A beta-1-6-glucanase mutant and use thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a beta-1-6-glucanase mutant and application thereof. The beta-1-6-glucanase FlGlu30 is obtained by gene synthesis, and the beta-1-6-glucanase mutant FlGlu30M is further obtained by using site-directed mutation and combination mutation. Compared with the starting template FlGlu30, the enzyme specific activity of the mutant is increased by 1.95 times. The application also realizes high-efficiency expression of the beta-1-6-glucanase mutant FlGlu30M in Pichia pastoris X33 through high-density fermentation. The highest enzyme activity of the recombinant Pichia pastoris engineering bacteria in a 7-liter fermentation tank is 125.8 U / mL, and the beta-1-6-glucanase can efficiently hydrolyze 1-6-glucan in the cell wall of yeast, thereby laying a foundation for enzymatic preparation of oligomeric glucose.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a β-1-6-glucanase mutant and its applications. Background Technology

[0002] Yeast cell walls, as an important byproduct of yeast processing, are mainly composed of structural polysaccharides such as β-1,3-glucan and β-1,6-glucan. Among them, β-1,6-glucan, as a core component, possesses good bioactivity potential. However, due to its large molecular weight, dense spatial structure, and poor water solubility, it is difficult to utilize directly and efficiently, leading to the frequent low-value treatment of this resource in actual production, resulting in a certain degree of resource waste.

[0003] Currently, the development of yeast cell walls mainly focuses on the research and application of β-1,3-glucan, while the targeted development of β-1,6-glucan is relatively insufficient, resulting in a limited variety of functional products and restricting the high-value utilization of yeast cell wall resources. In traditional dextran hydrolysis methods, acid-base hydrolysis easily leads to excessive degradation of the product, high levels of byproducts, and environmental pollution risks; physical fragmentation methods struggle to precisely break specific glycosidic bonds, making it impossible to prepare oligosaccharides with controllable polymerization degrees, thus limiting their application in precision agriculture. In contrast, β-1,6-glucanase can specifically cleave β-1,6-glycosidic bonds, directionally preparing oligosaccharides with significant advantages such as mild reaction conditions, environmental friendliness, and stable product structure, making it an important technical pathway for the efficient biotransformation of yeast cell wall resources.

[0004] Currently, agricultural production is facing prominent problems such as soil degradation, frequent occurrences of continuous cropping obstacles, and decreased crop resistance, urgently requiring the development of green and efficient biostimulant products to support sustainable agricultural development. However, the existing technological system lacks a mature process route for preparing agricultural-specific oligosaccharides based on the directed hydrolysis of yeast cell walls by β-1,6-glucanase, which limits the effective conversion and application of this bioactive substance in the agricultural field.

[0005] Therefore, developing and optimizing a process for β-1,6-glucanase is crucial for further expanding its application in yeast cell walls. Summary of the Invention

[0006] To address the aforementioned challenges, this invention provides a β-1-6-glucanase mutant and its applications. The β-1-6-glucanase mutant provided by this invention can efficiently hydrolyze yeast cell walls, and its products can be applied to crop disease resistance and growth promotion, soil improvement, and yield enhancement, aligning with the needs of green agricultural development.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A β-1-6-glucanase mutant FlGlu30M, the amino acid sequence of which is shown in SEQ ID NO.6.

[0008] SKNVTANSGKVESWITTTDESSKLKKQTDLAFSSETNSNQTIEVNPAEKFQTVEGFGFSLTGGSAQAIKKLDKAKREALLQELFSRQSNAIGLSYLRISIGASDLNEKVFSYD DMPEGQTDMNLEHFNLGPDLNDVIPVLKEILAINPKIKIMGSPWSPPVWMKDNGSSKGGSLQPKYYGVYAQYFVKYIQAMKSHGIVIDAITPQNEPLHPGNNPSMLMLAEQQA DFIGNHLGPAFAKAGIKTKIIVYDHNCNKPEYPLTILRDPKANPFVTGSAFHLYEGDISALTTVHNEFPNKDLYFTEQYTGSGTNFETDLKWSVKNVVIGSMRNWSKNALSWG LANDEFYKPFTPGGCSTCKGALMIDQNQNIKREVGWYIIGHASKFVPEGSVRIGSNIAGNLHNVAFKTPSGQTVLIVENDGASAETFNIKYNQKQTTTTLNAGAVATFVW (SEQ ID NO.6).

