Agarase YM01-3 Mutant and Its Application

Abstract

The present invention utilizes genetic engineering technology to perform truncation mutation on agarase YM01-3. By designing a pair of complementary sequence primers with opposite directions and using a high-fidelity DNA polymerase for amplification, only a part of the gene is amplified to construct a truncated recombinant DNA molecule, and finally a mutant of agarase YM01-3 is constructed. The thermostability of the agarase YM01-3 mutant of the present invention has been greatly improved compared to the wild-type YM01-3. It is a novel agarase with great potential and can be applied to the enzymatic industrial production of agar oligosaccharides, thereby promoting a significant reduction in the production cost of agar oligosaccharides.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial enzymes, and particularly relates to an agarase YM01-3 mutant and its application. Background Art

[0002] Agar, also known as agar-agar, is the main component of carbohydrates in red algae cells, with its content reaching 60% of the dry weight of red algae. Agar can be degraded into agar oligosaccharides. Agar oligosaccharides, also known as low molecular weight agar oligosaccharides, are oligosaccharides with a molecular weight between 300 and 2000. This kind of oligosaccharide has unique structures and properties, mainly composed of two repeating units: agarobiose and neoagarobiose. Agar oligosaccharides obtained by degrading agar not only retain some biological activities of agar but also have better solubility and bioavailability due to their low molecular weight characteristics. In the health product industry, agar oligosaccharides have attracted much attention for their whitening effect. Their unique molecular structure can tightly bind to skin cells, promote cell metabolism, and then improve skin color, making the skin more fair and bright. In addition, agar oligosaccharides also exhibit excellent antioxidant ability, helping to resist the damage of free radicals, delaying cell aging, and protecting physical health. In the medical field, the low-calorie property of agar oligosaccharides makes them an ideal adjuvant treatment method. For diabetic patients, agar oligosaccharides not only have the potential to fight diabetes but also can reduce the calories they intake while meeting the taste needs of patients, helping to control blood sugar levels. In addition, agar oligosaccharides also show antibacterial activity and play a positive role in preventing infections and promoting wound healing. In the cosmetics industry, the solubility and bioavailability of agar oligosaccharides make them a precious raw material. It can be easily incorporated into various skin care products and cosmetics to exert its whitening, antioxidant and other effects, improving the overall quality of the products. In summary, agar oligosaccharides obtained by degrading agar have broad application potential in industries such as health products, medicine, and cosmetics.

[0003] Agar is mainly degraded into agar oligosaccharides by chemical methods and enzymatic methods. Chemical methods are divided into acid degradation methods and redox methods. Chemical methods have high production costs, difficult product separation, and it is difficult to obtain high-purity products, while enzymatic methods are more mild and environmentally friendly, with a simple production process, easy purification of products, uniform degree of oligomerization of oligosaccharides, and thus stable activity. Enzymatic methods are more mild and environmentally friendly. Using enzymatic methods in the industrial production of oligosaccharides can improve production efficiency, simplify the production process, increase product activity and recovery rate, etc. Agarase, as an enzyme that can catalyze the degradation of agarose, belongs to five different glycoside hydrolase families: GH16, GH50, GH86, GH117, and GH118. And according to the type of glycosidic bond that cleaves agarose, agarase plays a key role in the green biodegradation process of agarose.

[0004] At present, agarase of the GH16 family is one of the most widely studied enzymes. This family has many members, but their thermal stability shows significant differences. In the GH16 family, the optimum temperature of most agarases is around 40-50°C. On the other hand, studies have shown that the catalytic efficiency of agarase in liquid agarose solution is significantly better than that in the gel state, which may be related to the freer movement of molecules in the liquid and the smaller specific surface area of the gel state. However, agarose solution can only present a fluid state at high temperature (≥50°C), which makes the thermal stability of agarase a key factor affecting the degradation efficiency of agarose. Exploring and modifying high-temperature resistant agarases has become the key to the high value-added utilization of agar and obtaining agar oligosaccharides with high purity and a specific degree of polymerization. It is a core issue that needs to be solved urgently. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide an agarase YM01-3 mutant with good thermal stability and its application.

[0006] In a first aspect of the present invention, a mutant of agarase YM01-3 is provided, which has higher thermal stability than the wild type.

