Alginate lyase mutant with improved thermal stability and application thereof

Abstract

The invention discloses an alginate lyase mutant with improved thermal stability and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. A series of alginate lyase mutants are constructed, the apparent melting temperature of the single mutant is increased by 2.27-8.34 DEG C, the relative enzyme activity is 96.77%-125.81% of that of a wild enzyme, and the half-life period is 2.79-40.77 times of that of the wild enzyme. The apparent melting temperature of the combined mutant is increased by 16.25 DEG C, the relative enzyme activity is 119.04% of that of a wild enzyme, the half-life period is 946.30 times of that of the wild enzyme, 53.44% of original enzyme activity can still be reserved after incubation for 185 h at 55 DEG C, and compared with the wild enzyme, the combined mutant shows excellent heat resistance and can be industrially produced at a high temperature, so that the utilization rate of algin is increased, and the combined mutant has a good industrial application prospect and is worthy of popularization and application. The efficient conversion of marine biomass resources can be better realized.

Description

Technical Field

[0001] The present invention relates to an alginate lyase mutant with improved thermal stability and its application, belonging to the technical fields of genetic engineering and enzyme engineering. Background Art

[0002] Alginate is a linear polysaccharide with certain viscosity, mainly extracted from the cell walls and intercellular substances of brown algae plants. It is formed by the random arrangement of two isomeric monomers, β-D-mannuronic acid (M) and α-L-guluronic acid (G), through α,β-(1,4)-glycosidic bonds. The main product types include homopolymeric mannuronic acid (Poly-M), homopolymeric guluronic acid (Poly-G), and heteropolymers randomly combined by M and G (Poly-MG, Poly-GM). Alginate oligosaccharides are oligomers with a degree of polymerization of 2-25 formed after the hydrolysis of alginate. Compared with alginate, alginate oligosaccharides have a small average molecular weight, low viscosity, and good water solubility, overcoming many deficiencies in the application of alginate. Some studies have shown that alginate oligosaccharides are a type of prebiotic with various physiological activities, such as antioxidant, antibacterial, immunomodulatory, and plant growth-promoting activities, and have great application potential in the fields of medicine, food, agriculture, etc. Alginate oligosaccharides are a type of functional oligosaccharide of marine origin and are currently a research hotspot, worthy of in-depth research and development.

[0003] The degradation methods of brown algae mainly include three categories: physical methods, chemical methods, and enzymatic methods. Physical methods usually use high-pressure homogenization, ultrasonic fragmentation, etc. to degrade the alginate polysaccharide chain. Although the operation is relatively simple, there are problems such as uneven distribution of the product degree of polymerization, low yield, and high energy consumption. Chemical methods mainly include acidolysis or oxidative degradation methods, which break the glycosidic bonds of the polysaccharide chain through strong acids (such as hydrochloric acid) or oxidants (such as hydrogen peroxide). However, this method has severe reaction conditions and is prone to chemical residues and by-products, which not only affect the product purity but may also cause environmental pollution. In contrast, the enzymatic method has gradually become a research hotspot due to its unique advantages. The alginate lyase is used to specifically degrade the polysaccharide chain, with mild reaction conditions (room temperature and normal pressure), controllable product degree of polymerization, and high uniformity, avoiding the introduction of chemical reagents and excessive energy consumption. In addition, specific-structured oligosaccharides can be obtained during the enzymatic production process, thus maximizing the retention of their antioxidant, immunomodulatory, and other biological activities, showing higher application potential in the fields of medicine, functional foods, etc. Therefore, the enzymatic method is an ideal choice for the industrial production of alginate oligosaccharides and is also a current research hotspot direction.

[0004] However, although alginate lyases have a wide range of sources, most natural alginate lyases are derived from marine bacteria, with an optimal temperature range of 30 - 55°C, making it difficult to be applied in high-temperature industries. Therefore, it is very necessary to improve the thermal stability of existing alginate lyases through protein engineering technology to meet the standard for continuous industrial production of alginate oligosaccharides. However, most existing alginate lyases still cannot maintain good activity and stability for a long time in high-temperature environments and meet industrial production conditions. Therefore, it is necessary to develop new alginate lyases with improved thermal stability through protein and enzyme engineering to meet the conditions for industrial production of alginate oligosaccharides, significantly improve the industrial application value of alginate lyases, and realize the high-value utilization of marine resources. Summary of the Invention

[0005] [Technical Problem]

[0006] Alginate shows low solubility under low-temperature conditions, and its high-concentration solution has a high viscosity at room temperature, which directly affects the enzymatic reaction efficiency. Although increasing the reaction temperature can effectively improve the dissolution state of alginate and reduce the solution viscosity, enabling alginate lyase to fully contact the substrate and thus improve the catalytic efficiency, currently, alginate lyases generally have low thermal stability and are difficult to adapt to the continuous high-temperature environment required for industrial production, which is also the main technical bottleneck restricting the large-scale preparation of alginate oligosaccharides by enzymatic methods.

[0007] [Technical Solution]

[0008] The present invention provides an alginate lyase mutant, which is obtained by mutating at least one amino acid at position 94, 136, 205, 255, 303, 324, 326, 353, 363, or 385 based on the alginate lyase shown in SEQ ID NO.1.

[0009] In one embodiment, the mutant is obtained by mutating the threonine at position 94 of the amino acid sequence shown in SEQ ID No.1 to valine, and the obtained mutant is named: T94V.

[0010] In one embodiment, the mutant is obtained by mutating the serine at position 136 of the amino acid sequence shown in SEQ ID No.1 to cysteine, and the obtained mutant is named: S136C.

[0011] In one embodiment, the mutant is obtained by mutating the alanine at position 205 of the amino acid sequence shown in SEQ ID No.1 to proline, and the obtained mutant is named: A205P.

[0012] In one embodiment, the mutant is obtained by mutating asparagine at position 255 in the amino acid sequence shown in SEQ ID No.1 to arginine, and the obtained mutant is named: N255R.

[0013] In one embodiment, the mutant is obtained by mutating glycine at position 303 in the amino acid sequence shown in SEQ ID No.1 to asparagine, and the obtained mutant is named: G303N.

