Alginate lyase mutant, recombinant expression vector, engineering bacterium and application thereof

Abstract

The invention provides an alginate lyase mutant, a recombinant expression vector, an engineering bacterium and application of the alginate lyase mutant, belongs to the technical field of gene engineering and enzyme engineering, and can solve the technical problem that alginate lyase which has high enzyme activity and good thermal stability and can be used for efficiently preparing alginate oligosaccharide needs to be developed. The alginate lyase mutant disclosed by the invention is an alginate lyase mutant Amut6, and the amino acid sequence of the alginate lyase mutant Amut6 is as shown in SEQ ID NO. 1. The alginate lyase mutant Amut6 is prepared from alginate lyase Algt1 through an error-prone PCR reaction, compared with the alginate lyase Algt1, the enzyme activity and the thermal stability of the alginate lyase mutant Amut6 are both improved, and the preparation efficiency of alginate oligosaccharide with the polymerization degree of 1-4 is higher.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and enzyme engineering, and in particular relates to an alginate lyase mutant, a recombinant expression vector, an engineered bacterium and applications thereof. Background Art

[0002] Alginate is a water-soluble acidic polysaccharide found in the cell walls and extracellular matrix of brown algae. It is composed of β-D-mannuronic acid (M) and its C5 diastereomer, α-L-guluronic acid (G), randomly polymerized through 1,4-glycosidic bonds. Due to its strong water absorption, alginate is often used for food thickening and emulsification. However, its large molecular weight results in low bioavailability, limiting its further application in functional foods. In contrast, alginate oligosaccharides, obtained by degradation of alginate, have a smaller molecular weight, are more readily absorbed by the body, and possess a wider range of physiological activities. Studies have shown that alginate oligosaccharides with a degree of polymerization of 1–4 exhibit significant effects in anti-obesity, antioxidant, and lipid metabolism regulation. Therefore, the efficient preparation of alginate oligosaccharides with a specific degree of polymerization is of great significance for their widespread application in related fields.

[0003] Currently, the preparation methods of alginate oligosaccharides mainly include chemical, physical, and enzymatic methods. Although chemical degradation can achieve initial cleavage, the reaction process is difficult to precisely control, takes a long time, and residual acid is difficult to remove. Physical degradation suffers from high energy consumption and large product molecular weight. In contrast, enzymatic hydrolysis has the advantages of being gentle and environmentally friendly, and can achieve the precise preparation of oligosaccharides with a specific degree of polymerization, making it widely used in the preparation of alginate oligosaccharides.

[0004] However, the high viscosity of alginate itself not only affects the diffusion efficiency of the lyase, but also reduces the binding ability of the enzyme to the substrate. Increasing the temperature can reduce the viscosity of alginate to a certain extent. Therefore, in order to improve the industrial lysis efficiency of alginate, it is particularly important to develop a lyase with high enzyme activity and good thermal stability. In addition, in the industrial process, high substrate concentration means less water consumption, electricity consumption and a more compact reaction system, which helps to reduce the overall production cost. Therefore, in order to reduce energy consumption and production costs, it is of great practical significance to develop an alginate lyase that can be used for the efficient preparation of alginate oligosaccharides. Summary of the Invention

[0005] In response to the above-mentioned technical problem of developing an alginate lyase with high enzymatic activity and good thermal stability and capable of efficiently preparing alginate oligosaccharides, the present invention proposes an alginate lyase mutant Amut6. Compared with the original sequence alginate lyase Algt1, the alginate lyase mutant Amut6 of the present invention has higher enzymatic activity and thermal stability, and has higher preparation efficiency when used to prepare alginate oligosaccharides with a degree of polymerization of 1-4 under the same conditions.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: an alginate lyase mutant, which is the alginate lyase mutant Amut6, and the amino acid sequence of the alginate lyase mutant Amut6 is shown in SEQ ID NO. 1.

