A beta-mannanase mutant, genes and uses thereof

Abstract

The application discloses a beta-mannanase mutant, a coding gene and a use thereof, and belongs to the technical field of enzyme engineering and feed additives.The wild-type beta-mannanase is used as a parent, and a novel beta-mannanase mutant M1 containing three-site synergistic mutations of L23A, E120Q and W181F is obtained through site-directed mutation modification.The beta-mannanase mutant M1 is perfectly adapted to the high-temperature granulation processing condition of feed, can significantly improve the feed utilization rate and animal growth performance, and has extremely high industrial application and popularization value in the field of livestock and poultry feed and aquatic feed.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering and feed additive technology, specifically to a β-mannanase mutant, its encoding gene, and its uses. Background Technology

[0002] β-Mannanase is a core anti-nutritional factor degrading enzyme in the feed industry. It specifically hydrolyzes the β-1,4-mannan glycosidic bonds in plant raw materials, breaking down large mannan molecules into small-molecule reducing sugars that can be absorbed by animals. In large-scale livestock, poultry, and aquaculture farming, the mainstream corn-soybean meal feed is rich in water-soluble mannans. These polysaccharides cannot be degraded by the animal's own digestive enzymes and are one of the most significant anti-nutritional factors in feed. Their high presence significantly increases the viscosity of intestinal digesta, hindering the transmembrane absorption of proteins, amino acids, starches, and minerals by intestinal epithelial cells, directly leading to decreased feed conversion ratio and stunted animal growth. Simultaneously, high-viscosity intestinal contents slow down digesta emptying, promoting the growth of harmful bacteria such as Escherichia coli and Salmonella, disrupting intestinal flora homeostasis, damaging the intestinal mucosal barrier function, reducing the animal's disease resistance, and significantly increasing farming losses and production costs.

[0003] Currently, most commercially available β-mannanases for feed are natural wild-type microbial enzymes, which generally have inherent performance defects and are difficult to adapt to modern feed processing and aquaculture applications. On the one hand, wild-type β-mannanases have low catalytic activity and limited degradation efficiency per unit enzyme activity, resulting in large feed addition dosages and high application costs. On the other hand, wild-type enzymes have poor structural stability, maintaining activity only within a narrow range of room temperature and slightly acidic / neutral pH. High-temperature conditioning, pelleting, and extrusion processes at 75–85°C during feed production cause rapid collapse of their protein conformation and significant loss of enzyme activity, resulting in extremely low residual effective activity after processing. Furthermore, wild-type enzymes cannot tolerate the highly acidic environment of livestock stomachs and the acid-base fluctuations during feed processing, easily becoming inactive after entering the animal's gastrointestinal tract. This leads to unstable synergistic effects in actual feeding, severely restricting the large-scale, industrial application of β-mannanases in the field of high-end green feed additives. Site-directed molecular mutagenesis is a core technology for targeted improvement of enzyme properties, precisely optimizing enzyme molecular structure and catalytic performance, and is a key technical means to overcome the application bottlenecks of wild-type enzymes.

[0004] Existing research largely focuses on single-site mutation modification, which suffers from limitations in improving performance in a limited way and stability, failing to simultaneously address enzyme activity, heat resistance, and acid-base tolerance. Therefore, this invention utilizes precise bioinformatics analysis to screen key functional sites affecting the catalytic efficiency and structural stability of wild-type β-mannanase. It innovatively employs a site-coordinated mutation strategy, combined with Pichia pastoris-specific codon optimization and homologous integration expression system modification, achieving a comprehensive upgrade of enzyme performance from multiple dimensions and effectively overcoming the shortcomings of existing technologies. Summary of the Invention

[0005] Based on the technical problems existing in the prior art, the technical solution of the present invention is implemented as follows: This invention provides a β-mannanase mutant, characterized in that the mutant is obtained by site-directed amino acid mutation of the wild-type β-mannanase shown in SEQ ID NO:1; the site-directed mutation is L23A, E120Q, or W181F.

[0006] Furthermore, the amino acid sequence of the β-mannanase mutant is shown in SEQ ID NO:2.

