A mutant for improving the thermal stability of α-agarase and its application

Abstract

The present invention discloses a mutant and its application for improving the thermal stability of α-agarase, belonging to the field of genetic engineering and enzyme engineering technology. Specifically, the present invention provides a directed modification of α-agarase derived from marine microorganism Catenovulum agarivorans STB13, mutating around the 398th, 467th or 470th amino acid, and finding that the mutated enzyme activity can be kept equivalent to the wild type, and the thermal stability of the mutant of the 467th or 470th amino acid is significantly improved in the heat resistance experiment, wherein the mutant D467S shows excellent heat resistance, which is about 1.32 times higher than the wild type, which is conducive to improving the production efficiency of continuous production of enzymes in actual industrial applications, has good industrial application prospects, and lays a foundation for further research on the structure of seaweed polysaccharides.

Description

Technical Field

[0001] The invention relates to a mutant for improving the thermal stability of alpha-agarase and application thereof, belonging to the technical field of genetic engineering and enzyme engineering. Background Art

[0002] Marine algae have attracted widespread attention as a source of food, materials and renewable energy. Among all commercial algae, red algae have the largest output. Agar is the main component of the cell wall of red algae and is usually used as a thickener and gelling agent. In the food industry, the added value is low. The oligosaccharides extracted from agar have a variety of beneficial biological activities, including anti-inflammatory, antioxidant, probiotic, antibacterial and skin whitening, which makes agar oligosaccharides have application potential in food, medicine, agriculture and other fields, greatly improving the economic benefits of agar.

[0003] The enzymatic method is considered to be the main method for sustainable commercial production of agar oligosaccharides because it can specifically hydrolyze agar and does not form harmful compounds. However, the research on agarase in my country is still in its infancy. Compared with β-agarase, α-agarase has fewer varieties, more complex structures, and generally poorer thermal stability, which seriously hinders the large-scale application of α-agarase. The α-agarases in the prior art include AgaA from Alteromonas agarlyticus GJ1B, AgaA33 from Thalassomonas sp.44JAMB-A3, AgaD and AgaE from Thalassomonas sp.LD5, CaLJ96 from Catenovulum agarivorans, AgaWS5 from Catenovulum sediminis WS1-A, OUC-GaJJ96 from Gilvimarinus agarilyticus, Ce2834 and Ce2835 from Colwellia echini A3T, and Cm-AGA from Catenovulum maritimum STB14. Among them, the problem of poor thermal stability is common, which makes it difficult to meet the needs of industrial production.

[0004] The present invention selects Ca-AGA from Catenovulum agarivorans STB13, which is the α-agarase with the highest enzyme activity expressed in the laboratory at present, and has strong substrate specificity, can produce agar oligosaccharides with different polymerization degrees, and has huge application potential. However, most of the agarases from natural sources have poor thermal stability, which greatly limits the hydrolysis efficiency and practical industrial application of agarase. Therefore, improving the thermal stability of α-agarase Ca-AGA by means of genetic engineering and enzyme engineering is of great significance for the directional molecular modification of α-agarase and high-efficiency, low-cost agar processing. Summary of the invention

[0005] To solve the above problems, the present invention obtains a series of mutants with significantly improved thermal stability by targeted molecular modification of α-agarase derived from the marine microorganism Catenovulumagarivorans STB13, and the enzyme activity of these α-agarase mutants is slightly improved compared with the wild type, making them more suitable for industrial production.

[0006] The first object of the present invention is to provide an α-agarase mutant, wherein the α-agarase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 or a sequence having a homology of not less than 90%, wherein the mutation includes any one of the following or a combination thereof:

[0007] Mutate aspartic acid at position 467 to serine;

[0008] or mutating asparagine at position 470 to glutamine;

[0009] Or mutate the asparagine at position 470 to serine.

[0010] Furthermore, the gene sequence encoding the wild-type enzyme is shown in SEQ ID NO.2.

[0011] The second object of the present invention is to provide a nucleic acid molecule encoding the α-agarase mutant.

[0012] The third object of the present invention is to provide a recombinant expression vector carrying the nucleic acid molecule.

[0013] Furthermore, the vector can be selected according to needs (such as host species), including but not limited to peT series plasmids.

[0014] The fourth object of the present invention is to provide an expression cassette containing the nucleic acid molecule.