[0014] In one embodiment, the mutant is obtained by mutating leucine at position 324 in the amino acid sequence shown in SEQ ID No.1 to valine, and the obtained mutant is named: L324V.

[0015] In one embodiment, the mutant is obtained by mutating lysine at position 326 in the amino acid sequence shown in SEQ ID No.1 to asparagine, and the obtained mutant is named: K326N.

[0016] In one embodiment, the mutant is obtained by mutating aspartic acid at position 353 in the amino acid sequence shown in SEQ ID No.1 to valine, and the obtained mutant is named: D353V.

[0017] In one embodiment, the mutant is obtained by mutating methionine at position 363 in the amino acid sequence shown in SEQ ID No.1 to threonine, and the obtained mutant is named: M363T.

[0018] In one embodiment, the mutant is obtained by mutating threonine at position 385 in the amino acid sequence shown in SEQ ID No.1 to valine, and the obtained mutant is named: T385V.

[0019] In one embodiment, the mutant is obtained by mutating leucine at position 324 in the amino acid sequence shown in SEQ ID No.1 to valine and mutating aspartic acid at position 353 to valine, and the obtained mutant is named: L324V-D353V.

[0020] In one embodiment, the mutant is obtained by mutating leucine at position 324 in the amino acid sequence shown in SEQ ID No.1 to valine and mutating methionine at position 363 to threonine, and the obtained mutant is named: L324V-M363T.

[0021] In one embodiment, the mutant is obtained by mutating leucine at position 324 in the amino acid sequence shown in SEQ ID No.1 to valine and mutating threonine at position 385 to valine, and the obtained mutant is named: L324V-T385V.

[0022] In one embodiment, the mutant is obtained by mutating the aspartic acid at position 353 in the amino acid sequence shown in SEQ ID No.1 to valine, and mutating the methionine at position 363 to threonine. The obtained mutant is named: D353V-M363T.

[0023] In one embodiment, the mutant is obtained by mutating the aspartic acid at position 353 in the amino acid sequence shown in SEQ ID No.1 to valine, and mutating the threonine at position 385 to valine. The obtained mutant is named: D353V-T385V.

[0024] In one embodiment, the mutant is obtained by mutating the methionine at position 363 in the amino acid sequence shown in SEQ ID No.1 to threonine, and mutating the threonine at position 385 to valine. The obtained mutant is named: M363T-T385V.

[0025] In one embodiment, the mutant is obtained by mutating the leucine at position 324 in the amino acid sequence shown in SEQ ID No.1 to valine, mutating the aspartic acid at position 353 to valine, and mutating the methionine at position 363 to threonine. The obtained mutant is named: L324V-D353V-M363T.

[0026] In one embodiment, the mutant is obtained by mutating the leucine at position 324 in the amino acid sequence shown in SEQ ID No.1 to valine, mutating the aspartic acid at position 353 to valine, and mutating the threonine at position 385 to valine. The obtained mutant is named: L324V-D353V-T385V.

[0027] In one embodiment, the mutant is obtained by mutating the leucine at position 324 in the amino acid sequence shown in SEQ ID No.1 to valine, mutating the methionine at position 363 to threonine, and mutating the threonine at position 385 to valine. The obtained mutant is named: L324V-M363T-T385V.

[0028] In one embodiment, the mutant is obtained by mutating the aspartic acid at position 353 in the amino acid sequence shown in SEQ ID No.1 to valine, mutating the methionine at position 363 to threonine, and mutating the threonine at position 385 to valine. The obtained mutant is named: D353V-M363T-T385V.

[0029] In one embodiment, the mutant is obtained by mutating the leucine at position 324, the aspartic acid at position 353, the methionine at position 363, and the threonine at position 385 in the amino acid sequence shown in SEQ ID No.1 to valine, threonine, and valine respectively. The obtained mutant is named: L324V-D353V-M363T-T385V.

[0030] The present invention also provides a gene encoding the mutant.

[0031] The present invention provides a recombinant vector carrying the gene.

[0032] In one embodiment, the vector includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series vectors.

[0033] In one embodiment, the expression vector is pET28a(+).

[0034] The present invention provides a recombinant cell expressing the mutant, or containing the gene, or containing the recombinant vector.

[0035] In one embodiment, the recombinant cell uses a prokaryotic cell or a eukaryotic cell as an expression host.

[0036] In one embodiment, the prokaryotic host cell can be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Pelobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma; the eukaryotic cell is a fungal cell.

[0037] In one embodiment, the recombinant cell uses Escherichia coli E.coli BL 21(DE3) as an expression host.

[0038] The present invention provides a method for preparing the mutant, and the steps of the method are as follows:

[0039] (1) According to the determined mutation sites, design site-directed mutagenesis primers, and perform site-directed mutagenesis using the vector carrying the alginate lyase encoding gene as a template to construct a vector containing the gene encoding the mutant;

[0040] (2) Transform the vector containing the gene encoding the mutant into the expression host of Escherichia coli E.coli BL(21)DE3;

[0041] (3) Select positive clones for fermentation culture, centrifuge to collect cells, and the supernatant obtained after disrupting the cells with an ultrasonic cell disruptor is the crude enzyme solution of the alginate lyase mutant;

[0042] (4) Purify the crude enzyme solution of the alginate lyase mutant by passing it through a nickel column to obtain the pure enzyme solution of the alginate lyase mutant.

[0043] The present invention also provides a method for improving the thermal stability of alginate lyase, which performs one or more of the following mutations on the alginate lyase with the amino acid sequence shown in SEQ ID NO.1:

[0044] (1) Mutate the threonine at position 94 to valine;

[0045] (2) Mutate the serine at position 136 to cysteine;

[0046] (3) Mutate the alanine at position 205 to proline;

[0047] (4) Mutate the asparagine at position 255 to arginine;

[0048] (5) Mutate the glycine at position 303 to asparagine;

[0049] (6) Mutate the leucine at position 324 to valine;

[0050] (7) Mutate the lysine at position 326 to asparagine;

[0051] (8) Mutate the aspartic acid at position 353 to valine;

[0052] (9) Mutate the methionine at position 363 to threonine;

[0053] (10) Mutate the threonine at position 385 to valine.

