advantages such as low cost and simple control of culture conditions, and can effectively achieve large-scale industrial preparation of β-agarase.

[0034] In some specific embodiments, the initial OD of the solution in the induction expression culture is... 600 The value is preferably 25 to 35, specifically 25, 27, 29, 30, 33, 35 or any value between them.

[0035] In some specific embodiments, the fermentation medium specifically includes: glucose at a concentration preferably 6 g / L to 10 g / L, specifically 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L or any value between them; magnesium sulfate at a concentration preferably 0.5 g / L to 2 g / L, specifically 0.5 g / L, 0.8 g / L, 1 g / L, 1.2 g / L, 1.5 g / L, 1.8 g / L, 2 g / L or any value between them; potassium dihydrogen phosphate at a concentration preferably 10 g / L to 16 g / L, specifically 10 g / L, 12 g / L, 14 g / L, 16 g / L or any value between them; and sodium sulfate at a concentration preferably 1 g / L to 4 g / L, specifically... The concentrations are 1 g / L, 1.5 g / L, 2 g / L, 2.3 g / L, 2.8 g / L, 3 g / L, 3.5 g / L, 4 g / L, or any value between them; the preferred concentration is 2 g / L to 6 g / L of diammonium hydrogen phosphate, specifically 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, or any value between them; the preferred concentration is 1 g / L to 3 g / L of sodium citrate, specifically 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, or any value between them; the preferred concentration is 0.05% (v / v) to 0.2% (v / v) of defoamer, specifically 0.05% (v / v), 0.08% (v / v), 0.1% (v / v). 0.12% (v / v), 0.15% (v / v), 0.18% (v / v), 0.2% (v / v), or any value between them; the preferred concentration is sodium citrate dihydrate of 30 mg / L to 60 mg / L, specifically 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 60 mg / L, or any value between them; the preferred concentration is cobalt chloride hexahydrate of 1 mg / L to 3 mg / L, specifically 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, or any value between them; the preferred concentration is sodium molybdate dihydrate of 0.8 mg / L to 1.5 mg / L, specifically 0.8 mg / L, The concentrations are preferably 0.9 mg / L, 1 mg / L, 1.2 mg / L, 1.5 mg / L, or any value between them; the preferred concentrations are copper sulfate pentahydrate (0.6 mg / L to 1 mg / L), specifically 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, or any value between them; the preferred concentrations are manganese chloride tetrahydrate (0.5 mg / L to 1 mg / L), specifically 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, or any value between them; the preferred concentrations are zinc chloride (0.5 mg / L to 1 mg / L), specifically 0.5 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, or any value between them; and the preferred concentrations are zinc chloride (0.5 mg / L to 1 mg / L), specifically 0.5 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, or any value between them.9 mg / L, 1 mg / L, or any value between them; and, preferably, boric acid with a concentration of 0.3 mg / L to 1 mg / L, specifically 0.3 mg / L, 0.5 mg / L, 0.8 mg / L, 1 mg / L, or any value between them.

[0036] In some specific embodiments, the supplemental culture medium specifically includes: glucose monohydrate with a concentration preferably between 400 g / L and 700 g / L, specifically 400 g / L, 450 g / L, 500 g / L, 600 g / L, 700 g / L, or any value between them; magnesium sulfate heptahydrate with a concentration preferably between 5 g / L and 10 g / L, specifically 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any value between them; and sodium citrate dihydrate with a concentration preferably between 5 mg / L and 15 mg / L, specifically 5 mg / L, 8 mg / L, 10 mg / L, or any value between them. The concentrations are: mg / L, 12 mg / L, 15 mg / L, or any value between them; the preferred concentrations are ferric chloride hexahydrate (1 mg / L to 10 mg / L), specifically 1 mg / L, 3 mg / L, 5 mg / L, 8 mg / L, 10 mg / L, or any value between them; the preferred concentrations are cobalt chloride hexahydrate (0.1 mg / L to 0.5 mg / L), specifically 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, or any value between them; the preferred concentrations are sodium molybdate dihydrate (0.1 mg / L to 0.5 mg / L), specifically... The concentrations are 0.1 mg / L, 0.3 mg / L, 0.5 mg / L, or any value between them; preferably, it is copper sulfate pentahydrate with a concentration of 0.1 mg / L to 1 mg / L, specifically 0.1 mg / L, 0.3 mg / L, 0.7 mg / L, 0.8 mg / L, 1 mg / L, or any value between them; preferably, it is manganese chloride tetrahydrate with a concentration of 1 mg / L to 3 mg / L, specifically 1 mg / L, 1.3 mg / L, 1.5 mg / L, 1.8 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, or any value between them; preferably, it is 1 mg / L to 3 mg / L. The zinc chloride concentration is preferably 1 mg / L, specifically 1 mg / L, 1.2 mg / L, 1.5 mg / L, 2 mg / L, 3 mg / L, or any value between them; the calcium chloride dihydrate concentration is preferably 0.5 mg / L to 1 mg / L, specifically 0.5 mg / L, 0.75 mg / L, 0.9 mg / L, 1 mg / L, or any value between them; and the boric acid concentration is preferably 1 mg / L to 3 mg / L, specifically 1 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, or any value between them.

