Marine-derived alpha-agarase gene for producing high-proportion agarotriose and application method of marine-derived alpha-agarase gene

Abstract

The invention discloses a marine-derived alpha-agarase gene for producing high-proportion agarotriose and an application method of the marine-derived alpha-agarase gene, and belongs to the technical field of gene engineering and enzyme engineering. The gene contains a nucleotide sequence as shown in SEQ ID NO.1, the gene is introduced into a genetically engineered bacterium E.coli BL21 (DE3) (pET-28a (+) / th-aga) for secretory expression, substrates such as agarose or agar and the like are hydrolyzed by using purified and recombinant alpha-agarase to generate corresponding functional agaro-oligosaccharides such as agarotriose, agarotetraose, agaropentaose and the like, and the obtained functional agaro-oligosaccharides can be directly used for producing foods, medicines and the like.

Description

Technical Field

[0001] The invention relates to a marine-derived alpha-agarase gene for producing agarotriose with a high ratio and an application method thereof, belonging to the technical field of gene engineering and enzyme engineering. Background Art

[0002] Agarase is an enzyme that degrades agaropolysaccharide. The agaro-oligosaccharides produced by its hydrolysis have multiple functional activities and have broad application prospects in the food, cosmetics and pharmaceutical industries. According to the different modes of agarase hydrolysis, it can be divided into two categories: α-agarase (EC 3.2.1.158) and β-agarase (EC 3.2.1.81). α-Agarase can hydrolyze the α-1,3 bonds of agarose to produce agaro-oligosaccharides (AOS) with D-galactose as the non-reducing end. The α-agarases reported so far include AgaA from Alteromonas agarolyticus, AgaA33 from Psychromonas psychrophilus, AgaD from Psychromonas psychrophilus, CaLJ96 from Streptomyces agarophilus, AgaWS5 from Catenovulum sediminis and OUC-GAJJ96 from Catenovulum sediminis. The research content mainly includes heterologous expression and enzymatic properties.

[0003] Agar oligosaccharides have rich physiological activities such as anti-oxidation, whitening and moisturizing, anti-inflammatory, anti-cancer, and immunity enhancement, and have application potential in the food, cosmetics, and pharmaceutical industries. At present, the preparation methods of agar oligosaccharides are mainly divided into acid hydrolysis and enzymatic hydrolysis. Compared with the enzymatic hydrolysis method, the acid hydrolysis method has difficulties such as easy loss of substrate activity, uneven products and difficult separation and recovery, and high energy consumption of high-temperature acid hydrolysis, which also makes the production of agar oligosaccharides difficult to achieve industrialization. Compared with the acid hydrolysis method, the enzymatic hydrolysis method for preparing agar oligosaccharides has extremely strong specificity and specificity. It can selectively cut glycosidic bonds and produce small molecular oligosaccharides with a specific degree of polymerization. It is easy to control the degree of degradation, the reaction conditions are mild, the energy consumption cost is low, and it is environmentally friendly and low-carbon. Microorganisms are the main source of α-agarase. As the largest and oldest habitat on earth, the ocean contains extremely rich carbohydrates and microbial resources. However, the current process for preparing high-value-added agar oligosaccharides by agar hydrolysis is still imperfect, mainly because of the lack of enzyme preparations that can efficiently hydrolyze agarose substrates. Therefore, it is necessary to develop a marine-derived α-agarase with stable properties and high catalytic efficiency, and to establish a stable and efficient α-agarase Escherichia coli expression system. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the present invention provides a marine-derived α-agarase gene for producing a high-proportion agarotriose and an application method thereof, and the secretory expression of the α-agarase is achieved by introducing a gene encoding α-agarase derived from Thalassomonas sp. into a genetically engineered bacterium, with the aim of solving the technical solution of the lack of a marine-derived α-agarase with stable properties and high catalytic efficiency in the prior art.

[0005] The first technology provided by the present invention is a gene encoding α-agarase, the nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0006] The second technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the first technical solution.

[0007] In certain embodiments, the recombinant plasmid uses pET-28a(+) vector as an expression vector.

[0008] The third technical solution provided by the present invention is a recombinant cell containing the gene described in the first technical solution or transformed with the recombinant plasmid described in the second technical solution.

[0009] The fourth technical solution provided by the present invention is a genetically engineered bacterium, which uses Escherichia coli as a host and expresses the gene described in the first technical solution.

[0010] In certain embodiments, the genetically engineered bacteria uses the Escherichia coli pET-28a(+) vector as an expression vector.

[0011] In certain embodiments, the genetically engineered bacteria uses Escherichia coli BL21 (DE3) as a host.

