A mutant of alginate lyase AlgL3199, a recombinant expression vector and a genetically engineered bacterium

Abstract

The present invention belongs to the field of enzymes, and specifically relates to a mutant of alginate lyase AlgL3199, a recombinant expression vector and a genetically engineered bacterium. The present invention provides an alginate lyase AlgL3199, and performs mutations on the basis of alginate lyase AlgL3199 to obtain mutants with mutation sites of K81A, D148P, V163T, I274G and combinations thereof. The specific activity of the mutant enzyme and the thermal stability of some mutants are improved, and the thermal stability of the mutation site K81A is improved most significantly. The residual activity after incubation at 60°C for 2h is retained by 52.97%, and the half-life is 123.12 min, which is 5.18 times that of the original enzyme. The present invention also provides alginate lyase AlgL3199 and its mutant coding genes, a recombinant expression vector for producing alginate lyase and a genetically engineered bacterium.

Description

Technical Field

[0001] The invention belongs to the field of enzymes, and specifically relates to an alginate lyase AlgL3199 mutant, a recombinant expression vector and a genetically engineered bacterium. Background Art

[0002] Alginate is a kind of polysaccharide substance widely found in various brown algae. It can be used in food, medicine, agriculture and other aspects. For example, it can be made into gel to maintain the original shape and quality of some foods; it can be used as an adhesive in the production process of various preparations, and has anti-coagulation and lipid-lowering effects. However, due to its complex structure, large molecular weight, high viscosity and high degree of polymerization, it is difficult to develop and utilize. The brown algae oligosaccharides obtained by the degradation of alginate have the advantages of simple structure and high solubility. At the same time, it has multiple biological activities such as anti-inflammatory, antioxidant, immunomodulatory, anti-tumor, and promotion of plant root growth. Compared with the physical and chemical methods for degrading alginate, the bioenzyme degradation method has the advantages of high efficiency, specificity, safety, and few by-products. Therefore, enzyme catalysis has become a green and efficient method for preparing brown algae oligosaccharides.

[0003] Alginate lyase generates an unsaturated C=C double bond between the non-reducing end C4 and C5 through a β-elimination reaction. Alginate lyase is widely found in animals, plants, bacteria and fungi, among which marine bacteria (Vibrio, Pseudomonas, Alteromonas, Bacillus, Flavobacterium, etc.) are the main source of alginate lyase. In previous studies (Liu, Lu, et al. Frontiers in Microbiology, 2021) cloned an alginate hydrolase (referred to as Alyw203) from Vibrio W2 and expressed it in food-grade Yarrowia lipolytica.

[0004] Since most marine bacteria are mesophilic or psychrophilic, the corresponding alginate lyase has inherent limitations in thermal stability. Most alginate lyases have poor thermal stability in environments above 40°C, and the residual enzyme activity after 0.5 hours of treatment is less than 50%. Poor thermal stability greatly limits the industrial application potential of alginate lyase. Using protein engineering methods such as directed evolution and rational design is an effective way to improve the thermal stability of alginate lyase. Summary of the invention

[0005] In order to solve the technical problem that alginate lyase has poor thermal stability, the present invention provides a new alginate lyase AlgL3199, and on the basis of the alginate lyase, a mutant of AlgL3199 with improved thermal stability is obtained by genetic engineering technology, and some amino acids are substituted on the basis of AlgL3199, such as lysine (K) at position 81 is replaced by alanine (A), and the activity can be retained by 52.97% after incubation at 60°C for 2h, and the half-life reaches 123.12 min, which is 5.18 times higher than that of the original enzyme. The improvement of thermal stability is conducive to the degradation of alginate in a higher temperature environment.

[0006] The present invention firstly provides an alginate lyase AlgL3199, whose amino acid sequence is shown in SEQ ID NO:1.

[0007] MKKVYLGLVVAGLLAGCSTAEQSNSTSTAMAAPTATQAELIKQLDVNKAPAENFDLSKWKINLPYPDDKPERQGKTMELSKEQLNNHVPPFSHPDWFFTDPESGAMVFSVPNHAVTTPNSKNARTELRAMLGTEYKAPDNNFAIAANDNADEYGSVGGHLSAVLKVDWVSTSGN DSKSGAHAVVIGQIHGSNNEPLKIIYRKLPNHDLGSLSWNYETNPENKADRKDIKHDVFGKNGLKQSDPSPKDGIALGEIFSYDVNVEGNIMHLTFKKNIGEPTEIVKTFDIDLSKPYPGEAKDTSYAQDWMYFKAGAYNQCNTGSSQCTNNGIEAGDYTKVSFYKLTLDQ (SEQ ID NO: 1).

