A beta-mannanase mutant with enhanced thermostability and application thereof

Abstract

The application discloses a kind of β-mannanase mutant with enhanced thermostability and application, method includes the following steps: S1, construct recombinant Pichia pastoris strain;S2, production and purification β-mannanase;S3, analysis β-mannanase;S4, determination β-mannanase enzymatic property.The beneficial effects of the present application are as follows: 1, mutant β-mannanase Y50G in the application compared with wild type PpRmMan134A optimum reaction temperature is increased from 40 DEG C to 60 DEG C, and thermal melting temperature (T m ) is increased from 59.9 DEG C to 72.3 DEG C, show better stability in high temperature environment, with higher potential suitable for high temperature industrial applications.2, mutant β-mannanase Y50G and wild type PpRmMan134A in the application respectively keep 75% and 65% or so enzyme activity after heating at 90 DEG C for 60 min.3, mutant β-mannanase Y50G in the application compared with wild type PpRmMan134A, its half-life (t 1 / 2 ) at 90 DEG C is increased from 122.8 min to 199.4 min, increases by 76.6 min.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a thermostable β-mannanase mutant and its applications. Background Technology

[0002] In industrial applications, β-mannanase has attracted significant attention due to its high demand. Existing technologies include several patents, such as CN116463320A, which involves using histidine tags for enzyme purification, and CN116218817A, which employs EcoRI and NotI restriction enzymes for double digestion to construct recombinant plasmids. While these methods have played a role in the production of recombinant proteins, they share a common limitation: the amino acid sequence of recombinant β-mannanase contains, in addition to the target protein itself, unnecessary amino acid sequences, such as additional cleavage sites or histidine tags. The addition of these extra sequences can potentially affect the enzymatic functional properties of recombinant β-mannanase. To overcome this limitation, researchers in this field have focused on optimizing the expression process of recombinant β-mannanase and modifying its thermostability to meet the demands of broader, high-temperature industrial applications. Summary of the Invention

[0003] To address the above problems, this invention provides a thermostability-enhanced β-mannanase mutant and its applications. This invention is achieved through the following technical solutions.

[0004] A thermostable β-mannanase mutant and its application, comprising the following steps:

[0005] S1, Constructing a recombinant Pichia pastoris strain, specifically includes the following sub-steps:

[0006] S11, construct the recombinant plasmid pPICZαA-RmMan134A containing the wild-type β-mannanase RmMan134A gene;

[0007] S12, using recombinant plasmid pPICZαA-RmMan134A as a template, construct a mutant recombinant plasmid;

[0008] S13, Pichia pastoris X-33 glycerol-preserved strain was recombinantly expressed with recombinant plasmid pPICZαA-RmMan134A and mutant recombinant plasmid to obtain recombinant Pichia pastoris strain;

[0009] S2, Production and purification of β-mannanase: Recombinant Pichia pastoris strain is inoculated, the produced β-mannanase is collected, and the β-mannanase is purified.

[0010] S3, β-mannanase was analyzed, and the β-mannanase produced by different recombinant Pichia pastoris strains was analyzed;

[0011] S4, determine the enzymatic properties of β-mannanase, and determine the enzymatic properties of β-mannanase produced by different recombinant Pichia pastoris strains.

[0012] Preferably, step S11 includes the following sub-steps:

[0013] S111, The gene sequence of RmMan134A was optimized. While maintaining the second and third preferred codon selection of Pichia pastoris, Pichia pastoris preferred codons with high GC content were selected from the RmMan134A gene sequence to replace codons with high AT content, so that the AT content was in the range of 30% to 55%. Then, the codons in the local GC regions of RmMan134A gene with GC content of more than 46% and less than 22% were adjusted to obtain the optimized RmMan134A gene, whose gene sequence is recorded as SEQ ID NO.1;

[0014] S112, synthesize the target gene. Add the gene AAAAGA corresponding to the KEX2 cleavage site before the gene sequence of SEQ ID NO.1, add the terminator TAA after the gene sequence of SEQ ID NO.1, and add XhoI and Not I restriction sites before and after the cleavage site and the terminator, respectively. XhoI is CTCGAG and Not I is GCGGCCGC, to obtain the synthesized target gene SEQ ID NO.2.

[0015] S113, construct the recombinant plasmid, and express SEQ ID NO.2 with Escherichia coli to obtain recombinant Escherichia coli EcRmMan134A, which contains the recombinant plasmid pPICZαA-RmMan134A.

[0016] Preferably, in step S12,

[0017] Using the AxyPrep plasmid DNA small quantity kit, the recombinant plasmid pPICZαA-PpRmMan134A was extracted from EcRmMan134A, and then pPICZαA-PpRmMan134A was linearized using the restriction endonuclease Sac I.

[0018] Using recombinant plasmid pPICZαA-PpRmMan134A as a template, single-point mutations of Q17A, V43P, and Y50G were constructed, as well as multi-point mutation combinations of Q17A / V43P, Q17A / Y50G, V43P / Y50G, and Q17A / V43P / Y50G.

[0019] The β-mannanase in the recombinant plasmid pPICZαA-PpRmMan134A is designated as PpRmMan134A, and its gene sequence is designated as SEQ ID NO.3. The β-mannanases in the mutant recombinant plasmids are designated as Q17A, V43P, Y50G, Q17A / V43P, Q17A / Y50G, V43P / Y50G, and Q17A / V43P / Y50G, respectively, and their gene sequences are designated as SEQ ID NO.4 to SEQ ID NO.10.

[0020] Preferably, step S13 includes the following sub-steps:

[0021] S131, preparation of Pichia pastoris X-33 competent cells.