[0007] In another preferred embodiment, the amino acid sequence of the agarase mutant has more than 95% homology with the amino acid sequence shown in SEQ ID NO.3, preferably 96% homology, more preferably 97%, 98%, 99% or 100% homology, and has higher thermal stability than the wild type.

[0008] In another preferred embodiment, the amino acid sequence of the agarase mutant is shown in SEQ ID NO.3.

[0009] In the second aspect of the present invention, a biological material related to the mutant of agarase according to the first aspect of the present invention is provided, wherein the biological material comprises at least one of the following 1)-5):

[0010] 1) A gene encoding the mutant of agarase according to the first aspect of the present invention;

[0011] 2) an expression cassette containing the coding gene described in 1);

[0012] 3) a recombinant vector containing the coding gene described in 1);

[0013] 4) A recombinant microorganism containing the coding gene described in 1);

[0014] 5) A recombinant microorganism containing the recombinant vector described in 3).

[0015] Further preferably, in the above (1), the nucleotide sequence of the coding gene has a homology of more than 95%, preferably 96%, more preferably 97%, 98%, 99% or 100% with the nucleotide sequence shown in SEQ ID NO.4.

[0016] Further preferably, in the above (1), the nucleotide sequence of the coding gene is as shown in SEQ ID NO.4.

[0017] Further preferably, in the above (3), the recombinant vector uses the pET-28a(+) vector as the expression vector.

[0018] Further preferably, in the above (4) or (5), the recombinant microorganism is recombinant Escherichia coli E.coli BL21(DE3).

[0019] In the third aspect of the present invention, there is provided the use of the mutant of the agarase described in the first aspect of the present invention in agar or agarose decomposition and the production of agar oligosaccharides.

[0020] It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here.

[0021] Compared with the prior art, the present invention has the following advantages and progressiveness:

[0022] In previous studies, the agarase YM01-3 of the GH16 family (GenBank sequence number: AGU13985) was isolated from Catenovulum agarivorans YM01. The present invention uses genetic engineering technology to truncate and mutate the above-mentioned agarase YM01-3. By designing a pair of complementary sequence primers with opposite directions and using a high-fidelity DNA polymerase for amplification, only a part of the gene is amplified to construct a truncated recombinant DNA molecule, and finally a mutant of agarase YM01-3 is constructed. The thermal stability of the mutant of agarase YM01-3 of the present invention has been greatly improved compared with the wild-type AgaCA233. It is a novel agarase with great potential and can be applied to the enzymatic industrial production of agar oligosaccharides, thereby promoting a significant reduction in the production cost of agar oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0024] Figure 1: PCR and double digestion agarose gel electrophoresis. Lane 1: PCR amplified fragment; Lane 2: Double digestion verification of recombinant plasmid pET-28a(+)-ym01-3m; Lane M: Marker.

[0025] Figure 2 : PCR verification of positive transformed colonies.

[0026] Figure 3 : SDS-PAGE electrophoresis detection of YM01-3 and YM01-3 mutants. (a) M: Marker; Lane 1: Crude enzyme of YM01-3; Lane 2: Enzyme of YM01-3; Lane 3: Protein of E. coli pET-28a(+) bacterial solution; (b) M: Marker; Lane 1: Crude enzyme of YM01-3 mutant; Lane 2: Pure enzyme of YM01-3 mutant; Lane 3: Protein of E. coli pET-28a(+) bacterial solution.

[0027] Figure 4 : Comparison of thermal stability of YM01-3 and YM01-3 mutants at 60°C.

[0028] Figure 5 : Galactose standard curve. Specific implementation mode

[0029] The present invention provides an agarase YM01-3 mutant and its application. The present invention will be specifically described below in conjunction with embodiments to facilitate the further understanding of the present invention by those skilled in the art. However, the embodiments described below are only a part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be noted that the adjustments and improvements made by those of ordinary skill in the art based on the concept of the present invention should be regarded as the protection scope of the present invention. For the specific technical operation steps and operators not specified in the embodiments, they are all carried out according to the general technical conditions described in the literature in this field or the relevant product specifications.

