CN113699140A - Alginate lyase and application thereof - Google Patents
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Abstract
The invention discloses an alginate lyase, the amino acid sequence of which is shown as SEQ ID NO. 1. The nucleotide sequence of the gene for coding the alginate lyase is shown as SEQ ID NO. 2. The application of the algin lyase in degrading algin or sodium alginate and the application in preparing alginate oligosaccharides. The invention also discloses a method for preparing the brown algae oligosaccharide, which comprises the following steps: the alginate lyase is adopted to degrade alginate to prepare alginate oligosaccharide, the optimum reaction pH is 9.0, the optimum reaction temperature is 60 ℃, the reaction time is 2 hours, and the polymerization degree of the alginate oligosaccharide is 2-4. The alginate lyase can cleave the alginate or the sodium alginate under mild conditions, has high enzyme activity and better temperature stability, can react at high temperature with better substrate solubility compared with the reported alginate lyase, and can efficiently degrade the alginate to prepare the alginate oligosaccharide.
Description
Technical Field
The invention relates to alginate lyase and application thereof, belonging to the technical field of functional lyase discovery.
Background
The brown algae in China are abundant in resources and have high development and application values. The brown algae is used as a raw material, a large amount of algin can be extracted, and the algin has strong hydration and cohesiveness and is widely applied to the industries of food, medicine and chemical industry. However, the algin is long-chain biological polysaccharide, so that the algin is difficult to be absorbed and utilized by plants after being directly eaten, and the application of the algin in the field of functional products is limited. The algin is hydrolyzed into the alginate oligosaccharide, so that the water solubility and the absorption and utilization of the algin oligosaccharide can be greatly increased, the alginate oligosaccharide also has the functions of reducing blood sugar, resisting inflammation, regulating immunity, promoting the growth of animals and plants and the like, the application value and the economic value of the brown algae can be further improved by hydrolyzing the algin into the alginate oligosaccharide, and the application range of seaweed resources is greatly widened.
The method for hydrolyzing algin into alginate oligosaccharide mainly comprises three methods, namely an acid hydrolysis method, an enzyme hydrolysis method and an oxidation degradation method, wherein the acid hydrolysis method needs to use a large amount of strong acid reagents, the oxidation degradation method needs to use strong oxidation reagents such as hydrogen peroxide, and the two methods have the problems of violent reaction process, easy loss of products, hidden danger of environmental pollution and the like. The enzymatic hydrolysis method mainly uses alginate hydrolase, carries out biological hydrolysis on the alginate by utilizing the efficient hydrolysis action of the alginate hydrolase, has mild hydrolysis process conditions and high biological safety of products, ensures that the hydrolysate has a certain polymerization degree range, and is a method for producing alginate oligosaccharides by using the alginate.
Disclosure of Invention
Aiming at the prior art, the invention provides an alginate lyase and application thereof in preparation of alginate oligosaccharides, and the alginate lyase is used for degrading high molecular weight alginate so as to prepare alginate oligosaccharides with single polymerization degree.
The invention is realized by the following technical scheme:
an alginate lyase with amino acid sequence shown in SEQ ID NO. 1 and derived from Haemophilus agar (Haemophilus agar) (A. agar)Catenovulum agarivorans）。
The amino acid sequence of alginate lyase (shown as SEQ ID NO: 1):
MYKSVLIGVS LAITSLTAAA GQQVGATLNH KQSLALADIP AGVIKQIAAV RPKFVAKEAE KEFKHGKVYI DVEGLDQYGN EIEFDMLQQD GTWKIVEIQR DLEMSQCPDS VVIALTKAHP DIQPKRIIES EQATGEIIYE FYTVDSAGQE AKYEVKLANG TAELLNQEWQ H。
the nucleotide sequence of the gene for coding the algin lyase is shown as SEQ ID NO. 2.