[0054] The present invention provides a composition containing the alginate lyase mutant.

[0055] In one embodiment, the composition includes, but is not limited to, enzyme preparations.

[0056] In one embodiment, the composition uses the alginate lyase as the main enzyme component and contains one or more adjuvants.

[0057] In one embodiment, the adjuvant includes, but is not limited to, enzyme stabilizers.

[0058] In one embodiment, the enzyme stabilizer includes mannitol, DTT, trehalose, or vitamin C.

[0059] The present invention also provides the application of the alginate lyase mutant in the fields of food, health products or medicine.

[0060] In one embodiment, the application includes but is not limited to hydrolyzing alginate or preparing products containing alginate oligosaccharides.

[0061] In one embodiment, the application includes but is not limited to reacting the alginate lyase mutant in an environment containing alginate at 50-65°C.

[0062] Beneficial effects:

[0063] Compared with the wild-type enzyme Pedsa0632 (the enzyme activity loss was 77.73% after incubation at 55°C for 15 min and it was basically inactivated after incubation for 30 min), the optimal reaction temperature and thermal stability of the mutant of the present invention have been significantly improved, specifically including:

[0064] 1. The mutants constructed and obtained in the present invention have significantly improved thermal stability while maintaining the original catalytic activity. Among them, the apparent melting temperatures of the single mutants T94V, S136C, A205P, N255R, G303N, L324V, K326N, D353V, M363T, T385V increased by 2.28 - 8.34°C, the relative enzyme activity was 96.77% - 125.81% of the wild type, and the half-life was 2.79 - 40.77 times that of the wild type. The apparent melting temperature of the combined mutant L324V-D353V-M363T-T385V increased by 16.25°C, the relative enzyme activity was 119.04% of the wild type, and the half-life at 55°C was 946.30 times that of the wild type.

[0065] 2. The optimal reaction temperatures of the mutants A205P and N255R provided by the present invention increased by 10°C compared with the wild type, reaching 65°C, and the thermal stability was significantly improved. Among them, the single mutants L324V, D353V, M363T, T385V all retained more than 50% of the original enzyme activity after incubation at 55°C for 120 min, and the combined mutants (except L324V-M363T and M363T-T385V) all maintained more than 85% of the original enzyme activity under the same conditions. When incubated at 60°C for 90 min, the combined mutants (except L324V-M363T and M363T-T385V) all maintained more than 55% of the original enzyme activity. When incubated at 65°C for 150 min, the combined mutant L324V-D353V-M363T-T385V could maintain more than 65% of the original enzyme activity.

[0066] 3. The mutant L324V-D353V-M363T-T385V provided by the present invention has an optimum temperature 10 °C higher than that of the wild-type enzyme Pedsa0632, and the relative enzyme activity is 119.04% of that of the wild type, and it shows good heat resistance and activity, and can still retain 53.44% of the original enzyme activity after incubation at 55 °C for 11140 min. Description of the Drawings

[0067] Figure 1 It is a schematic diagram of plasmid construction.

[0068] Figure 2 It is the SDS-PAGE diagram of the purified wild-type alginate lyase Pedsa0632 and mutants; where M in the figure: protein molecular weight standard (Marker), Figure A: 1: purified wild-type alginate lyase Pedsa0632; 2-11: the purified mutants are T94V, A205P, S136C, L324V, D353V, K326N, N255R, G303N, M363T, T385V in sequence; Figure B: 1-11: the purified mutants are L324V-D353V, L324V-M363T, L324V-T385V, D353V-M363T, D353V-T385V, M363T-T385V, L324V-D353V-M363T, L324V-D353V-T385V, L324V-M363T-T385V, D353V-M363T-T385V, L324V-D353V-M363T-T385V in sequence.

[0069] Figure 3 It is the enzyme activity and T characterization of the wild-type alginate lyase Pedsa0632 and single-point mutants m Characterization

[0070] Figure 4 It is the enzyme activity and T characterization of the wild-type alginate lyase Pedsa0632 and combined mutants m Characterization

[0071] Figure 5 It is the characterization of the optimum reaction temperature of the wild-type alginate lyase Pedsa0632 and single-point mutants.

[0072] Figure 6 It is the characterization of the optimum reaction temperature of the wild-type alginate lyase Pedsa0632 and combined mutants.

[0073] Figure 7 It is the characterization of the optimum reaction pH of the wild-type alginate lyase Pedsa0632 and single-point mutants.

[0074] Figure 8Characterization of the optimal reaction pH of wild-type alginate lyase Pedsa0632 and combinatorial mutants.

[0075] Figure 9 Characterization of the thermal stability of wild-type alginate lyase Pedsa0632, single mutants, and combinatorial mutants at 55 °C.

[0076] Figure 10 Characterization of the thermal stability of wild-type alginate lyase Pedsa0632, single mutants, and combinatorial mutants at 60 °C.

[0077] Figure 11 Characterization of the thermal stability of triple combinatorial mutants and quadruple combinatorial mutants at 65 °C. Detailed implementation

[0078] Technical terms:

[0079] Alginate lyase: The term "alginate lyase" refers to an enzyme in the EC 4.2.2.- class as defined by enzyme nomenclature. For the purposes of the present invention, the activity of "alginate lyase" is determined according to the detection procedures described in the detailed implementation of this application. The alginate lyase of the present invention is an alginate lyase having the amino acid sequence shown in SEQ ID NO.1.

[0080] Expression: The term "expression" includes any steps involved in the production of alginate lyase mutants, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0081] Expression vector: The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding an alginate lyase mutant of the present invention and is operably linked to a control sequence that provides for its expression.

[0082] Host cell: The term "host cell" means any cell type that is readily transformable, transfectable, transducible, etc. with a nucleic acid construct or expression vector containing a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parental cell that is not identical to the parental cell due to mutations that occur during replication.