[0030] Example 1 Construction of recombinant expression plasmid of agarase YM01-3

[0031] In previous studies, the agarase YM01-3 (GenBank accession number: AGU13985) of the GH16 family was isolated from Catenovulum agarivorans YM01. According to the agarase ym01-3 gene sequence, NcoI and XhoI restriction sites were added to both ends, and then it was synthesized by a gene company according to the method of chemical total synthesis. Then it was ligated to the pET-28a(+) expression vector linearized by double digestion to construct the recombinant expression plasmid pET-28a(+)-ym01-3 of agarase YM01-3, which was transferred into Escherichia coli Top10 for amplification. After plasmid extraction, it was verified to be completely correct by sequencing.

[0032] Amino acid sequence of wild-type agarase YM01-3:

[0033] MYAADWDGVPIPAPAGQNKTWQIQSVSDDFNYTASANNKPNAFTSRWNDSYINAWLGPGDTEFSAGHSYTNSGKLALQAAEKTGTDKVYAGIISSKQTFTYPLYIEARAKSTNNTMANAVWMLSADSTQELDAMEAYGSDRPGQEWFDRRMHVSHHVFIREPFQDYQPKDEGSWIYNEQEPWRVSYHNYGMHWKDPWNVDYYIDGVLVRSVSGPQMIDPNNFTNGTGINKPLHIIIDMEHQDWRDVKPTSAELADPAKSIFYVDWIRVYKPVDSGASAPTPPTGATSLQARHSSKCLDLSAGNSADGTNMQQWGCSATNTNQDITFVAKGDGYYEMKTKHNKCIDVAGKETTNGANLVQWSCYNGTNPQFKLLDKGNGWFQLQAKHSGKCLEVVNSATTNGANVQQWACGNGNNQQWKFQ(SEQ ID NO.1).

[0034] DNA sequence of wild-type agarase ym01-3 gene:

[0035]

[0036] Using pET-28a(+)-ym01-3 as a template, the mutant gene ym01-3m was amplified by PCR with primers F(-cbm) and R(-cbm) according to the following reaction system and reaction conditions.

[0037] Primer sequences for amplifying the mutant gene ym01-3m of agarase AgaCA233:

[0038] F(-cbm): TTTAAGAAGGAGATATACCATGGGC;

[0039] R(-cbm):

[0040] TGGTGGTGGTGGTGGTGCTCGAGTTTGTAGACCCTGATCCAATCTAC.

[0041] Table 1. PCR reaction system

[0042]

[0043] Table 2. PCR reaction process

[0044]

[0045] The target gene ym01-3m fragment amplified by the above PCR and the plasmid pET-28a(+) linearized by double digestion (NcoI and XhoI) were ligated according to the ligation system in Table 3 to ligate the target gene and the linearized vector, thereby obtaining the YM01-3 mutant recombinant expression plasmid pET-28a(+)-ym01-3m.

[0046] Amino acid sequence of agarase YM01-3 mutant:

[0047] MYAADWDGVPIPAPAGQNKTWQIQSVSDDFNYTASANNKPNAFTSRWNDSYINAWLGPGDTEFSAGHSYTNSGKLALQAAEKTGTDKVYAGIISSKQTFTYPLYIEARAKSTNNTMANAVWMLSADSTQELDAMEAYGSDRPGQEWFDRRMHVSHHVFIREPFQDYQPKDEGSWIYNEQEPWRVSYHNYGMHWKDPWNVDYYIDGVLVRSVSGPQMIDPNNFTNGTGINKPLHIIIDMEHQDWRDVKPTSAELADPAKSIFYVDWIRVYK (SEQ ID NO.3).