[0007] FELVWWYLIIPTLNCGNGKADSIKENELSGGYENSSYFYTASDGGMVFRCPIAGYKTSTNTSYTRTELREMLRRGDTSIDTQGVNKNNWVFGSAPSSARNAAGGVDGVLRATLAVNHVTTTGDSSQVGRVIIGQIHA NDDEPLRLYYRKLPGNSKGSIYFAHEPEGGSDQWYEMIGSRSSSASDPADGIALNEVFSYEIKVVGNTLTVTISRDGKDDVVKSVDMSDSGYDSSWQYQYFKAGVYNQNNTGLDDDYVQATFYALENSHDGYPY (SEQ ID NO. 1) In one embodiment, the alginate lyase mutant Amut6 was prepared from the alginate lyase Algt1 by error-prone PCR reaction, and the amino acid sequence of the alginate lyase Algt1 is shown in SEQ ID NO. 3. Specifically, a gene fragment of the alginate lyase Algt1 was used as a template and PCR amplified using Taq DNA polymerase. The resulting product was an error-prone PCR product, which was purified and sequenced to obtain the alginate lyase mutant Amut6 gene fragment.

[0008] FELVDWYLSIPTDNDNGKADSIKENELSGGYENSSYFYTASDGGMVFRCPIAGYKTSTNTSYTRTELREMLRRGDTSIDTQGVNKNNWVFGSAPSSARNAAGGVDGVLRATLAVNHVTTTGDSSQVGRVIIGQIHA NDDEPLRLYYRKLPGNSKGSIYFAHEPEGGSDQWYEMIGSRSSSASDPADGIALNEVFSYEIKVVGNTLTVTISRDGKDDVVKSVDMSDSGYDSSDQYQYFKAGVYNQNNTGDDDDYVQATFYALENSHDGYPY (SEQ ID NO. 3) In one embodiment, the mutation positions of the amino acid sequence as shown in SEQ ID NO. 3 include: the 5th amino acid mutates to a W amino acid, the 9th amino acid mutates to an I amino acid, the 13th amino acid mutates to an L amino acid, the 15th amino acid mutates to a C amino acid, the 233rd amino acid mutates to a W amino acid, and the 250th amino acid mutates to an L amino acid.

[0009] In one embodiment, the nucleotide sequence of the gene encoding the alginate lyase mutant Amut6 is shown as SEQ ID NO. 2.

[0010] (SEQ ID NO. 2) The present invention also provides a recombinant expression vector comprising a gene encoding the alginate lyase mutant Amut6.

[0011] The present invention also provides a recombinant engineered bacterium, comprising the recombinant expression vector, wherein the recombinant strain uses Pichia pastoris as a vector.

[0012] In one embodiment, the Pichia pastoris includes Pichia pastoris X33.

[0013] The present invention also provides the use of the alginate lyase mutant Amut6 in the preparation of alginate oligosaccharides.

[0014] In one embodiment, the preparation conditions are: substrate concentration 20% (W / V), temperature 40° C., and reaction time 4 h.

[0015] In one embodiment, the degree of polymerization of the alginate oligosaccharide is 1-4.

[0016] Compared with the prior art, the advantages and positive effects of the present invention are: the alginate lyase mutant Amut6 of the present invention has high enzymatic activity and good thermal stability, and has a higher production efficiency for preparing alginate oligosaccharides with a degree of polymerization of 1-4. The alginate lyase mutant Amut6 of the present invention is prepared from the alginate lyase Algt1 by error-prone PCR, and a recombinant expression vector mutant library thereof in Pichia pastoris is constructed. The alginate lyase mutant Amut6 of the present invention has higher enzymatic activity than the original alginate lyase Algt1. The alginate lyase mutant Amut6 of the present invention can be used to prepare alginate oligosaccharides with a degree of polymerization of 1-4 under the preparation conditions of a substrate concentration of 20% and a reaction temperature of 40°C for 4 hours. The preparation efficiency of alginate oligosaccharides with a degree of polymerization of 1-4 using the alginate lyase mutant Amut6 of the present invention is higher than that of the original alginate lyase Algt1. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of nucleic acid electrophoresis for the gene amplification of the alginate lyase mutant Amut6 of the present invention; Figure 2 Schematic diagram of the enzymatic activities of the alginate lyase mutant Amut6 and the original alginate lyase Algt1 of the present invention; Figure 3 This is a schematic diagram of the optimum temperature of the alginate lyase mutant Amut6 of the present invention; Figure 4 Schematic diagram of the thermal stability of the alginate lyase mutant Amut6 and the original alginate lyase Algt1 of the present invention; Figure 5 FPLC analysis of the products of 20% sodium alginate degradation by the alginate lyase mutant Amut6 and the original alginate lyase Algt1 at different degradation times. DETAILED DESCRIPTION

