CN114317636A - Preparation method of oligosaccharide - Google Patents

Preparation method of oligosaccharide Download PDF

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CN114317636A
CN114317636A CN202111636211.0A CN202111636211A CN114317636A CN 114317636 A CN114317636 A CN 114317636A CN 202111636211 A CN202111636211 A CN 202111636211A CN 114317636 A CN114317636 A CN 114317636A
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gly
beta
glucanase
val
ala
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CN114317636B (en
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孙科
沙凤
谭芳美
孙良
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Suzhou Koning Polyol Co ltd
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Suzhou Koning Polyol Co ltd
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Abstract

The invention discloses a preparation method of a glucan oligosaccharide, wherein the glucan oligosaccharide is beta-1, 3-glucan oligosaccharide, the glucan oligosaccharide is prepared by hydrolyzing curdlan with beta-glucanase in one step, and the sequence of the beta-glucanase is shown as SEQ ID NO. 1. The invention obtains a beta-glucanase mutant with improved activity by error-prone PCR screening, and the sequence of the mutant is shown as SEQ ID NO. 2. The hydrolysis rate of the beta-glucanase mutant is higher than 80%, and the polymerization degree of a hydrolysate is 5-7. The method has the advantages of high efficiency, mild reaction, low energy consumption, low cost, no pollution and the like, and can realize the industrial preparation of the beta-1, 3-glucan oligosaccharide.