[0022] S132, the recombinant plasmid pPICZαA-PpRmMan134A and seven mutant recombinant plasmids were recombinantly expressed in red yeast X-33 competent cells and transformed cells were grown.

[0023] S133. Colonies with good growth were selected from the transformants and sequenced. The strain with the correct sequencing and the largest colony was the recombinant Pichia pastoris strain.

[0024] Preferably, in step S3, the analytical methods include SDS-PAGE analysis and size exclusion chromatography.

[0025] Preferably, in step S4, the determination of enzymatic properties includes the optimal pH, optimal temperature, stability, substrate specificity, enzyme kinetic parameters, thermal stability, and thermal melting temperature of β-mannanase.

[0026] An application of a β-mannanase for the hydrolysis of glucomannan, konjac gum, locust bean gum, guar gum, xanthan gum, and coconut meat products.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. The optimal reaction temperature of the mutant β-mannanase Y50G in this invention is increased from 40℃ to 60℃ compared to the wild-type PpRmMan134A, and the thermal melting temperature (T) is also increased. m The temperature has increased from 59.9℃ to 72.3℃, demonstrating better stability in high-temperature environments and showing greater potential for high-temperature industrial applications.

[0029] 2. Compared to RmMan134A and mRmMan134A, which almost completely lose enzyme activity after heating at 65℃ to 80℃ for 30 min, the mutant β-mannanase Y50G and wild-type PpRmMan134A of this invention retain about 75% and 65% of their enzyme activity, respectively, after heating at 90℃ for 60 min.

[0030] 3. Compared with the wild-type PpRmMan134A, the mutant β-mannanase Y50G in this invention has a longer half-life (t) at 90℃. 1 / 2 The time increased from 122.8 min to 199.4 min, an increase of 76.6 min. Attached Figure Description

[0031] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 Flowchart for recombinant plasmid construction;

[0033] Figure 2 Purification and validation of wild-type and mutant β-mannanase;

[0034] Figure 3 The optimal pH for wild-type, mutant, and commercially available β-mannanases;

[0035] Figure 4 Optimal temperatures for wild-type, mutant, and commercially available β-mannanases;

[0036] Figure 5 pH stability of wild-type, mutant, and commercially available β-mannanases;

[0037] Figure 6 Substrate specificity of wild-type, mutant, and commercially available β-mannanases;

[0038] Figure 7 Comparison of relative enzyme activities between wild-type and mutant β-mannanases;

[0039] Figure 8 Thermostability of wild-type, mutant, and commercially available β-mannanases;

[0040] Figure 9 The half-life (t) of Y50G and PpRmMan134A at 90℃ 1 / 2 );

[0041] Figure 10 Morphological analysis of Y50G and PpRmMan134A on coconut flakes after treatment at 65℃;

[0042] Figure 11 The difference in amino acid sequences between mRmMan134A and PpRmMan134A. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] A thermostable β-mannanase mutant and its application, comprising the following steps:

[0045] S1, Constructing a recombinant Pichia pastoris strain, specifically includes the following sub-steps:

[0046] S11, construct the recombinant plasmid pPICZαA-RmMan134A containing the wild-type β-mannanase RmMan134A gene.

[0047] Includes the following sub-steps:

[0048] S111, the gene sequence of RmMan134A was optimized. While maintaining the second and third preferred codon selection of Pichia pastoris, Pichia pastoris preferred codons with high GC content were selected from the RmMan134A gene sequence to replace codons with high AT content, so that the AT content was in the range of 30% to 55%. Then, the codons in the local GC regions of RmMan134A gene with GC content of more than 46% and less than 22% were adjusted to obtain the optimized RmMan134A gene, the gene sequence of which is recorded as SEQ ID NO.1.

[0049] SEQ ID NO.1:

[0050] GCTGATAGAGGTACTGAAACTGTTCCTGGTTTGGGTCAAAGAAAGCAACAGATCTTGAACTCTGGTGGAGGTGTCTGGGACTTGGCTATCGCTATGTTGGAGACTAAGAACCTTGGTACTGACTACGTTTACGGAGATGGTAAGACTTACGATTCTGCTAACTTCGGTATCTTCAAGCAGAACTGGTTCATGTTGAGAACCTCTACTTCTCAGTTCAAGGGTCAGACTACTAACCAGTGGAAC AATGTGCTGTCTTGAACTCTAACTTGCAGCAAGATATCAAGGCTAGACAGGAGTCCCAGAACTACTACGGTCCAGACAAGTGGTTCGCTGGTCATAGAAACGGTGAGTCTGGATTGTCCAACCCATACACTCAGGACATCACTAACTACAAGGATGCTGTCAACTGGATCCATGATCAACTGGCTTCTGACCCAAAGTACCTGTCTGATGACACTAGATTCTGGGTTGATGTCACTGCTATC.

[0051] S112, synthesize the target gene. Add the gene AAAAGA corresponding to the KEX2 cleavage site before the gene sequence of SEQ ID NO.1, add the terminator TAA after the gene sequence of SEQ ID NO.1, and add XhoI and Not I restriction sites before and after the cleavage site and terminator, respectively. XhoI is CTCGAG and Not I is GCGGCCGC, to obtain the synthesized target gene SEQ ID NO.2.

[0052] SEQ ID NO.2:

[0053] CTCGAGAAAAGA-SEQ ID NO. 1-TAAGCGGCCGC.

[0054] S113, construct recombinant plasmids, such as Figure 1 As shown, SEQ ID NO.2 was recombinantly expressed with Escherichia coli to obtain recombinant Escherichia coli EcRmMan134A, which contains the recombinant plasmid pPICZαA-RmMan134A.

[0055] S12, using recombinant plasmid pPICZαA-RmMan134A as a template, constructs a mutant recombinant plasmid.

