CN119082156A - A marine-derived α-agarase gene for producing high-ratio agarotriose and its application method - Google Patents

A marine-derived α-agarase gene for producing high-ratio agarotriose and its application method Download PDF

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CN119082156A
CN119082156A CN202411283586.7A CN202411283586A CN119082156A CN 119082156 A CN119082156 A CN 119082156A CN 202411283586 A CN202411283586 A CN 202411283586A CN 119082156 A CN119082156 A CN 119082156A
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agarase
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班宵逢
曹语轩
李兆丰
李才明
顾正彪
程力
洪雁
徐恬
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Jiangnan University
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    • C12Y302/01158—Alpha-agarase (3.2.1.158)

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Abstract

本发明公开了一种生产高比例琼三糖的海洋来源α‑琼胶酶基因及其应用方法，属于基因工程和酶工程技术领域。该基因含有SEQ ID NO.1的核苷酸序列，将其基因导入基因工程菌E.coli BL21(DE3)(pET‑28a(+)/th‑aga)中进行分泌表达，利用纯化重组α‑琼胶酶水解琼脂糖或琼脂等底物，生成琼三糖、琼四糖、琼五糖等相应功能性琼寡糖的方法，所得功能性琼寡糖可直接用于食品、药品等生产。

The invention discloses a marine-derived α-agarase gene for producing a high-proportion agarotriose and an application method thereof, belonging to the technical fields of genetic engineering and enzyme engineering. The gene contains the nucleotide sequence of SEQ ID NO.1, and the gene is introduced into the genetic engineering bacterium E.coli BL21 (DE3) (pET-28a (+) / th-aga) for secretory expression, and a method for hydrolyzing substrates such as agarose or agar using purified recombinant α-agarase to generate agarotriose, agarotetraose, agarpentaose and other corresponding functional agar oligosaccharides, and the obtained functional agar oligosaccharides can be directly used in the production of food, medicine and the like.

Description

Marine source alpha-agarase gene for producing high-proportion agaragar and application method thereof
Technical Field
The invention relates to a marine source alpha-agarase gene for producing high-proportion agaragar and an application method thereof, belonging to the technical fields of genetic engineering and enzyme engineering.
Background
Agarase is an enzyme for degrading agarose, and the agarose produced by hydrolysis of agarase has multiple functional activities and has wide application prospect in the industries of foods, cosmetics and medicines. The types of agarose patterns can be classified into alpha-agarase (EC 3.2.1.158) and beta-agarase (EC 3.2.1.81). The alpha-agarase can hydrolyze the alpha-1, 3 bond of agarose to produce an Agaropectin Oligosaccharide (AOS) with D-galactose as a non-reducing end. The currently reported alpha-agarases include AgaA from alteromonas agaropectina, agaA33 from psychrophilia, agaD from psychrophilia, caLJ from Alternaria agaropectina, agaWS from Marine agaropectina (Catenovulum sediminis), OUC-GAJJ96 from marine agaropectina, and the like, and the research contents mainly comprise heterologous expression, enzymology and the like.
The agar oligosaccharides have rich physiological activities of resisting oxidation, whitening and moisturizing, resisting inflammation, resisting cancer, enhancing immunity and the like, and have application potential in the industries of foods, cosmetics and pharmacy. At present, the preparation methods of the agar oligosaccharides mainly comprise an acidolysis method and an enzymolysis method. Compared with the enzymolysis method, the acidolysis method has the difficulties of easy loss of substrate activity, nonuniform products, difficult separation and recovery, high energy consumption of high-temperature acidolysis and the like, and the industrialization of the production of the agar oligosaccharides is difficult to realize. Compared with acidolysis, the preparation of the agar oligosaccharide by the enzymolysis method has extremely strong specificity and specificity, can selectively cut off glycosidic bonds and produce small-molecule oligosaccharide with specific polymerization degree, is easy to control the degradation degree, has mild reaction conditions and lower energy consumption cost, and is environment-friendly and low-carbon. Microorganisms are the most major source of α -agarases, and the ocean serves as the largest, oldest habitat on earth, which is harboring extremely abundant sugars and microbial resources. However, the current technology for preparing high-added value agarose by agar hydrolysis is not perfect, and the main reason is the lack of an enzyme preparation capable of efficiently hydrolyzing agarose substrates. Therefore, it is necessary to develop a stable and efficient marine source alpha-agarase and to establish a stable and efficient alpha-agarase E.coli expression system.