[0083] A host cell can be any cell useful in the recombinant production of alginate lyase mutants, such as prokaryotic cells or eukaryotic cells.

[0084] The prokaryotic host cell can be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Pelobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0085] The host cell can also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

[0086] Alginic acid: It is a polysaccharide widely present in brown algae, formed by the random arrangement of β-D-mannuronic acid and α-L-guluronic acid through α,β-(1,4)-glycosidic bonds.

[0087] The technical solution of the present invention will be elaborated in detail below in combination with examples and drawings. The cited examples are only used to explain the present invention rather than to limit the scope of the present invention.

[0088] Unless otherwise specified, all kinds of reagents, experimental methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0089] The main reagents involved in the following examples:

[0090] The Escherichia coli E.coli BL21(DE3) strain and the BCA concentration assay kit were purchased from Beyotime Biotechnology Co., Ltd. Other commonly used reagents are all domestic analytical pure.

[0091] The gene of Pedsa0632, the pET28a-Pedsa0632 recombinant vector, and the mutant primers involved in the following examples were all synthesized by Suzhou Anshengda Biotechnology Co., Ltd. The mutant was prepared using the plasmid pET28a-Pedsa0632 of wild-type Pedsa0632 as a template, adding upstream and downstream primers for PCR reaction, and transferring the obtained gene into Escherichia coli E.coli BL21(DE3) to obtain the recombinant strain E.coli BL21(DE3) / pET28a-Pedsa0632 mutant 。

[0092] The media involved in the following examples are as follows:

[0093] LB liquid medium: Weigh 0.5 g of yeast extract, 1.0 g of tryptone, and 1.0 g of NaCl, dissolve with deionized water and make up the volume to 100 mL, then autoclave at 121 °C for 20 min in an autoclave.

[0094] LB solid medium: Based on LB liquid medium, add 1.5 g of agar powder per 100 mL.

[0095] LB liquid resistant medium: Based on LB liquid medium, add an appropriate amount of kanamycin to the liquid medium to make its final concentration 25 μg·mL -1 .

[0096] LB solid resistant medium: Based on LB solid medium, add an appropriate amount of kanamycin to the solid medium to make its final concentration 25 μg·mL -1 .

[0097] The purified solutions of the enzymes involved in the following examples are as follows:

[0098] (1) Resuspension buffer: Weigh 2.42 g of Tris-HCL and 29.22 g of NaCl, dissolve with ultrapure water and make up the volume to 1 L (pH = 8);

[0099] (2) Loading buffer: Weigh 2.42 g of Tris-HCL, 29.22 g of NaCl, and 1.36 g of imidazole, dissolve with ultrapure water and make up the volume to 1 L (pH = 8).

[0100] (3) Elution buffer: Weigh 2.42 g of Tris-HCL, 29.22 g of NaCl, and 34.04 g of imidazole, dissolve with ultrapure water and make up the volume to 1 L (pH = 8).

[0101] The resuspension buffer is used to equilibrate the nickel column, the loading buffer is used to remove impurities after loading, and the elution buffer is used to elute and collect the target protein, and then the purified enzyme solution can be obtained.

[0102] The detection methods involved in the following examples are as follows:

[0103] 1. Enzyme activity determination of wild type and mutant:

[0104] The 3,5-dinitrosalicylic acid method (DNS method) is used to determine the enzyme activity. Alginate lyase catalyzes the hydrolysis of alginate to produce reducing sugars under certain conditions, which can react with DNS in a boiling water bath to form a brownish-red amino complex. Within a certain range, the amount of reducing sugar produced is proportional to the color depth. The absorbance can be measured at a wavelength of 520 nm and the enzyme activity can be calculated.

[0105] Definition of enzyme activity unit: Under the optimal conditions, when the absorbance is 520 nm, the amount of enzyme required to catalyze the production of reducing sugar equivalent to 1 μmoL per minute is defined as one activity unit (U).

[0106] Steps for enzyme activity determination:

[0107] (1) Take 200 μL of the alginate solution (pH = 9.0) at 6 mg·mL -1 and preheat it in a 2 mL centrifuge tube;

[0108] (2) Add 10 μL of the enzyme solution, mix well by shaking, react for 5 min, and immediately add 150 μL of DNS. Terminate the reaction by boiling water bath for 5 min;

[0109] (3) After the sample is cooled to room temperature, make up the volume to 1 mL and measure the absorbance at a wavelength of 520 nm, and calculate the enzyme activity.

[0110] 2. Determination of thermodynamic stability (T m ) Measurement:

[0111] Use a fluorescence quantitative PCR instrument (Nano DSF) for measurement. Dilute the purified enzyme solution to 2.0 mg·mL -1 , take 20 μL of the enzyme solution and add it to a 96-well plate, mix well with 5 μL of the SYPRO Orange dye probe. The temperature gradient is 1 °C / min, from 25 °C to 95 °C. Observe the temperature corresponding to the peak of the sample to obtain the T m value.

[0112] Example 1: Construction of the recombinant vector pET28a - Pedsa0632 containing the wild-type alginate lyase Pedsa0632 gene

[0113] According to Pseudopedobacter saltans (UniProt accession number: F0S7Y7, amino acid sequence as shown in SEQ ID NO.1) disclosed in the database, add two amino acids GS at the N-terminus as a linker site to obtain the sequence shown in SEQ ID NO.2. In the present invention, the description of the mutation sites is based on the amino acid sequence shown in SEQ ID NO.1 without the above linker site.

[0114] The amino acid sequence shown in SEQ ID NO.1 was codon-optimized and synthesized by Suzhou Anshengda Biotechnology Co., Ltd. to obtain the nucleotide sequence encoding the wild-type alginate lyase Pedsa0632 of the present invention as shown in SEQ ID NO.3.

[0115] The nucleotide sequence shown in SEQ ID NO.3 was ligated with the vector pET28a(+) through the restriction enzyme sites BamH I and Xho I to obtain the recombinant plasmid pET28a - Pedsa0632.