[0009] Preferably, the sequence encoding the amino acid is a polynucleotide, the sequence of which is shown in SEQ ID NO. 5.

[0010]

[0011] The present invention also provides a recombinant expression vector pPICZαA-FlGlu30M, comprising polynucleotides of the β-1-6-glucanase mutant FlGlu30M.

[0012] Preferably, the C-terminus of the polynucleotide sequence of the β-1-6-glucanase mutant FlGlu30M is supplemented with a 6His-Tag nucleotide sequence.

[0013] The nucleotide sequence of 6His-Tag is: CACCACCACCATCACCAC.

[0014] The amino acid sequence of 6His-Tag is: HHHHHH.

[0015] The present invention also provides a recombinant bacterium containing the recombinant expression vector pPICZαA-FlGlu30M.

[0016] Preferably, the recombinant bacteria uses Pichia pastoris as the host.

[0017] Preferably, the engineered Pichia pastoris strain is Pichia pastoris X33.

[0018] The present invention also provides the application of the β-1-6-glucanase mutant FlGlu30M in the preparation of oligodextrose.

[0019] Preferably, the oligosaccharide is prepared by hydrolyzing and pretreating the cell wall of Saccharomyces cerevisiae using the β-1-6-glucanase mutant FlGlu30M.

[0020] Compared with the prior art, the technical advantages of the present invention are as follows: (1) Through codon optimization design, this invention obtains gene-synthesized bacteria derived from the genus Flavobacterium (Flavobacterium). Flavobacterium sp. The β-1,6-glucanase gene FlGlu30 was extracted and heterologously expressed in Pichia pastoris. (2) This invention successfully screened and obtained the mutant FlGlu30M with significantly enhanced enzyme specific activity through site-directed mutagenesis and combined mutagenesis. Furthermore, through high-density fermentation, the mutant was efficiently expressed in the Pichia pastoris system. (3) The mutant FlGlu30M obtained in this invention has a high efficiency in hydrolyzing yeast cell walls, good substrate specificity and catalytic efficiency, which lays the foundation for its industrial application in the fields of yeast cell wall resource utilization and agricultural biostimulant development. Attached Figure Description

[0021] Figure 1This is a three-dimensional conformational assessment diagram of β-1-6-glucanase FlGlu30; Figure 2 Three-dimensional conformation diagram of the docking of β-1-6-glucanase FlGlu30 and gentiotriose molecules; Figure 3 This is an SDS-PAGE electrophoresis image of the β-1-6-glucanase mutant FlGlu30M. Figure 4 The high-density fermentation curve of recombinant engineered strain 7 containing the β-1-6-glucanase mutant FlGlu30M; Figure 5 This diagram illustrates the process by which the β-1-6-glucanase mutant FlGlu30M hydrolyzes the yeast cell wall. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto. Molecular biology experimental methods not specifically described in the following embodiments are all performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions; the reagents and biological materials mentioned are commercially available unless otherwise specified.

[0023] The experimental materials and reagents involved in the experiment of this invention are as follows: 1. Strains and vectors The Pichia pastoris X33 (A333926) and Escherichia coli Top10 competent cells (B528412) used in this invention were purchased from Sangon Biotech (Shanghai) Co., Ltd., and the expression vector pPICZαA (VT000155-1EA) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0024] 2. Enzymes and kits PrimeSTAR, a high-fidelity DNA polymerase in this invention ®Max DNA Polymerase Ver.2 (R047A), restriction endonucleases SacI (1078S) and DpnI (1235S) were purchased from BioNTech Biotechnology (Beijing) Co., Ltd.; plasmid extraction kit (DP103-03), common DNA product purification kit (DP204-03), and gel purification kit (DP209-02) were purchased from Tiangen Biotech (Beijing) Co., Ltd.; 12.5% ​​SDS-PAGE denaturing acrylamide color gel rapid preparation kit (C671103-0125) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; Zeocin was purchased from Invitrogen. Yeast dextran (V87797) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; other chemical reagents were purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0025] 3. Culture medium The culture medium for Escherichia coli was LB liquid medium (1% (w / v) peptone, 0.5% (w / v) yeast extract (Shanghai Yuanye Biotechnology Co., Ltd., MLP0021B, the same below), 1% (w / v) NaCl, pH 7.0).

