Recombinant bacillus subtilis mutant strain for producing beta-mannase

Abstract

The invention belongs to the field of industrial microorganisms, and particularly relates to a recombinant bacillus subtilis mutant strain for producing beta-mannase. The mutant strain is bacillus subtilis 168 M2-SP26 and is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, and the preservation number of the mutant strain is CGMCC No.35508. The invention provides a brand-new high-quality mutant strain, the mutant strain has good antibiotic resistance, meanwhile, the specific recognition and binding capacity of the mutant strain and beta-mannase mRNA is enhanced, and the capacity and efficiency of producing beta-mannase can be greatly improved in cooperation with a specific production process.

Description

Technical Field

[0001] This invention belongs to the field of industrial microbiology, and specifically relates to a recombinant Bacillus subtilis mutant strain that produces β-mannanase. Background Technology

[0002] Mannans, the second largest component of hemicellulose, are widely found in various plant tissues, but their large molecular weight makes them difficult to utilize directly. β-Mannanases, capable of hydrolyzing mannan-based organic compounds, are widely used in medicine, food, feed, and fine chemicals. For example, β-Mannanases can hydrolyze mannans into mannooligosaccharides. Mannooligosaccharides, as a novel prebiotic product, have become a research hotspot in the pharmaceutical and food fields due to their multiple functions, including lowering blood lipids, controlling blood sugar, and inhibiting fat absorption.

[0003] β-Mannanases are widely found in nature, including legumes, snails and other lower animals, bacteria, fungi and other microorganisms. Among them, microbial β-mannanases are the most abundant, and have advantages such as high stability, easy culture, low production cost and easy gene manipulation. Summary of the Invention

[0004] The present invention addresses the technical challenges of low yield of traditional β-mannanase by providing a recombinant Bacillus subtilis mutant strain that produces β-mannanase and its production method.

[0005] The main objective of this invention is: 1. Provide a recombinant Bacillus subtilis mutant strain that produces β-mannanase, with its β-mannanase production increasing by 40% compared to the original strain; II. Provide preservation information for the above-mentioned mutant strains; III. A method for producing β-mannanase is provided.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] A recombinant Bacillus subtilis mutant strain producing β-mannanase, The mutant strain is Bacillus subtilis 168 M2-SP26, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35508 and deposit date of August 4, 2025.

[0008] As a preferred option The mutant strain is a streptomycin-paromomycin double-resistant mutant strain, in which the 221st base of the rpsL gene is changed from T to C, and the 271st base is changed from A to G.

[0009] As a preferred option The mutant strain was used to produce β-mannanase.

[0010] As a preferred option The method for using the mutant strain to produce β-mannanase includes: The mutant strain was inoculated into seed culture medium and cultured with shaking at 35–38 °C and 180–220 r / min for 10–14 h to obtain a viable bacterial concentration ≥1×10⁻⁶. 8 CFU / mL seed culture; The seed culture was inoculated into the fermentation medium at an inoculation rate of 3-7%, and cultured at a temperature of 35-38 ℃ and a rotation speed of 180-220 r / min for 10-14 h. Then, the temperature was lowered to 23-27 ℃ and cultured for another 22-26 h. After fermentation, the fermentation broth was centrifuged at 4 ℃ and 10000-12000 r / min for 8-12 min, and the supernatant was collected.

[0011] As a preferred option The seed culture medium consists of: 20-30 g / L glucose, 8-12 g / L peptone, 3-7 g / L yeast extract, 5-10 g / L sodium chloride, pH 6.8-7.2, and water as the solvent.

[0012] As a preferred option The fermentation medium consists of: 20-30 g / L glucose, 15-25 g / L konjac flour, 8-12 g / L peptone, 3-7 g / L yeast extract, 0.05-0.15 g / L MnCl2, 0.2-0.4 g / L CaCl2, pH 6.0-7.0, and water as the solvent.

