CN114621987B - Method for preparing arabinoxylan with different molecular weight distribution characteristics - Google Patents

Method for preparing arabinoxylan with different molecular weight distribution characteristics Download PDF

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CN114621987B
CN114621987B CN202210160967.0A CN202210160967A CN114621987B CN 114621987 B CN114621987 B CN 114621987B CN 202210160967 A CN202210160967 A CN 202210160967A CN 114621987 B CN114621987 B CN 114621987B
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刘玉春
王超
郭超
任菲
张维清
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Academy of National Food and Strategic Reserves Administration
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Abstract

The application relates to the field of bioengineering, in particular to a method for preparing arabinoxylan with different molecular weight distribution characteristics. The method comprises hydrolyzing corn fiber gum with xylanase at high salt concentration to prepare arabinoxylans with different molecular weight distributions. The application discovers that the physicochemical state of the natural substrate with complex structure in a high-salt system can be changed, thereby influencing the enzyme catalysis efficiency and the characteristics of enzymolysis products. In addition, the high-salt system can reduce microbial contamination and mixed bacteria growth in the process of preparing the oligosaccharide by the enzymolysis substrate, so that high-temperature sterilization or bacteriostat addition treatment is not needed for the substrate and the reaction system, the cost can be reduced, the damage of high temperature to bioactive substances can be prevented, and the use of other bacteriostat components can be reduced.

Description

Method for preparing arabinoxylan with different molecular weight distribution characteristics
Technical Field
The invention relates to the field of bioengineering, in particular to a method for preparing arabinoxylan with different molecular weight distribution characteristics.
Background
Corn is one of three main grains in China, corn husks are byproducts of corn deep processing, and account for 10% -15% of the total mass of the corn, and the hemicellulose content of the corn is about 70%. Corn husk fiber is the most complex plant hemicellulose known at present, the side chain degree of the xylan main chain is very high, more than 70% of main chain xylose residues in the corn husk fiber have one or more side chains, and the side chains contain various substituents such as arabinose, ferulic acid, coumaric acid and the like. The corn husk degradation product has high application value in the field of foods and medicines. The corn husk fiber has the highest content of arabinose in plant tissues, can be degraded into arabinoxylan (Arabinoxylan), and has multiple biological functions, such as promoting the growth of intestinal probiotics, improving the sterilizing capacity of cell interferon and leukocidin, and inhibiting the growth and attachment of cancer cells; ferulic acid (Ferulic Acid) has effects of resisting platelet aggregation, relieving pain, relieving vasospasm, and treating Alzheimer disease. Corn husks are currently used mainly in feeds and fermentation bases.
The preparation method of corn husk fiber oligosaccharide mainly comprises a chemical method, a physical method and an enzymatic method. The enzymatic method has the advantages of mild conditions, controllable enzymolysis specific products, capability of maximally recovering effective components, and the like.
Endo-beta-1, 4-xylanase (beta-1, 4-xylanase, EC 3.2.1.8) is a glycoside hydrolase, can catalyze and hydrolyze a xylan main chain beta-1, 4-D-xyloside bond to generate xylooligosaccharide, and is the most important glycoside hydrolase in a xylan degradation enzyme system. Currently, the reported xylanases are mostly distributed in the GH10 and GH11 families. Halophilic/salt tolerance is an important property of enzymes. Halophilic/salt tolerant xylanases generally refer to xylanases that retain catalytic activity at high salt concentrations (0.5 to 4.5M). The halophilic/salt-tolerant xylanase has great application potential in the processing and production of high-salt-content foods such as seafood, fermented bean curd, flour products and the like. The salt-tolerant xylanases reported so far belong to the GH10 family, whereas the salt-tolerant xylanases belonging to the GH11 family are very few.
There is no report of preparing arabinoxylans with different molecular weight distribution characteristics by hydrolyzing corn fiber gum with extreme halophilic/salt-tolerant xylanases of GH10 family and GH11 family in high salt system, and the difference makes the arabinoxylans possible to have different physicochemical properties and biological functions.
Disclosure of Invention
The present invention has been made in order to solve the above-mentioned problems.
The invention aims to provide a method for preparing arabinoxylans with different molecular weight distribution characteristics.