[0008] The present invention also provides a mutant of alginate lyase AlgL3199 with improved thermal stability. Compared with alginate lyase AlgL3199, the mutant of alginate lyase AlgL3199 comprises one or more of the following amino acid substitution mutations: K81A, D148P, V163T and I274G; and the mutant of alginate lyase AlgL3199 has improved thermal stability compared with alginate lyase AlgL3199. The mutant may also comprise other mutations, for example, further mutations are made on the basis of the mutations, but the thermal stability thereof is not significantly reduced. K81A represents that the lysine at the 81st position of SEQ ID NO: 1 is replaced by alanine, D148P represents that the aspartic acid at the 148th position of SEQ ID NO: 1 is replaced by proline, V163T represents that the valine at the 163rd position of SEQ ID NO: 1 is replaced by threonine, and I274G represents that the isoleucine at the 274th position of SEQ ID NO: 1 is replaced by glycine.

[0009] The improved thermal stability described in the present invention means that the enzyme activity is significantly improved compared with the original enzyme activity when incubated at a certain temperature (such as 40°C, 50°C or 60°C) for the same time, that is, the thermal stability is improved.

[0010] In some embodiments, the alginate lyase AlgL3199 mutant comprises any of the following amino acid substitution mutations compared to alginate lyase AlgL3199: K81A, D148P, V163T and I274G; or the alginate lyase AlgL3199 mutant comprises any of the following amino acid substitution mutation combinations compared to alginate lyase AlgL3199: K81A / D148P, V163T / I274G, D148P / V163T, K81A / D148P / V163T, K81A / D148P / V163T / I274G. Among them, K81A / D148P represents the simultaneous presence of mutation sites K81A and D148P, and other mutation combinations are similar.

[0011] The present invention also provides a coding gene of the alginate lyase AlgL3199 mutant. In the case of a known amino acid sequence, the coding gene can be modified on the basis of the AlgL3199 wild-type coding gene, or a new coding gene can be obtained by codon optimization.

[0012] The present invention also provides a recombinant expression vector of the coding gene, which contains an expression frame of the coding gene and can be used to express and produce the alginate lyase AlgL3199 mutant. The recombinant vector can be constructed by connecting the nucleotide sequence of the coding gene of the alginate lyase to various vectors by conventional methods in the art. These vectors can be selected from various conventional vectors in the art, such as various plasmids, phages or virus vectors, etc., preferably pET-24a (+) vector.

[0013] The present invention also provides a genetically engineered bacterium expressing the alginate lyase AlgL3199 mutant, wherein the genetically engineered bacterium contains the coding gene of the alginate lyase AlgL3199 mutant, or contains the above-mentioned recombinant expression vector. It can be obtained by transforming the recombinant expression vector of the present invention into a host microorganism. The host microorganism can be various conventional host microorganisms in the art, as long as the recombinant expression vector can stably replicate itself and the alginate lyase coding gene carried by it is effectively expressed, preferably Escherichia coli, more preferably Escherichia coli BL21.

[0014] The present invention also provides a method for preparing a thermally stable alginate lyase AlgL3199 mutant, comprising: inoculating the genetically engineered bacteria into a fermentation medium, culturing at 37°C and 200 rpm until OD 600 is 0.6-0.8; IPTG is added to a final concentration of 0.2 mM for induction, and cultured overnight at 16°C and 200 rpm. Preferably, the inoculation is 3-5% of the seed solution by volume.

[0015] Compared with the prior art, the beneficial effects of the present invention include at least:

[0016] The present invention obtains 4 single mutants with reduced free energy through virtual mutation, and 5 combined mutations are carried out with the single mutants as templates. The enzyme activity of all mutants is improved compared with the original enzyme. Among them, the thermal stability of the single mutants K81A, D148P and the combined mutants K81A / D148P, K81A / D148P / V163T is significantly improved. The most preferred mutant is the single mutant K81A, the residual activity of which is increased from the original 21.36% to 85.45% after incubation at 40°C for 2 h, and the residual activity of which is increased from the original 3.15% to 52.97% after incubation at 60°C for 2 h, and the half-life reaches 123.12 min, which is 5.18 times higher than that of the original enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The three-dimensional structure of alginate lyase AlgL3199.

[0018] Figure 2This is the SDS-PAGE protein electrophoresis analysis of AlgL3199 mutants; wherein, lane M refers to Marker; lanes 1-18, adjacent lanes are the crude enzymes and pure enzymes of K81A, D148P, V163T, I274G, K81A / D148P, V163T / I274G, D148P / V163T, K81A / D148P / V163T, K81A / D148P / V163T / I274G, respectively.

[0019] Figure 3 This is the optimal temperature diagram for AlgL3199 and its mutants.

[0020] Figure 4 This is the optimal pH diagram of the recombinant enzyme AlgL3199.

[0021] Figure 5 This is the optimal pH diagram of mutant K81A.

[0022] Figure 6 This is the optimal pH diagram of mutant D148P.