[0026] LBZ is LB medium with 25 μg / mL Zeocin.

[0027] The yeast culture medium was YPD (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose).

[0028] The yeast selection medium was YPDZ (YPD + 100 μg / mL zeocin).

[0029] Yeast induction medium BMGY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 0.00004% (w / v) Biotin, 1% glycerol (v / v)) and BMMY medium (the composition was the same as BMGY except that 0.5% (v / v) methanol was used instead of glycerol).

[0030] Note: YNB stands for Yeast Nitrogen Base; Biotin stands for Biotin.

[0031] Pichia pastoris high-density fermentation in BSM medium: 85% phosphoric acid: 26.7 mL / L, CaSO4: 0.93 g / L, K2SO4: 18.2 g / L, MgSO4·7H2O: 14.9 g / L, KOH: 4.13 g / L, defoamer: 0.5 mL / L.

[0032] 4. Reagents and methods used for the assay of β-1-6-glucanase FlGlu30 activity. Yeast dextran stock solution: Dissolve 5g of yeast dextran in 100mL of deionized water to prepare a 5% (w / v) yeast dextran stock solution.

[0033] Preparation of DNS reagent: 6.3‰ (w / v) 3,5-dinitrosalicylic acid; 18.2% (w / v) potassium sodium tartrate tetrahydrate; 5‰ (w / v) phenol; 5‰ (w / v) anhydrous sodium sulfite, add distilled water to 100%.

[0034] The method for determining the activity of β-1-6-glucanase FlGlu30 is as follows: First, the yeast glucan stock solution and the diluted enzyme solution (diluted according to enzyme activity) were preheated at 50℃. The 500μL reaction system included: 400μL of 50mM pH 6.0 dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, 50μL of enzyme solution, and 50μL of yeast glucan stock solution. After incubation at 50℃ and 200rpm for 10min, 500μL of DNS reagent was added to terminate the reaction. After cooling, the supernatant was centrifuged and the absorbance was measured at 540nm.

[0035] The enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of glucose per minute.

[0036] Example 1: Codon Optimization and Gene Synthesis of β-1-6-glucanase FlGlu30 A strain originating from the literature (https: / / doi.org / 10.1007 / s00253-024-13269-1) was found. Flavobacterium β-1-6-glucanase (FlGlu30) of sp. NAU1659 belongs to the GH30 family of glycoside hydrolases and can effectively degrade... Magnaporthe oryzae Based on the cell wall of Guy 11, it is speculated that this protein has potential hydrolytic activity against yeast cell walls. According to the FlGlu30 protein information provided in the literature, the nucleotide sequence encoding FlGlu30 was found in the NCBI database (GenBank: PP690410). The gene is 1425 bp in length, encoding 474 amino acids, of which the first 25 amino acids are its signal peptide. Its encoded nucleotide sequence is shown in SEQ ID NO.1 (underlined are the nucleotide sequences encoding the 25 leader peptides), and its amino acid sequence is shown in SEQ ID NO.2 (underlined are the amino acid sequences of the 25 leader peptides).