[0056] Using the AxyPrep plasmid DNA small-scale kit, recombinant plasmid pPICZαA-PpRmMan134A was extracted from EcRmMan134A. pPICZαA-PpRmMan134A was then linearized with the restriction endonuclease Sac I. 10 μL of the reaction solution was then subjected to 1.2% (w / v) agarose gel electrophoresis to determine complete digestion. The remaining digest was purified using a PCR purification kit / DNA purification kit, yielding 24 μL of purified product dissolved in DEPC water. This product was stored at -20°C for later use.

[0057] Using recombinant plasmid pPICZαA-PpRmMan134A as a template, single-point mutations of Q17A, V43P, and Y50G, as well as multi-point mutation combinations of Q17A / V43P, Q17A / Y50G, V43P / Y50G, and Q17A / V43P / Y50G were constructed.

[0058] The mutant recombinant plasmid was constructed using PCR technology. The PCR reaction system is shown in Table 1. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 30 cycles (98℃ denaturation for 10 s, 72℃ extension for 20 s); and a final extension at 72℃ for 5 min. After the reaction, an appropriate amount of PCR product was taken for verification by 1.2% (w / v) agarose gel electrophoresis.

[0059] Table 1

[0060] Template DNA 0.1 Primer-F 1.0 Primer-R 1.0 2×PCRMaster 25.0 Double-distilled sterile water (ddH2O) 22.9 total 50.0

[0061] The β-mannanase in the recombinant plasmid pPICZαA-PpRmMan134A is designated as PpRmMan134A, and its gene sequence is designated as SEQ ID NO.3. The β-mannanases in the mutant recombinant plasmids are designated as Q17A, V43P, Y50G, Q17A / V43P, Q17A / Y50G, V43P / Y50G, and Q17A / V43P / Y50G, and their amino acid sequences are designated as SEQ ID NO.4 to SEQ ID NO.10, respectively.

[0062] SEQ ID NO.3:

[0063] ADRGTETVPGLGQRKQQILNSGGGVWDLAIAMLETKNLGTDYVYGDGKTYDSANFGIFKQNW FMLRTSTSQFKGQTTNQWNNGAVLNSNLQQDIKARQESQNYYGPDKWFAGHRNGESGLSNPYTQDI TNYKDAVNWIHDQLASDPKYLSDDTRFWVDVTAI.

[0064] SEQ ID NO.4:

[0065] ADRGTETVPGLGQRKQAILNSGGGVWDLAIAMLETKNLGTDYVYGDGKTYDSANFGIFKQNW FMLRTSTSQFKGQTTNQWNNGAVLNSNLQQDIKARQESQNYYGPDKWFAGHRNGESGLSNPYTQDI TNYKDAVNWIHDQLASDPKYLSDDTRFWVDVTAI。

[0066] SEQ ID NO.5:

[0067] ADRGTETVPGLGQRKQQILNSGGGVWDLAIAMLETKNLGTDYPYGDGKTYDSANFGIFKQNW FMLRTSTSQFKGQTTNQWNNGAVLNSNLQQDIKARQESQNYYGPDKWFAGHRNGESGLSNPYTQDI TNYKDAVNWIHDQLASDPKYLSDDTRFWVDVTAI。

[0068] SEQ ID NO.6:

[0069] ADRGTETVPGLGQRKQQILNSGGGVWDLAIAMLETKNLGTDYVYGDGKTGDSANFGIFKQNW FMLRTSTSQFKGQTTNQWNNGAVLNSNLQQDIKARQESQNYYGPDKWFAGHRNGESGLSNPYTQDI TNYKDAVNWIHDQLASDPKYLSDDTRFWVDVTAI。

[0070] SEQ ID NO.7:

[0071] ADRGTETVPGLGQRKQAILNSGGGVWDLAIAMLETKNLGTDYPYGDGKTYDSANFGIFKQNW FMLRTSTSQFKGQTTNQWNNGAVLNSNLQQDIKARQESQNYYGPDKWFAGHRNGESGLSNPYTQDI TNYKDAVNWIHDQLASDPKYLSDDTRFWVDVTAI。

[0072] SEQ ID NO.8:

[0073] ADRGTETVPGLGQRKQAILNSGGGVWDLAIAMLETKNLGTDYVYGDGKTGDSANFGIFKQNW FMLRTSTSQFKGQTTNQWNNGAVLNSNLQQDIKARQESQNYYGPDKWFAGHRNGESGLSNPYTQDI TNYKDAVNWIHDQLASDPKYLSDDTRFWVDVTAI.

[0074] SEQ ID NO.9:

[0075] ADRGTETVPGLGQRKQQILNSGGGVWDLAIAMLETKNLGTDYPYGDGKTGDSANFGIFKQNW FMLRTSTSQFKGQTTNQWNNGAVLNSNLQQDIKARQESQNYYGPDKWFAGHRNGESGLSNPYTQDI TNYKDAVNWIHDQLASDPKYLSDDTRFWVDVTAI.

[0076] SEQ ID NO.10:

[0077] ADRGTETVPGLGQRKQAILNSGGGVWDLAIAMLETKNLGTDYPYGDGKTGDSANFGIFKQNW FMLRTSTSQFKGQTTNQWNNGAVLNSNLQQDIKARQESQNYYGPDKWFAGHRNGESGLSNPYTQDI TNYKDAVNWIHDQLASDPKYLSDDTRFWVDVTAI.

[0078] S13, the Pichia pastoris X-33 glycerol-preserved strain was recombinantly expressed with the recombinant plasmid pPICZαA-RmMan134A and the mutant recombinant plasmid to obtain recombinant Pichia pastoris strains. This includes the following sub-steps:

[0079] S131, to produce Pichia pastoris X-33 competent cells.