[0007] The present invention also provides a gene encoding the β-mannanase mutant.

[0008] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0009] The present invention also provides a recombinant expression vector comprising the aforementioned gene sequence.

[0010] Furthermore, the recombinant expression vector uses pPIC9K as its backbone vector.

[0011] The present invention also provides a recombinant engineered strain, characterized in that the recombinant engineered strain comprises the recombinant expression vector.

[0012] Furthermore, the host of the recombinant engineered strain is Pichia pastoris GS115.

[0013] The present invention also provides the application of the β-mannanase mutant in the preparation of feed additives.

[0014] Furthermore, the feed additive is suitable for the degradation and improvement of anti-nutritional factors in livestock, poultry, and aquaculture feeds.

[0015] The present invention also provides a feed additive, characterized in that the feed additive contains the β-mannanase mutant described above.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs an innovative three-site synergistic mutation strategy. The L23A mutation optimizes the substrate binding pocket spatial structure and enhances substrate affinity; the E120Q mutation optimizes the catalytic microenvironment of the active site and lowers the reaction energy barrier; and the W181F mutation strengthens the internal hydrophobic interaction of the protein and improves structural rigidity. The three-site synergistic effect results in a 59.9% increase in specific enzyme activity of mutant M1 compared to the wild-type enzyme, achieving a qualitative breakthrough in catalytic efficiency. At the same time, it completely solves the defect of easy inactivation at high temperatures in wild-type enzymes. Even after extreme high-temperature treatment at 85℃, it still retains 65.3% of high activity, which can fully withstand the high-temperature pelleting and extrusion processes in the feed industry, and the processing stability is greatly improved. It also significantly broadens the acid-base stability range, maintaining more than 85% residual enzyme activity in environments ranging from strongly acidic pH 2.0 to neutral pH 7.0, perfectly adapting to the acid-base fluctuations in feed processing and the gastrointestinal physiological environment of livestock, poultry, and aquatic animals. The mutant M1 of this invention can effectively degrade anti-nutritional factors in feed, optimize intestinal microecology, and improve feed utilization and animal growth performance. Its performance is superior to existing wild-type enzymes in all aspects, with outstanding advantages in stability, applicability and economy, and it has extremely high value for industrialization and promotion. Detailed Implementation

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to. Example 1: Design, mutation, and gene synthesis of β-mannanase mutant M1

[0021] This embodiment is derived from... Micromonospora humidisoli The wild-type β-mannanase (SEQ ID NO:1) from GenBank (GHJ05707.1) is the original sequence, containing a total of 332 amino acids. To achieve targeted improvement of its enzymatic properties, this invention utilizes the SWISS-MODEL online protein modeling platform to construct the complete three-dimensional structure of the wild-type β-mannanase. The active site, substrate-binding pocket, flexible loop region, and key structurally stable amino acid residues of the enzyme molecule are analyzed using PyMOL visualization software. Based on the mannan substrate binding mechanism, key mutation sites affecting the enzyme's catalytic activity and structural stability are screened.

[0022] Structural simulations and molecular dynamics analyses confirmed that leucine (L) at position 23, glutamate (E) at position 120, and tryptophan (W) at position 181 of the wild-type enzyme are located in the substrate binding pocket, the catalytic region of the active site, and the protein's flexible structure region, respectively. The amino acid polarity and steric hindrance at these sites are the core factors restricting the catalytic efficiency and thermal stability of the wild-type enzyme. This invention precisely optimizes these sites through site-directed mutagenesis: leucine at position 23 is mutated to alanine (L23A), glutamate at position 120 is mutated to glutamine (E120Q), and tryptophan at position 181 is mutated to phenylalanine (W181F), ultimately obtaining a performance-improved β-mannanase mutant M1 (SEQ ID NO:2). The specific sequence is as follows: Wild-type β-mannanase (SEQ ID NO:1): MRTKYLVTFLLVVALAFTEFAV L RRVDITVAGSAAGPGGVAATEEQVPAYDVTALKSPKGRYLGVTVTGGAEMARVRAFAEETGKTPNLVAIFESFDDRFAASEVRELHNYGALAVIRW E PYDVPLRDIAAGKHDAYVKEFATAVRTLNLPIALTFAHEMNGHWYSWGTKQTRATEYVAA W RHLHDIFTQQGATNVIWTWTPNVVNYLRKVSIRALYPGDAYVDWVGMDGYYTNRGPKTFRDLFVPTITEIRTFTRRPLLIVETGAEPGSRRPAQITDLLTNVAKRGDLLGVAYFNINGSGKWNIDRDDAALRAFRRAAAKDTFGFDIRTVK.