[0037] ATGAAAACATCAACAAAAAACTTCAAATCCTAGTTTTGCTGCCATTAATTGCGATGCAGTTAAACTG TGGATCT MKNINKKLQILVLLPLIAMQLNCGS SKNVTANSGKVESWITTTDESSKLKKQTDLAFSSETNSNQTIEVNPAEKFQTVEGFGFSLTGGSAQAIKKLDKAKREALLQELFSRKDNAIGLSYLRISIGASDLNEKVFSYD DMPEGQTDMNLEHFNLGPDLNDVIPVLKEILAINPKIKIMGSPWSPPVWMKDNGSSKGGSLQPKYYGVYAQYFVKYIQAMKSHGIVIDAITPQNEPLHPGNNPSMLMLAEQQA DFIGNHLGPAFAKAGIKTKIIVYDHNCNKPEYPLTILRDPKANPFVTGSAFHLYEGDISALTTVHNEFPNKDLYFTEQYTGSGTNFETDLKWSVKNVVIGSMRNWSKNALSWG LANDEFYKPFTPGGCSTCKGALMIDQNQNIKREVGYYIIGHASKFVPEGSVRIGSNIAGNLHNVAFKTPSGQTVLIVENDGASAETFNIKYNQKQTTTTLNAGAVATFVW (SEQ ID NO.2).

[0038] because Flavobacterium sp. NAU1659 and Pichia pastoris differ in their gene coding, therefore, FlGlu30 needs to be optimized based on the codon bias of Pichia pastoris. The optimized FlGlu30 coding gene is named FlGlu30-1. To facilitate affinity purification of recombinant FlGlu30, a 6His-Tag tag is introduced at the C-terminus of FlGlu30. The nucleotide sequence of FlGlu30-1 is shown in SEQ ID NO.3 (the underlined portion is the nucleotide sequence encoding the 6His-Tag), and the amino acid sequence is shown in SEQ ID NO.4 (the underlined portion is the 6His-Tag).

[0039] CACCACCACCATC ACCAC TAA (SEQ ID NO.3).

[0040] SKNVTANSGKVESWITTTDESSKLKKQTDLAFSSETNSNQTIEVNPAEKFQTVEGFGFSLTGGSAQAIKKLDKAKREALLQELFSRKDNAIGLSYLRISIGASDLNEKVFSY DDMPEGQTDMNLEHFNLGPDLNDVIPVLKEILAINPKIKIMGSPWSPPVWMKDNGSSKGGSLQPKYYGVYAQYFVKYIQAMKSHGIVIDAITPQNEPLHPGNNPSMLMLAEQ QADFIGNHLGPAKAGIKTKIIVYDHNCNKPEYPLTILRDPKANPFVTGSAFHLYEGDISALTTVHNEFPNKDLYFTEQYTGSGTNFETDLKWSVKNVVIGSMRNWSKNAL SWGLANDEFYKPFTPGGCSTCKGALMIDQNQNIKREVGYYIIGHASKFVPEGSVRIGSNIAGNLHNVAFKTPSGQTVLIVENDGASAETFNIKYNQKQTTTTLNAGAVATFVW HHHHHH (SEQ ID NO.4).

[0041] Example 2: Construction of the expression strain and expression and purification of recombinant FlGlu30 This invention uses pPICZαA as an expression vector, and the signal peptide used is the α signal peptide carried by pPICZαA. Therefore, the signal peptide of FlGlu30 itself needs to be removed during the construction process. The construction process of the expression vector is as follows: The original gene sequence (SEQ ID NO.1) of FlGlu30-1 without the signal peptide was given to Sangon Biotech (Shanghai) Co., Ltd. for sequence optimization. The optimized gene sequence of FlGlu30-1 without the signal peptide was obtained and the gene was synthesized. The synthesized FlGlu30-1 sequence was inserted into the pPICZαA vector to obtain the recombinant vector pPICZαA-FlGlu30-1. The recombinant vector pPICZαA-FlGlu30-1 was introduced into Escherichia coli Top10 to obtain the recombinant bacteria pPICZαA-FlGlu30-1-Top10. The recombinant plasmid pPICZαA-FlGlu30-1 was extracted from the recombinant bacteria pPICZαA-FlGlu30-1-Top10 using a plasmid extraction kit (#DP103-03).