A method for preparing arabinoxylans having a different molecular weight distribution according to the present invention, said method comprising the steps of:
the xylanase is used for hydrolyzing corn fiber gum under high salt concentration to prepare the arabinoxylans with different molecular weight distribution.
The method for preparing the arabinoxylans with different molecular weight distributions, disclosed by the invention, comprises the following steps of: 1 or SEQ ID NO: 3.
SEQ ID NO：1
LPKRQTTGLSAHSRQTTGLNTIAQAAGLKYLGSATDNPELTDTHYVAILSDSSEFGQLTPGNSMKWDATEPTQGQFSFDNADAIVELAQNNSQLIRGHTCVWYSQLPSWVSNGSWDADSLNVAMTTHTSTVVDHFKGKIYSWDVVNEAFEDDGSFRQNVFYTTIGEDYIANAFKAARAADPDAKLYINDYNIEGTGAKADALYTFVSSLLNASVPIDGIGMQAHLIVGSVPTTIQENIARFTALGLEVALTELDIRMPVPAAEADLEQQKADYEAVVGACAAVEGCVGVTVWDYTDKYSWVPSVFDGYGAALPWDENLEKKPAYDGIVSGLGA.
The gene sequence is shown in SEQ ID NO:2 is shown as follows:
CTCCCCAAGCGTCAGACCACTGGCCTCAGCGCCCACTCGCGGCAAACGACCGGCCTTAACACCATCGCGCAAGCCGCCGGCCTCAAGTACCTCGGCTCCGCGACGGACAACCCCGAATTGACGGACACGCACTACGTCGCGATCCTGAGCGACTCGAGTGAGTTTGGGCAGCTCACGCCGGGGAATAGTATGAAGTGGGACGCCACGGAGCCCACGCAGGGACAGTTCTCGTTCGACAATGCGGACGCGATCGTGGAGCTTGCTCAGAACAACAGCCAGCTCATTCGAGGTCACACCTGCGTTTGGTACAGTCAGCTGCCCAGCTGGGTCTCAAACGGGTCCTGGGACGCGGACTCGCTGAATGTGGCCATGACGACGCATACTTCGACGGTGGTCGATCATTTCAAGGGCAAAATATATAGCTGGGATGTAGTAAACGAGGCGTTTGAGGACGACGGCAGCTTCCGTCAGAACGTCTTCTACACAACCATCGGCGAGGATTACATCGCCAACGCGTTCAAAGCCGCCCGCGCGGCTGACCCTGATGCAAAACTTTATATCAACGACTACAACATCGAAGGCACCGGCGCCAAAGCCGACGCGCTCTACACCTTCGTCTCCTCCCTTCTCAACGCCTCCGTCCCCATCGACGGCATCGGCATGCAGGCGCACCTCATCGTCGGCTCTGTCCCAACGACCATCCAGGAGAACATCGCGCGCTTCACTGCTTTGGGCCTCGAGGTTGCGCTCACGGAGCTCGACATACGGATGCCTGTGCCCGCCGCCGAGGCGGATTTGGAGCAGCAGAAGGCGGATTACGAGGCCGTGGTGGGCGCGTGTGCGGCGGTGGAGGGGTGCGTGGGTGTGACGGTCTGGGATTATACGGATAAGTACTCCTGGGTTCCGAGTGTCTTCGATGGGTATGGAGCGGCTTTGCCGTGGGATGAGAACTTGGAAAAGAAGCCAGCTTACGACGGGATCGTGAGCGGCTTGGGTGCA..
SEQ ID NO：3
SPLNATDVVDVSARSGTPSSTGTDGGYYYSWWTDGAGDATYQNNGGGSYTLTWSGNNGNLVGGKGWNPGAASRSISYSGTYQPNGNSYLSVYGWTRSSLIEYYIVESYGSYDPSSAASHKGSVTCNGATYDILSTWRYNAPSIDGTQTFEQFWSVRNPKKAPGGSISGTVDVQCHFDAWKGLGMNLGSEHNYQIVATEGYQSSGTATITVT.
