Mannanases derived from space-mutagenized yeast strains, methods of making, products, and uses thereof

Abstract

The application belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a mannanase from a space mutagenized yeast strain, a preparation method, a product and an application thereof. The amino acid sequence of the mannanase is shown as SEQ ID NO. 2, and the nucleotide sequence thereof is shown as SEQ ID NO. 3. The mutant has outstanding performances at high temperature (70% enzyme activity is retained at 85 DEG C for 3 min, while 40% enzyme activity is retained for the original enzyme at 85 DEG C for 3 min) and acidic pH (the stability is increased from 3% to 25% after treatment at pH 3.0 for 180 min), and has low-temperature high-efficiency catalytic characteristics of optimal pH 7.0, optimal temperature 40 DEG C and 30% enzyme activity at 20 DEG C, and has significant application potential in the fields of feed and papermaking industry.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to mannanase derived from space-mutated yeast strains, its preparation method, products, and applications. Background Technology

[0002] Mannanase, a hydrolase that specifically degrades mannan and its derivatives, has been widely used in various fields such as food processing, feed industry, papermaking industry, and bioenergy development due to its highly efficient substrate degradation capabilities, making it one of the important categories in the industrial enzyme market. In the food industry, it can be used for juice clarification, improving coffee extraction efficiency, and preparing functional oligosaccharides; in the feed industry, it can break down the anti-nutritional factor β-mannan in plant-based feeds; and in the papermaking industry, it can assist in pulp bleaching, reducing the amount of chemical bleaching agents used.

[0003] Currently, most neutral mannanases used in industrial applications are derived from microbial fermentation. However, the enzymatic properties of these natural enzymes or enzyme preparations modified using conventional techniques are limited by their inherent protein structure, and they still face severe challenges in complex industrial environments: First, they lack heat resistance, making it difficult to withstand the high temperatures of processes such as feed pelleting and food sterilization; second, they have poor acid resistance, rapidly losing activity in acidic environments such as the stomach acid of piglets or fruit juice processing; and third, they lack overall stability, being sensitive to storage conditions and easily inhibited by components such as metal ions in industrial systems.

[0004] To overcome these bottlenecks, researchers typically employ protein engineering (such as rational design and directed evolution) or traditional physicochemical mutagenesis (such as ultraviolet light and chemical reagent treatment) to modify enzyme-producing strains or enzyme genes. However, protein engineering heavily relies on a deep understanding of the relationship between enzyme structure and function, and the modification process is complex and costly; while traditional mutagenesis methods have low mutation rates and a narrow range of positive mutations, making it difficult to efficiently generate mutants with disruptive and desirable traits.

[0005] Space-induced mutagenesis offers a novel and efficient strategy for addressing the aforementioned challenges. This method utilizes the unique environmental factors of space (such as microgravity, strong radiation, and weak geomagnetism) to induce mutagenesis in microbial strains. This unique environment can induce broader, deeper, and more frequent variations in the organism's genome, thereby significantly expanding the diversity of genetic mutations. Compared to conventional ground-based mutagenesis, space-induced mutagenesis promises to screen rare and superior strains with significantly improved traits (such as extreme temperature tolerance, excellent acid-base stability, and higher specific activity) from a large number of mutants in a shorter time.

[0006] Therefore, this invention innovatively uses a yeast strain obtained through space mutagenesis screening as a new source of mannanase, aiming to obtain a new enzyme whose enzymatic performance comprehensively surpasses that of existing neutral mannanases, so as to better meet the needs of demanding industrial applications. Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention provides a superior mannanase mutant, Man45, which exhibits significantly better activity retention than the original enzyme. It maintains high catalytic activity even after high-temperature treatment, effectively avoiding the problem of significant enzyme activity loss caused by high temperatures. Furthermore, its stability in acidic environments is greatly improved, maintaining a certain level of activity even after prolonged exposure to acidic conditions, a significant improvement compared to the fragile state of the original enzyme under acidic conditions.

[0008] The technical solution of this invention is as follows:

[0009] On the one hand, the present invention provides a mannanase Man45, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] In another aspect, the present invention provides a nucleic acid encoding the aforementioned mannanase Man45.

[0011] Specifically, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 60% sequence identity with SEQ ID NO.3.

[0012] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 65% sequence identity with SEQ ID NO.3.