Description

Preparation method of oligosaccharide
Technical Field
The invention relates to the technical field of bioengineering, relates to a preparation method of a glucan oligosaccharide, and particularly designs a method for preparing beta-1, 3-glucan oligosaccharide by degrading curdlan with a biological enzyme method.
Background
Curdlan is one of β -glucans, which are secreted mainly by microorganisms. Must be glued byThe D-glucose is connected by beta- (1,3) -glycosidic bond, has no branched chain, and has a molecular weight of 4.4 × 104～1×105. Curdlan has a triple helix structure, is insoluble in most organic reagents such as water and ethanol, and can be dissolved in a solution with hydrogen bond breaking ability, such as a strong alkaline solution with pH > 12, formic acid, dimethyl sulfoxide (DMSO), and the like. Meanwhile, the gel has the characteristic of cooling gel after heating. Furthermore, curdlan powder is often used in the food industry as a food additive, in combination with its colorless and odorless properties. In contrast, beta-1, 3-glucan oligosaccharides have better biological activity, such as inducing plant to generate defense response, immunological activity and the like.
At present, the method for preparing beta-1, 3-glucan oligosaccharides based on hydrolysis curdlan mainly comprises acid hydrolysis. However, the acid hydrolysis efficiency is low, the hydrolysis efficiency is only about 25%, and the polymerization degree span of the product is large; meanwhile, the problems of environmental pollution and the like are caused. The biological hydrolysis of curdlan to prepare beta-1, 3-glucan oligosaccharide is one of the effective, green and pollution-free methods. Research shows that commercial alpha-amylase can hydrolyze curdlan to prepare beta-1, 3-glucan oligosaccharide with the polymerization degree of 1-9, however, the wide polymerization degree of a hydrolysate causes great difficulty for subsequent separation and purification. Therefore, the construction of an efficient hydrolysis process with a narrow polymerization degree is very important for the application of beta-1, 3-glucan oligosaccharide from curdlan.
Disclosure of Invention
The invention aims to provide a method for preparing beta-1, 3-glucan oligosaccharide with narrow polymerization degree by high-efficiency enzymolysis of curdlan. A new beta-glucanase which can hydrolyze curdlan is adopted, and after mutation, the beta-glucanase which can efficiently degrade curdlan is obtained, the hydrolysis efficiency is 80-85%, and the polymerization degree of beta-1, 3-oligoglucan is 5-7.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a glucan oligosaccharide takes curdlan as a raw material, and the glucan oligosaccharide is prepared by carrying out enzymolysis on the curdlan through beta-glucanase.
The said oligose is beta-1, 3-oligose with polymerization degree of 5-7.
The reaction conditions of the beta-glucanase are as follows: 1-8mL of curdlan at 1-30g/L, 1-4mL of 1MPBS (pH 5.0-7.0), 0.01-2mL of 0.1-5g/L of beta-glucanase, and deionized water is added into the reaction system at 10 mL. The reaction solution is put in a water bath kettle at the temperature of 30-70 ℃ for reaction for 1-24 h. The hydrolysis rate was measured at 45% -50% after the reaction was completed.
Further preferably, the reaction temperature is 20 to 40 ℃, more preferably, the reaction temperature is 40 ℃.
The PBS solution is preferably pH 6.0;
further preferably, the reaction time is from 1 to 12 h.
The beta-glucanase is derived from Novosphingobium sp.P6W, the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 3.
The invention takes the beta-glucanase from Novosphingobium sp.P6W as a template, and carries out non-directed evolution on the sequence of the beta-glucanase to obtain the novel recombinant beta-glucanase, wherein the amino acid sequence of the beta-glucanase is shown as SEQ ID NO. 2. The reaction is faster after mutation, the reaction time is shorter, and the hydrolysis efficiency is higher.
The beta-glucanase mutant (SEQ ID NO.2) is obtained by replacing proline at position 112, leucine at position 300 and isoleucine at position 415 of an original beta-glucanase amino acid sequence shown in SEQ ID NO.1 with glycine, glutamic acid and tyrosine.
The amino acid sequence of the mutant of the beta-glucanase is shown as SEQ ID NO. 2.
A nucleotide sequence of a mutant of beta-glucanase shown in SEQ ID NO. 2.
An expression vector comprising the gene for the beta-glucanase or beta-glucanase mutant.
A recombinant bacterium obtained by transforming a host cell with the above expression vector.
The reaction conditions of the beta-glucanase mutant are as follows: 1-8mL of curdlan at 1-30g/L, 1-4mL of 1M PBS (pH 6.0-8.0), 0.01-2mL of 0.5g/L of beta-glucanase, and deionized water is added into the reaction system at 10 mL. The reaction solution is put in a water bath kettle at the temperature of 30-70 ℃ for reaction for 1-24 h. The hydrolysis rate was determined to be 80% -85% after the reaction was complete.
Preferably, the curdlan concentration is 20g/L, the beta-glucanase solution concentration is 0.5g/L, and the PBS is preferably pH 7.0.
Further preferably, the reaction temperature is 30 to 60 deg.C, more preferably, the reaction temperature is 40 to 50 deg.C.
Further preferably, the reaction time is 1-12 h; more preferably, the reaction time of the beta-glucanase mutant is 1-4 h.
In the invention, the beta-glucanase and the mutant thereof can efficiently carry out enzymolysis on curdlan to prepare beta-1, 3-glucan oligosaccharide with narrow polymerization degree (5-7), the separation of sugar is difficult in industry, and tens of millions of simulated fluid bed devices are usually needed, so the more complex the components of hydrolysate, the larger the polymerization degree span, the more unfavorable the subsequent separation and purification of products, the narrow the polymerization degree of the beta-1, 3-glucan oligosaccharide obtained by the enzymolysis of the invention, and the difficulty of the subsequent separation and purification is greatly reduced.