[0015] Furthermore, the expression cassette includes elements necessary for expressing the nucleic acid molecule and / or elements that assist in expression, such as a promoter, a terminator, a signal peptide, an RBS, and the like.

[0016] The fifth object of the present invention is to provide a recombinant cell expressing the α-agarase mutant.

[0017] Furthermore, the host cells of the recombinant cells include microorganisms (fungi or bacteria), such as common Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, etc.

[0018] The sixth object of the present invention is to provide a method for producing an α-agarase mutant, comprising the step of fermentation production using the recombinant cell.

[0019] The seventh object of the present invention is to provide the use of the α-agarase mutant, nucleic acid molecule, recombinant expression vector, expression cassette or recombinant cell in hydrolyzing agar.

[0020] Furthermore, the application includes using an α-agarase mutant or a recombinant cell as a catalyst to catalyze the conversion of agar into agar oligosaccharides having 3,6-endoether-α-L-galactose as a reducing end.

[0021] Beneficial effects of the present invention:

[0022] The present invention provides a mutant for improving the thermal stability of alpha-agarase, wherein the wild-type alpha-agarase is derived from the marine microorganism Catenovulum agarivorans STB13, and the present invention screens the mutation sites, and finally determines that positions 398, 467 and 470 are sites to be modified. After mutations are performed respectively, it is found that the enzyme activities of different mutants can still maintain the enzyme activities equivalent to the wild type, and the thermal stability of some mutants is improved to varying degrees, and the thermal stability of the optimal mutant is improved by 1.32 times compared with the wild type. The alpha-agarase mutant provided by the present invention can be more adaptable to temperature changes and produce agar oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is an agarose gel electrophoresis diagram of the wild-type Ca-AGA and its mutants; wherein, M is the standard molecular weight of DNA, 1 is the wild-type Ca-AGA plasmid, and 2-6 are the mutant plasmids, which are D398E, D467S, N470Q, N470W and N470S, respectively.

[0024] Figure 2 The SDS-PAGE image of wild-type Ca-AGA and its mutants; wherein, M: protein standard molecular weight, 1: wild-type Ca-AGA, 2-6: mutant D398E, mutant D467S, mutant N470Q, mutant N470W and mutant N470S.

[0025] Figure 3To characterize the enzymatic activity of the wild enzyme Ca-AGA and its mutants.

[0026] Figure 4 To characterize the thermal stability of the wild enzyme Ca-AGA and its mutants at 37°C. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0028] The culture medium involved in the following examples is as follows:

[0029] LB liquid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0.

[0030] LB solid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, pH 7.0, 1.5% (w / v) agar.

[0031] Fermentation liquid medium: yeast powder 12 g / L, trypsin 24 g / L, sucrose 5 g / L, KH2PO4 17 mM, K2HPO4 72 mM, pH 9.0.

[0032] The detection methods involved in the following embodiments are as follows:

[0033] Detection of α-agarase activity:

[0034] The 3,5-dinitrosalicylic acid (DNS) method was used to measure the changes in reducing sugar content to characterize the hydrolysis activity of α-agarase. A 0.15% (w / w) agarose substrate solution was prepared with Tris-HCl buffer (20mM, pH 8.0). 0.1mL of enzyme solution was directly added to 0.9mL of agarose solution, and 1mL of DNS was added to terminate the reaction after reacting at 37°C for 10min. After boiling for 5min, ice water bath was added, and 2mL of deionized water was added to dilute and the absorbance value was measured at 540nm. The inactivated enzyme was used as a control, and galactose was used as a standard to draw a standard curve.

[0035] Enzyme activity (U / mL) definition: Under the above conditions, the volume of enzyme required to catalyze the production of 1 μmol of reducing sugar (calculated as galactose) per minute is defined as one enzyme activity unit (U).

[0036] The specific activity of α-agarase refers to the number of enzyme activity units per unit weight (mg) of protein under specific conditions. Specific activity (U / mg) = enzyme activity (U / mL) / enzyme concentration (mg / mL).

