Recombinant alginate lyase with thermal stability and high enzyme activity and application thereof

文档序号：824759 发布日期：2021-03-30 浏览：49次中文

阅读说明：本技术 一种热稳定性和高酶活力的重组褐藻胶裂解酶及其应用 (Recombinant alginate lyase with thermal stability and high enzyme activity and application thereof ) 是由牟海津杨敏朱常亮于 2020-12-09 设计创作，主要内容包括：本发明是提供一种具有更好热稳定性和高酶活力的褐藻胶裂解酶,其氨基酸序列为SEQ ID NO:3或SEQ ID NO5；本发明所提供的突变酶用于制备褐藻胶寡糖。本发明利用基因改造的重组褐藻胶裂解酶,定向制备褐藻胶寡糖,与现有酸法水解制备褐藻胶寡糖的方法相比,克服了破坏性大的缺点,与现有的酶法水解制备褐藻胶寡糖的方法相比,运用改造的重组褐藻胶裂解酶,可减少加酶量、缩短酶解时间、提高酶解效率。本发明的酶具有高酶活力和高热稳定性,更适合应用于工业上酶解褐藻胶制备褐藻胶寡糖。所制备的褐藻胶寡糖有抗肿瘤、抗炎、降血脂和提高免疫力等功效,可用于食品及保健品领域,具有广泛的应用前景。(The invention provides an alginate lyase with better thermal stability and high enzyme activity, and the amino acid sequence of the alginate lyase is SEQ ID NO. 3 or SEQ ID NO 5; the mutant enzyme provided by the invention is used for preparing alginate oligosaccharides. Compared with the existing method for preparing the alginate oligosaccharides by enzymatic hydrolysis, the modified recombinant alginate lyase is used, so that the enzyme adding amount can be reduced, the enzymolysis time can be shortened, and the enzymolysis efficiency can be improved. The enzyme of the invention has high enzyme activity and high thermal stability, and is more suitable for preparing alginate oligosaccharides by industrially hydrolyzing algin. The prepared alginate oligosaccharide has the effects of resisting tumor, resisting inflammation, reducing blood fat, improving immunity and the like, can be used in the fields of food and health care products, and has wide application prospect.)

1. The algin lyase is characterized in that the amino acid sequence of the algin lyase is SEQ ID NO. 3.

2. The alginate lyase of claim 1 wherein the amino acid sequence of the alginate lyase is SEQ ID NO. 5.

3. A gene encoding the alginate lyase of claim 1 or 2.

4. The gene of claim 3, wherein the nucleotide sequence of the gene is SEQ ID NO. 4 or SEQ ID NO. 6.

5. A recombinant expression vector, wherein the nucleic acid fragment of the gene of claim 3 is inserted into said recombinant expression vector.

6. A genetically engineered bacterium for recombinant expression of the alginate lyase of claim 1 or 2.

7. Use of the alginate lyase of claim 1 or 2 in the preparation of alginate oligosaccharides.

8. A method for preparing alginate oligosaccharides, wherein the alginate lyase of claim 1 or 2 is used to carry out enzymatic hydrolysis of alginate raw materials.

9. The method of claim 8, wherein the method comprises the steps of:

1) preparing a substrate: mixing the algin raw material with water to prepare an enzymolysis substrate solution with the concentration of 2-8% and the pH of 7.0-8.0;

2) step-by-step enzymolysis: adding alginate lyase, stirring and performing enzymolysis for 1-2h at 45-55 ℃, adding 0.5-2% of the alginate lyase again, stirring and performing enzymolysis for 2-4 h;

3) preparing oligosaccharide: and after the enzymolysis is finished, filtering or centrifuging, concentrating supernate, and freeze-drying to obtain the algal oligosaccharide.

Technical Field

The invention belongs to the technical field of alginase, and particularly relates to an algin lyase with better thermal stability and high enzyme activity.

Background

Algin oligosaccharides (alginate oligosaccharides) are low molecular weight fragments obtained by hydrolysis of algin and have a small degree of polymerization and good water solubility. The algin oligosaccharide has multiple physiological activities of reducing blood fat, resisting virus, resisting tumor, resisting oxidation and the like, can be widely applied to the fields of food, medicine, cosmetics and the like, and has great application and development values. Recent studies have shown that: the poly-M-segment oligosaccharide drug '971' prepared from algin can inhibit the aggregation and cytotoxicity of beta amyloid cells, and is being used for the second-phase clinical research of anti-Alzheimer disease; the poly G oligosaccharide can inhibit clinical multi-drug resistant pathogenic bacteria in cooperation with antibiotics. Therefore, the alginate oligosaccharide with special composition and specific polymerization degree has important application value and economic value, and has important significance for realizing the high-efficiency preparation of the oligosaccharide.