It is to be noted that the "beta-glucanase gene" also includes a mutated form of SEQ ID No.3 encoding a protein having the same function as the beta-glucanase, said mutated form including: true, nonsense, insert, missense. One skilled in the art will appreciate that as a result of the degeneracy of the genetic code, many different polynucleotides are capable of encoding the same polypeptide. In addition, it will be appreciated that those skilled in the art are able to make nucleotide substitutions using conventional techniques, which substitutions do not affect the polypeptide sequence encoded by the polynucleotide used in the present invention. In addition, the polynucleotides may be modified using methods known in the art to enhance the activity or survival of the polynucleotides of the invention in vivo.
The nucleotide sequence and functional equivalents of the beta-glucanase provided by the invention can be self-adjusted by the skilled person according to the amino acids of the beta-glucanase, the degeneracy principle of the codon and the like, and the adjusted sequences are also within the protection scope of the invention.
Has the advantages that: the invention provides a method for preparing beta-1, 3-glucan oligosaccharide with the polymerization degree of 5-7 by degrading curdlan efficiently and greenly, and the hydrolysis rate is high.
Drawings
FIG. 1 determination of the hydrolysis rate of the wild-type beta-glucanase;
FIG. 2 shows the hydrolysate distribution of curdlan hydrolyzed by wild-type beta-glucanase;
FIG. 3 determination of the enzyme activities of wild type and mutant;
FIG. 4 pH optimum assay for beta-glucanase;
FIG. 5 temperature optima determination of beta-glucanase;
FIG. 6 determination of hydrolysis rate of beta-glucanase mutants;
FIG. 7 shows the hydrolysate distribution of beta-glucanase mutants in hydrolysis of curdlan.
Detailed Description
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments that can be derived by one skilled in the art from the embodiments given herein are intended to be within the scope of the invention.
In the following embodiments, the method for preparing curdlan is as follows: 20g of curdlan was dissolved in 1L of 1M NaOH solution and then dissolved by magnetic stirring overnight. The pH was then adjusted to 7 using 1M HCl and the precipitate was collected by centrifugation (6000g,20 min). After washing the precipitate three times with pure water, pure water was added thereto so that the curdlan final concentration was 20g/L, followed by homogenization to prepare a curdlan suspension.
Example 1 preparation of beta-glucanase and use thereof
The gene (sequence shown in SEQ ID NO. 3) of the beta-glucanase is synthesized by general biology (Anhui) GmbH, and the synthesized fragment is constructed into a pET-28a (+) vector (enzyme cutting sites are BamH I and EcoR I). Then it was transformed into E.coli BL21(DE3), and the resulting recombinant strain was cultured in LB medium (NaCl 1%, peptone 1%, yeast powder 0.5%) at 37 ℃ under conditions ofCulturing at 200rpm for 2-3 hours until OD600Adding IPTG with the final concentration of 1mM for induction expression between 0.6 and 0.8, wherein the conditions for induction expression are as follows: further culturing at 30 ℃ and 200rpm for 16 hours, homogenizing and crushing under high pressure, centrifuging at low temperature, and collecting supernatant to obtain the crude enzyme solution of the beta-glucanase.
The beta-glucanase crude enzyme liquid is purified by a high affinity Ni-NTA purification medium (purchased from Nanjing Jinslei Biotech Co., Ltd.), filtered by a dialysis bag and freeze-dried to obtain beta-glucanase powder.
The beta-glucanase powder is subjected to constant volume to 0.5g/L by deionized water, and the enzymolysis reaction is carried out according to the following system: 2.5mL of curdlan at 20g/L, 2mL of 1M citrate buffer (pH 5.0), 0.1mL of 0.5g/L of beta-glucanase, and an additional 5.4mL of water. The reaction solution is placed in a water bath kettle at 40 ℃ for reaction for 16 h. Samples were taken every two hours (20mL) to determine the curdlan content, and the oligosaccharide composition and conversion at the end of the reaction.
The content determination method of curdlan comprises the following steps: boiling 10mL of sample for 5min, centrifuging at high speed (20000g,20min), collecting precipitate, oven drying to heavy weight, weighing and recording mass as m1. In addition, 10mL of 0.5g/L beta-glucanase solution was boiled for 5min, followed by high speed centrifugation (20000g,20min), the precipitate was collected and dried to constant weight, and the recorded mass was weighed as m2。
Content (%) of curdlan ═ m1-m2)/10×100
Wherein, the method for measuring the composition of the oligosaccharide comprises the following steps: and (3) analyzing the polymerization degree of the enzymolysis oligosaccharide component by using the chitosan oligosaccharide with the polymerization degree of 2-10 as a standard substance by adopting HPLC. The chromatographic column is Shodex polyvinyl alcohol amino column (NH)2P-504E), the detector is an evaporative light detector, the mobile phase is acetonitrile/water (0-15 min: 70% -60% acetonitrile; 15-25 min: 60% -50%; acetonitrile; 25-30 min: 50% -70% acetonitrile), flow rate of 1mL/min, column temperature of 30 ℃.
The results are shown in figures 1 and 2, and the enzyme is stable after 12h of enzymolysis, the hydrolysis rate is 47.7%, and the oligosaccharide consists of glucan 5, glucan 6 and glucan 7.
Example 2 screening of beta-glucanase mutants
