CN115873912A - Method for preparing alginate oligosaccharides by using alginate lyase FaAly554 - Google Patents
Method for preparing alginate oligosaccharides by using alginate lyase FaAly554 Download PDFInfo
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- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 title claims abstract description 42
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Abstract
The invention discloses a method for preparing alginate oligosaccharides by using alginate lyase FaAly554, which comprises the following steps: sodium alginate is degraded by adopting alginate lyase FaAly87 under the following degradation conditions: the temperature is 30-40 ℃, the pH is 6.0-9.0, and the time is more than 30 minutes; the amino acid sequence of the alginate lyase FaAly554 is shown in SEQ ID NO. 1. The nucleotide sequence of the coding gene of the algin lyase FaAly554 is shown in SEQ ID NO. 2. The algin lyase FaAly554 disclosed by the invention belongs to a PL6 family, and can degrade sodium alginate. The sodium alginate is degraded to generate unsaturated 1-5 sugar, the main product of enzymolysis is unsaturated 3-4 sugar, and the biocatalysis efficiency is good.
Description
Technical Field
The invention relates to a method for preparing alginate oligosaccharides by using alginate lyase FaAly554, belonging to the technical field of functional enzymes.
Background
The polymerization Degree (DP) of alginate oligosaccharide generated by degradation of algin is generally between 2 and 20, the alginate oligosaccharide has low molecular weight and good water solubility, has various physiological activities such as immunoregulation, antibiosis, antioxidation, antihypertensive, antidiabetic, antitumor, anticoagulation and the like, and is widely applied to the fields of medicine, health products, food, agriculture and the like.
The preparation method of the brown algae oligosaccharide mainly comprises a chemical method, a physical method and a biological enzyme method. The preparation of the brown algae oligosaccharide by a chemical method and a physical method has the defects of violent reaction conditions, difficult control, large environmental pollution, uncontrollable and difficult separation of products, damage to oligosaccharide structures and the like. The biological enzyme method is to degrade the algin by specific or non-specific algin lyase to prepare the oligosaccharide, and has the advantages of mild reaction conditions, easy control of reaction process, high product purity and easy separation, and the obtained oligosaccharide has unsaturated double bonds and better biological activity. However, the prior alginate lyase has low activity and low degradation efficiency, and is difficult to meet the requirements of industrial production and application.
Disclosure of Invention
Aiming at the prior art, the invention provides a method for preparing alginate oligosaccharides by using alginate lyase FaAly554, and the method for preparing alginate oligosaccharides by degrading brown algae by using the alginate lyase FaAly554 has the advantages of long-term maintenance of high enzyme activity, high degradation efficiency and the like.
The invention is realized by the following technical scheme:
a method for preparing alginate oligosaccharide by using alginate lyase FaAly554 comprises the following steps: sodium alginate is degraded by adopting alginate lyase FaAly554 under the following degradation conditions: the temperature is 30-40 ℃, the pH is 6.0-9.0, the time is more than 30 minutes, and the time can reach 192 hours, preferably 12 hours; the amino acid sequence of the algin lyase FaAly554 is shown as the following SEQ ID NO. 1.
SEQ ID NO.1：
QVPADLIRNCKQWYITYPTGKNVNTICNEPSNEFYFVNKDKNAITFRVPIRSDNGTTPNTRNIRSELREKTADGKENIFWTTDGTHQIYVKQAITHLPLKHPQLVATQIHGDKAAGIDDAMVMRLEGKKLFLCFNGGKLHPNVMIKSDYVLGTVHEVIFKIVDGKHYCYYSEDGKLLSAYKNGTAERYLIKDGNNDFVMDKNYDKSYFKVGNYTQSNPTDEGDLTGDPNNYGEVVVYDFDVDHSGKGFTTAKSSK。
Further, the specific degradation mode is as follows: adding an enzyme solution containing algin lyase FaAly554 into the sodium alginate solution.
Further, the addition amount of the alginate lyase FaAly554 is 0.33U/ml.
Further, the concentration of the sodium alginate solution is 2-10 mg/ml, preferably 5mg/ml.
