Beta-1, 3-glucanase derived from Paenibacillus phocasia and mutant with improved enzyme activity and thermal stability of beta-1, 3-glucanase

Abstract

The invention discloses beta-1, 3-glucanase derived from Paenibacillus phocaensis and application of the beta-1, 3-glucanase, and belongs to the field of genetic engineering and modern enzyme technology engineering. The enzyme activity of the wild type beta-1, 3 glucanase provided by the invention is as high as 129.62 U / mg. A curdlan oligosaccharide product produced by adopting the enzyme mainly comprises DP4-6 oligosaccharides (accounting for 68.30%) and is concentrated in distribution (DP1-20), wild type beta-1, 3-glucanase is modified by utilizing a site-directed mutagenesis technology to obtain mutants N181M, L164E, N170E, P175T, G176I and G182H, compared with wild enzymes, the enzyme activity is improved by 1.25-1.62 times, the enzyme activity of the wild type beta-1, 3-glucanase is optimized and improved, and the enzyme activity of the wild type beta-1, 3-glucanase is improved. Better use conditions are created for the enzyme in practical application.

Description

Technical Field

[0001] This invention relates to a β-1,3-glucanase and its application in the production of glucan oligosaccharides, belonging to the fields of genetic engineering and modern enzyme technology engineering. Background Technology

[0002] Curdlan oligosaccharides (COS) are linear glucosinolates linked by β-1,3-glycosidic bonds, widely found in yeast, fungi, and some algae. Besides inducing plant resistance, COS has also been shown to possess various biological activities, including antitumor activity, immunomodulation, and wound healing promotion. Due to its low toxicity, high stability, and good biocompatibility, COS shows great application potential in medicine, functional foods, and agricultural production.

[0003] Currently, the main industrial methods for producing curdran oligosaccharides include chemical hydrolysis and enzymatic hydrolysis. While chemical methods can obtain oligosaccharides with specific degrees of polymerization by controlling reaction conditions, they generally suffer from high energy consumption, severe pollution, and low product purity, limiting their large-scale application. In contrast, enzymatic hydrolysis has become a research hotspot due to its advantages such as mild reaction conditions, high specificity, and environmental friendliness.

[0004] Currently, the enzymes used to produce glideran oligosaccharides are mainly endo-β-1,3-glucanases, among which the GH16 and GH17 family enzymes are the most studied. However, these enzymes often produce an excessively high proportion of glucose (DP1), reducing the yield of the target oligosaccharide, and their stability is insufficient, making them difficult to meet the needs of industrial production. These technical bottlenecks severely restrict the industrialization process of enzymatic production of glideran oligosaccharides. The GH64 family is a unique class of β-1,3-glucanases that mainly generate oligosaccharides with specific degrees of polymerization, such as DP3 to DP6, through an intrachain cleavage mechanism, exhibiting higher substrate specificity and controllability compared to other enzyme families. However, most of the reported GH64 family enzymes are derived from terrestrial actinomycetes or Bacillus, and their catalytic efficiency and adaptability to industrial substrates (such as glideran powder) still have considerable room for optimization. In addition, research on GH64 enzymes derived from extreme environments (such as high temperature, high salt, and acidic conditions) remains insufficient, limiting the widespread application of this family of enzymes in industrial production. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention is the first to identify a GH64 family β-1,3-glucanase from the marine microorganism *Paenibacillus phocaensis*, whose natural form exhibits excellent catalytic activity under acidic and high-temperature conditions. Furthermore, this invention significantly improves the specific activity and thermostability of this enzyme through key site mutations (such as modification of the hydrophobic core or flexible loop region adjacent to the catalytic residue), providing a novel solution for the efficient and green production of kerogen oligosaccharides.

[0006] To address the above problems, the present invention provides a GH64 family β-1,3-glucanase (Endo-β-1,3-glucanase, EC3.2.1.39) with high enzymatic activity against currant gum, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the above-mentioned β-1,3-glucanase, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] This invention involves molecularly modifying the β-1,3-glucanase gene (nucleotide sequence shown in SEQ ID NO.2, amino acid sequence shown in SEQ ID NO.1) from Paenibacillus phocaensis (accession number WP_110945382.1) and using site-directed mutagenesis to obtain a β-1,3-glucanase mutant with significantly improved enzyme activity and thermostability.

[0009] The first technical solution provided by the present invention is a β-1,3-glucanase mutant, which is obtained by mutating one or more of the amino acids at positions 181, 164, 170, 175, 176 and 182 of the β-1,3-glucanase parent amino acid sequence as shown in SEQ ID NO.1.

[0010] In one embodiment of the present invention, the mutant is obtained by mutating the asparagine at position 181, leucine at position 164, asparagine at position 170, proline at position 175, glycine at position 176, and / or glycine at position 182 of the β-1,3-glucanase parent whose amino acid sequence is as shown in SEQ ID NO.1.

[0011] In one embodiment of the present invention, the mutant is obtained by mutating asparagine at position 181 of the β-1,3-glucanase parent amino acid sequence as shown in SEQ ID NO.1 to methionine, thus obtaining mutant N181M.

[0012] In one embodiment of the present invention, the mutant is obtained by mutating leucine at position 164 of β-1,3-glucanase, as shown in SEQ ID NO.1, to glutamic acid, thus obtaining mutant L164E.

[0013] In one embodiment of the present invention, the mutant is obtained by mutating asparagine at position 170 of the β-1,3-glucanase parent amino acid sequence as shown in SEQ ID NO.1 to glutamic acid, thus obtaining mutant N170E.