The objective fragments were randomly mutated using the GeneMorph II Random Mutagenesis Kit (Agilent, USA). Among the primers used were GF (5'-cagcaaatgggtcgcggatccGTGACGTCCCCCGATTTCGGC-3') and GR (5'-ttgtcgacggagctcgaattcTCAGTTGCCGTCCGGCCACGCC-3'). The error-prone PCR system is: mu.L of recombinant plasmid, 1. mu.L each of primers GF and GR, 1. mu.L of Mutazyme II DNA polymerase, 1. mu.L of dNTPs, 5. mu.L of 10 XMutazyme II reaction buffer, made up to 50. mu.L with ddH 2O. The error-prone PCR reaction conditions were: 3min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃ and 1.8min at 72 ℃ (30 cycles); 5min at 72 ℃. Subsequently, after recovery through the column, the plasmid was ligated to pET-28a (+) digested by BamH I and EcoR I by In-fusion cloning to obtain a recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21(DE3) and screened using kanamycin-containing resistant plates.
All transformants were picked into a 96-deep well plate (1mL LB), cultured at 37 ℃ for 2 hours at 200rpm, then induced to express with 0.1M IPTG, and subsequently cultured for 8 hours at 37 ℃ for 200 rpm. 100 μ L of the broth was added to a common 96-well plate and 50mL of 10mM p-nitrophenyl- β -D-glucopyranoside (pNPG), 50mL of 0.2M citrate buffer (pH 5.0) was added. After 10min of reaction, the reaction was terminated by immediately adding a 2% trisodium phosphate solution (pH 12), and the absorption wavelength at 400nm was measured. And (3) taking pNPG solutions with different concentrations as standard curves, and calculating the pNPG content in the reaction solution. Wherein the definition of enzyme activity is as follows: the amount of enzyme required to hydrolyze 1. mu.M pNPG substrate at pH 5 and 40 ℃ per minute.
Relative enzyme activity (%) ═ beta-glucanase mutant enzyme activity multiplied by 100/beta-glucanase initial enzyme activity
The optimum pH test method comprises the following steps of putting 100 mu L of bacteria liquid for induced expression into a common 96-well plate, and additionally adding 50mL of 10mM p-nitrobenzene-beta-D-glucopyranoside (pNPG) and 50mL of 0.2M citrate or phosphate buffer to adjust the pH to 4, 5, 6, 7, 8, 9 and 10; after reacting for 10min at 30 ℃, measuring and calculating the enzyme activity.
The optimum temperature test method comprises the following steps of putting 100 mu L of bacteria liquid for induced expression into a common 96-well plate, and additionally adding 50mL of 10mM p-nitrobenzene-beta-D-glucopyranoside (pNPG) and 50mL of 0.2M citrate buffer (pH 6.0); after reaction at 20, 30, 40, 50, 60, 70 and 80 ℃ for 10min, the enzyme activity was measured and calculated.
The experimental results are shown in figure 3, and the enzyme activities of the mutants 1-63, the mutants 3-75 and the mutants 6-22 are obviously improved. The enzyme activity of the mutant 3-75 is improved most remarkably and is 272% of the initial enzyme activity of the beta-glucanase. Through the study on the optimum pH and the optimum temperature of the mutant 3-75, the mutant is found to be remarkably improved compared with the initial beta-glucanase (figure 4 and figure 5).
Example 3 preparation and use of beta-glucanase mutants
After overnight culture of mutants 3-75, the plasmids of mutants 3-75 were extracted using the Fastpure Plasmid Mini Kit (SoftNavovisa Biotech Co., Ltd., Nanjing) and sent to general-purpose organisms (Anhui) Co., Ltd., where the translated amino acid sequence was shown in SEQ ID NO. 2. Through comparison, the amino acid sequence shown in SEQ ID NO.1 of the original beta-glucanase shown in SEQ ID NO.2 has the proline at position 112 replaced by glycine, the leucine at position 300 replaced by glutamic acid, and the isoleucine at position 415 replaced by tyrosine.
And (3) carrying out IPTG induced expression on the mutant 3-75, homogenizing and crushing the mutant at high pressure, centrifuging the homogenate at low temperature, and collecting the supernatant to obtain the crude enzyme solution of the beta-glucanase mutant. Purifying the crude enzyme liquid of the beta-glucanase mutant by a high-affinity Ni-NTA purification medium, filtering by a dialysis bag, and freeze-drying to obtain powder of the beta-glucanase mutant.
The beta-glucanase mutant powder is subjected to constant volume to 0.5g/L by using deionized water, and the enzymolysis reaction is carried out according to the following system: 2.5mL of curdlan at 20g/L, 2mL of 1M phosphate buffer (pH 7.0), 0.1mL of 0.5g/L beta-glucanase, and an additional 5.4mL of water. The reaction solution is placed in a water bath kettle at 50 ℃ for reaction for 8 hours. Samples were taken every two hours (20mL) to determine the curdlan content, and the oligosaccharide composition and conversion at the end of the reaction. Curdlan content and oligosaccharide were determined as described in example 1.
The results of the experiments are shown in fig. 6 and 7, and the enzyme is stable after 4h enzymolysis, the hydrolysis rate is 82.3%, and the oligosaccharide consists of glucan 5, glucan 6 and glucan 7.
Sequence listing
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<120> preparation method of oligosaccharide
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aacggcgacg gcggccggac gatctttttc caatcagagc ctccccatga cgcgctgacc 1440
caatcggacc gcatgaacgg caccgaaaag ggctatgcat attacgagct cgcgccgacc 1500
gttcgaacgc acgagggcat cggcttcgct ctgtacggcg ccgcgagcaa gtcgaccgaa 1560
ccgatcatcc agatgagtga gatcaaggct cccgagcggc ccggcatttc attccgcagc 1620
gtcacaggag tatggctgtt cggtcacggc cagtatctga ataccttcaa ttccgatggg 1680
cgcgccgtcg gtaaggacat cgttccaagc ttcgtgccgg gcatcgcggc gacacggcag 1740
gtcgtggcgt ggccggacgg caactga 1767
<210> 4
<211> 42
<212> DNA
<213> Artificial synthesis (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 4
cagcaaatgg gtcgcggatc cgtgacgtcc cccgatttcg gc 42
<210> 5
<211> 43
<212> DNA
<213> Artificial synthesis (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 5
ttgtcgacgg agctcgaatt ctcagttgcc gtccggccac gcc 43