Further, the degradation conditions are: the temperature is 35 ℃, the pH value is 7.0, and the time is within 30 hours; or: the temperature is 4 ℃ or 30 ℃, the pH is 7.0, and the time is within 168 hours.
The amino acid sequence of the algin lyase FaAly554 is shown in SEQ ID NO. 1. It can be used for degrading brown algae or preparing brown algae oligosaccharide.
The nucleotide sequence of the coding gene of the algin lyase FaAly554 is shown as SEQ ID NO. 2.
The algin lyase FaAly554 disclosed by the invention belongs to a PL6 family, and can degrade sodium alginate. The sodium alginate is degraded to generate unsaturated 1-5 sugar, the main product of enzymolysis is unsaturated 3-4 sugar, and the biocatalysis efficiency is good.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: SDS-PAGE electrophoresis picture, wherein, M is a standard protein Marker;1 is purified alginate lyase protein; 2, transferring into no-load cell disruption solution; 3 is cell disruption liquid for transferring target gene.
FIG. 2: schematic diagram of optimal reaction temperature.
FIG. 3: pH diagram of optimal reaction.
FIG. 4 is a schematic view of: schematic diagram of temperature stability test.
FIG. 5: pH stability test schematic.
FIG. 6: schematic diagram of the effect of metal ions on alginate lyase.
FIG. 7: and (3) a mass spectrogram of a degraded sodium alginate product.
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 and materials used in the following examples are conventional instruments, reagents and materials known in the art and are commercially available. Unless otherwise specified, the experimental methods and the detection methods mentioned in the following examples are all conventional experimental methods and detection methods known in the art.
Example 1 cloning of expression Gene of alginate lyase FaAly554
In order to excavate the alginate lyase with high application value, the inventor excavates a gene fragment (nucleotide sequence is shown as SEQ ID NO. 2) of the alginate cleavage fragment of Flavobacterium Flavobacterium algicola with the strain number of CGMCC:1.12076, which is deposited in China General Microbiological Culture Collection Center (CGMCC), and the protein (amino acid sequence is shown as SEQ ID NO. 1) expressed by the gene fragment possibly has the activity of the alginate lyase, and the protein expressed by the gene fragment belongs to polysaccharide hydrolase family 6 (PL 6) according to the comparison of evolution trees and is named as the alginate lyase FaAly554.
SEQ ID NO.2：
5’-CAGGTTCCTGCAGATTTAATCCGAAATTGCAAACAGTGGTATATTACATATCCTAC AGGTAAAAATGTAAATACTATTTGTAACGAACCCAGTAACGAATTTTATTTTGTAAATAAAGATAAAAACGCAATCACTTTTCGTGTTCCTATCAGAAGTGACAATGGTACCACACCAAATACTAGAAACATTCGATCTGAATTGAGAGAAAAAACAGCCGATGGTAAAGAAAATATATTTTGGACTACAGATGGGACGCACCAAATTTATGTAAAACAAGCTATTACTCATTTACCATTAAAGCATCCACAACTTGTCGCCACCCAGATTCATGGTGATAAAGCTGCTGGTATCGATGATGCCATGGTCATGCGATTAGAAGGAAAAAAATTATTTCTATGTTTTAATGGAGGTAAATTACATCCCAATGTAATGATCAAATCTGATTATGTTCTGGGCACTGTTCATGAGGTTATTTTCAAGATAGTTGACGGAAAACATTATTGTTATTATTCCGAAGATGGAAAACTTTTGTCAGCTTACAAAAACGGAACTGCTGAACGTTATTTAATTAAGGACGGAAATAATGATTTTGTGATGGACAAAAATTATGATAAATCTTATTTTAAAGTGGGAAATTATACGCAAAGTAATCCGACCGATGAAGGTGACCTAACGGGTGACCCAAATAATTATGGTGAAGTTGTTGTTTACGATTTCGATGTAGATCATTCAGGTAAAGGCTTTACTACTGCAAAAAGTTCAAAA-3’。
The above gene fragment was synthesized in whole gene. Then, the synthesized gene fragment is used as a template, and PCR amplification is carried out by using a specific primer.