[0048] DNA sequence of the agarase ym01-3m mutant gene:

[0049] ATGTATGCAGCAGACTGGGATGGTGTACCGATTCCGGCACCAGCAGGTCAAAACAAAACTTGGCAAATACAATCTGTGTCAGATGATTTTAACTACACAGCTTCAGCCAATAATAAACCAAACGCTTTTACCAGTCGTTGGAACGACAGCTATATTAATGCGTGGTTAGGGCCTGGTGATACTGAATTTAGTGCAGGTCATTCGTACACAAATTCAGGCAAACTGGCTTTGCAAGCTGCTGAAAAAACAGGTACAGACAAAGTATACGCAGGTATTATTTCTTCAAAACAAACATTTACCTATCCGCTTTATATTGAAGCGCGGGCAAAATCAACCAACAACACTATGGCGAATGCAGTATGGATGTTAAGTGCTGATTCAACTCAAGAATTAGATGCAATGGAAGCTTATGGTAGTGATAGGCCAGGCCAAGAATGGTTTGATCGTCGTATGCATGTGAGCCATCATGTATTTATTCGCGAACCATTCCAAGATTATCAACCAAAAGACGAAGGCTCTTGGATATACAATGAGCAAGAGCCATGGCGCGTTTCTTATCACAACTATGGTATGCATTGGAAAGATCCTTGGAATGTTGATTATTACATTGATGGTGTTTTAGTTAGAAGTGTCTCTGGCCCTCAAATGATTGATCCAAATAACTTCACCAACGGCACAGGTATCAACAAACCATTACATATTATTATTGATATGGAACATCAAGACTGGCGCGATGTTAAACCAACGTCTGCTGAGCTTGCAGATCCTGCAAAAAGTATTTTTTATGTAGATTGGATCAGGGTCTACAAA(SEQ ID NO.4).

[0050] Table 3. Ligation system of the target gene and the vector

[0051]

[0052] The amplified product was detected by 1% agarose gel electrophoresis (Figure 1 ) showed that the size of the PCR product was consistent with that of the YM01-3 mutant, and the YM01-3 plasmid was double digested ( Figure 1 ) and gel recovered. The gel recovery product was of the size consistent with the target gene fragment. The PCR fragment and the digested plasmid were ligated, and screening was carried out on the LB medium containing kanamycin to obtain positive colonies. Agarose gel electrophoresis ( Figure 2 ) was performed on the positive colonies, which contained the target band, and the positive colonies were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing result was completely consistent with the mutant-designed gene sequence, determining that the construction of the mutant enzyme genetic engineering strain was successful.

[0053] Example 3 Induced expression of agarase YM01-3 and YM01-3 mutant in Escherichia coli

[0054] The plasmids pET-28a(+)-ym01-3 and pET-28a(+)-ym01-3m were transferred into the competent cells of Escherichia coli E.coli BL21(DE3) according to the following method to obtain the genetic engineering strains E.coli BL21(DE3) / pET-28a(+)-ym01-3 and E.coli BL21(DE3) / pET-28a(+)-ym01-3m of YM01-3 and YM01-3 mutant.

[0055] (1) Take 5 μL of the recombinant plasmid sample and add it to 50 - 100 μL of competent cells. After gently mixing, place it on ice for 30 min.

[0056] (2) Perform heat shock for 90 s (in a 42 °C water bath), then quickly place it back in the ice bath and wait for 3 - 5 min.

[0057] (3) Add 500 μL of LB liquid culture medium (without antibiotics), gently mix, and shake culture for 1 h (37 °C).

[0058] (4) Centrifuge the bacterial solution (5000 rpm, 1 min) to precipitate the bacteria, and aspirate most of the supernatant (leave about 50 - 100 μL to resuspend the bacteria).

[0059] (5) Spread the bacterial solution evenly on the LB plate (containing Kana antibiotic) and culture it overnight in a 37 °C incubator.

[0060] (6) Pick single colonies on the LB plate for culture. Take 50 μL of the bacterial solution for testing to identify whether the recombinant strain is correct.