[0037] The sequence involved in the present invention is as follows:

[0038] SEQ ID NO.1: α-Agarase amino acid

[0039]

[0040] SEQ ID NO.2: α-Agarase nucleotide

[0041]

[0042] Example 1: Preparation of mutants

[0043] The specific steps are as follows:

[0044] (1) chemically synthesizing α-agarase Ca-AGA as shown in SEQ ID NO.2, and cloning it into plasmid pET-28a(+) using restriction endonucleases Nco I and Xho I to obtain recombinant plasmid pET-28a(+)-ca-aga;

[0045] (2) Using the recombinant plasmid pET-28a(+)-ca-aga prepared in step (1) as a template, the complementary primer chains required for the experiment were designed (see Table 1). The primers were synthesized by GENEWISE Biotechnology Co., Ltd. and site-directed mutagenesis was performed according to the method described in the instructions of the PrimerSTARGXL kit of TaKaRa Company. Recombinant plasmids containing the genes of the mutants D398E, D467S, N470Q, N470W and N470S were prepared respectively.

[0046] Table 1 Introduction of mutation sites of α-agarase

[0047]

[0048] 1 The underlined bases correspond to the corresponding mutated amino acids

[0049] The PCR reaction system followed the conditions specified in the PrimerSTAR GXL kit instruction manual: 10 μL of 5× PrimeSTAR GXL Buffer, 4 μL of dNTP Mixture (2.5 mM each), 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 0.5 μL of DNA template, 1 μL of PrimeSTAR GXL DNA Polymerase, and 32.5 μL of ddH2O.

[0050] The PCR amplification conditions were as follows: pre-denaturation at 98°C for 3 min; followed by denaturation at 98°C for 10 s, annealing at 58°C for 15 s, and extension at 68°C for 10 min, for a total of 30 cycles; complete extension at 68°C for 10 min, and finally incubation at 4°C.

[0051] The PCR product obtained after the reaction was digested with DpnI at 37°C for 1-2h, and then the treated PCR product was transferred into E.coli JM109 according to the E.coli JM109 competent transformation method to prepare transformants. The transformants were spread on kanamycin (20μg / mL) LB solid medium and inverted at 37°C for 12h. The positive monoclonal clones were picked and transferred to LB liquid medium containing kanamycin (20μg / mL), and cultured at 37°C and 200rpm for 10-12h. The plasmids were extracted and sent to the company for sequencing. The recombinant plasmids containing mutant genes were obtained: pET-28a(+)-D398E, pET-28a(+)-D467S, pET-28a(+)-N470Q, pET-28a(+)-N470W, pET-28a(+)-N470S, and the plasmid extraction verification results were as follows Figure 1 shown.

[0052] Example 2: Construction of genetically engineered bacteria and expression of mutants

[0053] The specific steps are as follows:

[0054] (1) The recombinant plasmids pET-28a(+)-ca-aga, pET-28a(+)-D398E, pET-28a(+)-D467S, pET-28a(+)-N470Q, pET-28a(+)-N470W, and pET-28a(+)-N470S obtained in Example 1 were respectively transferred into E. coli BL21(DE3) to prepare E. coli BL21(DE3) / pET-28a(+)-ca-aga, E. coli BL21(DE3) /

[0055] pET-28a(+)-D398E, E.coliBL21(DE3) / pET-28a(+)-D467S, E.coliBL21(DE3) / pET-28a(+) -N470Q, E.coliBL21(DE3) / pET-28a(+)-N470W, E.coliBL21(DE3) / pET-28a(+)-N470S recombinant strain;

[0056] (2) Streak the recombinant strains on LB solid medium containing kanamycin (20 μg / mL) and culture them in a constant temperature incubator at 37°C for 12 h. Pick the positive single clones and transfer them to LB liquid medium containing kanamycin (20 μg / mL) and culture them at 37°C, 200 rpm for 10-12 h to prepare seed solution.

[0057] (3) The seed solution was inoculated at a 2% (v / v) inoculation volume into a fermentation liquid medium containing kanamycin (20 μg / mL) and fermented in a shake flask at 37°C until OD 600 When the value reaches 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG, 10 μL / 50 mL, final concentration 0.005 mM) is added, and the flask is cultured at 25°C and 200 rpm for 16 h to obtain a fermentation broth. The fermentation broth is centrifuged at 4°C and 10000 rpm for 20 min, the bacteria are collected, and lysis buffer (20 mM Tris-HCl, pH 8.0) is added in equal proportion to resuspend the bacteria. After ultrasonic disruption, the supernatant is collected by high-speed centrifugation to obtain the intracellular crude enzyme solution. The SDS-PAGE gel electrophoresis diagram is shown in the figure. Figure 2 As shown, the results showed that both the original enzyme and the mutant enzyme were expressed.