[0080] (1) Select Pichia pastoris X-33 glycerol-preserved strains and streak them on YPD plates.

[0081] (2) After the colonies grow, pick a spot and inoculate it into 45mL of YPD liquid medium, 27℃, 200r / min, overnight, shake for 12h-18h until OD600=1.8-2.0.

[0082] (3) Take 1.5 mL of bacterial solution, centrifuge at 5000×g at 4℃ for 1 min.

[0083] (4) Add 500 μL of sterile double-distilled water (ddH2O), centrifuge at 5000×g for 1 min, and repeat twice.

[0084] (5) Add 1 mL of yeast competent cell solution and mix well. Shake at 27℃ and 220 r / min for 1 h.

[0085] (6) Add 25 μL of 1M DTT, 27℃, 220 r / min, shake for 30 min.

[0086] (7) Centrifuge at 5000×g for 1 min, remove the supernatant, and wash twice with 500μL DEPC water.

[0087] (8) Add 0.5 mL of 1 M sorbitol, resuspend, and let stand on ice for 5 min.

[0088] (9) Prepare and use as soon as possible. If not used, store competent cells in a -80°C refrigerator for later use.

[0089] S132, the recombinant plasmid pPICZαA-PpRmMan134A and seven mutant recombinant plasmids were recombinantly expressed in red yeast X-33 competent cells, and transformants were grown.

[0090] The recombinant plasmid pPICZαA-PpRmMan134A is expressed in the same way as the seven mutant recombinant plasmids, and will be used as an example to illustrate the recombinant plasmid pPICZαA-PpRmMan134A.

[0091] (1) First, add 100 μL of Pichia pastoris competent cells to the electroporation cuvette, followed by 10 μL of linearized recombinant plasmid. Then, place on ice for 5 min before electroporation. Electroporation conditions: Select the Bio-Rad electroporator, and perform electroporation in Fungi mode with the Pic option.

[0092] (2) Add 0.6 mL of YPD liquid culture medium, 27℃, 220 r / min, shake for 3.5 h.

[0093] (3) Centrifuge at 12000×g, remove 1.2mL of supernatant, and then mix the remaining liquid.

[0094] (4) Take 300 μL and apply it to a YPD plate resistant to bleomycin (100 μg / mL).

[0095] (5) After strictly avoiding light, place in a 28°C incubator until transformants grow.

[0096] S133. Colonies with good growth were selected from the transformants and sequenced. The strain with the correct sequencing and the largest colony was the recombinant Pichia pastoris strain.

[0097] Using the recombinant plasmid pPICZαA-PpRmMan134A as an example, firstly, colonies with good growth were selected from the transformants and inoculated onto bleomycin-resistant YPD plates for secondary screening, followed by incubation at 30°C for 2–3 days. Subsequently, following the instructions of the yeast genomic DNA extraction kit, recombinant Pichia pastoris genomic DNA was extracted from 10 selected transformants for polymerase chain reaction (PCR) and sequencing analysis. The recombinant Pichia pastoris strain with the largest colonies and correct sequencing verification was selected.

[0098] S2, Production and Purification of β-Mannanase: Recombinant Pichia pastoris strain is inoculated, the produced β-mannanase is collected, and the β-mannanase is purified. This includes the following stages:

[0099] 1. Stages of bacterial growth:

[0100] Single colonies of recombinant Pichia pastoris strains selected from YPD solid plates were inoculated into 70 mL of modified BMGY medium and cultured at 30 °C and 200 r / min for 24 h. The colonies were then centrifuged at 25 °C and 5000 × g for 10 min to precipitate the cells.

[0101] 2. Enzyme production stage in bacteria:

[0102] The precipitated bacterial cells were resuspended in 100 mL of modified BMMY medium and cultured at 30 °C and 200 r / min for 24 h. After fermentation, the supernatant was collected by centrifugation at 25 °C and 5000 × g for 10 min.

[0103] 3. Enzyme purification stage:

[0104] (1) Centrifuge at 25°C and 5000×g for 40 min using a 3kDa ultrafiltration tube to concentrate the fermentation supernatant.

[0105] (2) Slowly add ammonium sulfate solid to the concentrate until the concentration reaches 5% to 10% (w / v). Stir until the turbidity of the solution stabilizes, then dispense into 2mL centrifuge tubes and centrifuge at 4℃ and 10000×g for 5min.

[0106] (3) After dissolving the precipitate in ultrapure water, desalination was performed again using a 3kDa ultrafiltration tube.

[0107] S3, Analysis of β-mannanase: The β-mannanase produced by different recombinant Pichia pastoris strains was analyzed. Analytical methods included:

[0108] SDS-PAGE analysis:

[0109] The protein concentration in the supernatant was determined using an enhanced BCA protein assay kit.

[0110] Follow the steps specified in the product manual to use BeyoGel TM Plus PAGE precast gel, SDS-PAGE electrophoresis buffer, 5×SDS-PAGE protein loading buffer, and BeyoBlue. TM SDS-PAGE analysis of concentrated fermentation supernatant and β-mannanase samples after salting out and desalting with Coomassie brilliant blue ultrafast staining solution.

[0111] Size exclusion chromatography (SEC) analysis:

[0112] 1) Protein elution was performed on a pre-equilibrated BioCore SEC-300 column (5 μm, 7.8 × 300 mm) using 100 mM sodium phosphate buffer at pH 7 as the mobile phase.

[0113] 2) Dissolve β-mannanase in 100mM potassium phosphate buffer at pH 7 and elute at 30°C with a flow rate of 0.5 mL / min.

[0114] 3) Follow the operating procedures in the product instructions for “69385 protein standard mixture 15–600 kDa for SEC / GFC column testing”.