[0061] The successfully constructed genetically engineered strains E. coli BL21(DE3) / pET-28a(+)-ym01-3 and E. coli BL21(DE3) / pET-28a(+)-ym01-3m were inoculated into 5 mL of LB medium and cultured in a shaker for 12 h (37 °C, 220 rpm) to obtain seed solutions. 200 μL of the seed solution was taken and inoculated into 200 mL of LB medium (containing Kana). After shaking culture for 2 h (37 °C, 220 rpm), 200 μL of IPTG (0.5 M) was added at a ratio of 1‰, and then the culture was continued with shaking for 20 - 24 h (16 °C, 220 rpm) to induce the expression of the target protein. The cells were collected and the supernatant was discarded after centrifugation. 15 mL of lysis buffer (20 mM Tris, 20 mM NaCl, 1 mM DTT, 20 mM imidazole, pH 7.0) was added to the cells, and the mixture was placed on an ice - water mixture for ultrasonic disruption (power 260 - 300 W, total disruption time 30 min, working time 3 s, interval time 3 s). After ultrasonic treatment, centrifugation was carried out again (4 °C, 8000 rpm) for 40 min, and the obtained supernatant was the crude enzyme solution. The crude enzyme solution was purified using a nickel column His Trap TM HP - 5 mL. The enzyme solution was added with 5×SDS loading buffer, mixed well, boiled for 10 min, then centrifuged at 12000 rpm for 10 min, and SDS - PAGE protein electrophoresis was performed on the supernatant using a 5% stacking gel and a 12% separating gel. Results( Figure 3 ) showed that YM01 - 3 and the YM01 - 3 mutant were successfully expressed, and their sizes were in line with expectations, approximately 50 kD and 30 kD respectively, and the purification degree was similar to the electrophoretic purity.

[0062] Example 4 Comparison of the thermal stabilities of agarase YM01 - 3 and the YM01 - 3 mutant and determination of the agar - degrading ability of the YM01 - 3 mutant

[0063] Method for measuring enzyme activity: The agarase was diluted to an appropriate concentration. The following reaction system (Table 4) was added to a 1.5 mL EP tube and reacted in a water bath at 60 °C for 20 min. After the reaction, 300 μL of DNS solution was added, and then it was placed in a boiling water bath for 7 min. (The experiment was performed in three parallel groups.) 200 μL was taken and placed in a 96 - well plate, and the OD value at 540 nm was measured using an enzyme - linked immunosorbent assay (ELISA) reader.

[0064] Table 4 Agarase reaction system

[0065]

[0066] At 60 °C, the YM01 - 3 mutant still had about 60% of its enzyme activity after incubation for 2.5 h, while the residual enzyme activity of YM01 - 3 was only about 33%( Figure 4)。The thermal stability of the YM01-3 mutants is better than that of YM01-3.

[0067] The agarase YM01-3 mutants degrade agarose to produce oligosaccharides. The production of the reaction products is determined by the DNS reducing sugar test method, and the production amount of the products is calculated using galactose as the standard reducing sugar. Prepare galactose solutions with different concentrations, take 300 μL and put it into a 1.5 mL EP tube, then add 300 μL DNS to the tube. After boiling water bath for 7 minutes, measure the OD values of different concentrations of galactose, and draw a standard curve based on the concentration and OD value ( Figure 5 ) as the standard curve for quantifying the oligosaccharides produced by the decomposition of agarose by agarase.

[0068] It is proposed that the amount of enzyme required to hydrolyze agarose to produce 1 μg of reducing sugar (galactose) per unit time (per minute) is defined as 1 enzyme activity unit (U). Specific enzyme activity = total activity (U) / [enzyme protein concentration (mg / mL) × enzyme solution volume].

[0069] The enzyme activity of the YM01-3 mutants of agarase is measured at 65 °C, and the specific enzyme activity of the YM01-3 mutants of agarase is determined according to its enzyme concentration. The results show that the specific enzyme activity of the YM01-3 mutants is 832 U / mg, indicating that the YM01-3 mutants of agarase have a high ability to degrade agar.

[0070] As described above, the above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. For those skilled in the art of this technology, any modifications and changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A mutant of agarase YM01-3, characterized in that, The amino acid sequence of the mutant is as shown in SEQ ID NO.

3.

2. A biomaterial related to the mutant of agarase YM01-3 according to claim 1, wherein the biomaterial comprises at least one of the following 1)-4): 1) The coding gene of the mutant of agarase YM01-3 according to claim 1; 2) An expression cassette containing the coding gene described in 1); 3) A recombinant vector containing the coding gene described in 1); 4) A recombinant microorganism containing the coding gene described in 1).

3. The biomaterial according to claim 2, wherein The nucleotide sequence of the coding gene in 1) is as shown in SEQ ID NO.

4.

4. Use of the mutant of agarase YM01-3 according to claim 1 in agar decomposition and the production of agar oligosaccharides.

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