[0023] β-Mannanase mutant M1 (SEQ ID NO:2): MRTKYLVTFLLVVALAFTEFAV A RRVDITVAGSAAGPGGVAATEEQVPAYDVTALKSPKGRYLGVTVTGGAEMARVRAFAEETGKTPNLVAIFESFDDRFAASEVRELHNYGALAVIRW Q PYDVPLRDIAAGKHDAYVKEFATAVRTLNLPIALTFAHEMNGHWYSWGTKQTRATEYVAA F RHLHDIFTQQGATNVIWTWTPNVVNYLRKVSIRALYPGDAYVDWVGMDGYYTNRGPKTFRDLFVPTITEIRTFTRRPLLIVETGAEPGSRRPAQITDLLTNVAKRGDLLGVAYFNINGSGKWNIDRDDAALRAFRRAAAKDTFGFDIRTVK.

[0024] Based on the codon usage preferences of Pichia pastoris, this invention performs global codon optimization on the full-length coding gene of the β-mannanase mutant M1, removing rare yeast codons, optimizing the GC content of the gene, eliminating internal termination signals, repetitive sequences, and secondary structures. Simultaneously, an EcoRI restriction endonuclease site is introduced at the 5' end and a NotI restriction endonuclease site is introduced at the 3' end of the optimized gene, adapting it for subsequent vector cloning and construction. The optimized complete nucleotide sequence is shown in SEQ ID NO:3: ATGCGAACTAAATACCTGGTAACTTTTTTGTTAGTAGTCGCACTGGCATTCACTGAATTCGCCGTGGCAAGAAGGGTAGATATAACCGTTGCCGGTTCTGCTGCTGGCCCAGGAGGTGTGGCCGCAACTGAAGAGCAAGTCCCAGCTTATGATGTAACAGCACTAAAGTCACCTAAAGGTAGATATCTTGGAGTAACGGTTACTGGTGGTGCTGAAATGGCAAGAGTTAGAGCATTTGCTGAAGAAACGGGAAAAACTCCAAACCTTGTTGCCATCTTCGAGTCCTTTGACGACAGATTCGCTGCCTCTGAGGTGAGAGAGTTGCACAACTACGGAGCCCTTGCAGTTATTCGTTGGCAGCCATACGACGTGCCTCTGAGAGACATAGCCGCAGGAAAGCACGATGCCTACGTGAAAGAATTTGCAACCGCTGTAAGAACCTTGAACCTGCCAATTGCACTGACTTTTGCCCATGAAATGAATGGTCACTGGTATTCATGGGGTACAAAACAAACACGAGCTACAGAATATGTGGCTGCCTTTAGACACCTTCATGACATTTTCACTCAGCAAGGTGCTACAAATGTGATATGGACATGGACCCCCAATGTTGTGAATTACCTTAGAAAAGTGTCCATCAGGGCTCTTTACCCCGGAGACGCCTACGTGGACTGGGTCGGTATGGACGGATACTACACAAACAGGGGACCTAAGACATTTAGGGATCTATTTGTTCCAACCATCACCGAGATTAGAACATTCACAAGGAGACCTCTGTTAATTGTTGAAACTGGCGCCGAACCAGGATCAAGGCGTCCCGCCCAAATAACCGACCTATTAACTAACGTTGCTAAGCGAGGAGACTTGTTGGGTGTCGCTTACTTCAACATCAACGGTTCAGGAAAATGGAACATTGATCGTGACGATGCTGCCCTGAGAGCCTTCAGACGTGCTGCTGCCAAGGACACTTTTGGCTTCGATATCAGAACAGTAAAG(SEQID NO:3).