[0115] exist Figure 2 In (a), this invention demonstrates that the wild-type PpRmMan134A and its mutants Q17A, V43P, Y50G, Q17A / Y50G, and Q17A / V43P / Y50G all have a molecular weight of 18.2 kDa, and SDS-PAGE analysis confirmed that these proteins can be used for subsequent experimental studies. On the other hand, mutants Q17A / V43P and V43P / Y50G exhibited significant aggregation and precipitation during the boiling step of denaturing protein electrophoresis in SDS-PAGE, failing to produce clear protein bands. Therefore, as... Figure 2 As shown in (b), the present invention employs size exclusion chromatography to verify the purity of these mutants to meet subsequent research needs. Sequence IDs SEQ ID NO.4 to SEQ ID NO.10 correspond to the amino acid sequences of the seven mutant β-mannanases, respectively.

[0116] S4, the enzymatic properties of wild-type and mutant β-mannanases were determined, as well as the enzymatic properties of β-mannanases produced by different recombinant Pichia pastoris strains. The determination of enzymatic properties included β-mannanase activity, optimal pH, optimal temperature, stability, substrate specificity, enzyme kinetic parameters, thermal stability, and thermal melting temperature.

[0117] 1. Method for measuring β-mannanase activity:

[0118] Substrate preparation: A 0.5% (w / v) locust bean gum solution (dissolved in 50 mM citric acid-sodium citrate solution, pH 5.5) was used as the substrate.

[0119] Preheating: Preheat 1 mL of locust bean gum solution at 40°C for 5 min.

[0120] Enzyme reaction: Add 0.1 mL of enzyme solution to the preheated substrate and incubate the mixture at 40 °C for 5 min.

[0121] Reaction termination: Add 1.5 mL of 3,5-dinitrosalicylic acid (DNS) and boil for 5 min to terminate the reaction.

[0122] Dilution and measurement: Dilute the reaction mixture to 10 mL with deionized water and measure the absorbance A (containing enzyme reaction mixture) and A1 (absorbance of the corresponding empty well) at 540 nm in a 96-well plate.

[0123] Blank control: Ultrapure water was used instead of β-mannanase solution as a blank control, and absorbance values ​​A0 (the reaction mixture containing the blank control) and A2 (the absorbance value of the corresponding empty well) were recorded. Each experiment was performed in triplicate.

[0124] Standard curve: A standard curve was plotted using D-mannose solutions with concentrations ranging from 0 mg / mL to 1.0 mg / mL (dissolved in 50 mM citric acid-sodium citrate solution at pH 5.0).

[0125] Enzyme activity calculation: The enzyme activity of β-mannanase is calculated according to the following formula:

[0126] Where M is the molar mass of D-mannose (180.2 g / mol) and n is the dilution factor of the enzyme.

[0127] Under these conditions, the amount of enzyme required to produce 1 μmol of reducing sugar per minute is defined as one unit of enzyme activity (U). Furthermore, enzyme activity measurements of wild-type, seven mutant, and commercially available β-mannanases were performed at their respective optimal pH and optimal temperature.

[0128] 2. Determine the optimal pH for β-mannanase:

[0129] A 0.5% (w / v) locust bean gum solution was prepared using 50 mM solutions at different pH values ​​(2.0–10.0) as substrates. The pH value at which the enzyme activity reached the highest value was defined as the optimum pH, and the enzyme activity at this value was defined as 100%.

[0130] exist Figure 3 This invention provides the following data regarding the optimal pH values ​​of different mutant β-mannanases: the optimal pH value for commercially available β-mannanase and mutant Q17A / Y50G is 4.5; the optimal pH value for mutant Q17A is 5.5, the same as the optimal pH value for wild-type PpRmMan134A; the optimal pH value for mutant V43P and Y50G is 5.0; the optimal pH value for mutant Q17A / V43P and V43P / Y50G is 6.0; and the optimal pH value for mutant Q17A / V43P / Y50G is 6.5. Regarding pH suitability, except for mutant Q17A / Y50G and the commercially available β-mannanase, the other six mutant β-mannanases can maintain at least 50% enzyme activity within a pH range of 2–10, indicating that these mutants have stability and potential application prospects under a wide range of pH conditions.

[0131] 3. Determine the optimal temperature for β-mannanase:

[0132] A 0.5% (w / v) locust bean gum solution was prepared using a 50 mM solution at the optimal pH as the substrate. The enzyme activity of β-mannanase was measured at different temperatures (20℃~80℃). The temperature at which the enzyme activity reached its maximum was defined as the optimum temperature, and the enzyme activity at this temperature was defined as 100%.

[0133] exist Figure 4 This invention provides experimental data on the optimal temperatures of different β-mannanase mutants and commercially available β-mannanases: the optimal temperature for commercially available β-mannanase, mutant Q17A / Y50G, and V43P / Y50G is 50°C; the optimal temperature for mutant V43P is 55°C; and the optimal temperatures for mutant Q17A, Y50G, Q17A / V43P, and Q17A / V43P / Y50G are 60°C. The data show that the optimal temperatures of all mutant β-mannanases of this invention and commercially available β-mannanases are 40°C higher than the optimal temperature of wild-type PpRmMan134A. Particularly noteworthy is that the optimal temperatures of the five mutant β-mannanases in this invention exceed the optimal temperature of 50°C for commercially available β-mannanases, indicating that these mutant enzymes may have superior temperature adaptability in specific applications.

[0134] 4. Determine the pH stability of β-mannanase:

[0135] β-Mannanase dissolved in 50 mM solutions at different pH values ​​(2.0–10.0) was treated at 37°C for 30 min, and its enzyme activity was then measured. The enzyme activity of β-Mannanase treated with ultrapure water was used as a control, and its activity was defined as 100%.