[0013] Based on the above, the complete production method is as follows: Specifically, The Bacillus subtilis 168 M2-SP26 mutant strain with accession number CGMCC No. 35508 was inoculated into seed culture medium and cultured with shaking at 35–38 ℃ and 180–220 r / min for 10–14 h to obtain a viable bacterial concentration ≥1×10⁻⁶. 8 The seed culture medium consists of CFU / mL of glucose, 8-12 g / L of peptone, 3-7 g / L of yeast extract, 5-10 g / L of sodium chloride, and a pH of 6.8-7.2. The seed culture was inoculated into the fermentation medium at an inoculum of 3-7%, and cultured at 35-38 ℃ and 180-220 r / min for 10-14 h. Then, the temperature was lowered to 23-27 ℃ and cultured for another 22-26 h. The fermentation medium consisted of: glucose 20-30 g / L, konjac flour 15-25 g / L, peptone 8-12 g / L, yeast extract 3-7 g / L, MnCl2 0.05-0.15 g / L, CaCl2 0.2-0.4 g / L, and pH 6.0-7.0. After fermentation, the fermentation broth was centrifuged at 4 ℃ and 10000-12000 r / min for 8-12 min, and the supernatant was collected. Subsequently, the enzyme activity was determined according to the "National Standard of the People's Republic of China for the Determination of β-Mannanase Activity in Feed Additives - Spectrophotometric Method" GB / T 36861-2018.

[0014] The core of this invention lies in obtaining a streptomycin-paromomycin resistant mutant strain through ribosome engineering mutagenesis. A specific point mutation occurs in the rpsL gene, resulting in the 74th amino acid changing from I to T and the 91st amino acid changing from S to G, thereby significantly increasing the yield of β-mannanase and achieving an enzyme activity of 7022 U / mL.

[0015] This invention integrates four core technologies—ribosome engineering mutagenesis, two-stage temperature control, culture medium synergistic induction, and standardized detection—to construct a complete high-efficiency β-mannanase production technology system, achieving technological innovation in strain improvement, metabolic regulation, nutrient optimization, and quality control.

[0016] Ribosome engineering technology achieves genetic improvement of strains through directed mutagenesis of the rpsL gene. A specific point mutation (T221C / A271G) leads to amino acid substitution in the S12 protein (Ile74Thr / Ser91Gly), a precise molecular modification that alters the conformational dynamics of the ribosome. The mutated ribosomes not only acquire antibiotic resistance but, more importantly, enhance their specific recognition and binding ability to β-mannanase mRNA. Molecular mechanism studies show that the conformational change of the S12 protein affects the interaction interface between ribosomal 16S rRNA and mRNA. The threonine and glycine residues introduced by the mutation alter the hydrophobicity and hydrogen bond network of the protein surface, optimizing the geometry and electrostatic environment of the mRNA binding pocket. This optimized binding environment improves the formation efficiency of the translation initiation complex and enhances the accuracy of translation elongation.

[0017] The resistance selection process utilizes dual antibiotic pressure to achieve targeted enrichment of target strains. The synergistic effect of streptomycin and paromomycin ensures that only strains carrying specific ribosomal mutations can survive, establishing a direct link between genotype and phenotype. Genetic stability verification, through multiple generations of propagation, demonstrates that the mutation is a stable genetic variation, providing a reliable strain basis for industrial applications.

[0018] In this invention, the two-stage temperature control strategy is based on the temperature-dependent differences in microbial growth and metabolism. At 37°C, the bacteria are in the exponential growth phase, with cellular metabolism primarily flowing towards biosynthetic pathways, supporting cell division and biomass accumulation. This high metabolic activity provides sufficient cellular basis and precursor substances for subsequent enzyme synthesis. When the temperature drops to 25°C, the bacterial growth rate significantly decreases, metabolic flow is redistributed, and activation of the cold shock response induces the expression of a series of stress proteins, including molecular chaperones and protein folding enzymes. These proteins significantly improve the correct folding efficiency and thermal stability of β-mannanase. Furthermore, metabolic reprogramming under low-temperature conditions also involves the reconstruction of the transcriptional regulatory network. The expression of the cold shock protein CspA family activates the transcription of secondary metabolism-related genes while inhibiting the expression of growth-related genes. This transcriptional regulation ensures the efficient transfer of metabolic resources to the synthesis of target products.