[0025] The optimized gene sequence was commissioned to Shanghai Sangon Biotech Co., Ltd. for whole-genome artificial synthesis. After synthesis, sequencing verified that the sequence was 100% correct and could be used for subsequent vector construction and expression. Example 2: Construction, transformation, expression, and purification of the β-mannanase mutant M1 vector.

[0026] In this embodiment, pPIC9K was used as the expression vector and Pichia pastoris GS115 was used as the expression host. Through PCR amplification, double enzyme digestion and ligation, electroporation, homology integration, resistance selection, induced expression, and protein purification, a recombinant yeast strain that efficiently secretes and expresses the β-mannanase mutant M1 was constructed, and the purified enzyme solution was prepared. The specific steps are as follows: 1. Target gene-specific PCR amplification Using the gene fragment SEQ ID NO:3, synthesized by Shanghai Sangon Biotech and pre-loaded with three amino acid mutations (L23A, E120Q, and W181F) and optimized codons, as a PCR amplification template, the full-length coding sequence of mutant M1 was amplified using upstream and downstream primers containing specific restriction enzyme sites. The primer sequences are strictly as follows: Forward outer primer F (containing EcoRI restriction site): 5'-GAGAATTCCGTACCAAGTACCTGGTGACCTT-3'; Reverse outer primer R (containing NotⅠ restriction site): 5'-GCGCGGCCGCTTACACGGTACCGTCTTGATCCAA-3'.

[0027] Prepare a 50 μL standard high-fidelity PCR amplification system: 25 μL 2×Phanta Max Buffer, 4 μL dNTP Mix (10 mM), 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 1 μL template DNA, 1 μL Phanta Max ultra-fidelity DNA polymerase, and 15 μL sterile ultrapure water.

[0028] PCR amplification program settings: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 90 s, 30 cycles; 72℃ final extension for 5 min, stored at 4℃. The purity of the amplified products was detected by 1% agarose gel electrophoresis. The target gene was purified using an Omega gel extraction kit to remove impurities and residual primers, obtaining a high-purity target gene fragment.

[0029] 2. Construction of recombinant expression vectors The purified target gene and pPIC9K empty vector plasmid were double-digested with EcoRI and Not I restriction endonucleases at 37℃ for 4 h. The 50 μL digestion system consisted of: 20 μL plasmid / target gene, 2 μL EcoRI, 2 μL Not I, 5 μL 10×CutSmart Buffer, and 21 μL sterile ultrapure water. After digestion, the linearized vector and target gene fragment were recovered by electrophoresis and ligated using T4 DNA ligase at 22℃ for 2 h. The ligation system consisted of: 4 μL vector fragment, 6 μL target gene fragment, 1 μL T4 ligase, and 1 μL 10× ligation buffer.

[0030] The ligation product was heat-shocked and transformed into *E. coli* DH5α competent cells. The cells were then incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and cooled on ice for 2 min. After recovery in antibiotic-free LB liquid medium for 1 h, the cells were plated onto LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C for 12 h. Single colonies were picked for colony PCR verification. Positive clones were sent to Shanghai Sangon Biotech for sequencing. After confirming the sequence was completely correct, high-purity recombinant plasmid pPIC9K-M1 was extracted.

[0031] 3. Linearization transformation of Pichia pastoris and screening of engineered strains The recombinant plasmid pPIC9K-M1 was linearized by single-enzyme digestion with Sac I restriction endonuclease, enabling the vector to integrate homologously at the AOX1 site. The linearized product was purified and used for yeast electroporation. 80 μL of Pichia pastoris GS115 competent cells were mixed with 10 μg of linearized plasmid, incubated on ice for 5 min, and then added to a pre-chilled electroporation cuvette. The electroporation parameters were set as follows: voltage 2.5 kV, capacitance 25 μF, and resistance 200 Ω. Immediately after electroporation, 1 mL of pre-chilled 1 M sorbitol was added to resuspend the cells.