The gene sequence is shown in SEQ ID NO:4, as follows:
TCGCCGCTCAATGCGACGGACGTCGTTGACGTCTCCGCTCGCTCCGGTACTCCTAGCTCGACCGGTACCGATGGTGGCTACTACTACTCTTGGTGGACGGACGGTGCTGGTGACGCCACCTACCAAAACAACGGCGGTGGATCGTACACCTTGACCTGGTCTGGCAACAACGGCAACCTCGTCGGCGGAAAGGGATGGAACCCAGGAGCTGCGTCCCGCTCCATCTCTTACTCCGGCACCTACCAGCCCAACGGCAACAGCTACCTCTCCGTCTACGGCTGGACGCGCAGCTCGCTCATCGAGTACTACATCGTCGAGTCCTACGGCTCCTACGACCCGTCCTCCGCGGCCAGCCACAAGGGCTCCGTCACCTGCAACGGCGCCACGTACGACATCCTCTCCACCTGGCGCTACAACGCGCCCTCCATCGACGGCACGCAGACCTTCGAGCAGTTCTGGAGCGTCCGGAACCCGAAGAAGGCGCCCGGAGGGTCGATCAGCGGGACCGTCGACGTCCAGTGCCACTTCGATGCTTGGAAGGGACTCGGGATGAACTTGGGTAGCGAGCACAACTACCAGATCGTGGCTACCGAAGGCTATCAGAGCAGCGGCACTGCGACCATCACGGTCACGTAA.
the method for preparing the arabinoxylans with different molecular weight distributions, provided by the invention, comprises the following steps of: 1 and SEQ ID NO: 3.
The method for preparing arabinoxylans having different molecular weight distributions according to the present invention, wherein arabinoxylans having different molecular weight distributions are prepared by hydrolyzing corn fiber gums with xylanases in an environment comprising 1-5M sodium salt.
The method for preparing arabinoxylans having different molecular weight distributions according to the present invention, wherein the corn fiber gum is prepared by:
1) Weighing corn husk powder, adding n-hexane, stirring, filtering, cleaning, drying, treating with alpha-amylase, adding saccharifying enzyme, adding neutral protease into the treating solution, performing enzymolysis, inactivating, and filtering.
2) Weighing the sample obtained in the step 1), adding NaOH, ca (OH) 2 and water, heating, and centrifugally separating supernatant and precipitate after reaction;
3) Adding H 2O2 into the supernatant obtained in the step 2), regulating the pH value of the solution to be alkaline, stirring at room temperature, regulating the pH value to be acidic, centrifugally separating the supernatant, and adding absolute ethyl alcohol into the supernatant to obtain white flocculent precipitate, namely the corn fiber gum.
4) Adding distilled water into the precipitate obtained in the step 2), adding H 2O2, regulating pH to be alkaline, heating, cooling, centrifuging to separate supernatant, regulating pH of the supernatant to be acidic, centrifuging to separate supernatant, adding absolute ethyl alcohol, and obtaining white flocculent precipitate, namely the corn fiber glue.
5) Mixing the corn husk fiber glue obtained in the step 3) and the step 4).
The application discovers that the physicochemical state of the natural substrate with complex structure in a high-salt system can be changed, thereby influencing the enzyme catalysis efficiency and the characteristics of enzymolysis products. In addition, the high-salt system can reduce microbial contamination and mixed bacteria growth in the process of preparing the oligosaccharide by the enzymolysis substrate, so that high-temperature sterilization or bacteriostat addition treatment is not needed for the substrate and the reaction system, the cost can be reduced, the damage of high temperature to bioactive substances can be prevented, and the use of other bacteriostat components can be reduced. The application provides a basis for industrially preparing the functional arabinoxylans with specific molecular weights by taking the corn husks as raw materials, is beneficial to the resource utilization of grain processing byproducts, prolongs the grain processing industry chain and drives the grain economy to develop.
Drawings
FIG. 1 is a picture of protein purification electrophoresis of recombinant xylanase Scxyn;
FIG. 2 is a photograph of an electrophoresis of protein purification of recombinant xylanase Scxyn;
FIG. 3 shows the optimal pH of recombinant xylanase Scxyn, 22;
FIG. 4 shows the optimal temperature of recombinant xylanase Scxyn, 22;
FIG. 5 shows the optimal pH of recombinant xylanase Scxyn;
FIG. 6 shows the optimal temperature of recombinant xylanase Scxyn;
FIG. 7 shows the effect of NaCl concentration on Scxyn hydrolytic activity;
FIG. 8 shows the effect of NaCl concentration on Scxyn hydrolysis activity;
FIG. 9 shows the effect of NaCl concentration on Scxyn hydrolysis CFG efficiency;
FIG. 10 shows the effect of NaCl concentration on Scxyn on the efficiency of CFG hydrolysis;
Figure 11 shows the effect of NaCl concentration on CFG rheology.