[0019] This invention selects konjac flour as a complex inducer to exert a dual function. Its β-1,4-mannoside bond not only serves as a natural substrate for β-mannanase but also activates the transcriptional expression of the enzyme gene through a substrate-inducible mechanism. Continuous substrate stimulation maintains the high activity state of the enzyme synthesis system. The synergistic effect of metal ions is based on the metal-dependent Mn content of the enzyme protein structure. 2+ As a cofactor, it participates in the coordination of the enzyme active site and stabilizes the spatial conformation of the catalytic triplet; Ca 2= By binding to negatively charged groups on the surface of enzyme molecules, the thermal stability and anti-denaturation ability of enzyme proteins are enhanced. The optimized ratio of the complex nitrogen source ensures the sufficiency and balance of amino acid supply. The organic nitrogen source provides readily available amino acid precursors, while the inorganic nitrogen source supplements the deficiency of specific amino acids. The synergistic effect of the two supports the efficient synthesis of enzyme proteins, and the precise control of the carbon-nitrogen ratio achieves the best balance between cell growth and enzyme synthesis.

[0020] This invention employs a multi-stage screening system to establish a complete evaluation framework from genotype to phenotype. Resistance screening ensures the success of genetic modification, primary screening rapidly assesses enzyme production potential using the enzymatic enzymatic folding method, and secondary screening verifies actual enzyme production capacity using shake-flask fermentation. This tiered screening strategy improves screening efficiency and reduces false positive rates. Real-time monitoring of process parameters is based on fermentation kinetics principles: pH changes reflect metabolic state transitions, dissolved oxygen levels affect enzyme protein oxidative folding, and cell concentration determines the absolute yield of the product. Collaborative monitoring of key parameters provides data support for process optimization. Terminal quality verification uses national standard methods to ensure the authority and comparability of test results. Standardized operation of enzyme activity assays eliminates detection errors, providing a reliable basis for product quality control and process improvement. The comprehensive quality control system ensures product stability and consistency.

[0021] The beneficial effects of this invention are: This invention provides a novel, high-quality mutant strain that exhibits excellent antibiotic resistance and enhances its specific recognition and binding ability to β-mannanase mRNA. When combined with a specific production process, it can significantly improve the production capacity and efficiency of β-mannanase. Attached Figure Description

[0022] Figure 1 The growth status of the mutant strain obtained in this invention on streptomycin-paromomycin double antibody LB plates; Figure 2 Comparison of hydrolysis zones formed by the strains on konjac flour plates; Figure 3 This is an electrophoresis image of PCR amplification. Detailed Implementation

[0023] The present invention will be further described clearly and in detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0024] Unless otherwise specified, all raw materials used in the embodiments of the present invention are commercially available or obtainable by those skilled in the art; unless otherwise specified, all methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0025] Example 1: Screening method for resistant mutants.

[0026] The method includes: Take 200 µL of each of the following 106 mL -1 Cell suspensions of the starting bacterial strain were plated onto LB agar plates containing different concentrations of single or double antibiotics and incubated at 37 °C for 24 h. Single colonies growing on the antibiotic-resistant plates were streaked onto fresh LB agar plates containing the corresponding antibiotic type and concentration and cultured. After five validations, stable, heritable single-resistance mutant strains were obtained. The mutant cultures were harvested with 20% (v / v) glycerol and stored at -80 °C.

[0027] A mutant strain that stably grew on streptomycin-paromomycin LB agar plates was obtained and named Bacillus subtilis 168 M2-SP26. The growth status of the obtained β-mannanase-producing recombinant Bacillus subtilis mutant strain (Bacillus subtilis) 168M2-SP26 on streptomycin-paromomycin LB agar plates is shown below. Figure 1 As shown.

[0028] Example 2: Method for initial screening of enzyme production activity of resistant mutant strains.

[0029] The method includes: The resistant mutant and the original strain were inoculated onto konjac flour plates (konjac flour 15 g / L, peptone 10 g / L, KH2PO4 0.2 g / L, MgSO4 0.1 g / L, agar 20 g / L) and incubated at 37 ℃ for 24 h. The diameter of the hydrolysis zone was measured and recorded using the cross-multiplication method, and three replicates were performed.

[0030] The materials prepared in the examples were subjected to performance testing, and the specific characterization results are as follows: Figure 2 As shown. Figure 2 The original strain Bacillus subtilis 168M2 is on the left, and the mutant strain Bacillus subtilis 168M2-SP26 is on the right.