[0014] In one embodiment of the present invention, the mutant is obtained by mutating proline at position 175 of the β-1,3-glucanase parent amino acid sequence as shown in SEQ ID NO.1 to threonine, thus obtaining mutant P175T.

[0015] In one embodiment of the present invention, the mutant is obtained by mutating glycine at position 176 of the β-1,3-glucanase parent amino acid sequence as shown in SEQ ID NO.1 to isoleucine, thus obtaining mutant G176I.

[0016] In one embodiment of the present invention, the mutant is obtained by mutating glycine at position 182 of the β-1,3-glucanase parent amino acid sequence as shown in SEQ ID NO.1 to histidine, thus obtaining mutant G182H.

[0017] The present invention also provides a second technical solution, which is a gene encoding the mutant described in the first technical solution.

[0018] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.

[0019] In one embodiment of the present invention, the expression vector of the recombinant plasmid includes, but is not limited to, pET series plasmids.

[0020] In one embodiment of the present invention, the pET series plasmids include pET-28a or pET-22b.

[0021] The fourth technical solution provided by the present invention is a recombinant microbial cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant plasmid described in the third technical solution.

[0022] In one embodiment of the present invention, the recombinant microbial cells include, but are not limited to, bacteria or fungi.

[0023] In one embodiment of the present invention, the microorganism is Escherichia coli.

[0024] In one embodiment of the present invention, the recombinant microbial cells use Escherichia coli BL21(DE3) as the host and pET-28a as the vector.

[0025] The fifth technical solution provided by the present invention is a method for improving the activity and / or thermal stability of β-1,3-glucanase, wherein the method involves mutating the asparagine at position 181, leucine at position 164, asparagine at position 170, proline at position 175, glycine at position 176, or glycine at position 182 of the β-1,3-glucanase parental amino acid sequence as shown in SEQ ID NO.1.

[0026] In one embodiment of the present invention, the method involves mutating asparagine at position 181 of the β-1,3-glucanase parent amino acid sequence as shown in SEQ ID NO.1 to methionine, or mutating leucine at position 164 to glutamic acid, or mutating asparagine at position 170 to glutamic acid, or mutating proline at position 175 to threonine, or mutating glycine at position 176 to isoleucine, or mutating glycine at position 182 to histidine.

[0027] The sixth technical solution provided by the present invention is a method for preparing β-1,3-glucan oligosaccharides, wherein the method uses β-glucan as a substrate and utilizes the β-1,3-glucanase mutant or β-1,3-glucanase parent described in the first technical solution to catalyze the generation of β-1,3-glucan oligosaccharides.

[0028] The present invention also provides a seventh technical solution, which is the application of the β-1,3-glucanase parent, or the β-1,3-glucanase mutant described in the first technical solution, or the gene described in the second technical solution, or the recombinant plasmid described in the third technical solution, or the recombinant microorganism described in the fourth technical solution, or the method described in the fifth technical solution, in the production of β-1,3-glucan oligosaccharides, wherein the amino acid sequence of the β-1,3-glucanase parent is shown in SEQ ID NO.1.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention provides a β-1,3-glucanase with an amino acid sequence as shown in SEQ ID NO.1. The molecular weight of this β-1,3-glucanase is about 50 kDa, and the enzyme activity is as high as 129.62 U / mg at a concentration of 4% (w / v) of guaran polysaccharide, at pH 4.5 and 70°C.

[0031] (2) The curdran oligosaccharide products produced by the method of this invention are mainly oligosaccharides with DP4-6 (accounting for 68.30%), and their distribution is concentrated (DP1-20), avoiding byproducts (such as monosaccharides or macromolecular fragments) generated by traditional chemical degradation or non-specific enzymatic hydrolysis, and significantly reducing subsequent purification costs; the reaction is carried out under acidic conditions, reducing the consumption of acid-base adjustment reagents and the pressure on wastewater treatment. Therefore, this β-1,3-glucanase has great application prospects in the production of curdran oligosaccharides.

[0032] (3) This invention uses β-1,3-glucanase from Paenibacillus phocaensis as the parent enzyme for molecular modification. Mutations were made at amino acids 181, 164, 170, 175, 176, and 182, resulting in a series of mutants with increased enzyme activity. The β-1,3-glucanase mutants provided by this invention show significantly increased enzyme activity compared to the wild-type enzyme. The mutant enzymes maintain an acidic pH and exhibit activities of 162.61 U / mg, 165.87 U / mg, 173.85 U / mg, 189.66 U / mg, 198.56 U / mg, and 209.57 U / mg, respectively, representing an increase of 1.25 to 1.62 times compared to the wild-type enzyme activity.

[0033] (4) The thermostability of the series of β-1,3-glucanase mutants provided by this invention is significantly improved compared with that of the wild-type enzyme. The Tm values ​​of mutants N181M, L164E, N170E, P175T, G176I, and G182H increased from 69.72℃ in the wild-type enzyme to 70.51℃, 70.42℃, 70.43℃, 70.55℃, 70.66℃, and 70.46℃, respectively. This invention optimizes and improves the enzyme activity and thermostability of wild-type β-1,3-glucanase, creating better conditions for its practical application. Attached Figure Description

[0034] Figure 1 SDS-PAGE analysis of recombinant Escherichia coli expression products; M: Marker; 1: PP whole cells; 2: PP lysate supernatant; 3: PP lysate; 4: PP purified enzyme.