The sequences of the primers are shown below:
an upstream primer: 5'-TTCGAGCTCCGTCAGGTTCCTGCAGATTTAATCC-3' as shown in SEQ ID No. 3;
a downstream primer: 5'-GTGTTTTGAACTTTTTGCAGTAGTAAAGCC-3' as shown in SEQ ID No. 4.
The PCR reaction system is as follows: 2 XPCR Buffer 25. Mu.l, dNTP 10. Mu.l, primers 1.5. Mu.l each, template 1. Mu.l, KOD Fx enzyme 1. Mu.l, sterile water 10. Mu.l, total 50. Mu.l.
The reaction conditions of PCR were: pre-denaturation at 94 ℃ for 5min, denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 60s, reaction for 30 cycles, and extension at 72 ℃ for 10min.
The 756bp PCR product fragment was recovered after agarose gel electrophoresis.
EXAMPLE 2 construction of recombinant expression vectors
The gene fragment obtained by PCR amplification in example 1 was ligated with pET-28a cloning vector using the seamless cloning technique, and the ligation product was transferred into E.coli DH 5. Alpha. Competent cells and plated on LB medium solid plate containing 50. Mu.g/m L kanamycin. After 16 hours of incubation at 37 ℃ in an incubator, the single clones were picked up in LB liquid medium containing 50. Mu.g/m L kanamycin and shake-cultured at 37 ℃ and 220rpm for 12 hours. After positive verification, the sequence was determined, and the plasmid was extracted and designated pET28a-FaAly554 and stored at-20 ℃ for further use.
EXAMPLE 3 construction of the engineered bacteria
The plasmid extracted in the embodiment 2 is transformed into a host E.coli BL21 competent cell, and a strain growing on a kanamycin sulfate resistant plate is the constructed engineering bacterium.
EXAMPLE 4 preparation of alginate lyase FaAly554
The engineered bacterial strain obtained in example 3 was inoculated into 5ml of LB liquid medium (containing 50. Mu.g/ml kanamycin) and activated (shaking culture at 37 ℃ and 220rpm for 12 hours); after activation, ZYP-5052 containing 50. Mu.g/ml kanamycin sulfate was inoculated at an inoculum size of 1% and shake-cultured at 20 ℃ for 48 hours at 200rpm to self-induce expression of alginate lyase FaAly554.
After the culture is finished, centrifuging the culture solution at 4 ℃ for 10min at 8000g, collecting thalli, suspending in Tirs-HCl buffer solution with the concentration of 50mmol/l and the pH =8.0, carrying out ultrasonic crushing in ice water bath for 30min (200W, 3s on and 3s off), centrifuging at 12000g for 15min, and collecting supernatant, namely the crude enzyme solution. Based on the His-tag fused protein, the crude enzyme solution is subjected to affinity chromatography purification by using a Ni-NTA column, 10mmol/l imidazole solution (containing 500mM NaCl,50mM Tris-HCl) is used for balancing the column, then the hybrid protein with weak binding force is eluted by using 20mmol/l imidazole solution (containing 500mM NaCl,50mM Tris-HCl), the target protein is eluted by using 50mmol/l imidazole solution (containing 500mM NaCl,50mM Tris-HCl), and the buffer elution component of the part is collected to obtain a solution containing the target protein, namely the enzyme solution. Protein purity and molecular weight were determined by SDS-PAGE (results are shown in FIG. 1) and protein concentration was determined by Bradford assay. The results show that: the enzyme solution obtained after the purification by the affinity column has the protein concentration of 0.168mg/ml and the molecular weight of 33.9kDa.
Example 5 determination of specific enzyme Activity
The standard assay method for the activity of alginate lyase FaAly554 is as follows:
in a reaction system of 200. Mu.l, 20. Mu.l of the enzyme solution (prepared in example 4) and 180. Mu.l of a 5mg/ml sodium alginate solution (pH =7.0, prepared from citrate buffer) were reacted at 35 ℃ for 10min, and the reaction was stopped by boiling for 5 min; after the reaction, the mixture was mixed with 300. Mu.l of DNS reagent, boiled in a boiling water bath for 10min for color development, and the absorbance was measured at 540 nm. Enzyme activity is defined as the amount of enzyme required to produce 1. Mu. Mol/l reducing sugar per min under standard conditions. The enzyme activity is 97.29U/mg by determination.