[0032] The bacterial cells were resuspended and incubated at 30°C for 2 hours to recover. They were then plated onto MD solid selection plates and incubated at 30°C inverted mode for 3 days to screen for positive transformants. To obtain high-copy-value, high-expression strains, single colonies were sequentially transferred to YPD gradient resistance plates containing 1 mg / mL, 2 mg / mL, 4 mg / mL, and 8 mg / mL genimycin. High-resistance strains were screened stepwise, ultimately yielding the stable homologous integration, Mut+ phenotype recombinant engineered bacterium GS115-pPIC9K-M1, which was then stored at low temperature on slant for later use.

[0033] At the same time, a control strain GS115-pPIC9K-WT (wild-type β-mannanase mutant) integrating wild-type genes was constructed.

[0034] 4. Seed culture of recombinant strains Single colonies of the recombinant strain GS115-pPIC9K-M1 and the wild-type control strain GS115-pPIC9K-WT were aseptically inoculated into 500 mL sterile shake flasks containing 100 mL of BMGY liquid medium. Culture conditions were set at a constant temperature of 30℃ and a shaking speed of 220 rpm for 18 hours, during which the bacterial growth status was monitored regularly. Culture was continued until the OD600 value of the bacterial solution stabilized in the range of 6.0–8.0, indicating that the bacteria were in the late logarithmic growth phase and at their optimal activity. Sufficient qualified seed culture was obtained for subsequent induction expression experiments. The BMGY medium formulation consisted of 1% yeast extract, 2% tryptone, 1.34% amino-free yeast nitrogen (YNB), 0.00004% biotin, and 1% glycerol (v / v). The medium was autoclaved at 121℃ for 20 min and then cooled before use.

[0035] 5. Methanol-induced expression of recombinant strains Take the above seed culture and centrifuge at 8000 rpm and 4℃ for 5 min using a high-speed refrigerated centrifuge. Completely discard the supernatant from the upper BMGY medium and collect the bacterial precipitate. Gently resuspend the bacterial precipitate in sterile BMMY induction medium, mix thoroughly, and measure the OD600 of the bacterial solution. Adjust the OD600 value to a uniform 0.8 by adding BMMY medium to ensure consistent initial induction concentrations for both groups of strains. Transfer the adjusted bacterial solution to a 500 mL sterile shake flask and incubate at 30℃ and 220 rpm for 72 h. Every 24 h, aseptically add 1% (V / V) anhydrous methanol to a final concentration. BMMY medium formulation: 1% yeast extract, 2% tryptone, 1.34% YNB, 0.00004% biotin, with 0.5% methanol (V / V) replacing glycerol. Adjust the pH to 4.0 and autoclave before use.

[0036] 6. Preparation and pretreatment of crude fermentation enzyme solution After 72 hours of induction culture, the culture was terminated, and all fermentation broth was collected. The mixture was centrifuged at 8000 rpm and 4°C for 10 minutes to thoroughly remove yeast cells, residues, and impurities. The clear supernatant was collected as the crude β-mannanase enzyme solution. The collected crude enzyme solution was thoroughly mixed and first filtered through a 0.45 μm sterile aqueous filter membrane to remove trace suspended impurities and cell fragments, preventing clogging of subsequent purification equipment. This pretreated crude enzyme solution was then prepared for use.

[0037] 7. Enzyme solution concentration and purification (1) Ultrafiltration desalting and concentration: The pretreated crude enzyme solution is ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove inorganic salts, small molecule proteins and other impurities from the enzyme solution. At the same time, the enzyme solution is gradient concentrated to reduce the volume of the enzyme solution to 1 / 10 of the original volume.