[0035] Figure 2 This is a standard curve for glucose concentration.

[0036] Figure 3 The effects of temperature and pH on β-1,3-glucanase activity.

[0037] Figure 4 The following are thin-layer chromatograms of the reaction solution; A: different reaction times, 5–180 min; B: different enzyme dosages, pure enzyme diluted 0.5–160 times.

[0038] Figure 5 The image shows the high-performance anion exchange chromatogram of the reaction solution; 1: mobile phase; 2: glucose; 3: β-1,3-glucobiose; 4: β-1,3-glucotriose; 5: β-1,3-glucotetraose; 6: β-1,3-glucpentose; 7: β-1,3-gluchexaose; 8: β-1,3-glucheptaose; 9: β-1,3-glucoctaose; 10: β-1,3-glucnonaose; 11: β-1,3-glucadecanose; 2: β-1,3-gluconeose; 13: β-1,3-glucodecaose; 14: β-1,3-glucetadexose; 15: β-1,3-glucotedaose; 16: β-1,3-glucadevinose; 17: β-1,3-glucadehexadecose; 18: β-1,3-glucadeheptadecose; 19: β-1,3-glucadeoctadecose; 20: β-1,3-glucadenodeaose; 21: β-1,3-glucotetidecose.

[0039] Figure 6 This is a construction map of the recombinant plasmid.

[0040] Figure 7 The relative enzyme activities of wild-type enzyme PpGH64 and mutant enzyme are given. Detailed Implementation

[0041] Reference Appendix Figures 1-7 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0042] Test method:

[0043] 1. Protein concentration was determined using the BCA protein kit. The specific steps are as follows:

[0044] (1) Preparation of BCA working solution. Based on the number of samples, BCA working solution was prepared by mixing BCA Reagent A and B at a volume ratio of 50:1 and then thoroughly mixed.

[0045] (2) Draw the standard curve.

[0046] (3) Sample preparation: Dilute the protein sample to be tested with deionized water to an appropriate concentration, and add 200 μL of LCA working solution to 20 μL of sample.

[0047] (4) After shaking and mixing, place at 37°C for 20 to 30 minutes.

[0048] (5) Use the absorbance value without BSA as a blank control, and use an ELISA reader to detect the sample in A. 562 Absorbance value at nm.

[0049] (6) Plot a standard curve with protein content (μg) on ​​the x-axis and absorbance on the y-axis.

[0050] (7) The protein content of the sample can be calculated by using a linear equation in two variables based on the measured absorbance value.

[0051] (8) Calculation of protein concentration: Divide the obtained protein content by the sample volume (20 μL) and multiply by the corresponding dilution factor to determine the true concentration of the sample.

[0052] 2. Enzyme activity assay of recombinant enzymes

[0053] Enzyme activity assays for wild-type PpGH64 and mutants were performed using the dinitrosalicylic acid (DNS) method. Standard reaction conditions were as follows: a total volume of 1 mL, with the reaction mixture consisting of 4% (w / v) quercetin polysaccharide, 50 mM citrate-sodium citrate buffer (pH 4.5), and an appropriate amount of fermentation broth or purified enzyme. The mixture was incubated at 70 °C and 220 rpm for 10 min in a water bath, centrifuged, and 300 μL was added to 500 μL of DNS reagent to stop the assay. The mixture was then boiled at 100 °C for 20 min to inactivate the enzyme. The reaction was measured spectrophotometrically at 540 nm. PpGH64 activity was determined by measuring the amount of reducing sugar released (based on glucose).

[0054] Recombinant PpGH64 enzyme activity definition (1U): The amount of enzyme required to catalyze the hydrolysis of 1 μmol of glucose per minute using 4% (w / v) kerogen polysaccharide as a substrate under the experimental test conditions. All experiments were repeated three times.

[0055] Raw materials used in the examples:

[0056] Materials and Reagents: Restriction endonucleases, Dpn I enzyme PCR reagents, etc., were purchased from TaKaRa Biotechnology Co., Ltd.; primers were purchased from Anshengda Biotechnology Co., Ltd.; plasmid extraction kit, genome extraction kit, agarose purification kit, E. coil DH5α, E. coil BL21(DE3) strains, yeast extract, tryptone, agar, sodium chloride, glucose, electrophoresis buffer (TAE), nucleic acid dye (4S Green Plus), kanamycin (Kan), isopropyl-β-D-thiogalactoside (IPTG), agarose, imidazole (C3H4N2), rapid competent cell preparation kit (one-step method), BCA method protein concentration assay kit for recombinant protein concentration determination, and nickel ion chelate affinity chromatography packing material were purchased from Sangon Biotech (Shanghai) Engineering Co., Ltd.; DNS reagent was purchased from Feijing Biotechnology (Fuzhou) Co., Ltd.; protein loading buffer (non-reducing 5×) and protein marker were purchased from Shanghai EpiZyme Biotechnology Co., Ltd. Disodium EDTA·2Na, citric acid (C6H8O7), sodium citrate (C6H7NaO7), tris(hydroxymethyl)aminomethane (Tris), sodium dodecyl sulfate (SDS), glycine, and other reagents of analytical grade or higher were purchased from Sinopharm Chemical Co., Ltd. Novozymes Biotechnology Co., Ltd. (Nanjing, China) provided chemical reagents and supplies for protein purification. Kederan polysaccharide was provided by Shandong Jinyang Pharmaceutical Co., Ltd. (Shandong, China). Agarose gel DNA recovery kits and plasmid extraction kits were purchased from Novozymes (Nanjing) Biotechnology Co., Ltd.