[0116] Example 2: Construction of the recombinant vector containing the mutant

[0117] (1) Construction of the recombinant plasmid containing the single mutant

[0118] Design site-directed mutagenesis primers, and use the recombinant plasmid pET28a-Pedsa0632 constructed in Example 1 as a template for site-directed mutagenesis to obtain recombinant plasmids pET28a-T94V, pET28a-S136C, pET28a-A205P, pET28a-N255R, pET28a-G303N, pET28a-L324V, pET28a-K326N, pET28a-D353V, pET28a-M363T, pET28a-T385V containing mutants T94V, S136C, A205P, N255R, G303N, L324V, K326N, D353V, M363T, T385V respectively. The primer sequences involved are as follows.

[0119] The primer for introducing the T94V mutation site is:

[0120] T94V-F: 5'-AAAAGGGCGGTGAAGTTATCTTCTCTGGCAACAG-3';

[0121] T94V-R: 5'-AACTTCACCGCCCTTTTCAGCTGCC-3'.

[0122] The primer for introducing the S136C mutation site is:

[0123] S136C-F: 5'-CCAAAAGCACCTCGAATTGTCGTATCACCAA-3';

[0124] S136C-R: 5'-ATTCGAGGTGCTTTTGGTAAACAAAATCACGTCC-3'.

[0125] The primer for introducing the A205P mutation site is:

[0126] A205P-F: 5'-GCCCACGTCCGCCTCTGGGTGTTAA-3';

[0127] A205P-R: 5'-AGGCGGACGTGGGCCGAAGTAGTTG-3'.

[0128] The primer for introducing the N255R mutation site is:

[0129] N255R-F: 5'-CTGGTCATAATACTGTGCGTAACAACTTGTT-3';

[0130] N255R-R: 5'-ACGCACAGTATTATGACCAGACTTAAGGGA-3'.

[0131] The primers for introducing the G303N mutation site are as follows:

[0132] G303N-F: 5’-TCGGCGAGAACCACAAAGTTTTTAACAACTACTTACAAGG-3’;

[0133] G303N-R: 5’-GTTAAAAACTTTGTGGTTCTCGCCGATGATGCGG-3’.

[0134] The primers for introducing the L324V mutation site are as follows:

[0135] L324V-F: 5’-TTATGAGCGCGGTTGAAAAGCCACA-3’;

[0136] L324V-R: 5’-CGCGCTCATAATTGAAATCGCTGCAC-3’.

[0137] The primers for introducing the K326N mutation site are as follows:

[0138] K326N-F: 5’-GCGCGCTGGAAAATCCACAACTGCA-3’;

[0139] K326N-R: 5’-TTCCAGCGCGCTCATAATTGAAATCGCT-3’.

[0140] The primers for introducing the D353V mutation site are as follows:

[0141] D353V-F: 5’-AAGAGGGCATCGTTATCGGCGCGGGTAAAAA-3’;

[0142] D353V-R: 5’-GATGCCCTCTTTGCTGTCGGCGATGATG-3’.

[0143] The primers for introducing the M363T mutation site are as follows:

[0144] M363T-F: 5’-AAAACGAGAAGCGCACTCTGCCGCCTAA-3’;

[0145] M363T-R: 5’-AGTGCGCTTCTCGTTTTTACCCGCG-3’.

[0146] The primers for introducing the T385V mutation site are as follows:

[0147] T385V-F: 5'-CCGTCATTAAAGTTGAAAACGAGCC-3';

[0148] T385V-R: 5'-TTTAATGACGGTACGCGTGTTAATC-3'.

[0149] The PCR reaction system is shown in Table 1.

[0150] Table 1 PCR reaction system (20 μL)

[0151]

[0152] Table 2 PCR reaction conditions (30 cycles in total for steps 2 - 4)

[0153]

[0154] (2) Construction of recombinant plasmids containing combinatorial mutants:

[0155] On the basis of the previously constructed recombinant plasmids containing single mutations, mutations were continuously introduced in the same method to obtain combinatorial mutant plasmids: pET28a - L324V - D353V, pET28a - L324V - M363T, pET28a - L324V - T385V, pET28a - D353V - M363T, pET28a - D353V - T385V, pET28a - M363T - T385V, pET28a - L324V - D353V - M363T, pET28a - L324V - D353V - T385V, pET28a - L324V - M363T - T385V, pET28a - D353V - M363T - T385V, pET28a - L324V - D353V - M363T - T385V. Taking pET28a - L324V - D353V as an example, using the single - mutant plasmid pET28a - L324V as a template and primers introducing the mutation site D353V for whole - plasmid PCR, the combinatorial mutant plasmid pET28a - L324V - D353V was obtained.

[0156] According to the above method, the wild - type engineering bacteria plasmids expressing mutants were extracted.

[0157] Example 3: Construction of recombinant bacteria expressing mutants

[0158] The recombinant plasmids constructed in Example 2 were transformed into competent Escherichia coli E.coli BL21(DE3), and the transformants were spread on the plates containing kanamycin (25 μg·mL -1) on LB solid medium and cultured overnight at 37°C in an inverted position. After colonies grew, pick a single colony and inoculate it into liquid LB medium containing kanamycin (25 μg·mL -1 ). Culture it at 180 r / min for 18 h, and then send the cultured bacterial liquid to Suzhou Anshengda Biotechnology Co., Ltd. for sequencing. Engineered bacteria of mutants containing the correct mutation sites were obtained respectively. Transform the recombinant plasmid pET28a-Pedsa0632 constructed in Example 2 into competent Escherichia coli E. coli BL21(DE3) by the same method as above to obtain recombinant bacteria expressing wild enzyme.

[0159] Streak the constructed recombinant Escherichia coli on LB solid medium containing kanamycin (25 μg·mL -1 ), and culture it at 37°C for 16 h. Pick a single colony and inoculate it into LB liquid medium containing kanamycin (25 μg·mL -1 ). Culture it at 37°C and 200 r / min for 12 - 16 h to obtain a seed liquid. Take an appropriate amount of the seed liquid according to an inoculation amount of 1% (V / V) and add it to LB liquid medium containing kanamycin (25 μg·mL -1 ), and perform scale-up culture at 37°C and 200 r / min. When the OD value of the scale-up culture liquid reaches 0.6 - 0.8, add an appropriate amount of IPTG to make its final concentration 0.2 mM, and induce enzyme production at 20°C for 18 h.