[0031] from Figure 2 It can be clearly seen that the diameter of the hydrolysis zone of the mutant strain Bacillus subtilis 168 M2-SP26 is significantly larger than that of the original strain, indicating that its β-mannanase production is increased.

[0032] Example 3: Method for rescreening enzyme production activity of resistant mutant strains.

[0033] The method includes: The initially screened resistant mutants and the original strain were inoculated separately into LB liquid medium, with a medium volume of 50 mL / 250 mL Erlenmeyer flask. The medium was cultured at 180 r / min and 37 ℃ for 12 h to prepare the seed culture. The seed culture was then inoculated into fermentation medium at a volume ratio of 5%, with a medium volume of 100 mL / 250 mL Erlenmeyer flask. The medium was cultured at 200 r / min and 37 ℃ for 12 h, then cooled to 25 ℃ and cultured for another 24 h. The fermentation broth was centrifuged, and the supernatant was collected for enzyme activity assay.

[0034] The fermentation medium consisted of 25 g / L glucose, 20 g / L konjac flour, 10 g / L peptone, 5 g / L yeast extract, 0.1 g / L MnCl2, 0.3 g / L CaCl2, and a pH of 6.5.

[0035] The β-mannanase activity of the mutant strain Bacillus subtilis 168 M2-SP26 reached 7022 U / mL, which was 40% higher than that of the original strain.

[0036] Example 4: Method for analyzing the mutation sites of the rpsL gene in mutant strains.

[0037] The method includes: Total DNA was extracted from the original strain and the mutant strain, and the rpsL gene of the strain was amplified using primers rpslF / rpslR. The 20 μL amplification reaction system contained 10 μL ddH2O, 2 μL 10×PCR Buffer, 1 μL each of primers rpslF / rpslR (20 μM), 4 μL dNTPs (2.5 mM each), 1 μL Taq enzyme (1 unit / μL), and 1 μL total DNA. PCR amplification conditions were: 95 ℃ pre-denaturation for 10 min, followed by 30 cycles of denaturation at 94 ℃ for 50 s, annealing at 55 ℃ for 90 s, and extension at 72 ℃ for 1 min, with a final extension at 72 ℃ for 10 min. The PCR products were separated by 1.2% agarose gel electrophoresis. The electrophoresis results are shown below. Figure 3 As shown, Figure 3 In the sample, 1 represents 168M2rpsL, M represents a RealBand 10kb (0.25~10kb) DNA molecular weight standard ladder, and 2 represents 168M2-SP26rpsL. After separation, the target band was excised and purified using a Gel Extraction Kit. The recovered fragments were then sent to a sequencing company for analysis.

[0038] The rpsL gene sequence of the original strain Bacillus subtilis 168 M2 is shown in SEQ ID NO.1.

[0039] The rpsL gene sequence of the mutant strain Bacillus subtilis 168 M2-SP26 is shown in SEQ ID NO.2.

[0040] Sequencing results showed that the mutant rpsL gene changed from T to C at position 221 and from A to G at position 271, resulting in amino acid I changing to T at position 74 and S changing to G at position 91.

[0041] Comparative Example 1: Based on Example 2, this example only modifies the screening method, omitting the konjac flour plate screening; the remaining steps are the same as in Example 2. The specific settings are as follows:

[0042] The performance testing method for the comparative product was exactly the same as that for Example 2. Screening efficiency was characterized, and the results are shown in the table below.

[0043]

[0044] Analysis of the above characterization results shows that the konjac flour plate screening method is significantly superior to other screening methods in terms of screening efficiency, accuracy, and time cost, proving its irreplaceable role in the efficient screening of high-yielding β-mannanase strains.

