Mannanase and its application in ramie degumming
Patent Information
- Application Number
- CN202311686999.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-08
- Publication Date
- 2026-10-27
- Estimated Expiration
- 2043-12-08
Smart Images
Figure CN117701533B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of enzyme engineering technology, and in particular to a mannanase and its application in ramie degumming. Background Technology
[0002] Ramie fiber is composed of cellulose and non-cellulose components. Ramie fiber is an important raw material in the textile industry. The non-cellulose component in ramie is called ramie gum. Ramie cellulose is bonded together with ramie gum in the raw ramie. The gum residue directly affects the quality of ramie fiber, such as fiber bonding, poor fiber bundle separation, and a rough hand feel. Therefore, one of the important steps before ramie fiber is put into textile production is degumming. However, traditional degumming requires conditions such as high temperature, high pressure, strong acid, and strong alkali, which not only damages the fiber but also causes great pollution to the environment.
[0003] The non-cellulose components of ramie include lipids, waxes, water-soluble substances, pectin, hemicellulose, and lignin, with hemicellulose being the most abundant. Furthermore, the hemicellulose in ramie bast mainly consists of galacto-glucomannan, glucomannan, and xylan, accounting for 45.5%, 23.2%, and 8.4%, respectively. Mannanase (EC 3.2.1.78) is one of the key enzymes for decomposing mannan, breaking it down into glucose, mannan oligosaccharides, and other oligosaccharides by randomly cleaving the β-1,4 glycosidic bonds in the mannan backbone. The combined use of mannanase with pectinase and xylanase can remove gums from ramie with minimal damage to the ramie fibers, thus improving the quality of ramie fabrics, reducing wastewater discharge, and lowering environmental pollution.
[0004] Textile processes are mostly carried out in high-temperature and highly alkaline environments, thus requiring thermophilic and alkali-resistant mannanases. However, most mannanases sold in the domestic market are for animal feed, while there are fewer types of mannanases suitable for textile use, and the effective pH of most industrial mannanases used in textiles is in the acidic range. For example, Xiasheng industrial-grade mannanase has an effective temperature range of 30-70℃ and an effective pH of 3.0-6.5, which cannot meet the high-temperature and highly alkaline environment of ramie degumming. Thermophilic and alkali-resistant mannanases have attracted much attention due to their excellent properties under extremely alkaline and high-temperature conditions and their important industrial applications. Summary of the Invention
[0005] Based on this, the present invention obtains a thermophilic and alkali-resistant mannanase through enzyme mining and modification technology, and uses it in the ramie degumming industry to degrade mannan in ramie, providing a new option for the demand of compound enzyme preparations in the ramie degumming industry.
[0006] The above-mentioned objective of this invention is achieved through the following technical solution: A mannanase, the amino acid sequence of which is shown in SEQ ID NO:2.
[0007] The thermophilic and alkali-resistant mannanase provided by this invention has an optimal temperature of 75°C and an optimal pH of 10, and remains stable within a pH range of 7-12. Under hot alkaline conditions of 70°C and pH 10, the half-life of the mannanase of this invention is 3.9 times that of its corresponding wild-type mannanase, making it more valuable for application in ramie degumming processes and providing a new and excellent option for ramie degumming complex enzyme preparations.
[0008] Furthermore, the gene encoding the mannanase.
[0009] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO:4.
[0010] Furthermore, a recombinant vector or recombinant strain containing the gene.
[0011] Furthermore, the expression vector of the recombinant vector includes pET28a.
[0012] Furthermore, the host of the recombinant strain includes Escherichia coli BL21(DE3).
[0013] Furthermore, the preparation method of the mannanase includes the following steps: constructing the recombinant expression vector and transforming it into a host cell to obtain a recombinant expression strain, culturing the recombinant expression strain, inducing expression, separating and purifying it to obtain the mannanase.
[0014] Furthermore, the construction of the recombinant expression vector includes the following steps: (1) Screening to obtain mannanase with an amino acid sequence as shown in SEQ ID NO:1, synthesizing the corresponding nucleotide sequence and ligating it into an expression vector to obtain a recombinant plasmid, the nucleotide sequence of which is shown in SEQ ID NO:3; (2) Using the recombinant plasmid as a template, design mutation primers and perform PCR amplification to obtain the ManB085M gene and pET-28a(+) backbone. Digest the backbone with DpnI enzyme and perform homologous recombination with the ManB085M gene to obtain the recombinant expression vector. The primer sequences of the mutation primers are as follows: ManB085M-F: 5'-AGCAAATGGGTCGCGGATCCCAGACCCATAGCGGCTTTTA-3'; ManB085M-R: 5'-TGGTGGTGGTGGTGCTCGAGATGAAAAATGCTGCTCGGAA-3'; pET-28a(+)-F: 5'-CTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTA-3'; pET-28a(+)-R: 5'-GCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATATGG-3'.