[0018] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0019] The present invention provides an alginate lyase mutant, a recombinant expression vector, an engineered bacterium, and uses thereof. The alginate lyase mutant Amut6 of the present invention is prepared from the alginate lyase Algt1 by error-prone PCR, and a recombinant expression vector mutant library thereof is constructed in Pichia pastoris. Compared with the original sequence alginate lyase Algt1, the alginate lyase mutant Amut6 of the present invention has higher enzymatic activity and thermal stability. Furthermore, the alginate lyase mutant Amut6 of the present invention is used to prepare alginate oligosaccharides with a degree of polymerization of 1-4 with higher production efficiency than the original alginate lyase Algt1.

[0020] In order to more clearly and in detail introduce the alginate lyase mutant, recombinant expression vector, engineered bacteria and their applications provided by the embodiments of the present invention, they will be described below in conjunction with specific examples.

[0021] In the following examples, molecular biology experimental methods not specifically described were performed with reference to the specific methods listed in the book Molecular Cloning Laboratory Manual, 3rd edition, by J. Sambrook, or in accordance with the kits and product instructions. The reagents and biological materials described above, unless otherwise specified, can be obtained from commercial sources.

[0022] Example 1 Error-prone PCR reaction using alginate lyase The amino acid sequence of the alginate lyase published in the DNA sequence database (GenBank) established by the National Center for Biotechnology Information of the United States with sequence number WP_231902423.1 was used as the amino acid sequence of the wild-type enzyme. The structure of the wild-type enzyme contains three domains, namely CBM_4_9, F5_F8_type_C, and Alginate lyase2. The amino acid sequence of the wild-type enzyme was truncated to retain only the Alginate lyase2 domain to obtain the original alginate lyase Algt1. The gene sequence of the original alginate lyase Algt1 was used as a template for error-prone PCR reaction. The predicted mutation points of the amino acid sequence of the original alginate lyase Algt1 include: amino acid position 5 mutated to W amino acid, amino acid position 9 mutated to I amino acid, amino acid position 13 mutated to L amino acid, amino acid position 15 mutated to C amino acid, amino acid position 233 mutated to W amino acid, and amino acid position 250 mutated to L amino acid. The DNA polymerase used in the reaction was low-fidelity Taq DNA polymerase. The reaction system was configured according to the enzyme instruction manual, specifically: 10 μL buffer, 4 μL dNTPs, 2 μL Taq DNA polymerase, 2 μL forward primer, 2 μL back primer, 1 μL synthetic template, and ddH2O to make up the system to 50 μL. The annealing temperature in the reaction conditions was set to 56°C, the extension time at 72°C was 7 min, and other reaction conditions were carried out according to the enzyme instruction manual.

[0023] After the reaction is completed, the PCR reaction products are tested using agarose gel electrophoresis. Figure 1 As shown, a DNA amplification band with the correct molecular weight was obtained. The verified PCR product was further digested with DpnI and purified using the CyclePure Kit PCR Purification Kit. Specific steps were followed according to the DpnI and CyclePure Kit instructions. The DNA fragment recovered after purification was the error-prone PCR product of the alginate lyase.

[0024] Example 2 Construction of recombinant expression vector 2 μL of Not I and EcoR I enzymes were added to the PCR product, and then 5 μL of enzyme reaction buffer was added. The reaction was incubated at 37°C for 6 h. The Pichia pastoris X33 expression vector was linearized using Not I and EcoR I enzymes. The error-prone PCR product of alginate lyase was ligated with the linearized expression vector by homologous recombination in vitro. The cells were transformed into Escherichia coli DH5α using the heat shock method and cultured at 37°C for 12-16 h. Single colonies were selected. After verification of positive clone transformants, the construction of the recombinant expression vector for alginate lyase was completed.

[0025] Example 3 Expression of the alginate lyase mutant Amut6 Add 2 μL of Sac I to the extracted recombinant expression vector, then add 5 μL of enzyme reaction buffer, and react at 37°C for 6 h. Use Sac I enzyme to linearize the constructed recombinant expression vector, and use electroporation to transform the linearized recombinant expression vector into Pichia pastoris X33 competent cells. The specific transformation method is as follows: transform the linearized recombinant expression vector into the competent cells, ice bath for 30 min, use electroporation to transform, add 1 mL of pre-cooled 1 M sorbitol solution, incubate in a 30°C incubator for 1 h, spread the bacteria onto LB plates containing Zeocin antibiotics, and culture at 30°C for 2-3 days.