[0057] The culture media involved in the following examples are as follows:

[0058] All culture media were prepared using ddH2O and sterilized at 121℃ for 15–20 min after preparation.

[0059] LB liquid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0060] LB solid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L.

[0061] Example 1: Production of β-1,3-glucanase

[0062] The specific steps are as follows:

[0063] 1. Construction of recombinant plasmids

[0064] (1) Screening enzymes

[0065] The target protein, β-1,3-glucanase, derived from Paenibacillus phocaensis (protein sequence accession number WP_110945382.1), was used.

[0066] (2) Synthesis of the target gene

[0067] The target protein gene fragment with the sequence shown in SEQ ID NO.2 was synthesized, and PCR amplification reaction was performed using pET-28a(+) as a vector and PpGH64-F and PpGH64-R as upstream and downstream primers.

[0068] The recombinant plasmid pET-28a(+)-PpGH64 was prepared.

[0069] PpGH64-F: 5'-TAATACGACTCACTATAGGG-3';

[0070] PpGH64-R: 5'-TGCTAGTTATTGCTCAGCGG-3'.

[0071] The PCR reaction conditions are shown in Tables 1 and 2.

[0072] Table 1 Reaction System

[0073]

[0074] Table 2 Reaction conditions

[0075]

[0076] 2. Construction of engineered bacteria

[0077] The recombinant plasmid pET-28a(+)-PpGH64 obtained in step 1 was transformed into E. coli BL21(DE3) competent cells by thermal shock chemical transformation to obtain the corresponding recombinant engineered bacteria E. coli BL21(DE3) / pET-28a(+)-PpGH64.

[0078] 3. Expression Induction

[0079] (1) The recombinant engineered bacteria E.coli BL21(DE3) / pET-28a(+)-PpGH64 obtained in step 2 was streaked from the glycerol tube to LB solid medium plates (containing 50 μg / mL Kan, and incubated upside down at 37℃ for 12 h).

[0080] (2) Pick a single colony from LB solid medium and culture it in 5 mL of LB liquid medium containing 50 μg / mL Kan for 12 h at 37 °C and 200 r / min to prepare seed liquid.

[0081] (3) The prepared seed culture was transferred to 50 mL of LB liquid medium containing Kan at an inoculation rate of 2% (v / v) and cultured at 37°C and 200 r / min until OD. 600 The pH value was around 0.6. IPTG was added to bring the final concentration of IPTG in the culture medium to 0.1 mmol / L. After induction for 24 h at 16℃ and 200 r / min, the fermentation broth was obtained.

[0082] (4) Centrifuge the fermentation broth at 4℃ and 6000rpm for 10min. Resuspend the cells in cell lysis buffer, then sonicate for 15min (1s sonication, 2s rest). Take 40μL of crude enzyme solution, i.e., whole cells, lysate supernatant, and precipitate, for protein gel electrophoresis at 120V for 1h. Figure 1 As shown, the molecular weight of β-1,3-glucanase is around 50 kDa.

[0083] 4. Protein purification

[0084] The fermentation broth obtained in step 3 (3) was centrifuged at 4℃ and 6000rpm for 10min, the supernatant was removed, and the broth was resuspended in an equal volume of 50mM phosphate buffer (pH=7.0). Then, it was sonicated for 15min (1s supernatant, 2s rest), centrifuged again, and the supernatant was collected. Impurities were removed by passing the supernatant through a 0.45μm aqueous membrane. The supernatant was then connected to the constant flow pump and Ni in the protein purification system. 2+ For the tubing connecting the affinity chromatography column, UV detector, and other components, set the constant flow pump to 1 mL / min and check for leaks using deionized water. After the protein purification system is ready, equilibrate Ni with two column volumes of binding buffer (50 mM Tris, 500 mM NaCl, pH = 7.0). 2+Affinity chromatography column was used, and the crude protein solution was pumped into the column at a flow rate of 0.5 mL / min. After all the crude protein solution had entered the column, unadsorbed protein and other impurities were washed away with binding buffer. After the detector reading stabilized, washing buffer (50 mM imidazole, 50 mM Tris, 500 mM NaCl, pH = 7.0) was pumped in to wash away weakly binding proteins. After the detector reading stabilized again, elution buffer (500 mM imidazole, 50 mM Tris, 500 mM NaCl, pH = 7.0) was pumped in to elute the adsorbed recombinant protein, and the eluent was collected according to the UV detector signal value, which is the target recombinant protein. The target recombinant protein solution obtained above was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa. After clamping with dialysis clamps, the bag was placed in dialysis solution A (10 mM EDTA·2Na, 50 mM Tris, pH = 7.0) and incubated in a chromatography cabinet at 4°C for 18 hours. The dialysis solution was replaced with fresh dialysis solution every 6 hours to remove imidazole and other metal ions from the target recombinant protein solution. The dialysis bag was then transferred to dialysis solution B (50 mM Tris, pH = 7.0) and dialyzed for another 18 hours, with fresh dialysis solution replaced every 6 hours. After dialysis, the target recombinant protein solution was collected into a tube, which is the pure enzyme solution.