[0012] The fifth technical solution provided by the present invention is a method for constructing an Escherichia coli expression system of marine-derived α-agarase, by inserting the gene described in the first technical solution into a vector plasmid to obtain a secretory expression vector, and transforming it into Escherichia coli to obtain a genetically engineered bacterium.

[0013] In certain embodiments, the genetically engineered bacteria uses the Escherichia coli pET-28a(+) vector as an expression vector.

[0014] In certain embodiments, the genetically engineered bacteria uses Escherichia coli BL21 (DE3) as a host.

[0015] The sixth technical scheme provided by the present invention is a method for preparing recombinant marine α-agarase, wherein the genetically engineered bacteria E. coli BL21 (DE3) (pET-28a (+) / th-aga) described in the fourth technical scheme are inoculated into an LB culture medium containing kanamycin, cultured in a 37°C shaking incubator (200 r / min) to a logarithmic growth phase, and the cultured seed culture solution is inoculated into a TB culture medium containing kanamycin at an inoculation rate of 4% (v / v), and fermented and cultured in a shaking incubator at 25 to 30°C for 16 to 24 hours to produce recombinant marine α-agarase.

[0016] The seventh technical scheme provided by the present invention is a method for producing functional agar-oligosaccharides using marine-derived α-agarase, using the α-agarase encoded by the gene described in the first technical scheme as a catalyst and agar-oligosaccharides or agar as a substrate to hydrolyze and generate corresponding functional agar-oligosaccharides such as agarotriose, agar-tetraose, and agar-pentaose. The obtained functional agar-oligosaccharides can be directly used in the production of food, medicine, etc.

[0017] In certain embodiments, the catalyst is a crude enzyme solution obtained by fermentation of genetically engineered bacteria or a pure enzyme obtained after separation and purification and is added to the substrate in the form of an enzyme preparation.

[0018] The eighth technical solution provided by the present invention is the use of the gene described in the first technical solution, or the recombinant plasmid described in the second technical solution, or the recombinant cell described in the third technical solution, or the genetically engineered bacteria described in the fourth technical solution, or the method described in the fifth technical solution, or the method described in the sixth technical solution, or the method described in the seventh technical solution in the preparation of agar-oligosaccharide-containing product.

[0019] The technical effects of the present invention are as follows:

[0020] 1) The secretory expression of marine-derived α-agarase in Escherichia coli was achieved. The method is stable and efficient, and the hydrolysis activity of the crude α-agarase solution reaches 15-20 U / mL;

[0021] 2) Compared with other similar enzymes reported, the biggest advantage of the marine-derived α-agarase of the present invention is that it can synthesize agarotriose (A3) in a large amount with a yield of about 40% to 50%, so the application field of the enzyme is very broad.

[0022] 3) The marine-derived α-agarase of the present invention has a high substrate conversion rate and product purity, which can effectively improve the utilization rate of polysaccharide resources in the ocean and reduce the production and processing cost of functional agar-oligosaccharides, thereby having greater industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Agarose gel electrophoresis detection of recombinant pET-28a(+) / th-aga positive clones; standard product: DNA standard; lane 1: recombinant pET-28a(+) / th-aga positive clones.

[0024] Figure 2 The effect of temperature on the activity of recombinant α-agarase.

[0025] Figure 3 The effect of pH on the activity of recombinant α-agarase.

[0026] Figure 4 Thermostability of recombinant α-agarase.

[0027] Figure 5 pH stability of recombinant α-agarase.

[0028] Figure 6 This is the SDS-PAGE analysis of recombinant α-agarase; wherein, standard product: protein standard; lane 1: recombinant α-agarase th-aga crude enzyme;

[0029] Figure 7 This is the result of HPAEC-PAD detection of recombinant α-agarase hydrolyzate;

[0030] Figure 8 The results of ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry detection of the hydrolysis products of recombinant α-agarase. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0032] Test method:

[0033] The detection conditions of high-performance anion exchange chromatography (HPAEC-PAD) are as follows: (1) chromatographic column: Dionex CarboPacPA-200 anion exchange column, including analytical column (3mm×250mm) and guard column (3mm×50mm); (2) mobile phase: 96% 100mM NaOH, 4% 100mM NaOH & 500mM NaAc, flow rate 0.5mL / min; (3) detection by four-potential pulse amperometry through amperometric detector; (4) column temperature 35°C, injection volume 10μL. The type of agar oligosaccharide in the sample was determined according to the retention time of the corresponding chromatographic peak.

[0034] The raw materials used in the embodiment:

[0035] 1. LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0.

[0036] 2. TB medium: 1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17mM KH2PO4, 72mM K2HPO4, pH 6.0.

[0037] 3. pET28a(+) vector, Escherichia coli JM109 competent cells, and Escherichia coli BL21(DE3) are commercial plasmids and commercial strains.