Disclosure of Invention
In order to solve the technical problems, the invention provides a marine source alpha-agarase gene for producing high-proportion agaragar and an application method thereof, and the secretory expression of the alpha-agarase is realized by introducing a Thalassomonas sp-derived gene for encoding the alpha-agarase into genetically engineered bacteria, so that the technical scheme of the marine source alpha-agarase with stable property and high catalysis efficiency is solved.
The first technology provided by the invention is a gene for encoding alpha-agarase, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
The second technical scheme provided by the invention is a recombinant plasmid carrying the gene described in the first technical scheme.
In certain embodiments, the recombinant plasmid uses pET-28a (+) vector as an expression vector.
The third technical scheme provided by the invention is a recombinant cell containing the gene of the first technical scheme or transformed with the recombinant plasmid of the second technical scheme.
The fourth technical scheme provided by the invention is a genetically engineered bacterium, which takes escherichia coli as a host to express the gene in the first technical scheme.
In certain embodiments, the genetically engineered bacterium uses an E.coli pET-28a (+) vector as an expression vector.
In certain embodiments, the genetically engineered bacterium hosts ESCHERICHIA COLI BL (DE 3).
The fifth technical scheme provided by the invention is a construction method of an escherichia coli expression system of ocean-derived alpha-agarase, and the gene engineering bacteria are obtained by inserting the gene in the first technical scheme into a vector plasmid to obtain a secretion type expression vector and transforming the secretion type expression vector into escherichia coli.
In certain embodiments, the genetically engineered bacterium uses an E.coli pET-28a (+) vector as an expression vector.
In certain embodiments, the genetically engineered bacterium hosts ESCHERICHIA COLI BL (DE 3).
The sixth technical scheme provided by the invention is a preparation method of recombinant marine source alpha-agarase, which comprises the steps of inoculating the genetically engineered bacterium E.coli BL21 (DE 3) (pET-28 a (+)/th-aga) in an LB medium containing kanamycin, culturing in a shaking table (200 r/min) at 37 ℃ to logarithmic growth phase, inoculating the cultured seed culture solution to a TB medium containing kanamycin according to an inoculum size of 4% (v/v), and fermenting and culturing in a shaking table at 25-30 ℃ for 16-24 hours to produce the recombinant marine source alpha-agarase.
The seventh technical scheme provided by the invention is a method for producing functional agarase by using ocean source alpha-agarase, wherein the alpha-agarase coded by the gene in the first technical scheme is used as a catalyst, agarase or agar is used as a substrate, and agaragar trisaccharide, agaragar tetrasaccharide, agaragar pentasaccharide and other functional agaragar oligosaccharides are generated by hydrolysis, and the obtained functional agaragar oligosaccharide can be directly used for production of foods, medicines and the like.
In certain embodiments, the catalyst is crude enzyme liquid obtained by fermenting genetically engineered bacteria or pure enzyme obtained by separation and purification, and is added into a substrate in the form of an enzyme preparation.
The eighth technical scheme provided by the invention is the gene of the first technical scheme, the recombinant plasmid of the second technical scheme, the recombinant cell of the third technical scheme, the genetically engineered bacterium of the fourth technical scheme, the method of the fifth technical scheme, the method of the sixth technical scheme or the application of the method of the seventh technical scheme in the preparation of the product containing the agaragar.
The invention has the following technical effects:
1) The secretory expression of the ocean source alpha-agarase in the escherichia coli is realized, the method is stable and efficient, and the hydrolysis activity of the alpha-agarase crude enzyme solution reaches 15-20U/mL;
2) Compared with other reported similar enzymes, the ocean source alpha-agarase has the greatest advantage of being capable of directionally synthesizing a large amount of agaragar (A3) with the yield of about 40% -50%, so that the application field of the enzyme is quite wide.
3) The substrate conversion rate and the product purity of the ocean source alpha-agarase are higher, the utilization rate of polysaccharide resources in the ocean can be effectively improved, and the production and processing cost of the functional agarase is reduced, so that the method has more industrial application value.
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FIG. 1 shows the agarose gel electrophoresis detection of recombinant pET-28a (+)/th-aga positive clones, wherein the standard is a DNA standard, and the lane 1 is recombinant pET-28a (+)/th-aga positive clones.