[0026] The grown yeast transformants were screened for positive transformants and inoculated into BMGY liquid medium for shake flask fermentation at 30°C and 200 rpm. Methanol solution was added once every 24 h to a final methanol concentration of 1% for induction, and this was added three times. After expression was completed, the bacteria were centrifuged and the supernatant was collected to obtain the prepared alginate lyase mutant Amut6.

[0027] Example 4 Determination of enzyme activity of alginate lyase mutant Amut6 The heterologously expressed alginate lyase mutant Amut6 and the original alginate lyase Algt1 were purified by Ni+ affinity chromatography, and the protein concentration of the purified enzyme samples was determined using a BCA kit. The enzymatic activities of the recombinant alginate lyase mutant and the original alginate lyase were determined by the DNS method, and the specific activity of the enzyme was calculated based on the protein concentration.

[0028] The enzyme activity determination method includes the following steps: 900 μL of 0.8% sodium alginate solution and 100 μL of appropriately diluted enzyme solution are added to a 10 mL test tube in sequence as the experimental group, where the enzyme solution needs to be diluted to the absorbance value between 0.2 and 1.0 when measuring the enzyme activity; 900 μL of 0.8% sodium alginate solution and 100 μL of inactivated enzyme solution are added to a 10 mL test tube in sequence as the control group; the experimental group and control group test tubes are vortex mixed respectively and reacted in a 40°C water bath for 30 minutes; after the water bath, 1 mL of DNS solution is added to the experimental group and control group test tubes respectively and boiled in a boiling water bath for 10 minutes, cooled immediately, and the experimental group and control group are diluted to 10 mL with distilled water, and the absorbance is measured at 520 nm.

[0029] The absorbance of the experimental group minus the absorbance of the control group is the absorbance of the enzymatic hydrolysis to generate reducing sugars. The enzyme activity unit of alginate lyase is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute. The enzyme activity calculation formula is enzyme activity = (the content of reducing sugars generated by enzymatic hydrolysis × enzyme solution dilution multiple) / (reaction time × amount of diluted enzyme solution added × M), where the unit of the content of reducing sugars generated by enzymatic hydrolysis is μg, the unit of reaction time is min, the unit of amount of diluted enzyme solution added is mL, and M refers to the molar mass of mannuronic acid and guluronic acid, which is 180.16. The enzymatic activity calculation results of the alginate lyase mutant Amut6 and the original alginate lyase Algt1 are shown in Figure 2. Figure 2 shown.

[0030] Depend on Figure 2 It can be seen that the specific activity of the recombinant alginate lyase mutant Amut6 is 298.53 U / mg, and the specific activity of the original alginate lyase Algt1 is 212.86 U / mg. The specific activity of the recombinant alginate lyase mutant Amut6 is higher than that of the original alginate lyase Algt1, indicating that the recombinant alginate lyase mutant Amut6 with improved enzyme activity was obtained by error-prone PCR technology.

[0031] Example 5 Optimal Temperature and Thermal Stability Determination of the Alginate Lyase Mutant Amut6 The enzyme activity of the alginate lyase mutant was determined at different temperatures (30℃, 35℃, 40℃, 45℃, 50℃, and 55℃) in a buffer system with pH 7.0 and 0.8% sodium alginate as substrate. The relative enzyme activity at different temperatures was calculated with the highest enzyme activity as 100% to obtain the optimal temperature of the alginate lyase mutant.

[0032] The calculation results of the relative enzyme activity of the alginate lyase mutant Amut6 at different temperatures are as follows Figure 3 As shown by Figure 3 It can be seen that under neutral buffer pH conditions, the optimal temperature of the alginate lyase mutant Amut6 is 40 ℃, which is not significantly different from the optimal temperature of the original alginate lyase.

[0033] A 0.8% sodium alginate solution was prepared in sodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.0 as a substrate and treated with the original alginate lyase Algt1 and the alginate lyase mutant Amut6 at 40°C for 2 h. Samples were taken every 0.5 h and the enzyme activity was determined by the DNS method. The enzyme activity of the enzyme solution without heat treatment was taken as 100%. The residual enzyme activity of the original alginate lyase and the alginate lyase mutant after treatment at the same temperature for different times was calculated. The thermal stability calculation results of the alginate lyase mutant Amut6 and the original alginate lyase Algt1 are shown in Figure 2. Figure 4 shown.