[0015] The present invention also provides the application of the mannanase in ramie degumming.
[0016] Furthermore, the mannanase is used for ramie degumming at a temperature of 60-80℃ and a pH of 7-12. Attached Figure Description
[0017] Figure 1 This is an SDS-PAGE protein electrophoresis image of the wild-type mannanase and the mutant mannanase of the present invention, wherein well 1 is a protein marker, well 2 is the wild-type mannanase ManB085, and well 3 is the mutant mannanase ManB085M.
[0018] Figure 2 This is the optimal pH curve for the wild-type and mutant mannanases of this invention.
[0019] Figure 3 The optimal reaction temperature curves for the wild-type and mutant mannanases of this invention are shown.
[0020] Figure 4 The pH stability curves of the wild-type and mutant mannanases of this invention are shown.
[0021] Figure 5 The half-life curves of the wild-type and mutant mannanases of this invention at 70°C are shown. Detailed Implementation
[0022] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.
[0023] To obtain a novel thermophilic and alkali-resistant mannanase for use in the ramie degumming industry to degrade ramie gums, thus providing an alternative to the demand for complex enzyme preparations in the ramie degumming industry, the inventors conducted the following enzyme discovery process: (1) Enzymes that meet the environmental requirements for ramie degumming were screened through literature retrieval, and enzymes derived from [unclear] were selected. Bacillus sp. The β-mannanase (ManA) of N16-5 has an optimal reaction temperature of 70℃, an optimal pH of 9.5, and a specific enzyme activity of 5065 U / mg.
[0024] (2) Using β-mannanase (ManA) as a probe, the amino acid sequences with similarity of 40% to 80% were screened in the NCBI database through sequence homology. The sequences were then subjected to phylogenetic analysis and a phylogenetic tree was drawn through molecular evolutionary genetic analysis.
[0025] (3) Divide the family domains of the branch where the probe enzyme is located and its nearby branches, and screen for candidate amino acid sequences among enzymes with the same family domain as the probe enzyme.
[0026] (4) Select the amino acid sequence closest to the probe enzyme, prepare the enzyme corresponding to the amino acid sequence and analyze its enzymatic properties, and screen for thermophilic and alkali-resistant enzymes that can decompose mannan. Wild-type mannanase (ManB085) with the amino acid sequence shown in SEQ ID NO:1 was obtained. There are currently no reports on its application in ramie degumming or mannan degradation.
[0027] The preparation process and enzymatic property analysis of the enzyme corresponding to the amino acid sequence are as follows: (1) Based on the amino acid sequence, synthesize the corresponding nucleotide sequence, link the nucleotide sequence to the expression vector, obtain the recombinant plasmid and transform it into the host cell to obtain the recombinant strain; (2) The recombinant strain obtained in step (1) was subjected to seed culture, fermentation culture and induced expression; (3) Centrifuge to collect the bacterial precipitate from step (2), and wash, sonicate to break it up, centrifuge to collect the supernatant, and use a Ni-NTA affinity chromatography column to obtain recombinant protein with high purity.
[0028] (4) Detect the enzyme activity, optimal pH, optimal temperature, pH stability and half-life of the recombinant protein mannanase obtained in step (3) and screen for thermophilic and alkali-resistant recombinant proteins.
[0029] Then, based on semi-rational design, the inventors modified the above-mentioned wild-type mannanase to enhance its enzymatic properties, enabling the modified enzyme to remove the gum from ramie bast under high temperature and strong alkaline conditions. The specific modification steps are as follows: (1) The structure of wild-type mannanase was predicted by alphafold2 and the predicted structure was evaluated on the UCLA-DOE LAB website to obtain a reliable structural model.
[0030] (2) Molecular dynamics simulations of wild-type mannanase were performed at coupled temperatures of 300K and 350K. It was found that the root mean square fluctuation value (RMSF) of mannanase was high, and the region with the largest fluctuation was concentrated in the non-catalytic functional structure of mannanase (i.e., amino acid residues 301 to 464 of ManB085). The higher the RMSF value, the greater the flexibility of the region, and the higher the flexibility, the lower the stability.