[0045] In terms of false positive rates, group D1-1, due to the lack of initial screening and direct fermentation verification of all strains, resulted in 86.7% of strains being misidentified as potentially high-yielding strains. Subsequent rescreening required significant time and resources, ultimately yielding only 45.3% accurate identification results. Group D1-2 used cellulose plates; however, since cellulose is not a specific substrate for β-mannanase, some strains secreted cellulase or other enzymes that formed hydrolysis zones, leading to a 52.4% false positive rate. Although the screening time was shorter than D1-1, the accuracy was still insufficient. Group D1-3 relied solely on colony morphology, ignoring the correlation between enzyme activity and colony morphology. The 73.8% false positive rate reflected the method's inherent bias, with an accuracy of only 58.2%. In Example 2, the konjac flour agar plate used β-mannan as the sole substrate. The size of the hydrolysis zone directly corresponded to the ability of the strain to secrete β-mannanase. The false positive rate of 15.2% was much lower than that of other groups, and the average screening time of 3 days was shortened by more than 90% compared with D1-1. The 92.5% accuracy rate in identifying high-yielding strains ensured the reliability of the screening results. For example, in group D1-1, a strain with an enzyme activity of only 1200 U / mL after fermentation testing was far lower than the 7022 U / mL of the mutant strain, but it was included in the high-yielding candidate because it was not initially screened. In group D1-2, a strain that could decompose cellulose formed a large hydrolysis zone on the cellulose plate, but its β-mannanase activity was only 2500 U / mL, which was a false positive. In group D1-3, a strain with a plump colony morphology had an enzyme activity of only 1800 U / mL, further verifying the limitations of morphological screening. Based on the comparison of the patent data, the present invention uses konjac flour plate primary screening to achieve rapid enrichment of high-yield strains through substrate specificity, effectively avoiding the defects of other methods, which is the key link of the efficient screening system of the present invention.

[0046] Comparative Example 2: Based on Example 3, this example makes systematic modifications to the fermentation process conditions, while the remaining steps are the same as in Example 3. The specific settings are as follows:

[0047] The performance testing method for the comparative product was exactly the same as that for Example 3. The fermentation performance was characterized, and the results are shown in the table below.

[0048]

[0049] Analysis of the above characterization results shows that the two-stage temperature control, while ensuring appropriate cell growth, maximizes the synthesis and accumulation of β-mannanase, demonstrating the key role of this temperature control strategy in optimizing enzyme yield.

[0050] Looking at the specific differences in the data from each group, although the bacterial biomass in group D2-1, under constant temperature culture at 37 ℃ throughout, reached 14.2±0.4 g / L, an increase of approximately 14% compared to Example 3, the enzyme activity was only 5265 U / mL, a decrease of 25% compared to Example 3, and the product yield also decreased to 0.214±0.015 g / g. This phenomenon indicates that although the high temperature environment of 37 ℃ can significantly promote the rapid proliferation of bacteria, the synthesis process of β-mannanase is more sensitive to temperature—sustained high temperature may inhibit the transcription efficiency of the man gene (the gene encoding β-mannanase), or cause partial denaturation of the synthesized enzyme protein due to insufficient thermostability, thereby reducing the enzyme activity and accumulation.

[0051] The results of the D2-2 group, cultured at a constant temperature of 25°C throughout, revealed another extreme: the bacterial biomass was only 8.3±0.2 g / L, less than 70% of that in Example 3, and the enzyme activity was even lower, at 3874 U / mL, only 55% of that in Example 3. This indicates that although the low temperature of 25°C is suitable for the stable existence of enzymes, it severely slows down the growth rate of bacteria, resulting in insufficient cell number and metabolic activity. This makes it impossible to provide enough energy and precursor substances (amino acids, nucleotides) for the large-scale synthesis of enzymes, ultimately limiting the level of enzyme production.

[0052] Group D2-3 adjusted the time allocation of the two-stage temperatures. Although the bacterial biomass was close to that of Example 3, the enzyme activity was still lower than that of Example 3. The reason for this is speculated to be that the initial 37°C culture time was too long, and after the bacteria entered the stationary phase due to excessive growth, the metabolic focus within the cells shifted from "growth" to "maintenance," leading to a decrease in the activity of enzyme-related regulatory factors and a reduction in the expression level of the man gene. Conversely, the subsequent 25°C culture time was insufficient to fully induce a large accumulation of enzyme, thus failing to reach the enzyme production level of Example 3.

[0053] Group D2-4, which used a temperature combination of "24 h at 30 ℃ + 24 h at 20 ℃", showed the lowest levels of both cell biomass and enzyme activity among all groups. This is because while the growth temperature of 30 ℃ is higher than 25 ℃, it is still lower than the optimal growth temperature for Bacillus subtilis, resulting in a slower cell growth rate and insufficient biomass accumulation. Meanwhile, the enzyme-producing temperature of 20 ℃ is too low, further inhibiting the translation process of the enzyme protein, leading to a significant decrease in enzyme activity.