[0042] The extracted expression vector pPICZαA-FlGlu30-1 was then linearized with the restriction endonuclease SacI. The linearization system was as follows: pPICZαA-FlGlu30-1: 5 μg, SacI: 5 μL, 10×QuickCut Buffer: 5 μL, and sterile water to a final volume of 50 μL. After incubation at 37°C for 30 min, the product was recovered using a gel purification kit (#DP209-02). The recovered product was then transformed into Pichia pastoris X33 via electroporation, following the Pichia pastoris instruction manual provided by Thermo Fisher Scientific (https: / / www.thermofisher.cn / order / catalog / product / V19520?ICID=search-product, hereinafter the same). The transformed product was evenly spread on YPDZ plates and incubated statically at 30°C for 3 to 5 days to obtain transformants.

[0043] Transformants grown from static culture were screened using the following procedure: Recombinant transformants from YPDZ plates were individually transferred using toothpicks to 24-well plates containing 3 mL of BMGY medium per well. After overnight incubation at 30°C and 200 rpm for 24 hours, the plate was centrifuged at 4000 rpm for 10 min to remove the supernatant. 3 mL of BMGY medium was added, and fermentation was induced by adding 30 μL of methanol every 24 hours at 30°C and 200 rpm. After 72 hours of induced fermentation, the activity of recombinant β-1-6-glucanase FlGlu30 was measured. A dominant enzyme strain, named FlGlu-15, was obtained from 24 transformants and stored at -80°C for later use.

[0044] The recombinant engineered bacteria FlGlu-15 was further cultured in a 500mL shake flask. The experimental procedure was as follows: (1) The yeast culture containing recombinant FlGlu-15 stored at -80℃ was inoculated into a 250mL shake flask containing 50mL BMGY medium and cultured at 30℃ and 200rpm until OD. 600 (2) Centrifuge the cultured bacterial solution at 4000 rpm for 10 min, remove the supernatant, and transfer it to a 500 mL shake flask containing 100 mL of BMMY medium. The initial OD of the bacterial solution is 3.0. 600 The culture was carried out at a value of 1.0, 30℃, and 200rpm. During the culture, methanol was added at a ratio of 0.5% (v / v) every 24 hours for induction culture. At the same time, samples were taken to determine the activity of β-1-6-glucanase FlGlu30.

[0045] The purification process of recombinant β-1-6-glucanase FlGlu30 is roughly as follows: (1) After centrifuging the fermentation broth (10000g, 10min), the supernatant was collected to obtain the crude fermentation enzyme solution; (2) The purification of the crude fermentation enzyme solution was carried out on the AKTA purification system, using the affinity chromatography principle of nickel column and 6His Tag: First, Binding Buffer (pH7.5, 20mM imidazole, 20mM Tris, 0.5M NaCl) was connected to the tubing, and the flow rate was set to 1.0mL / min to balance the nickel column. After the nickel column was completely balanced, the tubing was connected to the crude fermentation enzyme solution, and the flow rate was adjusted to 0.5mL / min to fully bind the target protein. Then, Wash Buffer (pH7.5, 50mM imidazole, 20mM Tris, 0.5M NaCl) was connected to the tubing, and the flow rate was set to 1.0mL / min to wash away the impurities that had a weak binding ability to the nickel column. Finally, the tubing was connected to Elution Buffer (pH 7.5, 500 mM imidazole, 20 mM Tris, 0.5 M NaCl) and the flow rate was set to 1.0 mL / min to collect the target protein. The specific activity of the purified β-1-6-glucanase FlGlu30 was 23.21 U / mg.

[0046] Example 3: Homology Modeling and Molecular Docking of β-1-6-glucanase FlGlu30 Example 2 revealed that the specific activity of recombinant β-1-6-glucanase FlGlu30-1 was 23.21 U / mg, indicating a need for further improvement to lay the foundation for its industrial application. This invention further enhances the specific activity of β-1-6-glucanase FlGlu30 through rational protein design. Rational protein design must be based on the three-dimensional structure of the enzyme protein; therefore, it is necessary to simulate and construct the three-dimensional conformation of recombinant β-1-6-glucanase FlGlu30.