[0013] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 70% sequence identity with SEQ ID NO.3.

[0014] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 75% sequence identity with SEQ ID NO.3.

[0015] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 80% sequence identity with SEQ ID NO.3.

[0016] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 85% sequence identity with SEQ ID NO.3.

[0017] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 90% sequence identity with SEQ ID NO.3.

[0018] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 95% sequence identity with SEQ ID NO.3.

[0019] The nucleic acid sequence is as shown in SEQ ID NO.3 or has 97% sequence identity with SEQ ID NO.3.

[0020] Preferably, the nucleic acid sequence is as shown in SEQ ID NO.3 or has 99% sequence identity with SEQ ID NO.3.

[0021] Preferably, the sequence of the nucleic acid is shown in SEQ ID NO.3.

[0022] In another aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid.

[0023] Specifically, the recombinant vector includes, but is not limited to, plasmids, bacteriophages, or viruses.

[0024] Preferably, the recombinant vector can be a plasmid.

[0025] Preferably, the vector is the pPIC9K plasmid.

[0026] In another aspect, the present invention provides a host cell comprising the aforementioned nucleic acid or recombinant vector.

[0027] Specifically, the host cell is a eukaryotic cell or a prokaryotic cell.

[0028] Preferably, the host cell can be Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Bacillus or Lactobacillus.

[0029] Preferably, the cells can be Pichia pastoris, brewer's yeast, or Hansenula polymorpha.

[0030] Preferably, the cells are Pichia pastoris.

[0031] Preferably, the cells are Pichia pastoris GS115.

[0032] In another aspect, the present invention provides a method for preparing the aforementioned mannanase Man45, comprising the following steps:

[0033] S1. Culture the aforementioned host cells to induce the expression of mannanase Man45;

[0034] S2. Collect and purify the culture supernatant to obtain mannanase Man45.

[0035] In another aspect, the present invention provides an enzyme preparation comprising the aforementioned mannanase Man45.

[0036] Specifically, the enzyme preparation also includes excipients.

[0037] Preferably, the excipients are selected from one or more of fillers, stabilizers, protectants, pH adjusters, preservatives, or flow aids.

[0038] Preferably, the filler is selected from maltodextrin, lactose, calcium carbonate, or silicon dioxide; the stabilizer is selected from sorbitol, glycerol, or EDTA; the protectant is selected from sucrose, trehalose, or bovine serum albumin; the pH adjuster is a citrate-sodium citrate buffer pair or a phosphate buffer pair; the preservative is selected from sodium benzoate, potassium sorbate, or propylene glycol; and the flow aid is silicon dioxide or talc.

[0039] Specifically, the enzyme preparation includes, but is not limited to, powder, granules, or liquid.

[0040] Specifically, the enzyme preparations include, but are not limited to, food-grade, feed-grade, or industrial-grade enzymes.

[0041] In another aspect, the present invention provides the application of the aforementioned mannanase Man45 or nucleic acid or recombinant vector or host cell or enzyme preparation in the preparation of feed additives or in the papermaking industry.

[0042] The beneficial effects of this invention are as follows:

[0043] (1) The recombinant mannanase Man45 provided by the present invention has better pH performance, with an optimal pH of 7.0, enzyme activity exceeding 50% at pH 4.0-10.0, and enzyme activity exceeding 40% after treatment at pH 4.0-9.0. Its tolerance and stability are significantly better than Man23 and Man01, and its application range is wider.

[0044] (2) The recombinant mannanase Man45 provided by the present invention has outstanding thermal stability, with an optimal temperature of 40℃ and high activity maintained at 20-90℃; after treatment at 85℃, the enzyme activity is retained by more than 70%, which is far superior to Man23 (40%) and Man01 (48%), making it suitable for high-temperature scenarios. Attached Figure Description

[0045] Figure 1 The optimal pH for the recombinant mannanase mutant.

[0046] Figure 2 pH stability of the recombinant mannanase mutant.

[0047] Figure 3 The optimal temperature for the recombinant mannanase mutant.

[0048] Figure 4 The thermostability of the recombinant mannanase mutant was measured. Detailed Implementation

[0049] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0050] The main experimental materials and reagents of this invention are:

[0051] 1. Strains and vectors: The mannanase gene of this invention man45 The Pichia pastoris expression vector was synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd. pPIC9K and strains GS115 Purchased from Invitrogen.