[0037] In some specific embodiments, the concentration of the ammonia solution is preferably 25% (w / v) to 30% (w / v), specifically 25% (w / v), 27% (w / v), 29% (w / v), 30% (w / v) or any value between them; the concentration of the phosphoric acid solution is preferably 15% (w / v) to 25% (w / v), specifically 15% (w / v), 18% (w / v), 20% (w / v), 22% (w / v), 25% (w / v) or any value between them.

[0038] In some specific embodiments, the conditions for induction culture include: the temperature is preferably 20℃~25℃, specifically 20℃, 23℃, 24℃, 25℃ or any value between them; the rotation speed is preferably 200rpm~220rpm, specifically 200rpm, 205rpm, 210rpm, 220rpm or any value between them; the time is preferably 8h~24h, specifically 8h, 9h, 12h, 14h, 16h, 18h, 20h, 24h or any value between them.

[0039] This invention provides the application of the above-mentioned β-agarase, nucleic acid molecules, or expression strains in the preparation of new agarobiose.

[0040] The method for preparing the new agaroblocene provided by the present invention specifically includes: mixing the above-mentioned β-agarase with the new agarobloc oligosaccharide, and carrying out an enzymatic hydrolysis reaction to obtain the new agaroblocene.

[0041] In some specific embodiments, the degree of polymerization of the neo-agar oligosaccharide is preferably 4 to 10, specifically 4, 5, 6, 8, 10 or any value between them.

[0042] In some specific embodiments, the concentration of the β-agarase is preferably 0.1% (w / v) to 5% (w / v), specifically 0.1% (w / v), 0.5% (w / v), 1% (w / v), 2% (w / v), 2.5% (w / v), 2.8% (w / v), 3% (w / v), 3.6% (w / v), 4% (w / v), 4.5% (w / v), 5% (w / v) or any value between them; the concentration of the new agar oligosaccharide is preferably 1% (w / v) to 20% (w / v), specifically 1% (w / v), 3% (w / v), 5% (w / v), 8% (w / v), 10% (w / v), 12% (w / v), 16% (w / v), 18% (w / v), 20% (w / v) or any value between them.

[0043] In some specific embodiments, the conditions for the enzymatic hydrolysis reaction specifically include: the temperature is preferably 30℃~40℃, specifically 30℃, 32℃, 35℃, 38℃, 40℃ or any value between them; the pH value is preferably 6.5~7.5, specifically 6.5, 6.8, 7, 7.2, 7.5 or any value between them; the time is preferably 0.1h~10h, specifically 0.1h, 0.8h, 1h, 1.5h, 2h, 3h, 5h, 8h, 10h or any value between them.

[0044] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0045] Example 1 This embodiment illustrates a β-agarase, AgaSE05, and its preparation method, which is a marine microbial strain capable of degrading agar, obtained by screening from a large number of marine bacteria. Marinimicrobium sp. Based on this, genomic analysis and screening were performed to obtain the β-agarase AgaSE05, which specifically includes the amino acid fragment with the sequence shown in SEQ ID NO:1. The preparation process includes the following steps: 1. Construction of expression vector: Based on the amino acid sequence of β-agarase AgaSE05, codons of E. coli were optimized to obtain the AgaSE05 encoding gene (SEQ ID NO:2), which was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and loaded onto pET28a plasmid to obtain the expression vector.