[0160] Centrifuge and collect the Escherichia coli that produces enzyme at low temperature above, resuspend the cells with resuspension buffer, and ultrasonically disrupt them in an ice-water bath at 35% power for 20 min. After disruption, centrifuge at 4°C and 8000 r / min to collect the supernatant, and then obtain the crude enzyme liquid after passing through a 0.45 μm filter membrane. Use a nickel column to purify the crude enzyme liquid of wild-type alginate lyase and mutants by affinity chromatography to obtain the corresponding pure enzyme liquid. Detection by SDS-PAGE electrophoresis shows the results as Figure 2 shown.

[0161] Pure enzyme liquids containing wild-type alginate lyase and mutants were prepared respectively.

[0162] Determine the enzyme activities of the above-mentioned wild-type alginate lyase WT and mutants T94V, S136C, A205P, N255R, G303N, L324V, K326N, D353V, M363T, T385V, L324V-D353V, L324V-M363T, L324V-T385V, D353V-M363T, D353V-T385V, M363T-T385V, L324V-D353V-M363T, L324V-D353V-T385V, L324V-M363T-T385V, D353V-M363T-T385V, and L324V-D353V-M363T-T385V. Define the enzyme activity of the wild-type alginate lyase as 100%, and detect the relative enzyme activities of the mutants. The results are shown in Table 3 and Figure 3 , Figure 4 as follows.

[0163] Table 3 Wild-type and its mutants T m and relative enzyme activities

[0164]

[0165]

[0166] The results show that: By Nano DSF, the T m values of 21 sites of the above-mentioned wild-type alginate lyase WT and mutants T94V, S136C, A205P, N255R, G303N, L324V, K326N, D353V, M363T, T385V, L324V-D353V, L324V-M363T, L324V-T385V, D353V-M363T, D353V-T385V, M363T-T385V, L324V-D353V-M363T, L324V-D353V-T385V, L324V-M363T-T385V, D353V-M363T-T385V, and L324V-D353V-M363T-T385V were determined. Among them, those with significant effects were A205P (ΔT m = 8.34 °C), D353V (ΔT m = 7.48 °C), T385V (ΔT m = 6.69 °C), L324V (ΔT m = 6.86 °C), S136C (ΔT m = 6.41 °C), L324V-D353V (ΔT m = 12.18 °C), L324V-T385V (ΔT m = 12.81 °C), D353V-T385V (ΔTm = 12.02 °C), M363T-T385V (ΔT m = 13.24 °C), L324V-D353V-T385V (ΔT m = 16.25 °C), D353V-M363T-T385V (ΔT m = 16.65 °C) and L324V-D353V-M363T-T385V (ΔT m = 16.25 °C). The ΔT of all mutants m has increased. At the same time, the optimum temperature of the remaining mutants except the A205P and N255R mutants has increased by 10 °C, and the heat resistance has been improved, as shown in Table 3 and Figure 3 、 4 shown.

[0167] Example 4: Determination of the optimum reaction temperature of mutants

[0168] To further explore the enzymatic properties of the mutants, further enzymatic property characterization was carried out on T94V, S136C, A205P, N255R, G303N, L324V, K326N, D353V, M363T, T385V, L324V-D353V, L324V-M363T, L324V-T385V, D353V-M363T, D353V-T385V, M363T-T385V, L324V-D353V-M363T, L324V-D353V-T385V, L324V-M363T-T385V, D353V-M363T-T385V and L324V-D353V-M363T-T385V. An alginate substrate solution with a concentration of 6 g / L was prepared with 50 mM glycine-sodium hydroxide buffer (pH = 9). An enzyme solution with a final concentration of 0.1 mg / mL was added and reacted at different temperatures for 5 min to measure the enzyme activity. From Figure 5 、 Figure 6 it can be seen that the optimum reaction temperature of the wild-type alginate lyase WT is 55 °C, and the optimum reaction temperature of all mutants except A205P and N255R has increased by 10 °C to reach 65 °C.

[0169] Example 5: Determination of the optimum reaction pH of mutants

[0170] An alginate substrate solution of 0.6% at pH 6 was prepared with citrate buffer; an alginate substrate solution of 0.6% in the range of pH 7 - 8 was prepared with Tris-HCL buffer; an alginate substrate solution of 0.6% in the range of pH 9 - 11 was prepared with glycine-sodium hydroxide buffer. An enzyme solution with a final concentration of 0.1 mg / mL was added and reacted at the optimum temperature for 5 min to measure the enzyme activity. From Figure 7, Figure 8 It can be seen that the optimal reaction pH of the wild-type enzyme is 9, the optimal reaction pH of mutants S136C, A205P, L324V and K326N is 8, the optimal reaction pH of mutants T94V and G303N is the same as that of the wild-type, which is 9, and the optimal pH of the remaining mutant sites is 10. Although there are slight differences from the wild-type, the differences are not significant.

[0171] Example 6: Determination of the kinetic stability of wild enzymes and mutants

[0172] To determine the kinetic stability of wild-type alginate lyase WT and mutants T94V, S136C, A205P, N255R, G303N, L324V, K326N, D353V, M363T, T385V, L324V-D353V, L324V-M363T, L324V-T385V, D353V-M363T, D353V-T385V, M363T-T385V, L324V-D353V-M363T, L324V-D353V-T385V, L324V-M363T-T385V, D353V-M363T-T385V and L324V-D353V-M363T-T385V, the specific steps are as follows:

[0173] (1) Heat an appropriate amount of enzyme solution at different temperatures for different times, and then quickly cool it in an ice bath for 10 min;

[0174] (2) Take 200 μL of 6 mg·mL -1 alginate solution (pH = 9.0) into a 2 mL centrifuge tube and preheat it at the optimal temperature;

[0175] (3) Add 10 μL of enzyme solution, shake well, react for 5 min, and immediately add 150 μL of DNS, and terminate the reaction in a boiling water bath for 5 min;

[0176] (4) After the sample is cooled to room temperature, make up the volume to 1 mL and measure the absorbance at a wavelength of 520 nm, and calculate the enzyme activity. Define the initial enzyme activity without heat treatment as 100%, and the residual enzyme activity is the percentage value of the enzyme activity at different times to the initial enzyme activity. According to the relationship between the natural logarithm of the relative residual enzyme activity and time (lnA = K d ×t, where A represents the residual enzyme activity, K d is the first-order inactivation rate constant, and t is the treatment time), calculate the first-order inactivation rate constant K d , and calculate t 1 / 2 = ln2 / K d . Thus, the time when the enzyme activity of Pedsa0632 wild-type and mutants is reduced to 50% of the initial enzyme activity is t 1 / 2。

[0177] The results are shown in Table 4.