[0085] Example 2: Performance of β-1,3-glucanase

[0086] The enzyme activity of the purified target recombinant protein β-1,3-glucanase prepared in Example 1 was determined, and the specific steps are as follows:

[0087] (1) Preparation method of colloidal kojantan polysaccharide:

[0088] 2g of commercially available kerogen polysaccharide was resuspended in 50mL of buffer (50mM citrate-sodium citrate buffer, pH=4.5) to form a 4% (w / v) kerogen polysaccharide suspension.

[0089] The kerogen polysaccharide suspension was placed in a magnetically stirred water bath and heated at 70°C for 4 hours. After this treatment, the kerogen polysaccharide suspension formed a homogeneous gel, and 4% (w / v) colloidal kerogen polysaccharide was prepared.

[0090] (2) The reducing sugar content in the enzyme solution prepared in Example 1 was determined using the DNS method:

[0091] To construct the DNS standard curve: First, prepare a 1 mg / mL glucose solution and dilute it with 50 mM citrate-sodium citrate buffer (pH = 4.5) to prepare glucose standard solutions with concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL.

[0092] Add 950 μL of the prepared 4% (w / v) colloidal koran polysaccharide and 50 μL of enzyme solution diluted 5 times to a 2 mL centrifuge tube, react at 70 °C for 10 min, boil to inactivate, centrifuge to obtain the reaction solution.

[0093] Take 300 μL of the reaction solution, add 500 μL of DNS and mix well. Incubate in a boiling water bath for 7 min. After cooling to room temperature, add 500 μL of distilled water. Measure the absorbance at 540 nm and plot a glucose standard curve. Figure 2 ).

[0094] The glucose concentration of the sample was calculated using the DNS glucose standard curve.

[0095] (3) Protein concentration was determined using the BCA method:

[0096] a. Preparation of BCA working solution. Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA Reagent B to 50 volumes of BCA Reagent A (50:1) and mix thoroughly.

[0097] b. Standard curve plotting. Take an ELISA plate and add reagents according to the data in Table 3.

[0098] Table 3 Reagents for ELISA plates

[0099]

[0100] c. Sample preparation: Dilute the protein sample to be tested with deionized water to an appropriate concentration, take 20 μL of sample and add 200 μL of BCA working solution.

[0101] d. After shaking and mixing, place at 37°C for 20–30 min.

[0102] e. Measure the absorbance at 562 nm using an ELISA reader, with the absorbance value without BSA as a blank control.

[0103] f. Plot a standard curve with protein content (μg) on ​​the x-axis and absorbance on the y-axis.

[0104] g. The protein content of the sample can be calculated from the standard curve based on the measured absorbance value.

[0105] h. Calculate protein concentration: Divide the protein content found by the enzyme solution volume of 20 μL, and then multiply by the corresponding dilution factor to obtain the actual concentration of the sample to be tested.

[0106] The enzyme activity unit (1U) is defined as: the amount of glucose required to produce 1 μmol per minute under the above reaction conditions.

[0107] The formula for calculating the enzyme activity of β-1,3-glucanase is as follows:

[0108]

[0109] The test results showed that the pure enzyme solution yielded the following results:

[0110] The protein concentration in the enzyme solution was 0.35 mg / mL, and the enzyme activity was 129.62 U / mg.

[0111] Example 3: Determination of the properties of β-1,3-glucanase

[0112] The activity of the recombinant enzyme according to the present invention was measured as a function of temperature in the pH range of 2.0 to 5.0 and in the temperature range of 40°C to 90°C using the DNS method.

[0113] The specific steps are as follows:

[0114] (1) The recombinant engineered strain E. coli BL21(DE3) / pET-28a(+)-PpGH64 obtained by Example 1 with codon-optimized enzyme was fermented according to the method of Example 1 to obtain the purified target protein β-1,3-glucanase.

[0115] 50 μL of the obtained pure enzyme solution was added to 950 μL of 4% (w / v) colloidal koran polysaccharide prepared at pH=2.0, pH=2.5, pH=3.0, pH=3.5, pH=4.0, pH=4.5, and pH=5.0, respectively. The reaction was carried out at 70℃ for 10 min, then boiled to inactivate the enzyme, centrifuged, and the reaction solution was obtained. The reducing sugar content and enzyme activity data in the crude enzyme solution were determined.

[0116] The preparation method of the 4% (w / v) colloidal kerogen polysaccharide at pH=2.0, pH=2.5, pH=3.0, pH=3.5, pH=4.0, pH=4.5, and pH=5.0 is as follows: 2g of commercially available kerogen polysaccharide is resuspended in 50mL of citrate-sodium citrate buffer (at pH=2.0, pH=2.5, pH=3.0, pH=3.5, pH=4.0, pH=4.5, and pH=5.0, respectively) to form a 4% (w / v) kerogen polysaccharide suspension; the kerogen polysaccharide suspension is placed in a magnetically stirred water bath and heated at 70°C for 4 hours. After this treatment, the kerogen polysaccharide suspension forms a uniform gel, and 4% (w / v) colloidal kerogen polysaccharides with pH=2.0, pH=2.5, pH=3.0, pH=3.5, pH=4.0, pH=4.5, and pH=5.0 are prepared respectively.

[0117] The results show:

[0118] At pH=2.0, the relative enzyme activity is 8.48%;

[0119] At pH = 2.5, the relative enzyme activity is 8.80%;

[0120] At pH=3.0, the relative enzyme activity is 39.49%;

[0121] At pH 3.5, the relative enzyme activity is 65.63%;

[0122] At pH 4.0, the relative enzyme activity is 85.39%;

[0123] At pH = 4.5, the relative enzyme activity is 100%;

[0124] At pH 5.0, the relative enzyme activity is 64.51%.