[0054] In contrast, the two-stage temperature strategy in Example 3 perfectly balances the temperature requirements for bacterial growth and enzyme synthesis: the high-temperature environment in the early stage rapidly propels the bacteria into the logarithmic growth phase, accumulating sufficient biomass; the subsequent cooling to 25 °C provides optimal conditions for efficient expression of the man gene and correct folding of the enzyme protein, resulting in peak product yield and enzyme activity. This result fully demonstrates that the two-stage temperature control is not a simple temperature switching, but a precise regulation based on the growth and metabolic patterns of Bacillus subtilis and the synthetic characteristics of β-mannanase—by distinguishing the temperature requirements of the "growth phase" and the "enzyme production phase," the synergistic optimization of bacterial growth and enzyme yield is achieved, which is the core technological innovation in this invention for improving the enzyme production performance of mutant strains.

[0055] In Example 3, after culturing the mutant strain at 37 °C for 12 h, the cell density (OD600) reached 6.8 ± 0.2, indicating the late logarithmic growth phase. Switching to 25 °C at this point resulted in an increased mRNA transcription level of the *man* gene compared to the 37 °C culture, and the accumulation of enzyme protein also reached its maximum after 24 h of culture at 25 °C. In contrast, after culturing the D2-1 group at 37 °C for 36 h, the *man* gene transcription level was only 50% of that in Example 3, and the proportion of soluble enzyme protein decreased from 85% in Example 3 to 62%, further validating the inhibitory effect of high temperature on enzyme synthesis.

[0056] The results of Comparative Example 2 clearly demonstrate that the two-stage temperature control strategy adopted in Example 3 maximizes the enzyme production potential of the mutant strain while ensuring appropriate cell growth. It is a key process parameter for achieving high yield of β-mannanase, and its effect is significantly better than single temperature or unreasonable two-stage temperature combinations.

Claims

1. A recombinant Bacillus subtilis mutant strain producing β-mannanase, characterized in that, The mutant strain is Bacillus subtilis 168 M2-SP26, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35508.

2. The recombinant Bacillus subtilis mutant strain producing β-mannanase according to claim 1, characterized in that, The mutant strain is a streptomycin-paromomycin double-resistant mutant strain, in which the 221st base of the rpsL gene is changed from T to C, and the 271st base is changed from A to G.

3. A recombinant Bacillus subtilis mutant strain producing β-mannanase according to claim 1 or 2, characterized in that, The mutant strain was used to produce β-mannanase.

4. The recombinant Bacillus subtilis mutant strain producing β-mannanase according to claim 4, characterized in that, The method for using the mutant strain to produce β-mannanase includes: The mutant strain was inoculated into seed culture medium and cultured with shaking at 35–38 °C and 180–220 r / min for 10–14 h to obtain a viable bacterial concentration ≥1×10⁻⁶. 8 CFU / mL seed culture; The seed culture was inoculated into the fermentation medium at an inoculation rate of 3-7%, and cultured at a temperature of 35-38 ℃ and a rotation speed of 180-220 r / min for 10-14 h. Then, the temperature was lowered to 23-27 ℃ and cultured for another 22-26 h. After fermentation, the fermentation broth was centrifuged at 4 ℃ and 10000-12000 r / min for 8-12 min, and the supernatant was collected.

5. The recombinant Bacillus subtilis mutant strain producing β-mannanase according to claim 4, characterized in that, The seed culture medium consists of: 20-30 g / L glucose, 8-12 g / L peptone, 3-7 g / L yeast extract, 5-10 g / L sodium chloride, pH 6.8-7.2, and water as the solvent.

6. The recombinant Bacillus subtilis mutant strain producing β-mannanase according to claim 4, characterized in that, The fermentation medium consists of: 20-30 g / L glucose, 15-25 g / L konjac flour, 8-12 g / L peptone, 3-7 g / L yeast extract, 0.05-0.15 g / L MnCl2, 0.2-0.4 g / L CaCl2, pH 6.0-7.0, and water as the solvent.

Images