[0046] 2. Construction of the expression strain: Following the instructions, the expression vector was transformed into BL21(DE3) chemocompetent cells (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and then inoculated into LB liquid medium. After culturing at 37℃ and 170 rpm for 1 h, the cells were centrifuged at 4500 rpm for 2 min, the supernatant was removed, and the cell pellet was inoculated into LB solid medium containing 50 μg / mL kanamycin. The cells were then cultured at 37℃ and 200 rpm for 8 h. Samples were taken for sequencing to verify that the expression vector had been successfully introduced, and the expression strain was obtained. This expression strain was preserved in 60% sterile glycerol.

[0047] 3. Expression and purification of β-agarase AgaSE05: (1) The expression strain was inoculated into LB liquid medium at an inoculation rate of 2% (v / v) and cultured overnight at 37℃ and 200 rpm to obtain the primary seed culture; the primary seed culture was inoculated into LB liquid medium at an inoculation rate of 1% and cultured at 37℃ and 200 rpm until the solution OD reached 100%. 600 A value of 3 indicates the production of a secondary seed solution.

[0048] (2) Take 1% (v / v) of the secondary seed culture and inoculate it into the fermentation medium. Fermentation is carried out at 37℃, 1000 rpm and pH=7.0 until the solution OD reaches 100%. 600 When the pH value reaches 40 (monitor the DO and pH values ​​of the solution during fermentation: when the DO value is greater than 60%, add feed medium until the DO value is lower than 35%; when the pH value is lower than 7.0, add 28% (w / v) ammonia solution until the pH value is 7.0; when the pH value is higher than 7.0, add 20% (w / v) phosphate solution until the pH value is 7.0), then add IPTG at a final concentration of 0.2 mM, and induce culture at 20℃ and 220 rpm for 16 h to obtain the induced expression solution.

[0049] The fermentation medium includes 8 g / L glucose, 1 g / L magnesium sulfate, 15 g / L potassium dihydrogen phosphate, 2 g / L sodium sulfate, 4 g / L diammonium hydrogen phosphate, 2 g / L sodium citrate, 1 mL / L defoamer (Maclean, catalog number P728883), and 10 mL / L 100× Trace Element I. 100× Trace Element I includes 4 g / L sodium citrate dihydrate, 0.2 g / L cobalt chloride hexahydrate, 0.1 g / L sodium molybdate dihydrate, 0.08 g / L copper sulfate pentahydrate, 0.1 g / L manganese chloride tetrahydrate, 0.1 g / L zinc chloride, and 0.1 g / L boric acid.

[0050] The supplemental culture medium consisted of 600 g / L glucose monohydrate, 8 g / L magnesium sulfate heptahydrate, and 10 mL / L 100× micronutrient II. 100× micronutrient II consisted of 1 g / L sodium citrate dihydrate, 0.5 g / L ferric chloride hexahydrate, 0.022 g / L cobalt chloride hexahydrate, 0.042 g / L sodium molybdate dihydrate, 0.054 g / L copper sulfate pentahydrate, 0.135 g / L manganese chloride tetrahydrate, 0.14 g / L zinc chloride, 0.08 g / L calcium chloride dihydrate, and 0.15 g / L boric acid.

[0051] (3) Take pre-cooled deionized water to resuspend the induced expression solution to a concentration of 20% (w / v) to obtain a resuspended bacterial solution; take the resuspended bacterial solution and let it stand in an ice-water bath for 30 min, filter it to obtain bacterial sludge; take the bacterial sludge and perform a first high-pressure homogenization treatment at 4℃ and 600 bar to obtain a first-stage lysate; take the first-stage lysate and perform a second high-pressure homogenization treatment at 4℃ and 900 bar to obtain a second-stage lysate; take the second-stage lysate and perform a third high-pressure homogenization treatment at 4℃ and 900 bar until no bacterial cell precipitation is observed, complete the cycle of high-pressure homogenization treatment, and obtain a bacterial cell lysate.

[0052] (4) Centrifuge the bacterial cell lysate at 15000×g for 5 min, collect the supernatant, and obtain β-agarase AgaSE05 enzyme solution.