[0125] (2) 50 μL of pure enzyme solution diluted 5 times obtained in Example 1 was added to 950 μL of 4% (w / v) colloidal polysaccharide with pH=4.5. The reaction was carried out at 40℃, 50℃, 60℃, 70℃, 80℃ and 90℃ for 10 min respectively, then boiled to inactivate, centrifuged to obtain the reaction solution, and the reducing sugar content and enzyme activity data in the crude enzyme solution were determined.

[0126] The results show:

[0127] When reacting at 40℃, the relative enzyme activity was 31.94%.

[0128] When reacting at 50℃, the relative enzyme activity was 53.77%.

[0129] When reacting at 60℃, the relative enzyme activity is 72.04%;

[0130] When reacting at 70℃, the relative enzyme activity is 100.00%;

[0131] When reacting at 80℃, the relative enzyme activity is 43.57%;

[0132] When reacting at 90℃, the relative enzyme activity is 31.57%.

[0133] The results show that ( Figure 3 The optimal pH for the production of kerogen oligosaccharides by recombinant β-1,3-glucanase is 4.5, and the optimal temperature is 70℃.

[0134] Example 4: Identification of Kordan Oligosaccharides

[0135] The specific steps are as follows:

[0136] 2g of commercially available kerogen polysaccharide was resuspended in 50mL of buffer (50mM citrate-sodium citrate buffer, pH=4.5) to form a 4% (w / v) kerogen polysaccharide suspension.

[0137] The kerogen polysaccharide suspension was placed in a magnetically stirred water bath and heated at 70°C for 4 hours. After this treatment, the kerogen polysaccharide suspension formed a homogeneous gel, and 4% (w / v) colloidal kerogen polysaccharide was prepared.

[0138] 50 μL of the pure enzyme solution obtained in Example 1 was added to 950 μL of 4% (w / v) colloidal kojantan polysaccharide prepared at pH 4.5. The mixture was reacted at the optimal conditions of pH 4.5 and 70°C for 3 h, followed by boiling for 20 min for inactivation. The supernatant was collected by centrifugation and filtered through a 0.22 μm membrane. The oligosaccharides in the reaction solution were determined by thin-layer chromatography and anion exchange chromatography, respectively.

[0139] (1) The specific steps of thin-layer chromatography detection are as follows:

[0140] High-performance silica gel thin-layer chromatography was performed using aluminum foil plates; the developing solvent was n-butanol-acetic acid-water (2:1:1, v / v / v). The reference standard was a 0.1% β-1,3-glucpentose standard solution. 1 μL of both the reference standard and the sample were spotted into the developing solvent for chromatography. After chromatography, the plates were dried and then sprayed with a colorimetric reagent (0.2% w / v lichenin, 75% v / v anhydrous ethanol, 10% v / v concentrated sulfuric acid) and developed at 105℃ for 5 min.

[0141] The distribution of kodran oligosaccharides produced by recombinant β-1,3-glucanase was detected as a function of reaction time and different enzyme dosages (pure enzyme diluted 1–160 times). Results are as follows: Figure 4 As shown.

[0142] The results showed that the product had a wide molecular weight distribution, with DP5 oligosaccharides being the main component. The product concentration gradually increased with increasing reaction time and enzyme dosage.

[0143] (2) Anion exchange chromatography:

[0144] HPAEC-PAD: Model DIONEX ICS-5000+SP-5, Thermo Fisher Scientific, USA; Analytical column: CarboPac PA1; Sample loading volume: 25 μL; Elution conditions: 250 mM sodium hydroxide solution containing 1 M sodium acetate for linear elution; Elution time: 20 min; Flow rate: 1 mL / min; Column temperature: 30 °C.

[0145] The results are as follows Figure 5As shown in the figure, the results indicated that the concentration of oligosaccharides produced by the recombinant β-1,3-glucanase reaction solution after 3 hours was detected. Among them, β-1,3-glucopentaose had the highest concentration, accounting for 35.06%, followed by DP1-2 at 5.54%, DP3 at 5.69%, DP4 at 11%, DP6 at 22.24%, DP7 at 8.97%, DP8 at 4.60%, and DP≥9 at 6.88%.

[0146] Example 5: Design of β-1,3-glucanase mutant sites

[0147] The protein structure of β-1,3-glucanase was simulated using SWISS-MODEL software to obtain a tertiary structural model of β-1,3-glucanase. Mutation sites were selected using multiple sequence alignment combined with the online server EVcouplings. Site-directed saturation mutagenesis simulation was performed on the Pp amino acid sequence. The resulting 8170 mutant sequences and wild-type sequences were input into the DLKcat model. The Kcat values ​​were sorted, and the top 100 sequences were selected, showing that they were distributed across 26 amino acid sites. The mutant with the highest Kcat value at each site was experimentally verified. The amino acid sites to be mutated were determined to be asparagine at position 181, leucine at position 164, asparagine at position 170, proline at position 175, glycine at position 176, and glycine at position 182.

[0148] Example 6: Site-directed mutagenesis of β-1,3-glucanase and construction of recombinant plasmids and recombinant Escherichia coli

[0149] Primers were designed based on the β-1,3-glucanase gene of Paenibacillus phocaensis (accession number WP_110945382.1) as shown in SEQ ID NO.2. A mutant plasmid was constructed by site-directed mutagenesis using pET-28a(+)-PpGH64, constructed in Example 1, as a template.