[0038] (2) Anion exchange column chromatography purification: A DEAE-Sepharose Fast Flow anion exchange column was used for fine purification. The column was equilibrated with pH 5.5 citrate-disodium hydrogen phosphate buffer until the baseline was stable. The ultrafiltration concentrated enzyme solution was slowly loaded onto the column. After loading, a linear gradient elution of 0–500 mmol / L NaCl was used, with the elution flow rate set to 1.0 mL / min. Elution peaks were collected stepwise. By enzyme activity tracking detection, the high enzyme activity elution fractions were combined to obtain a single electrophoretically pure β-mannanase protein solution, which yielded purified wild-type β-mannanase solution and β-mannan mutant M1 enzyme solution, respectively.

[0039] (3) Sample preservation: The purified enzyme solutions were stored at 4°C for a short period of time for subsequent experiments. Example 3 Enzymatic properties of β-mannanase mutant M1 and wild-type β-mannanase

[0040] This embodiment strictly follows the national standard GB / T 36861-2018 "Detection Method for Mannanase in Feed", employing the classic DNS colorimetric method to detect the enzymatic properties of wild-type β-mannanase and β-mannan mutant M1. All experiments were conducted in triplicate, and the experimental procedures and results are analyzed below.

[0041] I. Comparative Enzyme Activity Detection Test (1) Test conditions: uniform reaction pH 5.5, water bath reaction temperature 37℃, and precise reaction time 30min. The enzyme activity definition strictly follows the national standard: the amount of enzyme required to degrade and release 1μmol of reducing sugar per minute from a 3mg / mL mannan solution under the conditions of pH 5.5 and 37℃ is defined as 1 enzyme activity unit (U).

[0042] (2) Experimental procedure: Take 2 mL of wild-type enzyme solution and mutant M1 enzyme solution diluted appropriately and place them in graduated test tubes. Add 2 mL of 0.6% mannan substrate solution to each tube, shake thoroughly to mix, and incubate at 37℃ for 30 min. Immediately after the reaction, add 5 mL of DNS colorimetric reagent to terminate the enzymatic reaction. Shake thoroughly and heat in a boiling water bath for 10 min for color development. Remove the test tubes and allow them to cool naturally to room temperature. Add sterile ultrapure water to a final volume of 25 mL, mix thoroughly, and measure the absorbance of each group at a wavelength of 540 nm. Calculate the enzyme activity and specific enzyme activity according to the standard curve and take the average value.

[0043] (3) Experimental results and analysis Table 1. Enzyme activity detection results of β-mannanase and mutants of the present invention.

[0044] As shown in Table 1 above, the specific enzyme activity of the β-mannanase mutant M1 is as high as 2982 U / mg, which is nearly 60% higher than that of the wild-type enzyme, indicating a significant optimization of catalytic performance. The synergistic mutation at the L23A, E120Q, and W181F sites plays a crucial role in significantly enhancing the binding affinity of the enzyme to the mannan substrate and lowering the activation energy barrier of the enzymatic reaction. This greatly improves the catalytic capacity per unit mass of enzyme protein, effectively reducing feed additive dosage and production costs, and possesses significant industrialization advantages.

[0045] II. High-Temperature Thermal Stability Comparative Test (1) Experimental conditions: Typical high temperature gradients of 75℃, 80℃ and 85℃ were set for feed pelleting, and constant temperature heat treatment was performed for 3 min. The enzyme activity of the original enzyme solution that had not undergone high temperature treatment was used as a 100% blank control. The residual enzyme activity of each group was measured and the relative enzyme activity was calculated.

[0046] (2) Experimental Procedure: Equal volumes and concentrations of wild-type enzyme solution and mutant M1 enzyme solution were separately dispensed into sterile centrifuge tubes and precisely heat-treated in a constant temperature water bath for 3 minutes. After heat treatment, the tubes were immediately removed and rapidly cooled to room temperature in an ice-water bath to terminate the heat denaturation process. Subsequently, the enzyme activity of each group was measured under standard detection conditions, and the relative residual enzyme activity was calculated by comparing it with the blank control group. Six parallel experiments were set up for each group to eliminate random errors. (3) Test results Table 2. Thermostability results of β-mannanase and mutants of the present invention