Example 6 determination of optimum reaction conditions
The enzyme activity was measured by the measurement method of example 5 at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃, 55 ℃ and 60 ℃, and the relative enzyme activities at different temperatures were calculated with the highest enzyme activity being 100%.
At 35 ℃, buffers (citrate buffer, phosphate buffer, tris-hydrochloric acid buffer, glycine-sodium hydroxide buffer) with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 were selected instead of the citrate buffer (pH = 7) in example 5, the enzyme activity was measured according to the measurement method of example 5, and the relative enzyme activities at different pH values were calculated with the highest enzyme activity being 100%.
The buffer solution with the pH value of 3.0-10.0 is used as different pH buffer solutions for enzyme reaction determination, and the optimum pH value of the alginate lyase is determined according to the enzyme activity of the alginate lyase. As shown in FIGS. 2 and 3, the optimum reaction temperature for the alginate lyase FaAly554 was 35 ℃ and the optimum pH was 7.
Example 7 measurement of temperature stability and pH stability
Determination of temperature stability: the enzyme solutions (prepared in example 4) were stored at 4 ℃, room temperature (25 ℃), 30 ℃, 35 ℃, and 40 ℃ respectively, and samples were taken at different storage times to measure the enzyme activities (the relative enzyme activities at different storage temperatures were calculated with the highest enzyme activity being 100% according to the measurement method of example 5), with the results shown in fig. 4. As can be seen from FIG. 4, the alginate lyase FaAly554 has better temperature stability at normal temperature, 4 ℃ and 30 ℃, can still retain 80% of enzyme activity after being stored for 96 hours at 30 ℃, and has better temperature stability than the AlyPB1 cloned from Photobacterium sp.FC615 (the residual enzyme activity is 80% after being placed for 24 hours at 30 ℃) and the AlyPB2 cloned from Photobacterium sp.FC615 (the residual enzyme activity is 70% after being placed for 2 hours at 30 ℃). FaAly554 was stored at 35 ℃ for 24h with a residual enzyme activity of 72% which was more stable at 35 ℃ than the AlgS7h cloned from Microbulbifer sp.SH-1 (30 min at 35 ℃ with a residual enzyme activity of 80%). The enzyme activity of the FaAly554 is sharply reduced within 24-48 hours, and is reduced to be below 12% along with the prolonging of the storage time, which shows that although the optimal reaction temperature of the algin lyase FaAly554 is 35 ℃, the stability of the algin lyase FaAly554 at 35 ℃ is poor compared with the stability at 30 ℃, and if high-efficiency enzymolysis is to be maintained for a long time, the enzymolysis temperature is preferably 30 ℃.
Determination of pH stability: the enzyme solution (prepared in example 4) was divided into 4 portions, and the portions were incubated at 4 ℃ in an environment of pH 6.0, 7.0, 8.0, and 9.0 (phosphate buffer, tris-hydrochloric acid buffer) (180. Mu.l of buffer was added per 20. Mu.l of enzyme solution), followed by addition of sodium alginate (5 mg/ml sodium alginate concentration after addition), reaction at 35 ℃ for 10min, measurement of enzyme activity (according to the measurement method of example 5), and calculation of relative enzyme activity, the results of which are shown in FIG. 5. As can be seen from FIG. 5, the enzyme has good stability at pH =7.0, and can maintain over 93.27% of enzyme activity after 168 hours of storage compared with Alg823 (24 hours in pH 6-10 environment and 80% of residual enzyme activity) cloned from Pseudomonas carrageenovora ASY5 and Alg7A (24 hours in pH 6-10 environment and 50% of residual enzyme activity) cloned from Vibrio sp.W13, and FaAly554 PH has better stability.