[0053] Example 2 This embodiment uses the β-agarase AgaSE05 provided in Example 1 to demonstrate its relevant performance. PBS buffer (100mM, pH=7) was mixed with agar oligosaccharide (degree of polymerization 10) and β-agarase AgaSE05 enzyme solution to obtain agar oligosaccharide substrate solution and enzyme solution, both at a concentration of 5% (w / v). The following tests were then performed: 1. Optimal Temperature: Mix the substrate solution and the enzyme solution to be tested at a volume ratio of 20:1. React at 25℃~60℃ for 5 min each time. Then, mix 1 mL of the mixture with 3 mL of DNS reagent, heat in a boiling water bath for 15 min, cool to room temperature, and measure the absorbance at 550 nm. Use deionized water mixed with DNS reagent as a blank control. Substitute these values ​​into the working curve plotted with galactose as a standard to calculate the enzyme activity of β-agarase AgaSE05 at each temperature. The results are as follows: Figure 1 As shown.

[0054] The amount of enzyme required to produce 1 μmol of reducing sugar per minute is defined as one unit of enzyme activity (1 U).

[0055] Depend on Figure 1 The results show that the optimal temperature for the β-agarase AgaSE05 provided by this invention is 35℃, at which point the enzyme activity reaches 383.5 U / mL, exhibiting excellent catalytic activity.

[0056] 2. Optimal pH: After adjusting the pH of the substrate solution to 4-10 using hydrochloric acid or sodium hydroxide, mix the substrate solution and the enzyme solution to be tested at a volume ratio of 20:1. React at 35℃ for 5 min. Then, mix 1 mL of the mixture with 3 mL of DNS reagent, heat in a boiling water bath for 15 min, cool to room temperature, and measure the absorbance at 550 nm. Use deionized water mixed with DNS reagent as a blank control. Substitute these values ​​into the working curve plotted using galactose as a standard to calculate the enzyme activity of β-agarase AgaSE05 at each pH value. The results are as follows: Figure 2 As shown.

[0057] Depend on Figure 2 The results show that the optimal pH value of the β-agarase AgaSE05 provided by the present invention is 7, at which point the enzyme activity is 381 U / mL.

[0058] 3. Temperature stability: The enzyme solutions to be tested were stored at 25℃~60℃ for 4 hours. The enzyme activity of β-agarase AgaSE05 was then measured at each temperature using the method provided in "1. Optimal Temperature" after 4 hours of storage. The enzyme activity retention rate was calculated based on the enzyme activity of β-agarase AgaSE05 before storage. The results are shown below. Figure 3 As shown in Table 1.

[0059] Table 1.

[0060] Depend on Figure 3 As shown in Table 1, the β-agarase AgaSE05 provided by this invention has good temperature stability. After being stored at a temperature of 25℃ to 55℃ for 4 hours, the enzyme activity remained above 50%.

[0061] 4. Enzyme activity stability: Under optimal temperature and pH conditions, the enzyme activity of β-agarase AgaSE05 was measured after storage for different times. The enzyme activity retention rate was calculated based on the enzyme activity of β-agarase AgaSE05 before storage. The results are as follows: Figure 4 As shown in Table 2.

[0062] Table 2.

[0063] Depend on Figure 4 As shown in Table 2, the β-agarase AgaSE05 provided by this invention retains an enzyme activity of over 78% after being stored at 35°C and pH 7.0 for 24 hours, demonstrating good enzyme activity stability.

[0064] 5. Enzymatic hydrolysis products: The substrate solution and the enzyme solution to be tested were mixed thoroughly at a volume ratio of 20:1. The mixtures were incubated at 35℃ for 2 hours (Experimental Group 1) or 6 hours (Experimental Group 2), respectively. The results were then analyzed by thin-layer chromatography (TLC) using the developing solvent (containing n-butanol, glacial acetic acid, and water in a volume ratio of 2:1:1) and the colorimetric reagent (containing concentrated sulfuric acid and anhydrous ethanol in a volume ratio of 1:9). The results are shown below. Figure 5 As shown.

[0065] Depend on Figure 5 The results show that the enzymatic hydrolysis product of the β-agarase AgaSE05 provided by the present invention is neo-agarobiose (DP2), which has excellent homogeneity of enzymatic hydrolysis product.

[0066] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

[0067] The amino acid and nucleotide sequences involved in this invention are specifically shown in Table 3.

[0068] Table 3.

Claims

1. A β-agarase, characterized in that, The β-agarase is the amino acid fragment shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is the nucleotide fragment shown in SEQ ID NO:

2.