[0136] exist Figure 5 This invention discloses the relative enzyme activity variations of different mutant β-mannanases within a pH range of 2 to 10. Specifically, the mutant Q17A / Y50G exhibits the largest relative enzyme activity fluctuation within this pH range, ranging from 69.84% to 120.88%, indicating that this mutant enzyme is highly sensitive to pH changes. In contrast, while the mutants Q17A, V43P / Y50G, and Q17A / V43P / Y50G show some inhibition of relative enzyme activity within the same pH range, the fluctuations are smaller, ranging from 77.29% to 99.73%, 78.03% to 91.51%, and 78.29% to 97.07%, respectively.

[0137] Furthermore, mutant V43P, Y50G, Q17A / V43P, and commercially available β-mannanase showed relatively increased enzyme activity within the pH range of 2 to 10, with minimal fluctuations, ranging from 77.29% to 99.73%, 95.09% to 118.64%, and 90.50% to 110.05%, respectively. These data indicate that these mutant enzymes and commercially available β-mannanases are less sensitive to pH changes, similar to the properties of wild-type PpRmMan134A.

[0138] 5. Determine the substrate specificity of β-mannanase:

[0139] 0.5% (w / v) solutions of xanthan gum, guar gum, locust bean gum, glucomannan, and konjac gum were prepared using 50 mM buffer. Furthermore, the pH of the buffer was adjusted to the optimal pH for the respective β-mannanase. Enzyme activity was measured using the prepared substrate solutions at the optimal temperature for the respective β-mannanase. The β-mannanase activity measured using 0.5% (w / v) locust bean gum as a substrate was set as a control, and its activity was defined as 100%.

[0140] exist Figure 6 In this context, KGM stands for konjac gum; GB for guar gum; GM for glucomannan; XG for xanthan gum; and LBG for locust bean gum.

[0141] This invention discloses that mutant β-mannanases V43P / Y50G and Q17A / V43P / Y50G lack the ability to hydrolyze konjac gum, while mutants Q17A, V43P, Q17A / Y50G, and Q17A / V43P are more efficient at hydrolyzing konjac gum than at hydrolyzing locust bean gum. Conversely, mutant Y50G and commercially available β-mannanases are more efficient at hydrolyzing locust bean gum than at hydrolyzing konjac gum. Furthermore, V43P / Y50G and Q17A / V43P / Y50G were more efficient at hydrolyzing glucomannan than at hydrolyzing locust bean gum, while wild-type PpRmMan134A, mutant Q17A, V43P, Q17A / Y50G, Q17A / V43P, Y50G, and commercially available β-mannanase showed the opposite trend in their efficiency in hydrolyzing glucomannan and locust bean gum. Further, Q17A / V43P and commercially available β-mannanase were less efficient at hydrolyzing xanthan gum than at hydrolyzing locust bean gum, while mutant Q17A, V43P, Y50G, Q17A / Y50G, Q17A / V43P, and V43P / Y50G showed the opposite trend in their efficiency in hydrolyzing xanthan gum and locust bean gum. Among them, the efficiency of the wild-type PpRmMan134A in hydrolyzing xanthan gum was only 62.77% of that in locust bean gum, while the efficiency of the mutant Y50G in hydrolyzing xanthan gum was 423.35% of that in locust bean gum. It is worth noting that the efficiency of these eight β-mannanases in hydrolyzing guar gum was higher than that in hydrolyzing locust bean gum.

[0142] Figure 7 In Chinese, GB stands for guar gum; LBG stands for locust bean gum.

[0143] according to Figure 7 The experimental results showed that when guar gum and locust bean gum were used as hydrolysis substrates, the relative enzyme activities of mutant Y50G and Q17A were higher than those of wild-type PpRmMan134A. Specifically, when guar gum was used as a substrate, the relative enzyme activities of Y50G and Q17A were 124.32% and 112.15%, respectively, and when locust bean gum was used as a substrate, they were 104.36% and 115.68%, respectively. Therefore, mutant Y50G and Q17A exhibited higher enzyme activities in the hydrolysis of guar gum and locust bean gum, and have potential application value.

[0144] 6. Determine the enzyme kinetic parameters of β-mannanase:

[0145] Locust bean gum solutions with concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL were prepared using 50 mM citrate-sodium citrate solution. The pH of the buffer solution was adjusted to the optimal pH for the corresponding β-mannanase. Then, the β-mannanase activity at each concentration was measured. Each experiment was performed in triplicate. The enzyme activity value corresponding to each locust bean gum concentration was recorded. After data collection, outliers were excluded, and the remaining data points were nonlinearly fitted using the Hyperbl function in Origin 2021 software to obtain the kinetic parameters of β-mannanase, such as the maximum reaction rate (VL). max ), Michaelis constant (K) m The number of substrates that an enzyme can convert per second (k) cat ) and catalytic rate (k cat / K m ).

[0146] Table 2 provides the enzyme kinetic parameters of Q17A, V43P, and Y50G when locust bean gum is used as a hydrolysis substrate, including the maximum reaction rate V. max Michaelis constant K m and conversions per second k cat These parameters are all higher than those of the wild-type PpRmMan134A. This result indicates that these mutant enzymes have higher efficiency in catalyzing the conversion of substrates to products. In particular, the K of the mutant V43P / Y50G is significantly higher. m The value was 1.10 mg / mL, the lowest among all tested enzymes, indicating the highest affinity for locust bean gum. However, although V43P / Y50G had the strongest affinity for the substrate, its V... max and k cat However, the value is the lowest, indicating that its relative enzyme activity is low.