The nucleotide sequence of the gene for coding the algin lyase (shown as SEQ ID NO: 2):
5-ATGTACAAAA GCGTTCTGAT CGGTGTAAGC CTGGCGATTA CCTCTCTGAC TGCTGCTGCC GGTCAGCAGG TGGGTGCGAC CCTGAACCAC AAACAGAGCC TGGCGCTGGC TGATATCCCG GCAGGTGTGA TCAAGCAGAT TGCAGCAGTT CGCCCGAAAT TCGTGGCTAA AGAAGCCGAA AAAGAGTTCA AACACGGCAA AGTTTACATT GATGTGGAAG GCCTGGATCA GTACGGCAAC GAAATCGAAT TCGATATGCT GCAGCAGGAT GGCACCTGGA AAATCGTAGA GATCCAGCGT GATCTGGAAA TGTCTCAATG TCCGGATAGC GTTGTCATTG CGCTGACCAA AGCCCACCCG GACATTCAGC CGAAGCGTAT CATCGAAAGC GAACAGGCGA CTGGTGAAAT CATCTATGAA TTCTATACCG TGGACTCCGC TGGCCAGGAG GCCAAATACG AAGTAAAACT GGCAAATGGT ACCGCGGAGC TGCTGAACCA GGAATGGCAG CAC-3。
the application of the algin lyase in degrading algin or sodium alginate; application in preparing brown algae oligosaccharide.
A method for preparing brown algae oligosaccharide comprises the following steps: the algin lyase is adopted to degrade algin or sodium alginate to prepare alginate oligosaccharide, and the enzymolysis conditions are as follows: the pH value is 6.0-10.0, and the temperature is 30-80 ℃; the polymerization degree of the obtained brown algae oligosaccharide is 2-4.
Preferably, the optimum reaction pH is 9.0, the optimum reaction temperature is 60 ℃, and the reaction time is 2 h.
An enzyme preparation containing alginate lyase, which contains the alginate lyase.
The application of the enzyme preparation containing the alginate lyase in degrading the algin or the sodium alginate; application in preparing brown algae oligosaccharide.
The algin lyase of the invention has the enzyme activity of more than 60 percent of the highest enzyme activity when the pH value is between 6.0 and 10.0, has good pH stability between 7.0 and 10.0, and can keep more than 70 percent of the enzyme activity within 100 h. The enzyme activity can be kept above 60% of the highest enzyme activity at 30-80 ℃, and the enzyme has better temperature stability at 50 ℃ and 60 ℃, and can still keep above 80% of the enzyme activity after heat preservation for 12 hours in the temperature range. Fe3+、Mn2+、Ba2+、Zn2+Can promote enzyme activity of the enzyme (when preparing brown algae oligosaccharide, appropriate amount of Fe can be added into substrate3+、Mn2+、Ba2+、Zn2+，It is added in the form of metal salt, such as ferric chloride, manganese sulfate, barium chloride, zinc nitrate, etc.), Mg2+、Cu2+、K+、Ni2+SDS inhibits the enzyme activity of the enzyme, Ca2+Has little influence on the enzyme activity of the enzyme, Na2EDTA can inactivate the enzyme.
The alginate lyase of the invention can cleave the alginate under mild conditions, has high enzyme activity and better temperature stability, can react at high temperature with better substrate solubility compared with the reported alginate lyase, and can efficiently degrade the alginate to prepare the alginate oligosaccharide.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
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FIG. 1: a pure enzyme SDS-PAGE electrophoresis picture after the purification of the alginate lyase, wherein 1 is a standard protein Marker; 2 is purified alginate lyase.
FIG. 2: the results of the alginate lyase determination of optimal reaction temperature and temperature stability are shown in the figure 2A, optimal reaction pH; FIG. 2B, optimum reaction temperature; FIG. 2C, pH stability; figure 2D, temperature stability.
FIG. 3: schematic diagram of the influence of metal ions on the enzyme activity of the alginate lyase.
FIG. 4: and (3) a mass spectrogram of the product of degrading the algin by the algin lyase.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1 alginate lyase Source and sequence analysis
To find a novel enzyme gene for alginate lyase, researchers derived from Haematococcus agar: (Catenovulum agarivorans) A section of potential alginate lyase protein containing 171 amino acids is discovered from the encoded protein. According to the literature report, the strain is separated from the nine-pore spirochete intestinal tract of marine organisms, the marine algae polysaccharide can be efficiently degraded, and researchers guess that potential alginate lyase contained in the strain possibly has higher alginate hydrolysis activity. The amino acid sequence is shown in SEQ ID NO. 1 and is named Alca-171. Through Blast analysis, compared with the reported alginate lyase, the highest sequence similarity is only 54%, which indicates that the alginate lyase is a novel alginate lyase.