[0047] The three-dimensional conformation of β-1-6-glucanase FlGlu30 was obtained through analysis and modeling using the online website SWISS-MODEL server (https: / / swissmodel.expasy.org / interactive). The obtained three-dimensional conformation was evaluated using the online website SAVES v6.0 (https: / / saves.mbi.ucla.edu / ). Figure 1 The Laplace plot results show that 99.5% of the amino acids in the model are located in the optimal region and other allowed regions, and the GMQE value reaches 0.97, indicating that the obtained model has a good conformation and can be used for further bioinformatics analysis.

[0048] β-1-6-glucanase FlGlu30 and gentiotriose were molecularly docked using the molecular docking software Autodock, with a binding free energy of -4.71 kcal / mol. Visual analysis of the docking results using Pymol software revealed that β-1-6-glucanase FlGlu30 primarily binds gentiotriose to the catalytically active region via hydrogen bonds. Figure 2 For example, Lys87 forms two hydrogen bonds with gentiotriose, with bond lengths of 2.9 Å and 3.0 Å, respectively; Asp88 and Tyr375 each form one hydrogen bond with gentiotriose, with bond lengths of 3.2 Å and 1.9 Å, respectively. The analysis results indicate that Lys87, Asp88, and Tyr375 are key active amino acids for β-1-6-glucanase FlGlu30.

[0049] Based on this, the present invention rationally mutates key amino acids in the active region of β-1-6-glucanase FlGlu30 to increase hydrogen bonds between it and the substrate, thereby further enhancing its specific activity. The mutants to be constructed include: K87R, K87H, K87Q, D88N, D88S, D88E, Y375W, Y375S, and Y375T.

[0050] Example 4 Single-point mutation Primers were designed separately (primer sequences are shown in Table 1) to construct mutants. The construction process for different mutants is as follows (taking the K87R site as an example, and others are similar): Using the expression vector pPICZαA-FlGlu30-1 as a template, PCR amplification was performed using upstream and downstream primers D87N-fw and D87N-rev. The 50μL PCR system included: pPICZαA-FlGlu30-1: 50ng, 1μM D87N-fw and 1μM D87N-rev: 1μL each, PrimeSTAR... ®Max DNA Polymerase Ver.2: 25 μL, sterile water to make up to 50 μL. The PCR program was as follows: 98℃: 5 min, (98℃: 10 s; 60℃: 15 s; 72℃: 2 min) × 30 cycles, 72℃: 10 min. After PCR, the PCR amplification results were detected by agarose gel electrophoresis. The PCR product was purified and recovered using a gel purification kit. Trace amounts of the original plasmid (expression vector pPICZαA-FlGlu30-1) in the recovered PCR product were digested with restriction endonuclease DpnI. The 10 μL digestion system was as follows: PCR product: 200 ng, 10×T Buffer: 1 μL, DpnI: 1 μL, sterile water to make up to 10 μL. After incubating the reaction system at 37℃ for 30 min, the digested product was recovered using a standard DNA product purification kit (DP204-03). The recovered digestion product was then transformed into E. coli Top10 using the heat shock method. The specific process was as follows: 10 μL of the recovered digestion product and 100 μL of E. coli Top10 competent cells were gently mixed on ice, incubated on ice for 30 min, then heat-shocked at 42°C for 90 s, and incubated on ice again for 2 min. Then, 1 mL of antibiotic-free LB liquid medium was added, and the mixture was incubated at 37°C for 1 h. After centrifugation at 4000 rpm for 2 min, the cells were resuspended in 100 μL of LB medium. The resuspended bacterial solution was evenly spread onto LBZ medium using a sterile spreader. The plates were inverted and placed in a 37°C incubator. After culturing for 18 hours, transformants were picked and cultured, and sequencing was used to determine whether the expression vector pPICZαA-FlGlu30-1-1 corresponding to the mutant K87R was successfully constructed.

[0051] Nine mutant expression vectors were constructed using the same method: pPICZαA-FlGlu30-1-1 (corresponding to mutant K87R), pPICZαA-FlGlu30-1-2 (mutant K87H), pPICZαA-FlGlu30-1-3 (mutant K87Q), pPICZαA-FlGlu30-1-4 (mutant D88N), pPICZαA-FlGlu30-1-5 (mutant D88S), pPICZαA-FlGlu30-1-6 (mutant D88E), pPICZαA-FlGlu30-1-7 (mutant Y375W), pPICZαA-FlGlu30-1-8 (mutant Y375S), and pPICZαA-FlGlu30-1-9 (mutant Y375T).