[0047] As shown in Table 2 above, the activities of both wild-type and mutant enzymes decreased with increasing heat treatment temperature. However, the thermal stability advantage of mutant M1 became increasingly significant with rising temperature. Wild-type enzymes were almost completely inactivated after treatment at 85℃, retaining only 15.6% activity, making them unable to withstand high-temperature pelleting and extrusion processes in feed. In contrast, mutant M1 retained 65.3% high activity even after extreme high-temperature treatment at 85℃. The W181F mutation in mutant M1 of this invention strengthens the hydrophobic stacking interactions within the enzyme molecule, effectively constraining the conformational expansion of the protein's flexible loop region, improving the overall structural rigidity of the protein, inhibiting peptide chain unfolding and spatial conformational collapse under high-temperature conditions, and significantly enhancing the enzyme's high-temperature tolerance. This makes it fully adaptable to the high-temperature processing conditions in the feed industry, solving the industry pain point of wild-type enzymes being easily inactivated at high temperatures.

[0048] III. Comparative Test of Acid-Base Stability (1) Experimental conditions: A gradient acid-base buffer system with pH 2.0 to pH 8.0 was set up and left to stand at room temperature for 2 hours. The untreated enzyme solution was used as a 100% control. The residual enzyme activity was measured to evaluate the acid-base tolerance of the enzyme.

[0049] (2) Experimental procedure: Equal volumes of purified wild-type enzyme solution and mutant M1 enzyme solution were added to citrate-disodium hydrogen phosphate buffer systems with different pH values, mixed thoroughly, and then allowed to stand at room temperature for 2 hours. After treatment, all enzyme solutions were adjusted to the standard detection pH of 5.5 to eliminate the interference of pH value on enzyme activity detection. The residual enzyme activity of each group was determined by the DNS method, and the relative enzyme activity was calculated. Six parallel experiments were conducted for each group.

[0050] (3) Experimental results and analysis Table 3. pH stability results of β-mannanase and mutants of the present invention.

[0051] As shown in Table 3 above, the wild-type β-mannanase exhibits significant limitations in acid-base tolerance, with an extremely narrow stable activity range. It maintains over 90% high activity only in weakly acidic environments (pH 4.0–6.0). Under the typical strongly acidic environment of the livestock stomach (pH 2.0), the wild-type enzyme retains only 32.6% residual activity, essentially losing all activity. In neutral-to-alkaline environments (pH 7.0 and pH 8.0), enzyme activity drastically decreases to 71.2% and 43.5%, respectively, failing to adapt to pH fluctuations during feed processing and the neutral water environment of aquaculture. This results in short effective action time and unstable degradation efficiency in practical applications. In contrast, the mutant M1, modified by the three-point synergistic mutation of this invention, demonstrates a significant improvement in acid-base adaptability. It retains 86.2% high enzyme activity even under extreme strongly acidic conditions (pH 2.0), and its residual enzyme activity remains above 85% across a wide acid-base range (pH 2.0–7.0). It retains 62.4% effective activity only under weakly alkaline conditions (pH 8.0). The β-mannanase mutant M1 of this invention can perfectly adapt to the acid-base fluctuation environment during feed processing conditioning and pelleting, while also being tolerant to the highly acidic digestive environment of the broiler's gizzard and proventriculus. It can continuously exert degradation activity throughout the animal's digestive tract, completely solving the industry pain points of rapid inactivation and unstable application effects of wild-type enzymes. This greatly expands the feed application scenarios and suitable livestock and poultry varieties of the product, and significantly improves its industrial applicability. Example 4: Feed Application Trial of β-Mannanase Mutant M1

[0052] To verify the practical application effect of mutant M1 in feed additives, this example uses 5-day-old healthy white-feathered broilers as test subjects and conducts a 28-day feeding comparison experiment. The specific experimental procedure and results are as follows: (1) Experimental grouping and feeding conditions Sixty healthy, disease-free, 5-day-old white-feathered broiler chickens of uniform weight were randomly divided into two groups, with 5 replicates per group and 6 chickens per replicate. The control group received a basal diet plus an equal amount of wild-type β-mannanase preparation; the experimental group received a basal diet plus an equal amount of β-mannanase mutant M1 enzyme preparation. The basal diet formulation, rearing environment, temperature and humidity, light, and drinking water were identical for both groups for 28 days. The basal diet was a conventional corn-soybean meal broiler feed, without any added exogenous enzymes.