Claims (9)

1. A preparation method of a glucan oligosaccharide is characterized in that curdlan is used as a raw material, and the glucan oligosaccharide is prepared by carrying out enzymolysis on curdlan through beta-glucanase; the beta-glucanase is beta-glucanase with an amino acid sequence shown as SEQ ID NO.1 and a mutant thereof.
2. The method for preparing a glucan oligosaccharide according to claim 1, wherein the glucan oligosaccharide is a β -1, 3-glucan oligosaccharide, and the degree of polymerization is 5-7.
3. The method of claim 1, wherein the beta-glucanase is derived fromNovosphingobium sp. P6W。
4. The method of claim 1, wherein the beta-glucanase mutant has a substitution of proline at position 112, a substitution of leucine at position 300 with glutamic acid, and a substitution of isoleucine at position 415 with tyrosine, of the beta-glucanase represented by SEQ ID NO. 1.
5. The method of claim 1, wherein the curdlan and the beta-glucanase are used in a ratio of 50-5000:1 (w/w).
6. The method for preparing the oligo-saccharide according to claim 1, wherein the enzymatic reaction is carried out at 30-70 ℃ for 1-24 h.
7. The method for preparing glucan oligosaccharide according to claim 1, wherein the curdlan concentration is 1-30g/L, and the beta-glucanase solution concentration is 0.1-5 g/L.
8. A mutant β -glucanase characterized in that proline at position 112, leucine at position 300, and isoleucine at position 415 of the β -glucanase represented by SEQ ID NO.1 are replaced with glycine, glutamic acid, and tyrosine, respectively.
9. The beta-glucanase mutant according to claim 8, characterized in that the sequence of the beta-glucanase mutant is shown in SEQ ID No. 2.
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Citations (4)

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Publication number Priority date Publication date Assignee Title
JP2003164284A (en) * 2001-11-30 2003-06-10 Noda Inst For Scient Res New endoglucanase and endoglucanase gene
CN105431534A (en) * 2013-08-02 2016-03-23 优瑞纳股份有限公司 beta-1,3-glucanase, polynucleotide, recombinant vector, transformant, production method for beta-1,3-glucanase, enzyme preparation, and production method for paramylon having reduced molecular weight
CN106085989A (en) * 2016-06-14 2016-11-09 中国农业大学 One Bacillus species β 1,3 1,4 glucanase and encoding gene thereof and application
CN110036108A (en) * 2016-11-09 2019-07-19 中国农业大学 A kind of bacterium beta-1,3-glucanase and its encoding gene and application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003164284A (en) * 2001-11-30 2003-06-10 Noda Inst For Scient Res New endoglucanase and endoglucanase gene
CN105431534A (en) * 2013-08-02 2016-03-23 优瑞纳股份有限公司 beta-1,3-glucanase, polynucleotide, recombinant vector, transformant, production method for beta-1,3-glucanase, enzyme preparation, and production method for paramylon having reduced molecular weight
CN106085989A (en) * 2016-06-14 2016-11-09 中国农业大学 One Bacillus species β 1,3 1,4 glucanase and encoding gene thereof and application
CN110036108A (en) * 2016-11-09 2019-07-19 中国农业大学 A kind of bacterium beta-1,3-glucanase and its encoding gene and application

Non-Patent Citations (1)

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Title
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