Example 2 construction and expression of an expression vector containing alginate lyase Gene and purification of enzyme protein
The Alca-171 enzyme gene was codon optimized to obtain the nucleotide sequence shown in SEQ ID NO. 2, which was artificially synthesized and loaded into pET-28a vector using seamless ligation. The vector was transformed into E.coli DH 5. alpha. competent cells by heat shock. Screening by using an LB medium plate containing ampicillin, selecting grown positive clones, carrying out PCR verification and sequencing verification, wherein the sequencing result is consistent with the original sequence comparison, which shows that the alginate lyase gene sequence is cloned into an expression vector, and the recombinant plasmid is named as pET-Alca-171.
The recombinant plasmid pET-Alca-171 is transformed into host escherichia coli BL21 competent cells by a heat shock method, the transformed cells are screened and identified on a plate containing ampicillin resistance, and the positive transformant which is successfully identified is the heterologous expression host which is successfully constructed.
The heterologous expression host is cultured and activated in an LB culture medium containing ampicillin resistance, bacterial liquid is inoculated into a ZYP-5052 culture medium containing ampicillin resistance according to the inoculation amount of 1 percent (v/v) after being activated, and is cultured for 48 hours at the temperature of 20 ℃ and at the speed of 200 r/min to express alginate lyase Alca-171.
After the recombinant bacteria are expressed, the recombinant bacteria are centrifuged for 10 min at 8000 r/min and 4 ℃, and thallus precipitates are collected. The pellet was sonicated using 50 mM Tris-HCl buffer, pH 8.0. The crushed solution is centrifuged for 10 min at 8000 r/min and 4 ℃, and the supernatant is collected as a crude enzyme solution. The crude enzyme solution is purified by using nickel column affinity chromatography, imidazole solutions with different concentration gradients are used for balancing and eluting columns, 80 mM imidazole solution elution components are collected, the components are concentrated by using an ultrafiltration membrane with the molecular weight cutoff of 5 kDa to obtain the alginate lyase enzyme solution, the protein purity is detected by using SDS-PAGE electrophoresis, and the result is shown in figure 1, and the protein band size is about 19 kDa and is similar to the predicted molecular weight, which indicates that the enzyme is successfully purified.
EXAMPLE 3 investigation of the enzymatic Properties of alginate lyase
The enzyme activity determination method comprises the following steps: preparing 0.3% (w/v, unit g/ml) sodium alginate solution as a reaction substrate, adding 20 mu L of enzyme solution into 380 mu L of sodium alginate substrate solution, reacting at 50 ℃, pH 9.0 of the system, reacting for 20 min, inactivating the enzyme in boiling water bath for 10 min after the reaction is finished, measuring the generation amount of reducing sugar in the system by using a DNS method, and calculating the enzyme activity according to a D-galactose standard curve.
When the optimum reaction pH is measured, taking an equivalent enzyme solution, setting the temperature of a reaction system to be 50 ℃, respectively selecting different system buffer solutions with the pH of 5.0-10.0 to adjust the pH of the system, and measuring the enzyme activity of the alginate lyase under different pH values; when the optimal reaction temperature is measured, taking an equivalent enzyme solution, respectively placing the reaction system under the conditions of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for reaction under the condition that the pH value of a buffer solution is 9.0, and measuring the enzyme activity of the alginate lyase at different temperatures; when measuring the pH stability, respectively storing the enzyme in a buffer solution with the pH of 7.0-10.0 at the temperature of 4 ℃, and sampling at different intervals to measure the enzyme activity; when measuring the temperature stability, the enzyme solution is placed in a buffer solution with the pH value of 9.0, stored at the temperature of 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively, and sampled at different intervals to measure the enzyme activity. When the influence of metal ions and chemical reagents on the enzyme activity is measured, various metal ions and chemical reagents with the final concentration of 1 mM are respectively added into a reaction system, the reaction system is placed under the conditions of pH 9.0 and 60 ℃ for reaction, and a sample is taken to measure the enzyme activity.
As shown in FIGS. 2 and 3, it is understood from FIG. 2A that the optimum reaction pH of the enzyme is 9.0, and that the enzyme activity is maintained at 60% or more of the highest enzyme activity at a pH of 6.0 to 10.0. As can be seen from FIG. 2B, the optimum reaction temperature of the enzyme is 60 ℃, and the enzyme activity of the enzyme can be kept above 60% of the highest enzyme activity at the temperature of 30-80 ℃. As can be seen from FIG. 2C, the enzyme has good pH stability between pH 7.0 and 10.0, and can maintain more than 70% of enzyme activity within 100 h. As can be seen from FIG. 2D, the enzyme has good temperature stability at 50 ℃ and 60 ℃, and can still retain more than 80% of enzyme activity after being kept for 12 hours in the temperature range. As can be seen from FIG. 3, among various metal ions and chemical agents to be tested, Fe3+、Mn2+、Ba2+、Zn2+Can promote the enzyme activity of the enzyme, Mg2+、Cu2+、K+、Ni2+SDS inhibits the enzyme activity of the enzyme, Ca2+Has little influence on the enzyme activity of the enzyme, Na2EDTA can inactivate the enzyme.