[0023] Figure 7 This is the optimal pH diagram of mutant K81A / D148P.

[0024] Figure 8 This is the optimal pH diagram of mutant K81A / D148P / V163T.

[0025] Fig. 9 Residual activity of AlgL3199 and mutants at different temperatures.

[0026] Fig.10 The thermal stability of AlgL3199 and its mutants at 40°C.

[0027] Fig.11 The thermal stability of AlgL3199 and its mutants at 50°C.

[0028] Fig.12 The thermal stability of AlgL3199 and its mutants at 60°C. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0030] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0031] Example 1, selection of mutation sites

[0032] AlgL3199 contains typical conserved regions of the PL7 family, namely SA3 (RXEXR), SA4 (YXKAGXYXQ) and SA5 (QXH). Homology modeling of AlgL3199 was performed using PDB:5ZU5 (sequence homology %) as a template. Like most PL7 family alginate lyases, AlgL3199 has a small amount of α-helical structure and is mainly composed of β-folded structures (such as Figure 1 ). Through virtual mutation in Discovery Studio, four single mutants with reduced free energy were obtained, namely K81A, D148P, V163T, and I274G. Combination mutation was performed based on these four single mutants to obtain five combined mutants. The mutation sites were used as the names of the mutants, and they were named K81A, D148P, V163T, I274G, K81A / D148P, V163T / I274G, D148P / V163T, K81A / D148P / V163T, and K81A / D148P / V163T / I274G.

[0033] Example 2, Recombinant preparation and purification of AlgL3199 and mutants

[0034] The mutant with confirmed sequence was connected to the pET-24a(+) vector and transformed into Escherichia coli BL21 (DE3) competent cells, cultured at 37°C overnight, and single colonies were picked for further culture. The seed culture was inoculated into 100 mL of LB medium supplemented with kanamycin at a ratio of 3-5% (w / v) of the seed liquid, and cultured at 37°C and 200 rpm until OD 600 was 0.6-0.8. IPTG was added to a final concentration of 0.2 mM for induction, and cultured at 16°C and 200 rpm overnight. After induction, centrifuged at 8000 rpm for 10 min, and the cell pellet was resuspended in 10 mL of pH 7.0 PB buffer. Ultrasonic disruption was performed for 10 min, and then centrifuged at 8000 rpm for 10 min to obtain a supernatant containing crude enzyme. The crude enzyme was purified by nickel column to obtain pure enzyme solution, which was verified by SDS-PAGE protein electrophoresis. Figure 2As shown, the mutants all showed a clear band near 36.06 kDa, which was consistent with the band of AlgL3199, indicating that all single mutants were successfully induced. The enzyme activity of alginate lyase was determined. Compared with AlgL3199, the relative enzyme activities of single mutants K81A, D148P, V163T, and I274G were increased to 128.59%, 142.56%, 120.17%, and 104.30%, respectively. The relative enzyme activities of combined mutants K81A / D148P, V163T / I274G, D148P / V163T, K81A / D148P / V163T, and K81A / D148P / V163T / I274G were increased to 134.71%, 117.65%, 121.63%, 112.15%, and 111.00%, respectively (Table 1). The above results prove that replacing lysine (Lys) at position 81, aspartic acid (Asp) at position 148, valine (Val) at position 163, and isoleucine (Ile) at position 274 in the original AlgL3199 enzyme gene with alanine (Ala), proline (Pro), threonine (Thr), and glycine (Gly), respectively, can enhance the enzyme specific activity.

[0035] Table 1: Comparison of enzyme activities of AlgL3199 and mutants

[0036]

[0037] Example 3, Determination of the Optimal Reaction Temperature and pH of AlgL3199 and Mutants

[0038] The activity of alginate lyase was determined by DNS method. 1.0 mL of 10 mmol / L pH 10.0 Gly-NaOH buffer containing 1% (w / v) sodium alginate was mixed with 1.0 mL of enzyme solution and reacted at 40°C for 30 min. After the reaction, 1 mL of DNS reagent was added, boiled for 5 min, cooled on ice and made up to 25 mL with distilled water. The absorbance was measured at 520 nm, and the amount of reducing sugar produced was calculated based on the glucuronic acid standard curve. The enzyme activity was expressed in units (U), which was defined as the amount of enzyme required to produce 1 μg of reducing sugar per minute. 0.2 mL of 10 mmol / L pH10.0 Gly-NaOH buffer (containing 1% (w / v) sodium alginate) was incubated at different temperatures (10-55°C, 5°C interval) for 10 min, and then 0.2 mL of enzyme solution was added and reacted for 30 min. The optimal enzyme activity was defined as 100%, and the relative activity was calculated based on this. pH optimization: Prepare buffers with pH values ​​between 3.0 and 12.0 and perform the assay as described above at the optimal temperature.