Detailed Description
Test materials and reagents
1. Strains and vectors: expression host Pichiapastoris GS, expression plasmid vector pPIC9K;
2. Tool enzyme and biochemical reagent: endonucleases were purchased from NEW ENGLAND Biolabs, recombinases from holo gold, birchwood from Sigma, wheat arabinoxylans from Megazyme; corn husks were purchased from hebei guangyu starch company. The other are all domestic analytically pure reagents (all can be purchased from common biochemical reagent companies);
3. Culture medium:
(1) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (v/v), 1.34% YNB,0.00004% Biotin,
(2) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB,0.00004% biotin,0.5% methanol (v/v).
EXAMPLE 1 construction of xylanase recombinant vector
Cloning fragments Scxyn and Scxyn of xylanase genes Scxyn and Scxyn by PCR reaction with GH10 and GH11 family xylanases Scxyn-pET 28a and Scxyn-pET 28a from S.communication sp.DB1 as templates, wherein the amino acid sequence of xylanase Scxyn5 is shown as SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO:2, the amino acid sequence of xylanase Scxyn is shown as SEQ ID NO:3, the nucleotide sequence is shown as SEQ ID NO:4, the primers and reaction conditions are as follows:
Scxyn5-F：5′-CCGGAATTCCTCCCC AAGCGTCAGACCACT-3′；
Scxyn5-R：5′-CGCTAGCGGCCGCTGCACCCAAG CCGCTCAC-3′；
Scxyn22-F：5’-CCGGAATTC TCGCCGCTCAATGCGACG-3’；
Scxyn22-R：5’-CGCTA GCGGCCGCGTGACCGTGATGGTCGCA-3’。
The amplified product is directly connected with the cut pPIC9k plasmid after being respectively digested by EcoRI and NotI, is converted into TransI-T1 competence, and is picked up for sequencing and verification. Sequencing to verify that the correct transformants are recombinant yeast expression plasmids pPIC9K-Scxyn and pPIC 9K-Scxyn.
EXAMPLE 2 construction of Pichia pastoris engineering bacteria for recombinant expression of xylanase genes
The recombinant expression vectors pPIC9K-Scxyn and pPIC9K-Scxyn are linearized with the endonuclease SacI and transformed into Pichia pastoris GS115 to obtain recombinant yeast strains GS115/pPIC9K-Scxyn and GS115/pPIC 9K-Scxyn.
Selecting positive transformants, transferring the positive transformants into 50mL triangular flasks containing 5mL of BMGY culture medium, and placing the transformants in a shaking table at 30 ℃ and at 230rpm for 36h; 3000g of fermentation broth is centrifuged for 5min, the supernatant is discarded, and the precipitated thalli are resuspended in 5mL of BMMY culture medium and are subjected to shaking table induction culture at 30 ℃ for 72h at 230 rpm. As shown in FIGS. 1 and 2, the supernatant of the fermentation broth was used for enzyme activity detection and SDS-PAGE electrophoresis detection.
EXAMPLE 3 analysis of Activity of recombinant xylanase
The xylanase enzyme activity detection method comprises the following steps: 200. Mu.L of the enzymatic reaction system comprises 190. Mu.L of substrate and 10. Mu.L of an appropriate diluent, and is incubated at a given temperature and pH for 10min, followed by the addition of 300. Mu.L of DNS reagent and boiling in water for 5min. The absorbance at 540nm was measured after cooling the sample. 1 enzyme activity unit (U) is defined as the amount of enzyme required to produce 1. Mu. Mol of reducing end per minute under the given conditions.
1. The method for determining the optimal pH of the recombinant xylanase comprises the following steps:
The recombinant xylanase purified in example 2 was enzymatically reacted at different pH to determine its optimum pH. The xylanase activity was determined with different pH buffers (0.1 mol/L Gly-HCl buffer, 1.5-3.0;0.1mol/L citrate buffer, 3.0-7.0;0.1mol/LNaH 2PO4-Na2HPO4 buffer, 6.5-8.0;0.1mol/LTris-HCl buffer, 7.5-8.5;0.1mol/L Gly-NaOH buffer, 8.5-10.5) at 50 ℃. The results indicate that the optimal reaction pH of recombinant xylanase Scxyn is approximately 5.0-6.0 (FIG. 3); the optimal reaction pH of recombinant xylanase Scxyn was approximately 4.0-5.0 (FIG. 4).