[0031] (3) The non-catalytic functional structure with a high RMSF value of wild-type mannanase was removed, leaving only the catalytic functional domain, and the structure of the shortened protein was remodeled. The structure of the shortened protein is consistent with the functional domain of wild-type mannanase.
[0032] (4) Molecular dynamics simulations were performed on the truncated protein at a coupling temperature of 300K and 350K. It was found that its RMSF was smoother than that of wild-type mannanase, indicating that the truncated protein may have stronger rigidity, which means that the stability of the protein may be higher. The truncated protein was synthesized to obtain mutant mannanase (ManB085M).
[0033] Based on the above theoretical analysis, the inventors obtained a wild-type mannanase (ManB085) with the amino acid sequence shown in SEQ ID NO:1 through probe enzyme screening. Then, using this wild-type mannanase as a template, they obtained a mutation method that may improve the stability of the wild-type mannanase through molecular dynamics simulation, namely, truncating amino acid residues 301-464 of ManB085. The amino acid sequence of the mutated mannanase (ManB085M) is shown in SEQ ID NO:2. Its preparation method includes the following steps: (1) The wild-type mannanase ManB085 with the amino acid sequence shown in SEQ ID NO:1 was screened, the corresponding nucleotide sequence was synthesized and ligated into the expression vector to obtain the recombinant plasmid, the nucleotide sequence of which is shown in SEQ ID NO:3; (2) Using the recombinant plasmid obtained in step (1) as a template, design mutant primers ManB085M-F, ManB085M-R, pET-28a(+)-F and pET-28a(+)-R, perform PCR amplification to obtain the ManB085M gene and pET-28a(+) backbone, digest the backbone with DpnI enzyme, and perform homologous recombination between the digested backbone and the ManB085M gene to obtain the recombinant expression vector; (3) Transform the recombinant expression vector obtained in step (2) into host cells to obtain recombinant expression strains; (4) The recombinant strain obtained in step (3) was subjected to seed culture, fermentation culture and induced expression. The bacterial precipitate was collected by centrifugation, washed and sonicated, and the supernatant was collected by centrifugation. The recombinant protein with high purity, namely mannanase ManB085M, was obtained by Ni-NTA affinity chromatography column.
[0034] The present invention will be further described below with reference to specific embodiments.
[0035] All whole-genome synthesis in the embodiments of this invention was completed by Suzhou Genewise Biotech Co., Ltd.
[0036] The reagents used in the embodiments of this invention are as follows: LB seed culture medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl.
[0037] TB fermentation medium: 24 g / L yeast extract, 12 g / L tryptone, 4 mL / L glycerol, 125.4 g / L K2HPO4, 23.1 g / L KH2PO4.
[0038] Binding buffer (Buffer A): 100 mM Hepes, 50 mM NaCl.
[0039] Elution buffer (Buffer B): 100 mM Hepes, 50 mM NaCl, 300 mM imidazole.
[0040] Unless otherwise specified, the materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed according to the manufacturer's recommendations.
[0041] Example 1: Preparation of wild-type mannanase (1) Construction of recombinant expression vector pET28a-ManB085 The thermophilic and alkali-tolerant wild-type mannanase ManB085 was obtained via NCBI, and its amino acid sequence is shown in SEQ ID No:1. After codon optimization of ManB085, the nucleotide sequence shown in SEQ ID No:3 was obtained. The optimized nucleotide sequence was synthesized from the whole genome and subcloned into the *E. coli* gene expression vector pET-28a(+), resulting in the recombinant expression vector pET28a-ManB085.
[0042] (2) Colony PCR identification and sequencing The recombinant expression vector pET28a-ManB085 was transformed into Escherichia coli BL21(DE3) and plated onto agar plates containing kanamycin sulfate, then incubated overnight at 37°C. Single colonies on the antagonistic selection plates were identified by colony PCR to ensure successful integration of the exogenous DNA into the expression host.