FIG. 2 is a graph showing the effect of temperature on the activity of recombinant α -agarase.
FIG. 3 is a graph showing the effect of pH on recombinant α -agarase activity.
FIG. 4 is the thermostability of recombinant α -agarase.
FIG. 5 is a graph showing the pH stability of recombinant α -agarase.
FIG. 6 is an SDS-PAGE analysis of recombinant alpha-agarase, wherein the standard is a protein standard, lane 1 is a crude enzyme of recombinant alpha-agarase th-aga;
FIG. 7 shows the results of HPAEC-PAD assay for recombinant α -agarase hydrolysis products;
FIG. 8 shows the results of ultra-high performance liquid tandem quadrupole time-of-flight mass spectrometer for the hydrolysis product of recombinant α -agarase.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for better illustration of the invention, and should not be construed as limiting the invention.
The testing method comprises the following steps:
The high-efficiency anion exchange chromatograph (HPAEC-PAD) has the detection conditions of (1) a chromatographic column, dionex CarboPac PA-200 anion exchange columns, including an analysis column (3 mm multiplied by 250 mm) and a protection column (3 mm multiplied by 50 mm), (2) a mobile phase, 96%100mM NaOH and 4%100mM NaOH&500mM NaAc with the flow rate of 0.5mL/min, (3) detection by adopting a four-potential pulse amperometric method by using an amperometric detector, and (4) a column temperature of 35 ℃ and a sample injection volume of 10 mu L. The type of the agaropectin in the sample is determined according to the retention time of the corresponding chromatographic peak.
The raw materials used in the examples:
1. LB medium, 1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0.
2. TB medium 1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17mM KH 2PO4,72mM K2HPO4, pH 6.0.
3. PET28a (+) vector, ESCHERICHIA COLI JM109 competent cells, ESCHERICHIA COLI BL21 (DE 3) are commercial plasmids and commercial strains.
4. The raw materials comprise tryptone and yeast extract all purchased from Oxoid company in England, and sodium chloride, glycerol and KH 2PO4、K2HPO4 all purchased from chemical reagent company of national medicine group.
EXAMPLE 1 construction of E.coli secretory expression System
The upstream primer 5'-ATGGAAACACTGGTTCTGCAAGCAG-3' and the downstream primer 5 '-GTGGTGGTGCTCGAGTTAATGTGCCAGTTCCAGAATGCCC' are designed according to the gene of the alpha-agarase. The target gene SEQ ID NO.1 and pET28a (+) vector containing the coding signal peptide sequence is cloned by designing an upstream primer 5'-GACGGGCTTGTCTGCTCC-3', a downstream primer 5'-AACCAGTGTTTCC ATGGCCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATT-3' and an upstream primer 5'-CTCGAG CACCACCACCAC-3' and a downstream primer 5'-GCAGACAAGCCCGTCAGG-3' according to the pET28a (+) vector sequence. The PCR system of the α -agarase gene was 2X phanta M ax Master Mix (Dye plus) 25. Mu.L, 2. Mu.L of forward primer (20. Mu.M), 2. Mu.L of reverse primer (20. Mu.M), 1. Mu.L of template DNA, and double distilled water was added to 50. Mu.L. The PCR amplification conditions were 95℃for 3min of pre-denaturation, 30 cycles (95℃for 15s,60℃for 15s,72℃for 4.5 min) and finally 72℃for 5min of incubation. The PCR system for pET28a (+) vector was 2X phanta Max Master Mix (Dye plus) 25. Mu.L, forward primer (20. Mu.M) 2. Mu.L, reverse primer (20. Mu.M) 2. Mu.L, template DNA 1. Mu.L, and double distilled water was added to 50. Mu.L. The PCR amplification conditions were 95℃for 3min of pre-denaturation, 30 cycles (95℃for 15s,60℃for 15s,72℃for 3 min) and finally 72℃for 5min.