2. The method for determining the optimal temperature of the recombinant xylanase comprises the following steps:
The determination of the optimum temperature of the recombinant xylanase is that enzymatic reactions are carried out at different temperatures in each optimum pH buffer system of 0.1mol/L citric acid buffer. The results showed that the optimal temperature of recombinant xylanase Scxyn was around 65-75 ℃ (FIG. 5); the optimal temperature of recombinant xylanase Scxyn was around 45-55deg.C (FIG. 6).
3. Effect of NaCl concentration on enzyme Activity
Effect of NaCl concentration on enzyme activity: the hydrolysis activity of recombinant xylanase was determined by reference to the above with birchwood xylan as substrate in buffer system containing different concentrations of NaCl (1-5M) and at optimal pH and temperature. The enzyme activity of the hydrolyzed birch xylan is respectively improved by 1.18 times, 1.46 times, 1.88 times, 2 times and 2.13 times in a NaCl reaction system containing 1 to 5M by 100 percent of enzyme activity of the reaction system without adding NaCl (figure 7); scXyn22 in the NaCl reaction system containing 1-5M, the enzyme activity of hydrolyzed birchwood xylan is respectively improved by 1.64 times, 2.13 times, 2.31 times, 2.39 times and 2.3 times (figure 8).
EXAMPLE 4 preparation of arabinoxylans
1. Preparation of corn fiber gum
The corn fiber gum is extracted by the following steps:
1) Drying and pulverizing corn husk, and sieving with 60 mesh sieve. 50g of corn husk powder was weighed, 500mL of n-hexane was added, and after stirring for 2h, the mixture was filtered through a Buchner funnel. Washing a sample with a proper amount of absolute ethyl alcohol, filtering, drying, adding distilled water according to a solid-to-liquid ratio of 1:10 (w/v), regulating the pH value to 6.0, adding high temperature resistant alpha-amylase into the system, treating for 30min in a water bath at 95 ℃, reducing the water bath temperature to 55 ℃, adding saccharifying enzyme, reacting for 30min, and checking the starch hydrolysis condition by using an I 2 -KI solution. Adding neutral protease into the treatment liquid, reacting for 1h at the water bath temperature of 55 ℃ and the pH value of 7, heating to 100 ℃ to inactivate enzyme for 5min, filtering with gauze, washing filter residues with distilled water for 2 times, and drying at 60 ℃ for later use.
2) 50G of the sample obtained in 1) was weighed, 2g of NaOH and 1.9g of Ca (OH) 2 were added, 0.5L of distilled water was added, and after mixing, the mixture was boiled for 1 hour. The reacted solution was cooled to room temperature, centrifuged at 6000 Xg for 20min, and then the supernatant A1 and the precipitate B1 were separated.
3) 3G of H 2O2 (30%, w/w) was added to the supernatant A1, the pH of the solution was adjusted to 11.5, and the mixture was stirred at room temperature for 2 hours; adjusting pH to 4.0, centrifuging 10000 Xg for 30min, and separating supernatant; 2 times of absolute ethyl alcohol is added into the supernatant fluid to obtain white flocculent precipitate CFG1.
4) An appropriate amount of distilled water was added to precipitate B1, followed by 30% H 2O2 (added at 0.1% on a dry basis), pH adjusted to 11.5, and a boiling water bath for 1.5H. Centrifuging at 6000 Xg for 20min after cooling, and separating supernatant A2; regulating the pH of the A2 to 4.0-4.5, centrifuging for 30min at 10000 Xg, separating supernatant, and adding 2 times volume of absolute ethyl alcohol to obtain white flocculent precipitate CFG2.
5) Dissolving CFG1 and CFG2 in distilled water, mixing, and freeze drying to obtain corn husk fiber gum CFG.