3. An expression strain, characterized in that, The expression strain expresses the β-agarase of claim 1, and the chassis cells of the expression strain are Escherichia coli.

4. The method for preparing β-agarase according to claim 1, characterized in that, The encoding gene of the β-agarase was introduced into chassis cells to obtain an expression strain; the expression strain was then subjected to induction culture to obtain the β-agarase. The chassis cells are Escherichia coli.

5. The method for preparing β-agarase according to claim 4, characterized in that, The preparation method includes one or more of the following technical features: (1) The gene encoding the β-agarase is carried on a plasmid vector, wherein the plasmid vector is pET28a plasmid and / or pET29a plasmid; (2) The method of introducing the encoding gene of the β-agarase into the chassis cells includes one or more of the following: CaCl2 chemical transformation, electroconversion, conjugation transfer and phage transduction.

6. The method for preparing β-agarase according to claim 4, characterized in that, The induction culture includes: inoculating the expression strain into a fermentation medium and culturing for 8-24 hours, and adding feed medium when the DO value of the solution is greater than 60% until the DO value is lower than 35%, and adjusting the pH of the solution to 6.9-7.1 using ammonia and phosphoric acid solutions; wherein the induction culture includes at least one of the following technical features: (1) The initial OD of the solution in the induction culture 600 The value is between 25 and 35; (2) The fermentation medium includes 6 g / L to 10 g / L of glucose, 0.5 g / L to 2 g / L of magnesium sulfate, 10 g / L to 16 g / L of potassium dihydrogen phosphate, 1 g / L to 4 g / L of sodium sulfate, 2 g / L to 6 g / L of diammonium hydrogen phosphate, 1 g / L to 3 g / L of sodium citrate, 0.05% (v / v) to 0.2% (v / v) of defoamer, 30 mg / L to 60 mg / L of sodium citrate dihydrate, 1 mg / L to 3 mg / L of cobalt chloride hexahydrate, 0.8 mg / L to 1.5 mg / L of sodium molybdate dihydrate, 0.6 mg / L to 1 mg / L of copper sulfate pentahydrate, 0.5 mg / L to 1 mg / L of manganese chloride tetrahydrate, 0.5 mg / L to 1 mg / L of zinc chloride and 0.3 mg / L to 1 mg / L of boric acid; (3) The supplemental culture medium comprises 400 g / L to 700 g / L glucose monohydrate, 5 g / L to 10 g / L magnesium sulfate heptahydrate, 5 mg / L to 15 mg / L sodium citrate dihydrate, 1 mg / L to 10 mg / L ferric chloride hexahydrate, 0.1 mg / L to 0.5 mg / L cobalt chloride hexahydrate, 0.1 mg / L to 0.5 mg / L sodium molybdate dihydrate, 0.1 mg / L to 1 mg / L copper sulfate pentahydrate, 1 mg / L to 3 mg / L manganese chloride tetrahydrate, 1 mg / L to 3 mg / L zinc chloride, 0.5 mg / L to 1 mg / L calcium chloride dihydrate and 1 mg / L to 3 mg / L boric acid; (4) The concentration of the ammonia solution is 25% (w / v) to 30% (w / v), and the concentration of the phosphoric acid solution is 15% (w / v) to 25% (w / v); (5) 0.2 mM IPTG was added to the induction culture; (6) The induction culture temperature is 20℃~25℃, the rotation speed is 200rpm~220rpm, and the time is 8h~24h.

7. The use of the β-agarase of claim 1, the nucleic acid molecule of claim 2, or the expression strain of claim 3 in the preparation of new agarobiose.

8. A method for preparing a novel agarobionic sugar, characterized in that, The preparation method includes: taking the β-agarase of claim 1 and the neo-agar oligosaccharide and performing an enzymatic hydrolysis reaction to obtain the neo-agar disaccharide.

9. The method for preparing neo-agarbiose according to claim 8, characterized in that, The preparation method includes one or more of the following technical features: (1) The degree of polymerization of the new agar oligosaccharide is 4~10; (2) The concentration of the β-agarase added is 0.1% (w / v) to 5% (w / v), and the concentration of the new agar oligosaccharide added is 1% (w / v) to 20% (w / v); (3) The temperature of the enzymatic hydrolysis reaction is 30℃~40℃, the pH value is 6.5~7.5, and the time is 0.1h~10h.

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