[0034] Depend on Figure 4 It can be seen that the alginate lyase mutant Amut6 can maintain a high enzyme activity even after being treated at 40°C for a long time, indicating that the alginate lyase mutant can function at 40°C. In addition, a comparison found that the residual enzyme activity of the alginate lyase mutant Amut6 after being treated at 40°C for 2 hours was 79.32%, while the residual enzyme activity of the original alginate lyase after being treated at 40°C for 2 hours was only 70.36%, indicating that the thermal stability of the alginate lyase mutant is significantly improved compared to the thermal stability of the original alginate lyase.

[0035] Example 6 Preparation of alginate oligosaccharides using the alginate lyase mutant Amut6 200 g of sodium alginate was weighed and poured into 1000 mL of ultrapure water to prepare a substrate stock solution with a substrate concentration of 20%. 50 mL of the alginate lyase mutant and the original alginate lyase were added to the substrate stock solution, respectively. The solution was stirred at 40°C for 6 h, and samples were taken every 2 h for the determination of the enzymatic hydrolysis products. To remove impurities, the reactants were centrifuged at 4000 rpm for 10 min and then purified by freeze-drying.

[0036] Detection of purified product by FPLC: using Superdex TM 30Increase10 / 300GL column, the mobile phase was 0.2 M NH4HCO3, the flow rate was 0.3 mL / min, and the variable wavelength detector (VWD) was used for detection at 235 nm. The results are shown in Figure 5 shown.

[0037] Depend on Figure 5 It can be seen that under the same conditions, the alginate lyase mutant Amut6 can produce alginate oligosaccharides with a degree of polymerization of 1-4 after 4 h of enzymatic hydrolysis, while the original alginate lyase Algt1 can produce alginate oligosaccharides with a degree of polymerization of 1-4 after 6 h of enzymatic hydrolysis, indicating that the alginate lyase mutant Amut6 is more efficient in preparing alginate oligosaccharides.

[0038] The above-described embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, evolutions, and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. An alginate lyase mutant, characterized in that: The alginate lyase mutant is alginate lyase mutant Amut6, and the amino acid sequence of the alginate lyase mutant Amut6 is shown in SEQ ID NO.

1.

2. The alginate lyase mutant according to claim 1, wherein The alginate lyase mutant Amut6 was prepared by an error-prone PCR reaction of alginate lyase Algt1. The amino acid sequence of the alginate lyase Algt1 is shown in SEQ ID NO.

3.

3. The alginate lyase mutant according to claim 2, characterized in that As shown in SEQ ID NO. 3, the mutation points of the amino acid sequence include: the 5th amino acid mutates to a W amino acid, the 9th amino acid mutates to an I amino acid, the 13th amino acid mutates to an L amino acid, the 15th amino acid mutates to a C amino acid, the 233rd amino acid mutates to a W amino acid, and the 250th amino acid mutates to an L amino acid.

4. The gene encoding the alginate lyase mutant according to claim 1, characterized in that The nucleotide sequence of the alginate lyase mutant Amut6 is shown in SEQ ID NO.

2.

5. A recombinant expression vector, characterized in that: The invention also comprises a gene encoding the alginate lyase mutant according to claim 4.

6. A recombinant engineered bacterium, characterized in that: The invention comprises the recombinant expression vector according to claim 5, wherein the recombinant engineered bacteria uses Pichia pastoris as a vector.

7. The recombinant engineered bacterium according to claim 6, characterized in that The Pichia pastoris includes Pichia pastoris X33.

8. Application of alginate lyase mutant Amut6 in the preparation of alginate oligosaccharides.

9. Use of the alginate lyase mutant Amut6 according to claim 8 in the preparation of alginate oligosaccharides, characterized in that: The preparation conditions were as follows: substrate concentration 20% (W / V), temperature 40°C, and reaction time 4 h.

10. Use of the alginate lyase mutant Amut6 according to claim 8 in the preparation of alginate oligosaccharides, characterized in that: The polymerization degree of the alginate oligosaccharide is 1-4.

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