[0053] (2) Detection indicators and test methods During the experiment, daily feed intake and mortality were recorded. After the experiment, the average weight of each group of broilers was measured, and the average daily weight gain, average daily feed intake, and feed conversion ratio were calculated. At the same time, duodenal chyme of each group of broilers was collected, and the viscosity of the intestinal chyme was measured. The number of probiotics such as Bifidobacterium and Lactobacillus in the intestine was detected, and the effect of enzyme preparation application was comprehensively evaluated.

[0054] (3) Results of feed application trials Table 4. Results of the application of the β-mannanase and mutant of the present invention in feed.

[0055] Table 4 above summarizes the results of a 28-day broiler chicken breeding experiment. The mutant M1 exhibits higher catalytic activity and broader environmental adaptability. It maintains high catalytic efficiency even during high-temperature feed processing and in the acidic / alkaline environment of the animal's gastrointestinal tract, effectively degrading the large-molecule polysaccharide mannan in corn-soybean meal feed. This significantly reduces the viscosity of duodenal digesta in broilers (by 18.0% compared to the control group), relieving the inhibitory effect of high-viscosity digesta on intestinal nutrient absorption and eliminating the negative nutritional effects of mannan. From a growth performance perspective, the reduction in intestinal digesta viscosity significantly accelerates digesta motility and digestion rate, improving the absorption and utilization rate of protein, carbohydrates, and trace elements in the feed. Ultimately, this results in an 8.3% increase in average daily weight gain and a 7.6% decrease in feed conversion ratio, significantly saving feed costs and improving the economic benefits of broiler farming. From the perspective of gut health, the decrease in chyme viscosity reduces the environment for harmful bacteria to grow. Simultaneously, the small-molecule oligosaccharides produced by mannan degradation can serve as substrates for probiotic proliferation, leading to a 15.0% increase in the number of beneficial bacteria such as Bifidobacteria and Lactobacillus in the gut. This effectively optimizes the gut microecological structure, repairs intestinal mucosal health, enhances animal immunity, and reduces the incidence of diseases during the breeding period. In summary, the mutant M1 obtained through multi-site targeted synergistic mutagenesis overcomes the shortcomings of wild-type enzymes, such as poor adaptability to working conditions, unstable in vivo effects, and weak breeding efficiency. It possesses excellent processing tolerance and in vivo bioactivity, and can be widely applied to the green efficiency improvement of livestock and aquatic feeds. It is compatible with modern large-scale feed production and breeding systems, possessing strong practicality and broad industrial application prospects.

[0056] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A β-mannanase mutant, characterized in that, The mutant was obtained by site-directed amino acid mutation of the wild-type β-mannanase shown in SEQ ID NO:1; the site-directed mutations were L23A, E120Q, and W181F; the amino acid sequence of the β-mannanase mutant is shown in SEQ ID NO:

2.

2. The gene encoding the β-mannanase mutant of claim 1.

3. The gene according to claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

3.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the gene sequence as described in any one of claims 2-3.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector uses pPIC9K as its backbone vector.

6. A recombinant engineered bacterial strain, characterized in that, The recombinant engineered strain comprises the recombinant expression vector according to any one of claims 4-5.

7. The recombinant engineered strain according to claim 6, characterized in that, The host of the recombinant engineered strain is Pichia pastoris GS115.

8. The use of the β-mannanase mutant of claim 1, the gene of any one of claims 2-3, the recombinant expression vector of any one of claims 4-5, or the recombinant engineered bacteria of any one of claims 6-7 in the preparation of feed additives.

9. The application according to claim 8, characterized in that, The feed additive is suitable for the degradation and improvement of anti-nutritional factors in livestock, poultry, and aquaculture feeds.

10. A feed additive, characterized in that, The feed additive contains the β-mannanase mutant as described in claim 1.

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