[0038] 4. Source of raw materials: Tryptone and yeast extract were purchased from Oxoid Company, UK; sodium chloride, glycerol, KH2PO4 and K2HPO4 were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0039] Example 1 Construction of Escherichia coli secretion expression system

[0040] According to the gene of α-agarase, the upstream primer was designed as follows: 5'-ATGGAAACACTGGTTCTGCAAGCAG-3'; the downstream primer was designed as follows: 5'-GTGGTGGTGCTCGAGTTAATGTGCCAGTTCCAGAATGCCC3'. According to the pET28a(+) vector sequence, the upstream primer was designed as follows: 5'-GACGGGCTTGTCTGCTCC-3'; the downstream primer was designed as follows: 5'-AACCAGTGTTTCC ATGGCCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATT-3' and the upstream primer was designed as follows: 5'-CTCGAG CACCACCACCAC-3'; the downstream primer was designed as follows: 5'-GCAGACAAGCCCGTCAGG-3', and the target gene SEQ ID NO.1 containing the coding signal peptide sequence and the pET28a(+) vector were cloned. The PCR system of α-agarase gene is: 2×phanta Max Master Mix (Dye plus) 25μL, forward primer (20μM) 2μL, reverse primer (20μM) 2μL, template DNA 1μL, add double distilled water to 50μL. PCR amplification conditions are: 95℃ pre-denaturation for 3min; then 30 cycles (95℃15s, 60℃15s, 72℃4.5min); finally 72℃ insulation for 5min. The PCR system of pET28a (+) vector is 2×phanta Max Master Mix (Dye plus) 25μL, forward primer (20μM) 2μL, reverse primer (20μM) 2μL, template DNA 1μL, add double distilled water to 50μL. The PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; 30 cycles (95°C for 15 s, 60°C for 15 s, 72°C for 3 min); and finally incubation at 72°C for 5 min.

[0041] The PCR products of the agarase gene and the pET28a(+) vector were subjected to nucleic acid electrophoresis and then cut and recovered, and the agarase gene and the pET28a(+) vector were connected using the homologous recombination method. The connection system is: 2μL of purified agarase PCR fragment (50ng / μL), 1μL of purified pET28a(+) vector PCR fragment (180ng / μL), 4μL of 5×CEⅡBuffer, 2μL of ExnaseⅡ, and 10μL of ddH2O. The homologous recombination conditions are: incubation at 37℃ for 30min, then transformation of Escherichia coli JM109, coating of LB plates with 20μg / mL kanamycin, picking transformants for sequencing and nucleic acid electrophoresis verification, and obtaining the expression vector pET-28a(+) / th-aga containing the α-agarase gene. The expression vector was transformed into Escherichia coli BL21(DE3) to obtain genetically engineered bacteria.

[0042] The expression vector pET-28a(+) / th-aga containing the α-agarase gene was verified by nucleic acid electrophoresis. Figure 1 The target gene fragment of pET-28a(+) / th-aga was sequenced, and the sequencing results were correct.

[0043] Example 2 Fermentation production of recombinant α-agarase

[0044] 100 μL of the glycerol tube stock solution containing the genetically engineered bacteria E. coli BL21 (DE3) (pET-28a (+) / th-aga) constructed in Example 1 was inoculated into 50 mL of LB medium containing 20 μg / mL kanamycin, and cultured overnight at 37 ° C and 200 rpm; 4% of the inoculum was transferred to 50 mL of TB medium containing 20 μg / mL kanamycin, and the enrichment culture was carried out at 37 ° C and 200 rpm for 2-4 hours, and then IPTG with a final concentration of 0.05 mM was added, and the induction was continued at 25 ° C (200 r / min) shaking table for 16-24 hours. After the fermentation was completed, the fermentation broth was centrifuged to collect the cells, and 20 mM Tris-HCl (pH 8.0) buffer was added in equal proportions to resuspend the cells. After ultrasonic disruption, the supernatant collected by high-speed centrifugation was the crude enzyme solution of recombinant α-agarase.

[0045] Example 3 Determination of the hydrolytic activity of recombinant α-agarase at different temperatures and pH

[0046] Prepare 0.25% (w / v) agarose substrate with deionized water, add 0.1 mL enzyme solution to 0.9 mL substrate, react at 37°C for 10 min, add 1 mL 3,5-dinitrosalicylic acid (DNS) solution to terminate the enzymatic reaction, boil in water for 5 min, cool in ice bath, measure absorbance at 540 nm, and compare with D-galactose standard curve. Define the amount of enzyme required to generate 1 μmol reducing sugar (in terms of D-galactose) per minute as 1 enzyme activity unit (U).