[0043] (3) Enzyme induction, expression and purification Positive single colonies were selected and inoculated into LB seed culture medium, and cultured overnight at 37°C with shaking at 220 rpm to obtain seed culture. The seed culture was then inoculated into TB fermentation medium, and the initial OD of the TB fermentation medium was controlled. 600 The concentration was 0.1, and the culture time was 1.5 h to 2 h (i.e., OD). 600 To reach a concentration of 0.6–0.8, add IPTG inducer to a final concentration of 0.5 mM, then incubate the culture medium at 16°C with shaking at 180 rpm for 16 h. After fermentation, collect the cells by centrifugation at 6000 rpm for 3 min at room temperature, wash with water, add Buffer A for ultrasonic disruption, and then centrifuge at 4°C and 10000 rpm for 30 min to collect the supernatant.
[0044] The collected supernatant was purified by nickel column affinity chromatography using an ATKA protein purifier. Poorly affinity proteins were removed using 10% Buffer B, and the obtained wild-type mannanase ManB085 was eluted with 100% Buffer B. The purity of the target protein in the eluent was analyzed by SDS-PAGE electrophoresis.
[0045] Example 2: Preparation of mutant mannanase (1) Construction of recombinant expression vector pET28a-ManB085M Using pET28a-ManB085 prepared in Example 1 as a template, primers as shown in Table 1 were designed. The ManB085M gene and pET-28a(+) backbone were amplified by PCR. The amplification products were recovered to obtain the target gene fragment and pET-28a(+) backbone. The PCR amplification reaction system is shown in Table 2. The PCR amplification reaction conditions are as follows: (1) 94℃ pre-denaturation for 2 min; (2) 98℃ denaturation for 10 s, 55℃ annealing for 30 s, and 68℃ extension; a total of 30 cycles were performed; (3) 68℃ annealing for 7 min, and incubation at 16℃.
[0046] Table 1 Primers and their sequences for introducing mutations
[0047] The pET-28a(+) backbone was digested with DpnI, and then homologous recombination was performed between the digested backbone and the target gene fragment using a homologous recombination enzyme to obtain the recombinant expression vector pET28a-ManB085M. The digestion system is shown in Table 3. The homologous recombination system consisted of 4 μL of target gene, 1 μL of pET-28a(+) backbone, and 5 μL of homologous recombination enzyme.
[0048] Table 2 PCR reaction system
[0049] Table 3 Enzyme digestion system
[0050] (2) Colony PCR identification and sequencing The recombinant expression vector pET28a-ManB085M was transformed into Escherichia coli BL21(DE3), plated on a plate containing kanamycin sulfate, and incubated overnight at 37°C. Single colonies on the antagonistic selection plate were identified by colony PCR to ensure successful integration of exogenous DNA into the expression host.
[0051] (3) Enzyme induction, expression and purification Positive single colonies were selected and inoculated into LB seed culture medium, and cultured overnight at 37°C with shaking to obtain the seed culture. The seed culture was then inoculated into TB fermentation medium, and the initial OD of the TB fermentation medium was controlled. 600 The concentration was 0.1, and the culture was carried out until the OD value was 0.1. 600 When the culture temperature reaches 0.6–0.8, IPTG inducing agent is added and the mixture is cultured at 16°C and 180 rpm for 16 h with shaking. After fermentation, the cells are collected by centrifugation at room temperature, washed with water, and then subjected to ultrasonic disruption after adding Buffer A. The supernatant is collected by centrifugation at 4°C after disruption.
[0052] The collected supernatant was purified by nickel column affinity chromatography using an ATKA protein purifier. Poorly bound proteins were removed using 10% Buffer B, and the target protein was obtained by elution with 100% Buffer B. The purity of the target protein in the eluent was analyzed by SDS-PAGE electrophoresis. The target protein was identified as the mutant mannanase ManB085M, with the amino acid sequence SEQ ID NO:2 and the encoding nucleotide sequence SEQ ID NO:4.
[0053] Example 1: Determination of the enzymatic properties of wild-type and mutant mannanases (1) Determination of enzyme activity One unit of mannanase activity (U) is defined as the amount of mannanase required per minute to hydrolyze mannan substrate to produce 1 μmol of mannose.
[0054] The activity of mannanase was determined using the DNS method. 40 μL of appropriately diluted enzyme solution and 160 μL of 0.2% substrate (konjac mannan solution prepared with 50 mM Gly-NaOH at pH 10) were mixed and reacted at 70℃ for 10 min. Then, 200 μL of DNS was added to terminate the reaction. After boiling in a water bath for 5 min, the reaction solution was cooled to room temperature, and the absorbance was measured at 540 nm. For the blank control group, 40 μL of buffer and 160 μL of 0.2% substrate were added, reacted at 70℃ for 10 min, and then 200 μL of DNS was added to terminate the reaction. After boiling in a water bath for 5 min, the reaction solution was cooled to room temperature, and the absorbance was measured at 540 nm.