[0147] Although the catalytic efficiency (kJ) of mutant Q17A and Y50G cat / K m The catalytic efficiency of V43P, V43P / Y50G, and Q17A / V43P / Y50G is higher than that of PpRmMan134A, but the catalytic efficiency of mutant V43P, V43P / Y50G, and Q17A / V43P / Y50G is lower than that of PpRmMan134A. This is because... Figure 7 The results shown are consistent.

[0148] Due to the lack of detailed amino acid sequence information for commercially available β-mannanases, they could not be included in the comparison of enzyme kinetic parameters. Based on the data in Table 2, it can be concluded that mutant Q17A and Y50G have potential advantages in application.

[0149] Table 2

[0150]

[0151] 8. Determination of the thermal stability of β-mannanase:

[0152] A. Thermal stability of wild-type β-mannanase at different temperatures:

[0153] (1) The PpRmMan134A enzyme solution was dispensed into several test tubes and incubated in a water bath at 40℃, 50℃, 60℃ and 70℃. The enzyme activity was measured at 0 min, 30 min, 70 min, 100 min, 130 min, 180 min and 360 min, respectively. The enzyme activity at 0 min was defined as 100%. It was incubated at 70℃ for only 130 min.

[0154] (2) Dispense the PpRmMan134A enzyme solution into several test tubes and keep them in a water bath at 80℃ and 90℃. Take them out at 0 min, 30 min, 60 min, 90 min and 120 min respectively to measure the enzyme activity. Define the enzyme activity at 0 min as 100%.

[0155] (3) Dispense the PpRmMan134A enzyme solution into several test tubes, keep them in a water bath at 100℃, and take them out at 0 min, 1 min, 2 min, 3 min and 4 min respectively to measure the enzyme activity. Define the enzyme activity at 0 min as 100%.

[0156] (4) Dispense the PpRmMan134A enzyme solution into several test tubes, incubate in a water bath at 100℃, remove the tubes at 0 min, 1 min, 2 min, 3 min and 4 min respectively, incubate at 25℃ for 5 min and then measure the enzyme activity. Define the enzyme activity at 0 min as 100%.

[0157] B. Thermal stability of commercial β-mannanase at different temperatures:

[0158] (1) The commercial β-mannanase solution was dispensed into several test tubes and kept in a water bath at 50℃, 60℃ and 70℃. The enzyme activity was measured at 0 min, 30 min, 70 min, 100 min, 130 min, 180 min and 360 min respectively. The enzyme activity at 0 min was defined as 100%.

[0159] (2) The commercial β-mannanase solution was dispensed into several test tubes and kept in a water bath at 80℃ and 90℃. The enzyme activity was measured at 0 min, 40 min and 90 min respectively. The enzyme activity at 0 min was defined as 100%.

[0160] (3) Determine the thermal stability of commercial β-mannanase at 100 °C according to methods (3) and (4) in step A.

[0161] C. Thermal stability of mutant β-mannanase at different temperatures

[0162] (1) Dispense the V43P enzyme solution into several test tubes and keep them in a water bath at 45℃, 55℃ and 65℃. Take them out at 0 min, 30 min, 70 min, 100 min, 130 min, 180 min and 360 min respectively to measure the enzyme activity. Define the enzyme activity at 0 min as 100%.

[0163] (2) Dispense the Q17A / Y50G enzyme solution into several test tubes and keep them in a water bath at 40℃, 50℃ and 60℃. Take them out at 0 min, 30 min, 70 min, 100 min, 130 min, 180 min and 360 min respectively to measure the enzyme activity. Define the enzyme activity at 0 min as 100%.

[0164] (3) Dispense the Q17A / V43P / Y50G enzyme solution into several test tubes and incubate them in a water bath at 60℃, 70℃ and 80℃. Take them out at 0 min, 30 min, 70 min, 100 min, 130 min, 180 min and 360 min respectively to measure the enzyme activity. Define the enzyme activity at 0 min as 100%.

[0165] (4) Dispense the Q17A, Q17A / V43P and V43P / Y50G enzyme solutions into several test tubes, incubate them in a water bath at 50℃, 60℃ and 70℃, and take them out at 0 min, 30 min, 70 min, 100 min, 130 min, 180 min and 360 min respectively to measure the enzyme activity. Define the enzyme activity at 0 min as 100%.

[0166] (5) Following the method in step A, dispense the Y50G enzyme solution into several test tubes and measure its enzyme activity at 50℃, 60℃, 70℃, 80℃, 90℃ and 100℃.

[0167] exist Figure 8This invention provides experimental data on the thermostability of different mutant β-mannanases. Specifically, mutants Q17A / V43P and V43P / Y50G retained 90% of their enzyme activity after treatment at 50°C, 60°C, and 70°C for 360 min. Similarly, mutant V43P retained 90% of its enzyme activity after treatment at 45°C, 55°C, and 65°C, and mutant Q17A / Y50G retained 90% of its enzyme activity after treatment at 40°C, 50°C, and 60°C for 360 min. However, mutant Q17A / V43P / Y50G retained only 80% of its enzyme activity after treatment at 60°C, 70°C, and 80°C for 360 min. Although mutant Q17A retained 90% of its enzyme activity after treatment at 50°C and 60°C for 360 min, it was inactivated after treatment at 70°C for 180 min. The mutant Y50G retained 72.33% of its enzyme activity after treatment at 70℃ for 180 min.