[0039] like Figure 3As shown in Figure 2, the optimum temperature of AlgL3199 is 40°C, the optimum temperatures of mutants K81A and K81A / D148P are the same as AlgL3199, and the optimum temperatures of mutants D148P and K81A / D148P / V163T are increased to 45°C. Figure 4-8 As shown, the optimal pH of AlgL3199 is 10.0, and the mutants are consistent with AlgL3199. Algin dissolves better in a weakly alkaline environment, so mutants with a preference for alkalinity can be more effectively used in industrial production.

[0040] Example 4, Determination of thermal stability of AlgL3199 and mutants

[0041] AlgL3199 and mutants were incubated at 40°C, 50°C, and 60°C for 0.5, 1 h, 1.5, and 2 h, respectively, and immediately cooled in an ice bath. The residual enzyme activity was determined under the optimal conditions.

[0042] AlgL3199 showed good thermal stability after being cultured at 10-35°C for 1 h, and the relative enzyme activity remained above 80% ( Fig. 9 ), but after incubation at 40℃ for 1h, the relative enzyme activity dropped to 50.96%, and the enzyme activity decreased significantly with increasing temperature. Fig.10 As shown, the thermal stability of mutants K81A, D148P, K81A / D148P, and K81A / D148P / V163T at 40°C was higher than that of AlgL3199. After incubation for 2 h, the residual enzyme activities were 85.45%, 90.45%, 71.37%, and 81.96%, respectively, which were significantly improved compared with the residual activity of the original enzyme (21.36%); Fig.11 As shown, when the temperature was raised to 50°C and incubated for 2 h, AlgL3199 was completely inactivated, and the four mutants still retained more than 60% of their activity; Fig.12 As shown in the figure, after incubation at 60℃ for 2h, K81A can still retain more than 50% of its activity. Table 2 shows the half-life of the original enzyme and mutants at 60℃. Compared with the original enzyme, K81A increased by 5.18 times, D148P increased by 4.66 times, K81A / D148P increased by 3.22 times, and K81A / D148P / V163T increased by 3.53 times.

[0043] Table 2: Comparison of half-life at 60°C between the original enzyme and the mutant enzyme of AlgL3199

[0044]

[0045] Example 5, Comparison of kinetic parameters of AlgL3199 and mutants

[0046] Sodium alginate solutions of different concentrations were prepared in 10 mM pH 10.0 Gly-NaOH buffer. After the substrate solution was preheated at the optimal temperature for 10 minutes, the enzyme solution was added and reacted for 30 minutes. Then, the reducing sugar was quantified by adding the DNS reagent, and the Michaelis-Menten equation was fitted to draw a graph. The Michaelis constant (Km), maximum reaction rate (Vmax), calculated turnover number (Kcat) and catalytic efficiency (Kcat / Km) were obtained from the graph. As shown in Table 3, compared with AlgL3199, the Km values ​​of mutants K81A, D148P, K81A / D148P, and K81A / D148P / V163T decreased, while the Vmax and Kcat / Km values ​​increased. The Km value of mutant enzyme K81A decreased to 6.59 mg / m, the maximum reaction rate increased to 45.22μg / min·mL-1, and the Kcat / Km value increased by 1.11 times compared with AlgL3199. The Km value of the mutant enzyme D148P was reduced to 5.79 mg / mL, which was 1.15 times lower than that of AlgL3199 (6.66 mg / mL), and the Kcat / Km value was 1.16 times higher than that of AlgL3199. The Km value of the mutant K81A / D148P was reduced to 5.91 mg / mL, and the Kcat / Km value was 1.17 times higher than that of AlgL3199, indicating that the mutation of lysine (Lys) to alanine (Ala) at position 81 and the mutation of aspartic acid (Asp) to proline (Pro) at position 148 not only improved the thermal stability of the enzyme, but also had a certain effect on the catalytic efficiency. In summary, K81A is a mutant with significantly improved thermal stability, and D148P is a mutant with significantly improved catalytic efficiency.

[0047] Table 3: Kinetic parameters of AlgL3199 and mutants

[0048]

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A mutant of alginate lyase AlgL3199 with improved thermal stability, characterized in that: Compared with alginate lyase AlgL3199, the mutation sites of the alginate lyase AlgL3199 mutant are K81A, D148P, K81A / D148P or K81A / D148P / V163T. The amino acid sequence of the alginate lyase AlgL3199 is shown in SEQ ID NO:

1.

2. A recombinant expression vector, characterized in that: The recombinant expression vector contains: an expression frame of the gene encoding the alginate lyase AlgL3199 mutant with improved thermal stability as claimed in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The recombinant expression vector is a plasmid vector, a phage or a virus vector.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contains the recombinant expression vector according to claim 2 or 3.

5. The genetically engineered bacterium according to claim 4, characterized in that The genetically engineered bacteria is Escherichia coli.

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