2. Effect of NaCl concentration on recombinant xylanase catalytic hydrolysis CFG enzyme Activity
The effect of NaCl concentration on the activity of recombinant xylanase-catalyzed hydrolysis CFG was determined with reference to example 3: 200. Mu.L of the enzymatic hydrolysis reaction system comprises 180. Mu.L of substrate (CFG substrate is dissolved in buffer containing 1-5M NaCl) and 20. Mu.L of proper diluted enzyme solution, and the reaction is carried out for 1h under the optimal pH and temperature conditions, 300. Mu.L of DNS reagent is added, and boiling is carried out for 5min. After cooling the sample, the absorbance at 540nm was measured and the amount of hydrolyzed CFG of the recombinant xylanase to generate the reducing end was calculated. The reduction end yield is 100% when xylanase is used for catalyzing and hydrolyzing CFG in a 0M NaCl reaction system; and under the same reaction conditions, the reduction end generation amount of xylanase in the catalytic hydrolysis of CFG in a NaCl reaction system of 1-5M is plotted against the reduction end generation amount in a NaCl reaction system of 0M, so that the influence of NaCl on xylanase activity is illustrated. As shown in FIGS. 9 and 10, scXyn increases the reduction end production amount of hydrolyzed CFG by 1.41, 1.56, 1.66, 2.03, 1.3 times in NaCl reaction system containing 1-5M (FIG. 9); scXyn22 in the NaCl reaction system containing 1-5M, the reduction end production of the hydrolyzed CFG is respectively increased by 1.75 times, 3.63 times, 3.66 times, 3.46 times and 3.86 times (figure 10).
3. Analysis of CFG products of recombinant xylanase catalytic hydrolysis at high NaCl concentration
Referring to the above-determined optimal reaction conditions, oligosaccharides were prepared by hydrolyzing CFG substrates under optimal conditions using recombinant xylanases Scxyn and Scxyn, respectively, and the hydrolysate molecular weight distribution characteristics were analyzed using a gel chromatograph (ELEOS System, wyatt). The calculated results are shown in Table 1, with 70.8% arabinoxylans in the recombinant xylanase Scxyn hydrolysate having a molecular weight between 2.6 and 22kDa and 73.4% arabinoxylans in the recombinant xylanase Scxyn hydrolysate having a molecular weight between 16 and 33kDa.
TABLE 1 CFG and recombinant xylanase hydrolysate molecular weight distribution characterization
4. CFG property analysis at high NaCl concentration
A1% (w/v) CFG solution was prepared with 1-5M NaCl solution. The light transmittance (%) of the CFG solution at a wavelength of 620nm was measured using distilled water as a reference, and the result is shown in Table 2, in which the light transmittance of the CFG solution was decreased as the concentration of the NaCl solution was increased.
Table 2 CFG transmittance of NaCl solution
NaCl concentration 0M 1M 2M 3M 4M 5M
Transmittance (%) 85.51 67.09 54.33 41.46 33.86 26.82
。
CFG solution rheology was measured with a rotameter (AR 2000 ex). A parallel plate measuring system with the diameter of 40mm is selected, the temperature of a sample table is set to 25 ℃, the plate spacing is 1.0mm, the shearing rate is increased from 0.1s -1 to 200s -1, and the result is shown in FIG. 11, and in a solution of NaCl with the concentration of 1-5M, the solution viscosity of CFG is obviously increased along with the increase of the concentration of NaCl.
The physicochemical state of the CFG substrate in a high-salt system is changed, so that the catalytic efficiency of the enzyme and the characteristics of an enzymolysis product are affected.
The foregoing is only for explaining the technical solution of the present application, and does not limit the protection scope of the present application.