[0150] N181M-F:5'-ACCAAAatgGGCGCCAATTACGCGAACATTTA-3';

[0151] N181M-R:5'-TTGGCGCCcatTTTGGTCAGATGACCCGGCGC-3';

[0152] L164E-F:5'-CCGTgagATTCCGATCAGTTCCAATTTGAACG-3';

[0153] L164E-R:5'-TGATCGGAATctcACGGCCCGACAAATCCAGA-3';

[0154] N170E-F:5'-CAGTTCCgaaTTGAACGATGCGCCGGGTCATC-3';

[0155] N170E-R:5'-CGTTCAAttcGGAACTGATCGGAATCAGACGG-3';

[0156] P175T-F:5'-TTTGAACGATGCGacgGGTCATCTGACCAAAAACGGC-3';

[0157] P175T-R:5'-CcgtCGCATCGTTCAAATTGGAACTGATCGGA-3';

[0158] G176I-F:5'-TGCGCCGattCATCTGACCAAAAACGGCGCCA-3';

[0159] G176I-R:5'-TCAGATGaatCGGCGCATCGTTCAAATTGGAA-3';

[0160] G182H-F:5'-CCAAAAACcacGCCAATTACGCGAACATTTATCA-3';

[0161] G182H-R:5'-ATTGGCgtgGTTTTTGGTCAGATGACCCGGCG-3'.

[0162] The lowercase part represents the codons corresponding to asparagine at position 181, leucine at position 164, asparagine at position 170, proline at position 175, glycine at position 176, and glycine at position 182 encoded by the mutant gene.

[0163] After amplification using the PCR system described in Example 1, 2 μL of DpnⅠ restriction endonuclease (10 U / μL) was added to the reaction solution, and the sample was incubated at 37°C for 2 hours to eliminate the template. The PCR product was transformed into E. coil DH5α cells, plated on LB agar plates, and single colonies were picked and transferred to LB liquid medium. The plasmid was extracted, and sequencing yielded the correct single-point mutant plasmid pET-28a(+)-PpGH64. N181M pET-28a(+)-PpGH64 L164E pET-28a(+)-PpGH64 N170E pET-28a(+)-PpGH64 P175T pET-28a(+)-PpGH64 G176IpET-28a(+)-PpGH64 G182H The successfully constructed mutant plasmid and the wild-type plasmid from step (1) were transformed into E. coil BL21(DE3) to obtain the single-point mutant BL21(DE3) / pET-28a(+)-PpGH64. N181M , BL21(DE3) / pET-28a(+)-PpGH64 L164E , BL21(DE3) / pET-28a(+)-PpGH64 N170E , BL21(DE3) / pET-28a(+)-PpGH64 P175T , BL21(DE3) / pET-28a(+)-PpGH64 G176I , BL21(DE3) / pET-28a(+)-PpGH64 G182H And the strain BL21(DE3) / pET-28a(+)-PpGH64 that expresses wild-type enzymes.

[0164] Example 7: Expression and purification of wild-type and mutant enzymes

[0165] Following the enzyme purification method in Example 1, the specific activity of the obtained pure enzyme solutions was measured. Under these conditions, the original enzyme activity was defined as 100%, and a graph was plotted against mutant types using the percentage of relative enzyme activity. The results of evaluating enzyme activity are as follows: Figure 7 Compared to the wild-type enzyme PpGH64, which has a specific enzyme activity of 129.62 U / mg, the specific enzyme activities of the mutants are as follows: N181M 162.61 U / mg, L164E 165.87 U / mg, N170E 173.85 U / mg, P175T 189.66 U / mg, G176I 198.56 U / mg, and G182H 209.57 U / mg.

[0166] Comparative Example 1:

[0167] Following the same strategy as in Examples 3-5, conserved sites R207 and Y403 were screened, and mutants R207A and Y403A were constructed respectively, along with plasmid pET-28a(+)-PpGH64. R207A pET-28a(+)-PpGH64 Y403A And construct the recombinant strain BL21(DE3) / pET-28a(+)-PpGH64 R207A , BL21(DE3) / pET-28a(+)-PpGH64 Y403AThe recombinant bacteria were cultured using the same method as in Example 5, and the crude enzyme solution and the purified enzyme activity were tested respectively. The results showed that the crude enzyme activity and the enzyme activity of R207A and Y403A were almost undetectable, indicating that the mutants R207A and Y403A lost their enzyme activity. R207A and Y403A are important residues that enable the enzyme to catalyze. After mutation, the enzyme activity was not improved but was lost instead.

[0168] The primers involved in this site-directed mutagenesis example are:

[0169] R207A-F: 5'-TATTACCTCGGGTgctCTGTTCCTGAGCGTAGACTCTCC-3';

[0170] R207A-R: 5'-AGagcACCCGAGGTAATACGCGGCAGGTTCACC-3';

[0171] Y403A-F: 5'-GCTTAGCTgctGGTTTCTGCTATGACGACGTGA-3';

[0172] Y403A-R: 5'-AGAAACCagcAGCTAAGCCATCTATGCTATGATCG-3'.