[0168] Although the optimal temperature for both mutant Q17A and Y50G is 60℃, and their enzyme activities are both higher than those of wild-type PpRmMan134A (e.g., Figure 4 and Figure 7 (As shown in the figure), but the thermostability of mutant Q17A is significantly lower than that of mutant Y50G. Mutant Y50G retained 72.33% and 65.97% of its enzyme activity after treatment at 80℃ and 90℃ for 120 min, respectively, while the relative enzyme activity of commercial β-mannanase decreased to 9.19% and 0%, respectively, after treatment at 80℃ and 90℃ for 90 min. Furthermore, mutant Y50G and PpRmMan134A retained over 80% of their enzyme activity after treatment at 100℃ for 4 min, while commercial β-mannanase retained only about 9% of its enzyme activity. The relative enzyme activity of mutant Y50G after heating at 100℃ for 2–4 min, followed by treatment at 25℃ for 5 min, was significantly lower than the relative enzyme activity measured directly at 60℃ (the optimum temperature of Y50G) after heating at 100℃ for only 2–4 min. This result indicates that the negative impact of 100℃ treatment on the activity of mutant Y50G enzyme can be partially repaired at the optimal temperature of 60℃.

[0169] like Figure 9 As shown, when PpRmMan134A mutates to Y50G, its half-life at 90℃ (t) 1 / 2 The half-life increased from 122.8 min to 199.4 min, an increase of 76.6 min. These experimental data indicate that the mutant Y50G exhibits significantly better thermal stability at high temperatures than commercially available β-mannanase, demonstrating its potential industrial application value.

[0170] 8. Determination of the thermal melting temperature of β-mannanase:

[0171] The thermal melting temperature (T0) of β-mannanase was measured using a Chirascan V100 instrument. m ):

[0172] First, the measurement parameters were set as follows: wavelength 180nm–260nm, bandwidth 1nm, and sampling time for a single data point 0.5s. Then, the initial temperature was set to 30℃, the final temperature to 90℃, the step size to 2℃, the heating rate to 1℃ / min, a stepped heating mode was selected, and the temperature stabilization time was 100s. After the experiment, the temperature was returned to 30℃. Finally, the sample was placed in a 0.5mm optical path cuvette, and a needle thermometer was inserted. Measurements were taken after the temperature stabilized at approximately 30℃. The corresponding thermal melting temperature (T0) was determined. m The result was obtained by calculation using the instrument's built-in Global3 software.

[0173] Table 3

[0174] Q17A 60 55.0±0.2 -4.9±0.5 V43P 55 76.7±0.2 16.8±0.5 Y50G 60 72.3±0.2 12.4±0.5 Q17A / V43P 60 74.3±0.2 14.4±0.5 Q17A / Y50G 50 66.5±0.4 6.6±0.3 V43P / Y50G 50 73.6±0.3 13.7±0.4 Q17A / V43P / Y50G 60 78.1±0.8 18.2±0.1 PpRmMan134A 40 59.9±0.7 0

[0175] The relative enzyme activities listed in the table above are measured based on locust bean gum as a substrate, with the enzyme activity of PpRmMan134A set as a reference, i.e., the enzyme activity of PpRmMan134A is 100%.

[0176] Table 3 provides the optimal temperature and thermal melting temperature (T) for β-mannanase. m ), ΔT m Comparison of values. The hot melt temperature of mutant Q17A is lower than that of PpRmMan134A, which is consistent with... Figure 9 The results show that it becomes inactive after treatment at 70℃ for 180 min (e.g. Figure 8 As shown in the figure, this indicates that the mutant Q17A has poor thermal stability. In contrast, the mutant Y50G has a melting temperature 12.4℃ higher than the wild type PpRmMan134A, its optimum temperature is increased by 20℃, and it exhibits excellent thermal stability in the temperature range of 50℃~100℃ (e.g., as shown in the figure). Figure 8 (As shown).

[0177] An application of a β-mannanase for the hydrolysis of glucomannan, konjac gum, locust bean gum, guar gum, xanthan gum, and coconut meat products.

[0178] Let's take its application in desiccated coconut as an example.

[0179] Sample preparation for the experimental group: Place an appropriate amount of shredded coconut into a 2.0 mL centrifuge tube to make the volume approximately 0.5 mL. Add 0.5 mL of 50 mM citric acid solution with pH 5.5 and 5.0 to each centrifuge tube respectively. Then, add 0.5 mL of 2.5 mg / mL PpRmMan134A or Y50G mutant enzyme solution to each centrifuge tube and mix well.

[0180] Control group setup: Untreated coconut flakes served as control 1. Another portion of coconut flakes was mixed with 1.0 mL of 50 mM citric acid solution (pH 5.5) to serve as control 2.

[0181] Enzyme treatment: The above experimental group samples and control 2 were treated at 65℃ for 30 min.

[0182] Post-processing: After processing, the sample was filtered to remove liquid. The filtered solid sample was then dried at 50°C for 90 min.

[0183] Scanning electron microscopy (SEM) observation: The dried sample, control 2, and control 1 were coated onto conductive adhesive and then sputtered with gold. The surface structure of the coconut flakes before and after treatment was observed using a scanning electron microscope (SEM) SU8010.

[0184] exist Figure 10 In this invention, the surface of untreated coconut flakes is smooth and intact; however, after treatment with a citric acid-sodium citrate solution, slight wrinkling occurs on the surface. Coconut flakes treated with mutant Y50G and wild-type PpRmMan134A exhibit obvious porous structures, with the surface damage from Y50G treatment being significantly greater than that from PpRmMan134A treatment. Therefore, it can be inferred that mutant Y50G exhibits superior stability under high-temperature conditions compared to PpRmMan134A, demonstrating greater potential for high-temperature industrial applications.

[0185] This invention reveals that mutant Y50G exhibits superior stability at high temperatures compared to wild-type PpRmMan134A and commercially available β-mannanase, and has greater potential for high-temperature industrial applications.

[0186] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to any specific implementation. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A thermostable β-mannanase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

6.

2. The application of the thermostability-enhanced β-mannanase mutant as described in claim 1, characterized in that, It is used in hydrolyzed glucomannan, locust bean gum, guar gum, xanthan gum, and coconut meat products.

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