Sequence listing
<110> National institute of food and material reserve science
<120> A method for preparing arabinoxylans having different molecular weight distribution characteristics
<160> 4
<170> SIPOSequenceListing 1.0
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Ser Ala Thr Asp Asn Pro Glu Leu Thr Asp Thr His Tyr Val Ala Ile
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Leu Ser Asp Ser Ser Glu Phe Gly Gln Leu Thr Pro Gly Asn Ser Met
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Lys Trp Asp Ala Thr Glu Pro Thr Gln Gly Gln Phe Ser Phe Asp Asn
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Gly His Thr Cys Val Trp Tyr Ser Gln Leu Pro Ser Trp Val Ser Asn
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Gly Ser Trp Asp Ala Asp Ser Leu Asn Val Ala Met Thr Thr His Thr
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Ser Thr Val Val Asp His Phe Lys Gly Lys Ile Tyr Ser Trp Asp Val
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Val Asn Glu Ala Phe Glu Asp Asp Gly Ser Phe Arg Gln Asn Val Phe
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Tyr Thr Thr Ile Gly Glu Asp Tyr Ile Ala Asn Ala Phe Lys Ala Ala
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Arg Ala Ala Asp Pro Asp Ala Lys Leu Tyr Ile Asn Asp Tyr Asn Ile
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Glu Gly Thr Gly Ala Lys Ala Asp Ala Leu Tyr Thr Phe Val Ser Ser
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Leu Leu Asn Ala Ser Val Pro Ile Asp Gly Ile Gly Met Gln Ala His
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Met Pro Val Pro Ala Ala Glu Ala Asp Leu Glu Gln Gln Lys Ala Asp
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<213> S. commune sp. DB1
<400> 2
ctccccaagc gtcagaccac tggcctcagc gcccactcgc ggcaaacgac cggccttaac 60
accatcgcgc aagccgccgg cctcaagtac ctcggctccg cgacggacaa ccccgaattg 120
acggacacgc actacgtcgc gatcctgagc gactcgagtg agtttgggca gctcacgccg 180
gggaatagta tgaagtggga cgccacggag cccacgcagg gacagttctc gttcgacaat 240
gcggacgcga tcgtggagct tgctcagaac aacagccagc tcattcgagg tcacacctgc 300
gtttggtaca gtcagctgcc cagctgggtc tcaaacgggt cctgggacgc ggactcgctg 360
aatgtggcca tgacgacgca tacttcgacg gtggtcgatc atttcaaggg caaaatatat 420
agctgggatg tagtaaacga ggcgtttgag gacgacggca gcttccgtca gaacgtcttc 480
tacacaacca tcggcgagga ttacatcgcc aacgcgttca aagccgcccg cgcggctgac 540
cctgatgcaa aactttatat caacgactac aacatcgaag gcaccggcgc caaagccgac 600
gcgctctaca ccttcgtctc ctcccttctc aacgcctccg tccccatcga cggcatcggc 660
atgcaggcgc acctcatcgt cggctctgtc ccaacgacca tccaggagaa catcgcgcgc 720
ttcactgctt tgggcctcga ggttgcgctc acggagctcg acatacggat gcctgtgccc 780
gccgccgagg cggatttgga gcagcagaag gcggattacg aggccgtggt gggcgcgtgt 840
gcggcggtgg aggggtgcgt gggtgtgacg gtctgggatt atacggataa gtactcctgg 900
gttccgagtg tcttcgatgg gtatggagcg gctttgccgt gggatgagaa cttggaaaag 960
aagccagctt acgacgggat cgtgagcggc ttgggtgca 999
<210> 3
<211> 211
<212> PRT
<213> S. commune sp. DB1
<400> 3
Ser Pro Leu Asn Ala Thr Asp Val Val Asp Val Ser Ala Arg Ser Gly
1 5 10 15
Thr Pro Ser Ser Thr Gly Thr Asp Gly Gly Tyr Tyr Tyr Ser Trp Trp
20 25 30
Thr Asp Gly Ala Gly Asp Ala Thr Tyr Gln Asn Asn Gly Gly Gly Ser
35 40 45
Tyr Thr Leu Thr Trp Ser Gly Asn Asn Gly Asn Leu Val Gly Gly Lys
50 55 60
Gly Trp Asn Pro Gly Ala Ala Ser Arg Ser Ile Ser Tyr Ser Gly Thr
65 70 75 80
Tyr Gln Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Thr Arg
85 90 95
Ser Ser Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly Ser Tyr Asp
100 105 110
Pro Ser Ser Ala Ala Ser His Lys Gly Ser Val Thr Cys Asn Gly Ala
115 120 125
Thr Tyr Asp Ile Leu Ser Thr Trp Arg Tyr Asn Ala Pro Ser Ile Asp
130 135 140
Gly Thr Gln Thr Phe Glu Gln Phe Trp Ser Val Arg Asn Pro Lys Lys
145 150 155 160
Ala Pro Gly Gly Ser Ile Ser Gly Thr Val Asp Val Gln Cys His Phe
165 170 175