[0052] Table 1 Primers for single-point mutation

[0053] Nine constructed mutant expression vectors were linearized with SacI (linearization process as in Example 2). The transformation concentration of different mutant plasmids was controlled at 80 ng / μL, and they were transformed into Pichia pastoris X33 by electroporation. The transformants were plated on YPDZ plates and screened after 4 days at 30°C (same as in Example 2). Because the plasmid concentration was controlled at 80 ng, only a few positive transformants (about 2-4) grew on each YPDZ plate. The screening of positive transformants was the same as in Example 2, and the enzyme activities of the recombinant bacteria corresponding to the nine mutants are shown in Table 2.

[0054] As shown in Table 2, among the nine mutants constructed, the fermentation enzyme activity of the recombinant engineered bacteria corresponding to mutants K87R, K87Q, D88N, D88S, and Y375W was significantly higher than that of the control group.

[0055] Table 2 Fermentation enzyme activities of different mutant recombinant bacteria

[0056] Example 5 Combined Mutations Combination mutations were performed using the positive mutants K87R, K87Q, D88N, D88S, and Y375W obtained in Example 4 to further enhance the specific enzyme activity of β-1-6-glucanase FlGlu30. The primers used in the experiment are shown in Table 3. In the table below, for cases involving two primer pairs, two rounds of PCR amplification are required during construction. The second round of amplification uses the PCR product from the first round as a template, and the PCR system and amplification process are the same as in Example 4. The construction and screening processes of the mutants are the same as in Example 4. Experiments showed that the recombinant strain corresponding to the combined mutant K87Q / D88S / Y375W had the best effect, with a fermentation enzyme activity of 2.52 U / mL, significantly higher than the wild-type enzyme activity. For ease of labeling, the mutant K87Q / D88S / Y375W was named mutant FlGlu30M.

[0057] Table 3 Primer information corresponding to combined mutations

[0058] Table 4. Fermentation enzyme activity of recombinant bacteria with combined mutants

[0059] Example 6: High-density fermentation culture of mutant FlGlu30M High-density fermentation of recombinant engineered bacteria was carried out in a 7-liter fermenter. The specific process is as follows: A single colony of recombinant yeast was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD medium and cultured overnight at 30°C with shaking at 200 rpm. Then, the overnight cultured recombinant yeast was inoculated at a rate of 1% (v / v) into a 500 mL Erlenmeyer flask containing 100 mL of YPD medium and cultured overnight at 30°C with shaking at 200 rpm until OD reached... 600 Greater than 10. The recombinant yeast strains, cultured overnight twice, were inoculated at a rate of 10% (v / v) into a 7L fermenter containing 2.5L of BSM medium. The culture conditions for the recombinant yeast strains in the 7L fermenter were: temperature 30℃, pH 5.0, stirring speed 700 rpm, and air flow rate 40 L / min. In the initial stage of culture, glycerol was used as the carbon source for cell growth. When the cell wet weight reached a certain amount (approximately 200 g / L), the addition of glycerol was stopped, and after the glycerol was completely absorbed by the cells (dissolved oxygen rose rapidly), methanol induction was initiated. The amount of methanol added was adjusted according to dissolved oxygen levels. The specific adjustment process was as follows: During methanol induction, dissolved oxygen was controlled at 10-30%. When dissolved oxygen fell below 10% and continued to decrease, methanol feeding was stopped. When dissolved oxygen rose above 30% and continued to rise, methanol feeding was restarted (the actual methanol flow rate was adjusted according to the fermentation cycle, specifically: at 24 hours of fermentation, the methanol flow rate was 2 g / L / h, increasing by 0.4 g / L / h every 2 hours; from 40 to 72 hours, the methanol flow rate was set to 5.5 g / L / h; from 72 to 96 hours, the methanol flow rate was set to 6 g / L / h; from 96 to 120 hours, the methanol flow rate was set to 6.5 g / L / h; and from 120 to 168 hours, the methanol flow rate was set to 7 g / L / h). During the cultivation process, samples were taken every 24 hours to determine the cell wet weight, enzyme activity, and total protein concentration. The purification of the mutant FlGlu30M was carried out according to Example 2.