[0055] Preparation of mannose standard curve: Prepare mannose standard solutions with concentrations of 0, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL using distilled water. Take 400 μL of each mannose standard solution and add 400 μL of DNS solution. Mix well and boil for 5 min. Cool the reaction solution to room temperature and measure its absorbance at 540 nm.
[0056] The formula for calculating mannanase activity is as follows: Enzyme activity (U / mg) = (W Df 1000) / (180.16) 10 V C), where C (mg / mL) represents the enzyme concentration, W (mg) represents the mass of mannose produced by enzymatic hydrolysis, Df represents the dilution factor, 180.16 represents the molecular weight of mannose, 1000 represents the coefficient for converting mmol to μmol, 10 (min) represents the time of enzymatic hydrolysis, and V (mL) represents the volume of enzyme solution added during the reaction.
[0057] (2) Determination of optimal pH Prepare 0.2% substrate solutions in buffers (Gly-NaOH buffer) ranging from pH 8 to 12. Take 160 μL of substrate and add 40 μL of appropriately diluted enzyme solution. Incubate at 70℃ for 10 min and measure mannanase activity. Calculate the relative enzyme activity of mannanase under each pH condition, taking the enzyme activity at the optimum pH as 100%.
[0058] (3) Determination of the optimal temperature Under optimal pH conditions, mannanase was mixed with its substrate, and the enzyme activity of wild-type and mutant mannanase was measured after reacting for 10 min at a temperature of 60℃~85℃ according to the reaction system described in step (1). The enzyme activity at the optimal temperature was taken as 100%, and the relative enzyme activity of mannanase at different temperatures was calculated.
[0059] (4) Determination of pH stability Enzyme solutions were prepared using buffer solutions with pH values of 7–12, and incubated overnight at 4°C. The mannanase activity was then measured under optimal reaction conditions according to the reaction system described in step (1). The highest calculated enzyme activity was taken as 100%, and the relative enzyme activity of mannanase under each pH condition was calculated.
[0060] (5) Determination of half-life The appropriately diluted enzyme solution was placed in an environment of 70°C, and its relative enzyme activity was measured every 15 minutes. The enzyme activity before heating was taken as 100%, and the relative activity of mannanase after incubation for different times was calculated.
[0061] participate Figures 2-3 The optimal pH for the wild-type mannanase ManB085 and the mutant mannanase ManB085M prepared in Example 1 was 10, and the optimal temperature was 75°C. (See also...) Figure 4 The wild-type and mutant mannanases showed enzyme activities of 983 U / mg and 1176 U / mg, respectively, under optimal conditions. Furthermore, their activities did not decrease significantly after overnight treatment in buffer solutions ranging from pH 7 to 12, demonstrating strong pH stability. (See also...) Figure 5 The half-life of wild-type mannanase at 70°C is 15 min, while that of mutant mannanase is 58 min. Therefore, through the modification of this invention, the mutant mannanase significantly improves the thermal stability of the enzyme under alkaline conditions compared to wild-type mannanase.
[0062] Example 2: Analysis of ramie degumming effects of wild-type and mutant mannanases The raw ramie was quantitatively analyzed according to GB 5889—88 "Quantitative Analysis Method for Chemical Composition of Ramie". After calculating the oil content of ramie according to GB / T 18147.1—2008 "Test Methods for Hemp Fiber Part 1: Oil Content Test Method", the ramie was degummed according to the following method: ① Mechanically crush the ramie until it becomes soft and loose.
[0063] ②Pretreatment: Soak 5 g of ramie in a solution containing 5 g / L urea and 3 g / L penetrant JFC-6 for 24 h, with a solid-bath ratio of 1:10.
[0064] ③ After cleaning the ramie with water, keep the ramie moist and beat it 200 times.
[0065] ④ Enzyme treatment: Solid bath ratio 1:10 (50mM Gly-NaOH, 1g / L JFC-6), add 800 U mannanase and compound with commercial enzyme, and perform enzymatic degumming treatment in a 60℃ water bath for 16 h. The commercial enzymes used in enzymatic degumming are Xiasheng textile xylanase and Novozymes compound refining enzyme (main component is pectinase), added at amounts of 800U and 3800U respectively.