The alginate lyase action substrate is sodium alginate, the sodium alginate solution is in a sticky state and has low solubility, the viscosity of the solution is reduced along with the increase of the temperature, and the solubility is increased, so the reaction at high temperature is more favorable for the degradation of the sodium alginate substrate. However, most of the alginate lyase found in the literature has an optimum temperature of 40 ℃ or lower (Cheng et al, International Journal of Biological Macromolecules),2020,164:1304-1320). Alginate lyase having a high optimum temperature has also been reported, for example, fromSphingomonassp, A1 alginate lyase A1-II, the optimum reaction temperature is 70 ℃, the hydrolysate is alginate oligosaccharide with the degree of polymerization of 3-4, but the alginate oligosaccharide loses 50% of activity after being kept for 10 min at 50 ℃ (Yoon et al, Protein Expression and Purification, 2000, 19: 84-91); derived fromNitratiruptorsp, SB 155-2 algin lyase Nitaly, the optimum reaction temperature is 70 ℃, the hydrolysate is alginate oligosaccharide with the polymerization degree of 3-5, and the alginate lyase Nitaly loses 50% of activity after being preserved for 30 min at 67 ℃; derived fromDefluviitalea phaphyphilaThe optimum reaction temperature of the alginate lyase DP0100 is 65 ℃, the hydrolysate is alginate oligosaccharide with the polymerization degree of 2-3, but 50 percent of activity of the alginate lyase is lost after the alginate oligosaccharide is kept for 45 min at the temperature of 65 ℃. Therefore, the reported alginate lyase with higher activity at high temperature has poor stability at high temperature, and the use of the alginate lyase at high temperature is limited. The alginate lyase Alca-171 can still retain more than 80% of enzyme activity after being insulated for 12 hours at the temperature of 60 ℃, and can also retain more than 70% of enzyme activity after being insulated for 4 hours at the temperature of 70 ℃, which shows that the alginate lyase Alca-171 can retain higher enzyme activity at high temperature and can be used for preparing alginate oligosaccharides at high temperature.
Example 4 preparation of alginate oligosaccharides Using alginate lyase
Alginate oligosaccharides were prepared by hydrolyzing sodium alginate with the alginate lyase prepared in example 2. Preparing a 0.3% sodium alginate solution, adding 1 mL of diluted Alca-171 enzyme solution (50U/mL) into 19 mL of substrate solution, reacting for 2 h at the conditions of pH 9.0 and 60 ℃, centrifuging the reaction solution at 10000 r/min for 10 min after the reaction is finished, taking supernatant, and freeze-drying to obtain the freeze-dried product, namely the brown algae oligosaccharide. The product identification is carried out on the brown alga oligosaccharide by utilizing a mass spectrum, and the analysis result is shown in figure 4, so that the product is the oligosaccharide with the polymerization degree of 2-4, the brown alga oligosaccharides with other polymerization degrees are very few, and the product purity is higher. The optimum reaction temperature of the alginate lyase is 60 ℃, the alginate lyase has good stability at the temperature, the reaction at the high temperature is favorable for fully dissolving a substrate and keeping the sterility in the reaction process, the reaction process does not generate environmental pollution, a tool enzyme with good property is provided for preparing the alginate oligosaccharide by a green method, and the alginate lyase has the potential of industrial application for producing the alginate oligosaccharide.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Sequence listing
<110> China oceanic university
<120> alginate lyase and application thereof
<141> 2021-10-19