[0052] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, ligases from Invitrogen, mannan from Sigma, and all other reagents were domestically produced (available from general biochemical reagent companies).

[0053] 3. Culture medium:

[0054] (1) Yeast culture medium YPD: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 20 g / L agar, pH 7.0.

[0055] (2) Escherichia coli culture medium LB: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0.

[0056] (3) BMGY medium: 20 g / L peptone, 10 g / L yeast extract, 1 g / L glycerol, 1.34% YNB (V / V), 0.00004% Biotin (V / V).

[0057] (4) BMMY medium: except that 0.5% (V / V) methanol is used instead of glycerol, the other components are the same as those of BMGY.

[0058] Note: Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0059] Example 1 Bacillus subtilis ( Bacillus subtilis Mannanase mutant gene man45 Acquisition and synthesis

[0060] Using the encoding gene of mannanase Man23 from Bacillus subtilis (its amino acid sequence is shown in SEQ ID NO.1) as a template, its mature peptide encoding sequence was amplified by PCR, and EcoRI and NotI restriction enzyme sites were introduced at the 5' and 3' ends of the amplified product, respectively. This fragment was cloned into the Pichia pastoris expression vector pPIC9K to construct a recombinant plasmid, which was then transformed into Pichia pastoris GS115 competent cells by electroporation. Homologous recombination was used to integrate the expression cassette into the yeast genome, and a positive recombinant strain, GS115-Man23, was obtained after antibiotic resistance selection.

[0061] The recombinant strain GS115-Man23 was subjected to space-borne mutagenesis. After mutagenesis, single colonies were isolated by streak plating, and small-scale fermentation and enzyme activity assays were performed to screen for mutants with superior performance. A mutant strain with significantly increased enzyme activity was obtained, and its mannanase gene was sequenced. Sequence analysis showed that this mutant gene (named...) man45 Compared to the original man23 The gene underwent an amino acid change that resulted in the deletion of amino acids at positions 19-30 and the mutation of aspartic acid (D) at position 69 into asparagine (N). The mutant protein it encodes is named Man45, and its amino acid sequence is shown in SEQ ID NO.2.

[0062] Beijing Ruiboxingke Biotechnology Co., Ltd. was commissioned to perform gene optimization and full-sequence chemical synthesis based on the sequenced amino acid sequence of Man45 (SEQ ID NO.2). EcoRI and Not I restriction enzyme sites were introduced at the 5' and 3' ends of the synthesized gene to facilitate subsequent vector construction. The nucleotide sequence of the synthesized mannanase mutant gene man45 is shown in SEQ ID NO.3.

[0063] SEQ ID NO.1:

[0064] EAHTVSPVNPNAQQTTKAVINWLAHLPNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLETANIEDTIDYSCNSNLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTVEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVDIVGLDAYFQDPYSINGYDQLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQRYPKTIYFLAWNDEWSPAVNKGASALYNDSWTLNKGEIWNGDSLTPIVE;

[0065] SEQ ID NO.2:

[0066] EAHTVSPVNPNAQQTTKAENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCNYARGWLETANIEDTIDYSCNSNLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTVEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVDIVGLDAYFQDPYSINGYDQLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQRYPKTIYFLAWNDEWSPAVNKGASALYNDSWTLNKGEIWNGDSLTPIVE;

[0067] SEQ ID NO.3:

[0068] GAAGCGCATACTGTGTCGCCTGTGAATCCTAATGCACAGCAGACAACAAAAGCGGAAAACAGAGTCCTTTCCGGAGCGTTCGGAGGTTACAGCCATGACACATTTTCTATGGCTGAGGCTGATAGAATCCGAAGCGCCACCGGGCAATCGCCTGCTATTTACGGCTGCAATTATGCCAGAGGATGGCTTGAAACAGCAAATATTGAAGATACAATAGATTACAGCTGCAACAGCAATTTAATATCGTATTGGAAAAATGGTGGAATCCCGCAAATCAGCTTGCACCTGGCGAATCCTGCTTTTCAGTCAGGGCATTTTAAAACACCGATTACAAATGATCAGTATAAAAAAATACTAGATTCTTCAACAGTAGAAGGAAAGCGGCTAAATGCCATGCTCAGCAAAATTGCTGACGGACTTCAAGAGTTGGAGAACCAAGGTGTGCCTGTTCTGTTCAGGCCGCTGCATGAAATGAACGGCGAATGGTTTTGGTGGGGACTCACATCATATAACCAAAAGGATAATGAAAGAATCTCTCTATATAAACAGCTCTACAAGAAAATCTATCATTATATGACCGACACAAGAGGACTTGATCATTTGATTTGGGTTTACTCTCCCGACGCCAACCGAGATTTTAAAACTGATTTTTACCCGGGCGCGTCTTACGTGGATATTGTCGGATTAGATGCGTATTTTCAAGATCCCTACTCGATCAATGGATACGATCAGCTAACAGCGCTTAATAAACCTTTTGCTTTTACAGAAGTCGGCCCGCAAACAGCAAACGGCAGCTTTGATTACAGCCTATTTATCAACGCAATAAAACAAAGATATCCTAAAACCATTTACTTTCTGGCATGGAATGATGAATGGAGCCCAGCTGTAAACAAGGGGGCTTCAGCTTTATATAACGACAGCTGGACACTCAATAAGGGAGAAATATGGAATGGCGATTCTTTAACGCCAATCGTTGAG。

[0069] Example 2 Mannanase Gene man45 Cloning

[0070] The synthesized gene vector was preserved as a puncture culture. The puncture culture was picked up with a sterile toothpick in a clean bench and placed in an LB shaker containing antibiotic Amp (working concentration: 100 μg / mL). It was cultured overnight at 37°C and 220 rpm. The next day, the gene-containing vector was extracted according to the instructions of the Kangwei Century Plasmid Extraction Kit PurePlasmid Mini Kit (CW0500).

[0071] Based on the mannanase gene sequence, the following primers were designed and synthesized:

[0072] P1 (SEQ ID NO.4):

[0073] 5'-GAATTCAGATCCCGGTCAGAAGCGCATACTGTGTCG-3';

[0074] P2 (SEQ ID NO.5):

[0075] 5'-GCGGCCGCTCACTCAACGATTGGCGTTAAAGAAT-3'.

[0076] PCR amplification was performed using the extracted vector as a template. The PCR reaction parameters were: denaturation at 94℃ for 5 min; followed by denaturation at 94℃ for 30 sec, annealing at 55℃ for 30 sec, extension at 72℃ for 1.5 min, and 30 cycles, followed by incubation at 72℃ for 10 min. A fragment of approximately 1084 bp was obtained. This fragment was recovered, ligated into the pMD19 vector, and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 36.9 kDa.

[0077] Based on the nucleotide sequences obtained from sequencing, the DNAMan software was used to compare the obtained nucleotide sequences with... man45 The sequences were compared and confirmed to be correct.

[0078] Example 3 Preparation of recombinant mannanase Man45

[0079] expression carrier pPIC9K Double enzyme digestion ( Eco R I+ Not I), and simultaneously the gene encoding mannanase man45 Double enzyme digestion ( Eco R I+ Not I) The gene fragment encoding mature mannanase and its expression vector were digested using enzymes. pPIC9K Linkage was performed to obtain the mannanase gene. man45recombinant plasmid pPIC9K- Man45 And transform Pichia pastoris GS115 Recombinant Pichia pastoris strain was obtained GS115 / Man45 .

[0080] Take the sample containing the recombinant plasmid. GS115 Strains and control strains (i.e., unmutated strains) GS115 / MAN23 The cells were inoculated into 300 mL of BMGY medium and cultured at 30°C with shaking at 200 rpm for 48 h. The cells were then collected by centrifugation. The cells were then resuspended in 150 mL of BMGY medium and cultured at 30°C with shaking at 200 rpm. After induction for 72 h, the supernatant was collected by centrifugation, and the mannanase activity was measured.

[0081] Example 4 Activity analysis of recombinant mannanase Man45

[0082] DNS method: The specific method is as follows: Under pH 7.0 and 37℃ conditions, a 1 mL reaction system includes 100 μL of appropriately diluted enzyme solution and 900 μL of substrate. The reaction is allowed to proceed for 10 min, then 1.5 mL of DNS is added to terminate the reaction. The mixture is boiled in water for 5 min, cooled, and the OD value is measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

[0083] Example 5: Determination of the properties of recombinant mannanase Man45

[0084] The enzymatic properties of recombinant mannanase Man45 and original mannanase Man23 were determined and compared.