Example 8 Effect of Metal ions and chemical reagents on enzymatic Activity
The enzyme solution (prepared in example 4) was added with 5Mg/ml sodium alginate solution (pH =7.0, prepared from phosphate buffer) (180. Mu.l sodium alginate solution was added per 20. Mu.l enzyme solution) and divided into 10 portions, and each of various metal ions and chemical reagents (Mg) was added to the solution to a final concentration of 1mmol/l 2+ 、Na + 、Fe 2+ 、Cu 2+ 、Ba 2+ 、Zn 2+ 、Mn 2+ 、Na 2 EDTA, SDS) (for metal ions, added in the form of hydrochloride) (1 part of which was a control group, and no metal ions or chemical agent was added), reacted at 35 ℃ for 10min, the enzyme activity was measured (according to the measurement method of example 5), and the relative enzyme activity was calculated, and the results are shown in FIG. 6.
From FIG. 6, mg is shown 2+ 、Na + 、Fe 2+ 、Cu 2+ 、Ba 2+ 、Zn 2+ 、Mn 2+ 、Na 2 Both EDTA and SDS can inhibit the activity of algin lyase FaAly554, fe 3+ 、Zn 2+ Can make the algin lyase FaAly554 lose enzyme activity.
Example 9 determination of degradation products of alginate lyase FaAly554
The enzyme solution (prepared in example 4) was added with 5mg/ml sodium alginate solution (pH =7.0, prepared from citrate buffer) (180. Mu.l sodium alginate solution was added to 20. Mu.l enzyme solution), reacted at 35 ℃ for 2 hours, and boiled for 5min to terminate the reaction. Centrifuging at 10000rpm for 10min to remove protein and other impurities, collecting supernatant, filtering with 0.22 μm filter membrane, and lyophilizing to obtain brown algae oligosaccharide. The brown algae oligosaccharide is subjected to product identification by using a mass spectrum, and the analysis result is shown in figure 7, so that the product is mainly unsaturated 1-5 sugar.
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.
Claims (10)
1. A method for preparing alginate oligosaccharides by using alginate lyase FaAly554 is characterized in that: sodium alginate is degraded by adopting alginate lyase FaAly87 under the following degradation conditions: the temperature is 30-40 ℃, the pH is 6.0-9.0, and the time is more than 30 minutes; the amino acid sequence of the alginate lyase FaAly554 is shown in SEQ ID NO. 1.
2. The method for preparing alginate oligosaccharides by using the alginate lyase FaAly554 according to the claim 1, wherein the degradation is carried out in the following way: adding an enzyme solution containing algin lyase FaAly554 into the sodium alginate solution.
3. The method for preparing alginate oligosaccharides by using the alginate lyase FaAly554 as claimed in claim 2, wherein the alginate lyase FaAly554 is selected from the group consisting of: the addition amount of the alginate lyase FaAly554 is 0.33U/ml.
4. The method for preparing alginate oligosaccharides by using the alginate lyase FaAly554 as claimed in claim 1, wherein the alginate lyase FaAly554 is selected from the group consisting of: the concentration of the sodium alginate solution is 2-10 mg/ml.
5. The method for preparing alginate oligosaccharides by using the alginate lyase FaAly554 as claimed in claim 4, wherein the alginate lyase FaAly554 is selected from the group consisting of: the concentration of the sodium alginate solution is 5mg/ml.
6. The method for preparing alginate oligosaccharides by using the alginate lyase FaAly554 as claimed in claim 1, wherein the degradation conditions are as follows: the temperature is 35 ℃, the pH value is 7.0, and the time is within 30 hours.
7. The method for preparing alginate oligosaccharides by using the alginate lyase FaAly554 as claimed in claim 1, wherein the degradation conditions are as follows: the temperature is 4 ℃ or 30 ℃, the pH is 7.0, and the time is within 168 hours.
8. Alginate lyase FaAly554, characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.
9. Use of the alginate lyase FaAly554 according to claim 8 for degrading brown algae or for preparing alginate oligosaccharides.
10. The gene encoding alginate lyase FaAly554 according to claim 8, wherein: the nucleotide sequence is shown in SEQ ID NO. 2.
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| CN112725319A (en) * | 2020-12-25 | 2021-04-30 | 中国海洋大学 | Alginate lyase FaAly7 with polyG substrate specificity and application thereof |
| CN113699140A (en) * | 2021-10-28 | 2021-11-26 | 中国海洋大学 | Alginate lyase and application thereof |
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| CN113699140A (en) * | 2021-10-28 | 2021-11-26 | 中国海洋大学 | Alginate lyase and application thereof |
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