[0058] Example 3: Enzyme purification and enzyme activity determination

[0059] The specific steps are as follows:

[0060] (1) Protein purification and ultrafiltration concentration:

[0061] Using HisTrap TM HP column (5mL) was used for nickel column affinity chromatography purification: ① Enzyme solution pretreatment: 500mM NaCl, 20mM imidazole were added to 50mL crude enzyme solution, and passed through a 0.45μm water filter to remove macromolecular impurities; ② First, the pump was washed with ultrapure water to clean the pipeline; ③ Buffer A (500mM NaCl, 50mM Tris-HCl, 20mM imidazole, pH 8.0) was used to balance the nickel column (about 5 column volumes) at a flow rate of 2mL / min, and then the sample was loaded at a speed of 1.5mL / min. After the loading was completed, the nickel column was balanced with buffer A again. ④ Nickel column elution: The sample was eluted with eluent B (500mM NaCl, 50mM Tris-HCl, 500mM imidazole, pH 8.0) in the best isocratic elution mode (80% A, 20% B, 2mL / min), and the eluent (containing the target protein) of the peak was collected in parts.

[0062] The ultrafiltration concentration steps are as follows: add the purified enzyme solution into an ultrafiltration centrifuge tube and centrifuge at a low speed, add an appropriate amount of Tris-HCl buffer (pH 8.0), and repeat 5 times to remove imidazole and NaCl.

[0063] Pure enzyme solutions containing wild-type agarase and mutant enzymes D398E, D467S, N470Q, N470W and N470S were prepared respectively.

[0064] (2) Enzyme activity determination:

[0065] The enzyme activities of the wild-type agarase and the mutant enzymes D398E, D467S, N470Q, N470W and N470S were measured, and the wild-type enzyme activity was set as 100%, and the relative enzyme activities of the mutant enzymes were detected. The results are as follows: Figure 3 As shown, the enzyme activities of the mutants were similar to or improved compared with the wild type.

[0066] Example 4: Enzyme stability determination

[0067] The specific steps are as follows:

[0068] The pure enzyme solution obtained in Example 3 was incubated at 37°C for 30 min and then the incubation was terminated. The enzyme activity of the enzyme solution before and after incubation was detected. The results were as follows: Figure 4 shown.

[0069] The experimental results showed that after incubation at 37°C for 30 minutes, the wild enzyme retained only 41.67% of the original enzyme activity; while the mutant D467S retained 56.12% of the original enzyme activity (1.32 times that of the wild enzyme) after incubation at 37°C for 30 minutes; the thermal stability of the mutants N470Q and N470S was also improved compared with the wild type, while the mutants D398 and N470W performed generally. In summary, the mutants D467S, N470Q and N470S meet the purpose of mutation design, that is, to ensure that the catalytic activity of the enzyme is not reduced while the thermal stability is also improved.

[0070] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. An α-agarase mutant, characterized in that The α-agarase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1, and the mutation is any one of the following: Mutate aspartic acid at position 467 to serine; or mutating asparagine at position 470 to glutamine; Or mutate the asparagine at position 470 to serine.

2. A nucleic acid molecule encoding the α-agarase mutant according to claim 1.

3. A recombinant expression vector carrying the nucleic acid molecule of claim 2.

4. An expression cassette containing the nucleic acid molecule of claim 2.

5. The expression frame according to claim 4, characterized in that: The expression cassette includes elements necessary for expressing the nucleic acid molecule and / or elements that assist in expression.

6. A recombinant cell expressing the α-agarase mutant according to claim 1.

7. The recombinant cell according to claim 6, characterized in that The host cell of the recombinant cell includes a microorganism.

8. A method for producing an α-agarase mutant, characterized in that: The method comprises the step of using the recombinant cell according to claim 6 or 7 for fermentation production.

9. Use of the α-agarase mutant according to claim 1, the nucleic acid molecule according to claim 2, the recombinant expression vector according to claim 3, the expression cassette according to claim 4 or 5, or the recombinant cell according to claim 6 or 7 in hydrolyzing agar.

10. The use according to claim 9, characterized in that: The application comprises: using alpha-agarase mutant or recombinant cell as catalyst to catalyze agar to be converted into agar oligosaccharide with 3,6-endoether-alpha-L-galactose as reducing end.

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