[0085] Meanwhile, the mutant recombinant mannanase Man01 was added for enzymatic property comparison. The Man01 sequence is based on SEQ ID NO.1 with an I55R mutation. The specific preparation methods of Man23 mutant and Man01 recombinant enzyme are the same as those of mannanase Man45.

[0086] 1. The method for determining the optimal pH and pH stability of recombinant mannanase Man45 is as follows:

[0087] Purified mannanases Man23, Man45, and Man01 were subjected to enzymatic reactions at different pH values ​​to determine their optimal pH. The substrate mannan was used in 0.1 mol / L citrate-disodium hydrogen phosphate buffer at different pH values ​​(1.0-10.0) for mannanase activity assays at 37°C. Results ( Figure 1The results showed that the optimal pH for all three was 7.0, but the recombinant enzyme Man45 had a relative enzyme activity of 50% or more at pH 4.0-10.0, while Man23 and Man01 had less than 30% enzyme activity at pH 3.0. This indicates that the recombinant enzyme Man45, which has undergone genetic recombination, has superior pH tolerance and may have a wider applicable pH range in practical applications.

[0088] Purified mannanases Man23, Man45, and Man01 were treated at 37°C for 180 min in various buffer solutions with different pH values, and then their activity was measured at 40°C in a pH 7.0 buffer system to study the pH tolerance of the enzymes. Results ( Figure 2 The results showed that the mannanase Man45 was very stable between pH 4.0 and 9.0. After treatment within this pH range for 180 min, the remaining enzyme activity was more than 40%, which was significantly higher than that of Man23 and Man01. This indicates that the recombinant enzyme has good pH stability over a wide pH range.

[0089] 2. The optimal temperature and thermal stability determination method for recombinant mannanase Man23 are as follows:

[0090] The optimal temperature for mannanase was determined by conducting enzymatic reactions in a citrate-disodium hydrogen phosphate buffer (pH 7.0) at different temperatures. Thermostability was determined by treating mannanase at different temperatures for different times, followed by enzyme activity assays at 40°C. Results of the optimal temperature determination for recombinant mannanase Man45 (…) Figure 3 This indicates that its optimal temperature is 40℃, and it maintains high enzyme activity at temperatures ranging from 20℃ to 90℃.

[0091] Thermostability tests of the enzyme showed that ( Figure 4 Man45 exhibits excellent thermal stability, retaining over 70% of its enzyme activity after incubation at 85°C for 3 minutes. In contrast, Man23 retains only 40% of its enzyme activity, and Man01 retains only 48%. Compared to these two, Man45 demonstrates significantly improved thermal stability and excellent heat resistance.

[0092] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A mannanase Man45, characterized in that, The amino acid sequence of the mannanase Man45 is shown as SEQ ID NO.

2.

2. A nucleic acid encoding the mannanase Man45 of claim 1.

3. The nucleic acid of claim 2, wherein, The sequence of the nucleic acid is shown as SEQ ID NO.

3.

4. A recombinant vector, characterized in that, comprising the nucleic acid of claim 2 or 3.

5. The recombinant vector of claim 4, wherein, The recombinant vector comprises a plasmid, a bacteriophage or a virus.

6. A host cell, characterized in that, comprising the nucleic acid of any one of claims 2-3 or the recombinant vector of any one of claims 4-5.

7. The host cell of claim 6, wherein, The host cell is a eukaryotic cell or a prokaryotic cell.

8. The method of producing the mannanase Man45 according to claim 1, characterized in that, comprising the following steps: S1, culturing the host cell of claim 6 or 7 to induce expression of the mannanase Man45; S2, collecting and purifying the supernatant of the culture solution to obtain the mannanase Man45.

9. An enzyme preparation, characterized in that, The enzyme preparation comprises the mannanase Man45 of claim 1.

10. Use of the mannanase Man45 of claim 1 or the nucleic acid of any one of claims 2-3 or the recombinant vector of any one of claims 4-5 or the host cell of claim 6 or 7 or the enzyme preparation of claim 9 in the preparation of a feed additive or in the papermaking industry.

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