[0066] ⑤ Alkali-oxygen treatment: Soak ramie in a solution containing 6 mL / L 30% H2O2 and 4 g / L NaOH in a 40℃ water bath; raise the temperature to 60℃ and keep it warm for 10 min, then add 6 mL / L 30% H2O2; raise the temperature to 98℃ and keep it warm for 30 min; then wash the ramie with hot water at 80~85℃, then soak the ramie in 2 g / L acetic acid for 10 min and wash it with water; soak it in a solution containing 2 g / L dehydrogenase JMN-6 at 50℃ for 10 min and wash it with water.
[0067] ⑥ Oiling: Soak in a solution containing 5 g / L emulsified oil JM-D1 and 5 g / L emulsified oil JM-D2 at 40°C for 45 min.
[0068] ⑦ Dry in an oven at 60℃.
[0069] The raw ramie was dried to constant weight, cooled and weighed (recorded as m0). After degumming, it was dried again and the dry weight of the degummed ramie was recorded (recorded as m1). The weight loss rate was calculated as (m0-m1) / m0×100%.
[0070] The residual gum content, blue light whiteness, and bundle fiber strength of ramie after enzymatic degumming were determined with reference to GB / T 18147.2-2008 "Test Methods for Hemp Fiber Part 2: Test Method for Residual Gummed Content", GB / T 5885-1986 "Test Method for Whiteness of Ramie Fiber", and GB / T 5882-1986 "Test Method for Breaking Strength of Ramie Bundle Fiber".
[0071] Referring to Table 4, the weight loss rate, residual gum rate, and blue light whiteness of ramie samples were improved after adding mannanase to the commercial enzyme compound. The weight loss rate of ramie samples treated with wild-type mannanase ManB085 and mutant mannanase ManB085M increased by 1.71% and 2.71%, respectively, the residual gum rate decreased by 2.59% and 2.65%, respectively, and the blue light whiteness increased by 21.34% and 9.76%, respectively. This indicates that adding mannanase helps to remove gum from ramie.
[0072] Table 4. Effects of different enzyme combinations on ramie fiber properties
[0073] In summary, the wild-type and mutant mannanases provided by this invention are thermophilic and alkali-resistant enzymes with an optimal temperature of 75℃ and an optimal pH of 10. They remain stable over a wide pH range (pH 7-12). The mutant mannanase exhibits significantly improved thermal stability under hot alkaline conditions compared to the wild-type mannanase. Therefore, the wild-type and mutant mannanases of this invention can be used in high-temperature, high-alkali processes such as ramie degumming. Furthermore, when combined with commercial enzymes such as xylanase and pectin lyase, they can reduce ramie residual gum content and increase ramie whiteness, thus aiding in ramie degumming.
[0074] In summary, this invention screened a thermophilic and alkali-resistant mannanase using gene mining techniques. By mutating the screened mannanase, its thermal stability was improved, enhancing its application value in ramie degumming. Currently, there are few commercially available textile mannanase varieties, both domestically and internationally. Therefore, this invention provides a high-quality mannanase for ramie degumming compound enzyme preparations.
[0075] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.
Claims
1. A mannanase, characterized in that, Its amino acid sequence is shown in SEQ ID NO:
2.
2. The gene encoding the mannanase of claim 1.
3. The gene as described in claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:
4.
4. A recombinant expression vector comprising the gene as described in claim 2.
5. The recombinant expression vector as described in claim 4, characterized in that, The recombinant expression vector includes pET28a.
6. A recombinant expression strain comprising the gene as described in claim 2.
7. The recombinant expression strain according to claim 6, characterized in that, The host cells of the recombinant expression strain include Escherichia coli BL21(DE3).
8. The method for preparing mannanase as described in claim 1, characterized in that, The process includes the following steps: constructing the recombinant expression vector as described in claim 4 or 5 and transforming it into a host cell to obtain a recombinant expression strain; culturing, inducing expression, separating and purifying the recombinant expression strain to obtain the mannanase.
9. The application of mannanase as described in claim 1 in ramie degumming, characterized in that, A compound enzyme, consisting of mannanase, xylanase, and pectinase, was used for ramie degumming.
10. The application as described in claim 9, characterized in that, The mannanase is used for ramie degumming at a temperature of 60-80℃ and a pH of 7-12.