[0173] Example 8: Characterization of the thermodynamic stability of wild-type and mutant enzymes

[0174] The thermodynamic stability of the recombinase was characterized by measuring its unfolding temperature (Tm) using Nano DSC. The specific steps are as follows:

[0175] (1) After degassing the recombinant enzyme purified dialysate from the last step of Example 3 for 10 min, the dialysate was added to the sample cell and the reference cell. After repeatedly blowing and aspirating to remove air bubbles, a baseline scan was performed. The operating program was set as follows: temperature scan range 20–100 °C, pressure 3 atm, heating rate 1 °C / min, and number of repetitions 5.

[0176] (2) After dialysis, the fresh recombinant enzyme solution was diluted to 1 mg / mL and degassed for 10 min. The dialysate in the sample cell was completely aspirated, and the degassed recombinant enzyme solution was added. After repeatedly blowing and aspirating to remove air bubbles, the sample was scanned. The operating program was set as follows: temperature scan range 20-100℃, pressure 3 atm, and heating rate 1℃ / min.

[0177] (3) The results were processed using NanoAnalyze software, and the data were fitted using a Two-State Scaled model to obtain the Tm value of the recombinase.

[0178] The Tm values ​​of mutants N181M, L164E, N170E, P175T, G176I, and G182H increased from 69.72℃ for the wild-type enzyme to 70.51℃, 70.42℃, 70.43℃, 70.55℃, 70.66℃, and 70.46℃, respectively.

[0179] Under these conditions, compared to the Tm value of wild-type enzyme PpGH64 (69.72℃), the Tm values ​​of the mutants were as follows: N181M 70.51℃ (an increase of 0.79℃), L164E 70.42℃ (an increase of 0.70℃), N170E 70.43℃ (an increase of 0.71℃), P175T 70.55℃ (an increase of 0.83℃), G176I 70.66℃ (an increase of 0.94℃), and G182H 70.46℃ (an increase of 0.74℃).

[0180] Comparative Example 2:

[0181] Following the same strategy as in Examples 3-6, conserved sites M218 and Y254 were screened, and mutants M218I and Y254W were constructed, respectively, along with plasmid pET-28a(+)-PpGH64. M218I pET-28a(+)-PpGH64 Y254W And construct the recombinant strain BL21(DE3) / pET-28a(+)-PpGH64 M218I , BL21(DE3) / pET-28a(+)-PpGH64 Y254W The recombinant bacteria were cultured using the same method as in Example 5, and their thermodynamic stability was characterized using the same method as in Example 6. The results showed that, compared with the Tm value of 69.72℃ for the wild-type enzyme PpGH64, the Tm values ​​of the mutants were: M218I 68.61℃ (a decrease of 1.11℃) and Y254W 67.52℃ (a decrease of 2.20℃), indicating that these two mutants would affect the enzyme structure and even have a negative impact on the enzyme's thermostability.

[0182] The primers involved in this site-directed mutagenesis example are:

[0183] M218I-F: 5'-CGCTGTATAtaAAGACCTTCGATGACGGCTTT-3';

[0184] M218I-R: 5'-GGTCTTtatATACAGCGGAGAGTCTACGCTCA-3';

[0185] Y254W-F: 5'-CtgcCACGGTAACACGACTCGCGTGGACCAGT-3';

[0186] Y254W-R: 5'-TCGTGTTACCGTGgcaGCCGCTGTCATCAACGGT-3'.

[0187] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A β-1,3-glucanase mutant, characterized in that, The mutant is a mutation performed on the β-1,3-glucanase parent with the amino acid sequence shown in SEQ ID NO. 1, involving at least one of the following mutations: (1) Asparagine at position 181 is mutated to methionine; (2) Leucine at position 164 is mutated to glutamic acid; (3) Asparagine at position 170 is mutated to glutamic acid; (4) Proline at position 175 is mutated to threonine; (5) Glycine at position 176 is mutated to isoleucine; (6) Glycine at position 182 is mutated to histidine.

2. The gene encoding the mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The expression vector for the recombinant plasmid is plasmid pET-28a or pET-22b.

5. Recombinant microbial cells expressing the β-1,3-glucanase mutant of claim 1, or containing the gene of claim 2, or transformed with the recombinant plasmid of claim 3 or 4.

6. The recombinant microbial cell according to claim 5, characterized in that, The recombinant microbial cells include, but are not limited to, bacteria or fungi.

7. The recombinant microbial cell according to claim 5, characterized in that, The recombinant microbial cells used Escherichia coli BL21(DE3) as the host and pET-28a as the vector.

8. A method for improving the activity and / or thermal stability of β-1,3-glucanase, characterized in that, The method involves mutating asparagine at position 181 of the β-1,3-glucanase parent amino acid sequence as shown in SEQ ID NO.1 to methionine, or mutating leucine at position 164 to glutamic acid, or mutating asparagine at position 170 to glutamic acid, or mutating proline at position 175 to threonine, or mutating glycine at position 176 to isoleucine, or mutating glycine at position 182 to histidine.

9. A method for preparing β-1,3-glucan oligosaccharides, characterized in that, The method involves using β-glucan as a substrate and catalyzing the generation of β-1,3-glucan oligosaccharides using the β-1,3-glucanase mutant or the β-1,3-glucanase parent described in claim 1.

10. The use of the β-1,3-glucanase parent, the β-1,3-glucanase mutant of claim 1, the gene of claim 2, the recombinant plasmid of claim 3 or 4, the recombinant microorganism of any one of claims 5 to 7, or the method of claim 8 in the production of β-1,3-glucan oligosaccharides, characterized in that, The amino acid sequence of the β-1,3-glucanase parent is shown in SEQ ID NO.1.

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