Asp Ala Trp Lys Gly Leu Gly Met Asn Leu Gly Ser Glu His Asn Tyr
180 185 190
Gln Ile Val Ala Thr Glu Gly Tyr Gln Ser Ser Gly Thr Ala Thr Ile
195 200 205
Thr Val Thr
210
<210> 4
<211> 636
<212> DNA
<213> S. commune sp. DB1
<400> 4
tcgccgctca atgcgacgga cgtcgttgac gtctccgctc gctccggtac tcctagctcg 60
accggtaccg atggtggcta ctactactct tggtggacgg acggtgctgg tgacgccacc 120
taccaaaaca acggcggtgg atcgtacacc ttgacctggt ctggcaacaa cggcaacctc 180
gtcggcggaa agggatggaa cccaggagct gcgtcccgct ccatctctta ctccggcacc 240
taccagccca acggcaacag ctacctctcc gtctacggct ggacgcgcag ctcgctcatc 300
gagtactaca tcgtcgagtc ctacggctcc tacgacccgt cctccgcggc cagccacaag 360
ggctccgtca cctgcaacgg cgccacgtac gacatcctct ccacctggcg ctacaacgcg 420
ccctccatcg acggcacgca gaccttcgag cagttctgga gcgtccggaa cccgaagaag 480
gcgcccggag ggtcgatcag cgggaccgtc gacgtccagt gccacttcga tgcttggaag 540
ggactcggga tgaacttggg tagcgagcac aactaccaga tcgtggctac cgaaggctat 600
cagagcagcg gcactgcgac catcacggtc acgtaa 636

Claims (3)

1. A method for preparing arabinoxylans having a different molecular weight distribution, comprising the steps of: hydrolyzing corn fiber glue with xylanase in an environment containing 1-5M sodium salt to prepare arabinoxylans with different molecular weight distributions, wherein the amino acid sequence of the xylanase is shown in SEQ ID NO: 3.
2. The method for preparing arabinoxylans having a different molecular weight distribution according to claim 1, wherein the corn fiber gum is prepared by:
1) Weighing corn husk powder, adding n-hexane, stirring, filtering, cleaning, drying, treating with alpha-amylase, adding saccharifying enzyme, adding neutral protease into the treating solution, performing enzymolysis, inactivating, and filtering;
2) Weighing the sample obtained in the step 1), adding NaOH, ca (OH) 2 and water, heating, and centrifugally separating supernatant and precipitate after reaction;
3) Adding H 2O2 into the supernatant obtained in the step 2), regulating the pH value of the solution to be alkaline, stirring at room temperature, regulating the pH value to be acidic, centrifugally separating the supernatant, and adding absolute ethyl alcohol into the supernatant to obtain white flocculent precipitate, namely the corn fiber gum.
3. The method of preparing arabinoxylans having a different molecular weight distribution according to claim 2, wherein the method further comprises the steps of:
Adding distilled water into the precipitate obtained in the step 2), adding H 2O2, regulating pH to be alkaline, heating, cooling, centrifuging to separate supernatant, regulating pH of the supernatant to be acidic, centrifuging to separate supernatant, adding absolute ethyl alcohol to obtain white flocculent precipitate, and mixing with the corn husk fiber gum obtained in the step 3).
CN202210160967.0A 2022-02-22 2022-02-22 Method for preparing arabinoxylan with different molecular weight distribution characteristics Active CN114621987B (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN108588144A (en) * 2018-03-29 2018-09-28 中国科学院广州能源研究所 A method of preparing xylo-oligosaccharide and fermentable sugars using lignocellulose-like biomass
CN114317500A (en) * 2022-02-21 2022-04-12 国家粮食和物资储备局科学研究院 Xylanase Scxyn5, and coding gene and application thereof

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CN108588144A (en) * 2018-03-29 2018-09-28 中国科学院广州能源研究所 A method of preparing xylo-oligosaccharide and fermentable sugars using lignocellulose-like biomass
CN114317500A (en) * 2022-02-21 2022-04-12 国家粮食和物资储备局科学研究院 Xylanase Scxyn5, and coding gene and application thereof

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谷春梅 等.酶法提取对玉米皮***木聚糖组成及分子质量分布的影响.《食品科学》.2019,第40卷(第6期),摘要、第1.3.1-1.3.3节. *
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