And (3) performing nucleic acid electrophoresis on PCR products of the agarase gene and the pET28a (+) vector, then cutting and recovering the cut rubber, and connecting the agarase gene and the pET28a (+) vector by using a homologous recombination method. The ligation system was 2. Mu.L of purified agarase PCR fragment (50 ng/. Mu.L), 1. Mu.L of purified pET28a (+) vector PCR fragment (180 ng/. Mu.L), 5X CEIIBuffer. Mu.L, exnaseII. Mu.L, ddH 2 O10. Mu.L. The homologous recombination conditions are that the bacillus coli JM109 is transformed after incubation for 30min at 37 ℃, LB plates with 20 mug/mL kanamycin are coated, transformants are selected for sequencing and nucleic acid electrophoresis verification, and an expression vector pET-28a (+)/th-aga containing the alpha-agarase gene is obtained. And (3) transforming ESCHERICHIA COLI BL (DE 3) the expression vector to obtain the genetically engineered bacterium.
The result of the nucleic acid electrophoresis verification of the expression vector pET-28a (+)/th-aga containing the alpha-agarase gene is shown in FIG. 1. The target gene fragment of pET-28a (+)/th-aga is sequenced, and the sequencing result is correct.
EXAMPLE 2 fermentative production of recombinant alpha-agarase
100. Mu.L of glycerol stock containing the genetically engineered bacterium E.coli BL21 (DE 3) (pET-28 a (+)/th-aga) constructed in example 1 was inoculated into 50mL of LB medium containing 20. Mu.g/mL kanamycin, shake-flask cultured overnight at 37℃and 200rpm, transferred into 50mL of TB medium containing 20. Mu.g/mL kanamycin at 4% of inoculum size, shake-flask enriched at 37℃and 200rpm for 2-4 h, then IPTG with a final concentration of 0.05mM was added, and the induction was continued on a 25℃ (200 r/min) shaker for 16-24 h. After fermentation, centrifugally collecting thalli from the fermentation liquid, adding 20mM Tris-HCl (pH 8.0) buffer solution in equal proportion to re-suspend thalli, and after ultrasonic crushing, centrifugally collecting supernatant at high speed to obtain crude enzyme liquid of the recombinant alpha-agarase.
EXAMPLE 3 determination of the hydrolytic Activity of recombinant alpha-Agave-Agents at different temperatures and pH
The agarose substrate of 0.25% (w/v) is prepared by deionized water, 0.1mL of enzyme solution is added into 0.9mL of substrate, the reaction is carried out for 10min at 37 ℃, 1mL of 3, 5-dinitrosalicylic acid (DNS) solution is added to terminate the enzymatic reaction, the boiling water bath is cooled by ice bath after 5min, and the absorbance is measured at 540nm and compared with the D-galactose standard curve. The amount of enzyme required to produce 1. Mu. Mol of reducing sugar (in terms of D-galactose) per minute was defined as 1 enzyme activity unit (U).
A crude enzyme broth of recombinant alpha-agarase was prepared as in example 2, wherein the induction temperature was 25℃and the fermentation time was 20 hours. The agarose activity of the recombinant alpha-agarase at different temperatures and pH values is shown in figures 2 and 3, and the activity of the recombinant alpha-agarase is highest at 30 ℃, and the activity of the recombinant alpha-agarase at 20-30 ℃ can also ensure higher enzyme activity, so that the recombinant alpha-agarase has cold adaptability, and the activity of the recombinant alpha-agarase for hydrolyzing agarose is highest when the pH value is 9, and has higher enzyme activity in the range of pH value of 8-9.
Example 4 determination of the stability of recombinant α -agarase at different temperatures and pH
A crude enzyme broth of recombinant alpha-agarase was prepared as in example 2, wherein the induction temperature was 25℃and the fermentation time was 20 hours. The agarose activity of the recombinant alpha-agarase at different temperatures and pH values is shown in figures 4 and 5, and the recombinant alpha-agarase has good stability at 30 ℃ and has stronger stability in a buffer solution with pH value of 8-9.