[0178] Table 4 Kinetic stability parameters of wild type and its mutants

[0179]

[0180] The results showed that the t 1 / 2，60℃ , and the t 1 / 2，65℃ of triple- and quadruple-site combinatorial mutants, and the half-life of single mutants at 55 °C was 2.79 - 40.77 times that of the wild type, the half-life of combinatorial mutants at 55 °C was 15.60 - 946.30 times that of the wild type, and the half-life of combinatorial mutants at 60 °C was 18.39 - 686.83 times that of the wild type. Among them, those with significant effects were L324V (t 1 / 2，55℃ increased by 40.77 times), D353V (t 1 / 2，55℃ increased by 33.12 times), M363T (t 1 / 2，55℃ increased by 21.20 times), T385V (t 1 / 2，55℃ increased by 26.50 times), D353V-T385V (t 1 / 2，55℃ increased by 278.91 times, t 1 / 2，60℃ increased by 145.74 times), L324V-D353V-M363T (t 1 / 2，55℃ increased by 588.81 times, t 1 / 2，60℃ increased by 263.23 times), D353V-M363T-T385V (t 1 / 2，55℃ increased by 529.93 times, t 1 / 2，60℃ increased by 304.87 times) and L324V-D353V-M363T-T385V (t 1 / 2，55℃ increased by 946.30 times, t 1 / 2，60℃ increased by 686.83 times). The t 1 / 2 of all mutants was increased, and the heat resistance was improved, as shown in Table 4.

[0181] Example 7: Determination of thermal stability of mutant wild enzyme and mutants

[0182] The thermal stability was determined according to the following steps:

[0183] (1) Take 200 μL of 6 mg·mL -1 alginate solution (pH = 9.0) in a 2 mL centrifuge tube and preheat it at the optimal temperature. Incubate an appropriate amount of enzyme in a water bath at 55 °C, 60 °C, and 65 °C for different times;

[0184] (2) Add 10 μL of enzyme solution, mix well by shaking, react for 5 min, and immediately add 150 μL of DNS. Terminate the reaction in a boiling water bath for 5 min.

[0185] (3) After the sample is cooled to room temperature, make up the volume to 1 mL and measure the absorbance at a wavelength of 520 nm, and calculate the enzyme activity.

[0186] The results are as Figure 9 shown. The thermal stability of the mutants is significantly enhanced at 55 °C. After the wild-type enzyme Pedsa0632 is incubated at 55 °C for 15 min, the enzyme activity loss is 77.73%, and it is basically inactivated after incubation for 30 min.

[0187] The mutants S136C, N255R, G303N, A205P, K326N, and T94V can respectively retain 71.49%, 77.77%, 78.79%, 87.08%, 71.04%, and 85.89% of the original enzyme activity after incubation at 55 °C for 10 min, and can respectively retain 33.73%, 51.23%, 44.42%, 29.79%, 61.98%, and 72.35% of the original enzyme activity after incubation for 30 min.

[0188] The mutants M363T, T385V, L324V, and D353V can respectively retain 96.99%, 94.91%, 95.56%, and 96.21% of the original enzyme activity after incubation at 55 °C for 10 min, and can respectively retain 94.30%, 93.00%, 93.30%, and 94.80% of the original enzyme activity after incubation for 30 min, and can respectively retain 50.16%, 59.45%, 71.59%, and 72.72% of the original enzyme activity after incubation for 120 min.

[0189] The thermal stability of the combined mutants is significantly enhanced at 55 °C; the double-point combined mutants L324V-D353V, L324V-M363T, L324V-T385V, D353V-M363T, D353V-T385V, and M363T-T385V can respectively retain 93.08%, 57.46%, 85.19%, 89.78%, 96.10%, and 51.22% of the original enzyme activity after incubation for 120 min under the same conditions.

[0190] The three-point combined mutants L324V-D353V-M363T, L324V-D353V-T385V, L324V-M363T-T385V, and D353V-M363T-T385V can respectively retain 96.76%, 109.94%, 21.83%, and 97.17% of the original enzyme activity after incubation for 1270 min under the same conditions.

[0191] The four-point combinatorial mutant L324V-D353V-M363T-T385V can retain 91.54% of its activity after incubation for 2400 min and 67.26% of its activity after incubation for 9700 min under the same conditions.

[0192] The results of the enzyme thermal stability experiment at 60 °C are as Figure 10 shown, and the thermal stability of the mutant is significantly enhanced. After incubation of the wild-type enzyme Pedsa0632 at 60 °C for 2 min, only 40.74% of the original enzyme activity is retained, and after incubation for 5 min, the activity is almost completely lost.

[0193] The combinatorial mutants L324V-D353V, L324V-M363T, L324V-T385V, D353V-M363T, D353V-T385V, and M363T-T385V can retain 86.89%, 42.70%, 89.18%, 94.85%, 91.35%, and 36.78% of the original enzyme activity respectively after incubation for 40 min under the same conditions. Among them, L324V-D353V and D353V-T385V can retain 57.71% and 64.78% of the original enzyme activity respectively after incubation for 120 min under the same conditions.