[0060] Depend on Figure 3 It can be seen that the molecular weight of the mutant FlGlu30M is around 55 kDa, which is basically consistent with its theoretical molecular weight. The specific enzyme activity of the purified mutant FlGlu30M is 45.25 U / mg. Figure 4 It can be seen that when induced and cultured for 144 hours, the fermentation enzyme activity reached its maximum (125.8 U / mL), the total protein concentration reached its maximum of 4.98 g / L, and the cell wet weight reached its maximum (386 g / L) after 168 hours of induction.

[0061] Example 7: Recombinant β-1-6-glucanase mutant FlGlu30M hydrolyzes the cell wall of Saccharomyces cerevisiae. The recombinant β-1-6-glucanase mutant FlGlu30M was used to hydrolyze the cell wall of Saccharomyces cerevisiae as follows: 1.0 g of yeast cell wall was weighed and suspended in 50 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (50 mM, pH 6.0). 100 U of recombinant β-1-6-glucanase mutant FlGlu30M was added, and the hydrolysis reaction was carried out at 50 °C and 200 rpm. 500 μL samples were taken after 0, 0.5, 1, 1.5, and 2 hours of hydrolysis, and the content of oligosaccharides produced by hydrolysis was determined using DNS reagent.

[0062] Depend on Figure 5 It is known that after one hour of hydrolysis, the reaction almost reaches equilibrium, at which point the oligosaccharide content in the reaction system is approximately 0.65 g / L, and the oligosaccharide hydrolysis efficiency is approximately 72.2% (hydrolysis efficiency = oligosaccharide content produced by hydrolysis / theoretical β-1,6-glucan content in the cell wall). Oligosaccharides can be used as novel green biostimulants for crop disease resistance and growth promotion, soil improvement, and quality and yield enhancement. In this invention, the recombinant β-1,6-glucanase mutant FlGlu30M can efficiently hydrolyze yeast cell walls to produce oligosaccharides, indicating that the recombinant β-1,6-glucanase mutant FlGlu30M has broad application prospects in the enzymatic preparation of oligosaccharides.

Claims

1. A β-1-6-glucanase mutant FlGlu30M, characterized in that, The amino acid sequence of the β-1-6-glucanase mutant FlGlu30M is shown in SEQ ID NO.

6.

2. The β-1-6-glucanase mutant FlGlu30M as described in claim 1, characterized in that, The sequence encoding the amino acid is a polynucleotide, and the sequence of the polynucleotide is shown in SEQ ID NO.

5.

3. A recombinant expression vector pPICZαA-FlGlu30M, characterized in that, Polynucleotides comprising the β-1-6-glucanase mutant FlGlu30M as described in claim 2.

4. The recombinant expression vector pPICZαA-FlGlu30M as described in claim 3, characterized in that, The polynucleotide of the β-1-6-glucanase mutant FlGlu30M has a 6His-Tag nucleotide sequence added to its C-terminus.

5. A recombinant bacterium, characterized in that, It contains the recombinant expression vector pPICZαA-FlGlu30M as described in any one of claims 3 to 4.

6. The recombinant bacteria according to claim 5, characterized in that, The recombinant bacteria used Pichia pastoris as the host.

7. The recombinant bacteria according to claim 6, characterized in that, The engineered Pichia pastoris strain is Pichia pastoris X33.

8. The use of the β-1-6-glucanase mutant FlGlu30M as described in claim 1 in the preparation of oligodextrose.

9. The application according to claim 8, characterized in that, The oligosaccharides were prepared by hydrolyzing and pretreating the cell walls of Saccharomyces cerevisiae using the β-1-6-glucanase mutant FlGlu30M.

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