[0047] The fermentation crude enzyme solution of recombinant α-agarase was prepared according to Example 2, wherein the induction temperature was 25°C and the fermentation time was 20 h. The agarose activity of recombinant α-agarase at different temperatures and pH values ​​is shown in FIG. Figure 2 and Figure 3 As shown, the enzyme has the highest activity at 30°C and can maintain a high enzyme activity at 20-30°C, indicating that it has cold adaptability; the enzyme has the highest activity in hydrolyzing agarose when the pH is 9, and has a high enzyme activity in the pH range of 8-9.

[0048] Example 4 Determination of the stability of recombinant α-agarase at different temperatures and pH

[0049] The fermentation crude enzyme solution of recombinant α-agarase was prepared according to Example 2, wherein the induction temperature was 25°C and the fermentation time was 20 h. The agarose activity of recombinant α-agarase at different temperatures and pH values ​​is shown in FIG. Figure 4 and Figure 5 As shown, the enzyme has good stability at 30°C and is more stable in a buffer solution of pH 8 to 9.

[0050] Example 5 Analysis of hydrolysis products of recombinant α-agarase

[0051] The method for determining the agarase hydrolysis product of recombinant α-agarase is as follows: using 0.5% (w / v) agarose solution as the substrate, add an appropriate amount of the th-aga crude enzyme solution prepared in Example 2 (the volume ratio of substrate to enzyme solution is 10:1), place it in a 35°C constant temperature oscillating water bath for reaction for 24 hours, then take a sample, boil it at high temperature for 10 minutes to inactivate the enzyme, and centrifuge to obtain the supernatant. Then dilute it, filter it through a 0.22μm water filter membrane, and use HPAEC-PAD to detect it. Using agarose trisaccharide (A3), agar pentose (A5), and agar heptaose (A7) standards as quantitative and qualitative standards, use HPAEC-PAD to detect the monosaccharide, disaccharide and agar oligosaccharide components in the product, and the results are as follows: Figure 7 As shown. After 12 hours of reaction, the recombinant α-agarase hydrolyzed agarose to produce four small molecule sugars, of which agarose (A3) was the main hydrolysis product with a yield of about 40% to 50%. Ultra-high performance liquid chromatography-electrospray-quadrupole-time of flight mass spectrometry determined that the products of enzymatic degradation of agarose were mainly agarose (A3), agarotetraose (A4), agarpentaose (A5) (as shown in Figure 2). Figure 8 and Table 2).

[0052] Table 1 Hydrolysis products of recombinant α-agarase

[0053]

[0054] Table 2 Molecular weight of agar oligosaccharides with different polymerization degrees

[0055]

[0056]

[0057] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A gene encoding α-agarase, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A recombinant plasmid carrying the gene according to claim 1.

3. A recombinant cell containing the gene according to claim 1 or transformed with the recombinant plasmid according to claim 2.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria uses Escherichia coli as a host and expresses the gene described in claim 1.

5. The genetically engineered bacterium according to claim 4, characterized in that: The genetically engineered bacteria uses pET-28a(+) plasmid as an expression vector; and the genetically engineered bacteria uses Escherichia coli BL21(DE3) as a host.

6. A method for constructing a genetically engineered bacterium, characterized in that: The gene of claim 1 is inserted into a vector pET-28a(+) plasmid to obtain a recombinant plasmid, and the recombinant plasmid is transformed into Escherichia coli BL21 (DE3) to obtain a genetically engineered bacterium.

7. A method for preparing recombinant marine-derived α-agarase, characterized in that: The method comprises the following steps: inoculating the genetically engineered bacteria described in claim 4 or 5 into an LB medium containing kanamycin, culturing the culture to a logarithmic growth phase, inoculating the cultured seed culture solution into a TB medium containing kanamycin at an inoculation rate of 4% (v / v), fermenting and culturing the culture at 25 to 30° C. for 16 to 24 hours, and producing recombinant marine-derived α-agarase.

8. A method for producing functional agar-oligosaccharide, characterized in that: The α-agarase encoded by the gene of claim 1 is used as a catalyst, and agar oligosaccharides or agar is used as a substrate to hydrolyze and generate functional agar oligosaccharides including agarotriose, agarotetraose and agaropentaose.

9. The method according to claim 8, characterized in that The catalyst is a crude enzyme solution obtained by fermentation of the genetically engineered bacteria as claimed in claim 4 or 5, or a pure enzyme obtained after separation and purification, which is added to the substrate in the form of an enzyme preparation.

10. Use of the gene according to claim 1, or the recombinant plasmid according to claim 2, or the recombinant cell according to claim 3, or the genetically engineered bacteria according to claim 4 or 5, or the method according to claim 6, or the method according to claim 7, or the method according to claim 8 or 9 in the preparation of agar-oligosaccharide-containing products.

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