[0194] The combinatorial mutants L324V-D353V-M363T, L324V-D353V-T385V, L324V-M363T-T385V, D353V-M363T-T385V, and L324V-D353V-M363T-T385V can retain 94.90%, 92.76%, 67.16%, 96.25%, and 100.64% of the original enzyme activity respectively after incubation for 120 min under the same conditions. Among them, L324V-D353V-M363T, L324V-D353V-T385V, D353V-M363T-T385V, and L324V-D353V-M363T-T385V can retain 60.64%, 77.64%, 59.33%, and 92.29% of the original enzyme activity respectively after incubation for 300 min under the same conditions.

[0195] The results of the enzyme thermal stability experiment at 65 °C are as Figure 11As shown, the wild-type enzyme Pedsa0632 was inactivated too quickly to be measured at 65°C. After incubation for 30 min under the same conditions, the combined mutants L324V-D353V-M363T, L324V-D353V-T385V, L324V-M363T-T385V, D353V-M363T-T385V, and L324V-D353V-M363T-T385V retained 72.30%, 78.21%, 25.05%, 68.80%, and 90.59% of their original enzyme activities, respectively. Among them, L324V-D353V-M363T-T385V still retained 66.54% of its original enzyme activity after incubation for 150 min under the same conditions.

[0196] Example 8: Determination of kinetic parameters of mutants

[0197] Prepare alginate substrate solutions with concentrations of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, and 1.4% using glycine-sodium hydroxide buffer (pH = 9) and glycine-sodium hydroxide buffer (pH = 10). Add enzyme solution with a final concentration of 0.1 mg / mL and react under optimal conditions to measure the amount of reducing sugar produced. Perform non-linear fitting to obtain the kinetic parameters of the wild-type and four-point combined mutants as shown in Table 5.

[0198] Table 5 Kinetic parameters of wild-type and its mutants

[0199]

[0200] From the data in Table 5, it can be seen that the K m decreases, and the K cat value increases, indicating that after mutation, L324V-D353V-M363T-T385V has enhanced affinity for the substrate and increased catalytic rate. Finally, the catalytic efficiency K cat / K m fluctuates little but is higher than that of the wild-type, which is consistent with the conclusion of relative enzyme activity.

[0201] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An alginate lyase mutant, characterized in that, Based on the alginate lyase shown in SEQ ID NO.1, at least one amino acid at position 94, 136, 205, 255, 303, 324, 326, 353, 363 or 385 is mutated.

2. The alginate lyase mutant according to claim 1, wherein Based on the alginate lyase shown in SEQ ID NO.1, having any one of the mutations (a) to (l): (a) Mutating the threonine at position 94 to valine, or mutating the serine at position 136 to cysteine, or mutating the alanine at position 205 to proline, or mutating the asparagine at position 255 to arginine, or mutating the glycine at position 303 to asparagine, or mutating the leucine at position 324 to valine, or mutating the lysine at position 326 to asparagine, or mutating the aspartic acid at position 353 to valine, or mutating the methionine at position 363 to threonine, or mutating the threonine at position 385 to valine; (b) Mutating the leucine at position 324 to valine and mutating the aspartic acid at position 353 to valine; (c) Mutating the leucine at position 324 to valine and mutating the methionine at position 363 to threonine; (d) Mutating the leucine at position 324 to valine and mutating the threonine at position 385 to valine; (e) Mutating the aspartic acid at position 353 to valine and mutating the methionine at position 363 to threonine; (f) Mutating the aspartic acid at position 353 to valine and mutating the threonine at position 385 to valine; (g) Mutating the methionine at position 363 to threonine and mutating the threonine at position 385 to valine; (h) Mutating the leucine at position 324 to valine, mutating the aspartic acid at position 353 to valine, and mutating the methionine at position 363 to threonine; (i) Mutating the leucine at position 324 to valine, mutating the aspartic acid at position 353 to valine, and mutating the threonine at position 385 to valine; (j) Mutating the leucine at position 324 to valine, mutating the methionine at position 363 to threonine, and mutating the threonine at position 385 to valine; (k) Mutating the aspartic acid at position 353 to valine, mutating the methionine at position 363 to threonine, and mutating the threonine at position 385 to valine; (l) Mutating the leucine at position 324 to valine, mutating the aspartic acid at position 353 to valine, mutating the methionine at position 363 to threonine, and mutating the threonine at position 385 to valine.

3. A gene encoding the alginate lyase mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. The recombinant vector according to claim 4, wherein The expression vector includes, but is not limited to, pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series vectors.

6. A recombinant microbial cell expressing the alginate lyase mutant according to claim 1 or 2, or containing the gene according to claim 3, or containing the recombinant vector according to claim 4 or 5.

7. Recombinant Escherichia coli, characterized in that, Express the alginate lyase mutant described in claim 1 or 2 in Escherichia coli E. coli BL21(DE3).

8. A method for improving the thermal stability of alginate lyase, characterized in that, Perform one or more of the following mutations on the alginate lyase with the amino acid sequence shown in SEQ ID NO.1: (1) Mutate the threonine at position 94 to valine; (2) Mutate the serine at position 136 to cysteine; (3) Mutate the alanine at position 205 to proline; (4) Mutate the asparagine at position 255 to arginine; (5) Mutate the glycine at position 303 to asparagine; (6) Mutate the leucine at position 324 to valine; (7) Mutate the lysine at position 326 to asparagine; (8) Mutate the aspartic acid at position 353 to valine; (9) Mutate the methionine at position 363 to threonine; (10) Mutate the threonine at position 385 to valine.

9. A composition containing the alginate lyase mutant described in claim 1 or 2.

10. An enzyme preparation, characterized in that, Using the alginate lyase mutant described in claim 1 or 2 as the main enzyme component and containing one or more adjuvants.

11. The application of the alginate lyase mutant described in claim 1 or 2, or the gene described in claim 3, or the recombinant microbial cell described in claim 6, or the recombinant Escherichia coli described in claim 7 in the fields of food, health products or medicine.

12. The application according to claim 11, characterized in that, The application includes, but is not limited to, hydrolyzing alginate or preparing products containing alginate oligosaccharides.

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