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 171
<212> PRT
<213> Catenovulum agarivorans
<400> 1
Met Tyr Lys Ser Val Leu Ile Gly Val Ser Leu Ala Ile Thr Ser Leu
1 5 10 15
Thr Ala Ala Ala Gly Gln Gln Val Gly Ala Thr Leu Asn His Lys Gln
20 25 30
Ser Leu Ala Leu Ala Asp Ile Pro Ala Gly Val Ile Lys Gln Ile Ala
35 40 45
Ala Val Arg Pro Lys Phe Val Ala Lys Glu Ala Glu Lys Glu Phe Lys
50 55 60
His Gly Lys Val Tyr Ile Asp Val Glu Gly Leu Asp Gln Tyr Gly Asn
65 70 75 80
Glu Ile Glu Phe Asp Met Leu Gln Gln Asp Gly Thr Trp Lys Ile Val
85 90 95
Glu Ile Gln Arg Asp Leu Glu Met Ser Gln Cys Pro Asp Ser Val Val
100 105 110
Ile Ala Leu Thr Lys Ala His Pro Asp Ile Gln Pro Lys Arg Ile Ile
115 120 125
Glu Ser Glu Gln Ala Thr Gly Glu Ile Ile Tyr Glu Phe Tyr Thr Val
130 135 140
Asp Ser Ala Gly Gln Glu Ala Lys Tyr Glu Val Lys Leu Ala Asn Gly
145 150 155 160
Thr Ala Glu Leu Leu Asn Gln Glu Trp Gln His
165 170
<210> 2
<211> 513
<212> DNA
<213> Artificial Sequence
<400> 2
atgtacaaaa gcgttctgat cggtgtaagc ctggcgatta cctctctgac tgctgctgcc 60
ggtcagcagg tgggtgcgac cctgaaccac aaacagagcc tggcgctggc tgatatcccg 120
gcaggtgtga tcaagcagat tgcagcagtt cgcccgaaat tcgtggctaa agaagccgaa 180
aaagagttca aacacggcaa agtttacatt gatgtggaag gcctggatca gtacggcaac 240
gaaatcgaat tcgatatgct gcagcaggat ggcacctgga aaatcgtaga gatccagcgt 300
gatctggaaa tgtctcaatg tccggatagc gttgtcattg cgctgaccaa agcccacccg 360
gacattcagc cgaagcgtat catcgaaagc gaacaggcga ctggtgaaat catctatgaa 420
ttctataccg tggactccgc tggccaggag gccaaatacg aagtaaaact ggcaaatggt 480
accgcggagc tgctgaacca ggaatggcag cac 513
Claims (10)
1. An alginate lyase, the amino acid sequence of which is shown in SEQ ID NO 1.
2. The gene of alginate lyase of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO. 2.
3. The use of the alginate lyase of claim 1 for degrading algin or sodium alginate; and/or: application in preparing brown algae oligosaccharide.
4. A method for preparing brown algae oligosaccharide is characterized in that: the alginate-derived oligosaccharide is prepared by degrading algin or sodium alginate by using the alginate lyase of claim 1.
5. The method for preparing brown alginate oligosaccharides according to claim 4, wherein: the enzymolysis conditions are as follows: the pH value is 6.0-10.0, and the temperature is 30-80 ℃.
6. The method for preparing brown alginate oligosaccharides according to claim 4, wherein: adding Fe to the substrate during enzymolysis3+、Mn2+、Ba2+、Zn2+Any one or two or more of them.
7. The method for preparing brown alginate oligosaccharides according to any one of claims 4 to 6, wherein: the polymerization degree of the brown algae oligosaccharide is 2-4.
8. An enzyme preparation comprising an alginate lyase, which comprises the alginate lyase of claim 1.
9. The use of the enzyme preparation containing alginate lyase of claim 8 for degrading algin or sodium alginate; and/or: application in preparing brown algae oligosaccharide.
10. The use of the alginate lyase gene of claim 2 in the preparation of recombinant engineered bacteria expressing alginate lyase.
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| CN115786318A (en) * | 2022-12-20 | 2023-03-14 | 中国海洋大学 | Alginate lyase truncation Algt1 and application thereof |
| CN115873912A (en) * | 2022-11-22 | 2023-03-31 | 中国海洋大学 | Method for preparing alginate oligosaccharides by using alginate lyase FaAly554 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115873912B (en) * | 2022-11-22 | 2024-04-05 | 中国海洋大学 | Method for preparing alginate oligosaccharides by utilizing alginate lyase FaAly554 |
| CN115786318A (en) * | 2022-12-20 | 2023-03-14 | 中国海洋大学 | Alginate lyase truncation Algt1 and application thereof |
| CN115786318B (en) * | 2022-12-20 | 2024-05-07 | 中国海洋大学 | Algin lyase truncated Algt1 and application thereof |
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