EXAMPLE 5 analysis of hydrolysis products of recombinant alpha-agarase
The method for measuring the agarase hydrolysate of the recombinant alpha-agarase comprises the steps of taking 0.5% (w/v) agarose solution as a substrate, adding a proper amount of th-aga crude enzyme liquid (the volume ratio of the substrate to the enzyme liquid is 10:1) prepared in the embodiment 2, placing the mixture in a 35 ℃ constant-temperature oscillating water bath kettle for reaction for 24 hours, sampling, boiling the mixture at a high temperature for 10 minutes to inactivate enzyme, and centrifuging to obtain supernatant. Then diluting the sample, passing the sample through a 0.22 mu m water-based filter membrane, and detecting the sample by adopting HPAEC-PAD. The mono-disaccharide and the agaro-oligosaccharide components in the products were detected with HPAEC-PAD using the agaro-trisaccharide (A3), agaro-pentasaccharide (A5) and agaro-heptasaccharide (A7) standards as quantitative and qualitative standards, and the results are shown in FIG. 7. And in the reaction for 12 hours, the recombinant alpha-agarase hydrolyzes agarose to generate four small molecular saccharides, wherein the agareose (A3) is a main hydrolysate, and the yield is about 40% -50%. The enzymatic degradation of agarose was determined to be predominantly agalloch (A3), agalloch (A4), agalloch (A5) using ultra-high performance liquid chromatography-electrospray-quadrupole-time-of-flight mass spectrometry (as shown in fig. 8 and table 2).
TABLE 1 hydrolysis products of recombinant alpha-agarase
TABLE 2 molecular weight of agar oligosaccharides with different degrees of polymerization
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1.一种编码α-琼胶酶的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO:1所示。1. A gene encoding α-agarase, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO: 1. 2.携带有权利要求1所述基因的重组质粒。2. A recombinant plasmid carrying the gene according to claim 1. 3.含有权利要求1所述基因或者转化有权利要求2所述重组质粒的重组细胞。3. A recombinant cell containing the gene according to claim 1 or transformed with the recombinant plasmid according to claim 2. 4.一种基因工程菌，其特征在于，所述基因工程菌以大肠杆菌为宿主，表达权利要求1所述的基因。4. A genetically engineered bacterium, characterized in that the genetically engineered bacterium uses Escherichia coli as a host and expresses the gene according to claim 1. 5.根据权利要求4所述的基因工程菌，其特征在于，所述基因工程菌以pET-28a(+)质粒为表达载体；所述基因工程菌以Escherichia coli BL21(DE3)为宿主。5. The genetically engineered bacterium according to claim 4, characterized in that the genetically engineered bacterium uses pET-28a(+) plasmid as an expression vector; and the genetically engineered bacterium uses Escherichia coli BL21(DE3) as a host. 6.一种基因工程菌的构建方法，其特征在于，通过将权利要求1所述的基因插入载体pET-28a(+)质粒中获得重组质粒，并将所述重组质粒转化至大肠杆菌Escherichia coliBL21(DE3)中，获得基因工程菌。6. A method for constructing a genetically engineered bacterium, characterized in that the gene described in 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. 7.一种重组海洋来源α-琼胶酶的制备方法，其特征在于，所述方法是将权利要求4或5所述的基因工程菌接入含有卡那霉素的LB培养基，培养至对数生长期，将培养好的种子培养液按4％(v/v)的接种量接种至含有卡那霉素的TB培养基，于25～30℃下发酵培养16～24h，生产重组海洋来源α-琼胶酶。7. A method for preparing recombinant marine α-agarase, characterized in that the method comprises inoculating the genetically engineered bacteria according to claim 4 or 5 into an LB medium containing kanamycin, culturing to the logarithmic growth phase, inoculating the cultured seed culture solution into a TB medium containing kanamycin at an inoculum rate of 4% (v/v), and fermenting and culturing at 25 to 30° C. for 16 to 24 hours to produce recombinant marine α-agarase. 8.一种生产功能性琼寡糖的方法，其特征在于，以权利要求1所述基因编码的α-琼胶酶为催化剂，以琼寡糖或琼脂为底物，水解生成包括琼三糖、琼四糖、琼五糖的功能性琼寡糖。8. A method for producing functional agar-oligosaccharides, characterized in that the α-agarase encoded by the gene according to 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.根据权利要求8所述的方法，其特征在于，所述催化剂是权利要求4或5所述的基因工程菌发酵生产得到的粗酶液或经分离纯化后得到的纯酶以酶制剂的形式添加到底物中的。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 according to 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.权利要求1所述的基因，或者权利要求2所述的重组质粒，或者权利要求3所述重组细胞，或者权利要求4或5所述的基因工程菌，或者权利要求6所述的方法，或者权利要求7所述的方法，或者第权利要求8或9所述的方法在制备含琼寡糖产品中的应用。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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Citations (4)

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