The preparation method of the algin oligosaccharide mainly comprises acid hydrolysis and enzyme hydrolysis. Acid hydrolysis can destroy the structure of alginate oligosaccharides to different degrees, and enzymatic hydrolysis has the advantages of mild reaction conditions and the like, so that the preparation of alginate oligosaccharides by enzymatic hydrolysis is widely concerned. The marine microorganism is an important source of alginate lyase, and some of the alginate lyase are separated from vibrio, alteromonas and pseudoalteromonas in sequence. However, due to the particularity of the marine environment, the yield of the alginate lyase produced by marine microorganisms is low, and the enzyme activity cannot meet the requirement of preparing alginate oligosaccharides. At present, many algin lyase are cloned and expressed, the expression level and the enzyme activity of the algin lyase are improved compared with those of the original enzyme, but the enzymological properties, such as thermal stability and the enzyme activity, of the algin lyase still need to be further improved. When the algin oligosaccharide is industrially prepared, the dosage of enzyme can be reduced by improving the thermal stability of the enzyme, the enzymolysis efficiency is improved, and the oligosaccharide yield is increased. Therefore, the focus of research is to find alginate lyase that has excellent enzymatic properties and can degrade alginate efficiently.

Disclosure of Invention

The invention aims to provide the alginate lyase with thermal stability and high enzyme activity and the method for preparing the alginate oligosaccharide by applying the alginate lyase, thereby making up the defects of the prior art.

The amino acid sequence of the alginate lyase D59C-A267C provided by the invention is MKVSCAVVLSACIASANADNNGDGKADSIKENDLNAGYADGTYFYTAADGGMVFRCPICGYKTSTNTSYTRTELREMLRRGDTSIATQGVNGNNWVFGSAPASAREAAGGVDGVLRATLAVNHVTTTGDSGQVGRVIVGQIHANNDEPLRLYYRKLPGHSKGSVYIAHEPNGGSDSWYDMIGSRSSSASDPSDGIALDEVWSYEVKVVGNTLTVTIFRAGKDDVVQVVDMGNSGYDVADQYQYFKAGVYNQNNTGNASDYVQVTFYCLEQSHD (SEQ ID NO: 3);

the coding gene sequence is as follows:

ATGAAAGTAAGTTGCGCTGTCGTACTGTCTGCTTGTATTGCCAGTGCCAACGCAGACAACAATGGCGATGGCAAGGCCGACTCCATCAAGGAAAATGACCTGAATGCAGGCTATGCAGATGGCACCTACTTCTATACTGCTGCCGATGGCGGCATGGTGTTCCGCTGCCCGATCTGTGGCTATAAAACATCGACCAACACGTCCTATACCCGCACCGAGCTGCGCGAGATGCTACGTCGTGGCGACACCAGCATTGCCACCCAGGGGGTCAATGGAAACAACTGGGTATTCGGCTCCGCACCCGCTTCGGCACGTGAAGCAGCCGGCGGTGTCGACGGTGTTTTACGCGCAACCCTCGCGGTAAACCATGTCACCACTACCGGAGATAGCGGCCAGGTTGGACGGGTGATTGTTGGACAGATTCACGCCAACAACGACGAACCGCTGCGTCTTTACTACCGCAAGTTACCGGGCCACAGCAAAGGTTCTGTGTATATCGCCCATGAGCCAAACGGCGGCAGCGACAGCTGGTACGACATGATTGGCAGCCGTTCCAGCAGCGCCTCGGACCCGTCCGACGGTATCGCACTGGATGAAGTCTGGAGCTACGAGGTCAAGGTTGTCGGTAACACCCTCACCGTGACCATCTTCCGTGCTGGTAAAGACGATGTGGTACAGGTTGTGGATATGGGCAACAGCGGTTACGACGTCGCCGACCAGTACCAGTACTTCAAGGCCGGGGTGTACAACCAGAACAACACCGGCAATGCCAGTGACTATGTCCAGGTGACCTTCTACTGCCTGGAGCAGTCGCACGATTAA(SEQ ID NO:4)；

the invention also provides another alginate lyase G60C-T70C, the amino acid sequence of which is MKVSCAVVLSACIASANADNNGDGKADSIKENDLNAGYADGTYFYTAADGGMVFRCPIDCYKTSTNTSYCRTELREMLRRGDTSIATQGVNGNNWVFGSAPASAREAAGGVDGVLRATLAVNHVTTTGDSGQVGRVIVGQIHANNDEPLRLYYRKLPGHSKGSVYIAHEPNGGSDSWYDMIGSRSSSASDPSDGIALDEVWSYEVKVVGNTLTVTIFRAGKDDVVQVVDMGNSGYDVADQYQYFKAGVYNQNNTGNASDYVQVTFYALEQSHD (SEQ ID NO 5);

the sequence of the coding gene is as follows:

ATGAAAGTAAGTTGCGCTGTCGTACTGTCTGCTTGTATTGCCAGTGCCAACGCAGACAACAATGGCGATGGCAAGGCCGACTCCATCAAGGAAAATGACCTGAATGCAGGCTATGCAGATGGCACCTACTTCTATACTGCTGCCGATGGCGGCATGGTGTTCCGCTGCCCGATCGATTGCTATAAAACATCGACCAACACGTCCTATTGCCGCACCGAGCTGCGCGAGATGCTACGTCGTGGCGACACCAGCATTGCCACCCAGGGGGTCAATGGAAACAACTGGGTATTCGGCTCCGCACCCGCTTCGGCACGTGAAGCAGCCGGCGGTGTCGACGGTGTTTTACGCGCAACCCTCGCGGTAAACCATGTCACCACTACCGGAGATAGCGGCCAGGTTGGACGGGTGATTGTTGGACAGATTCACGCCAACAACGACGAACCGCTGCGTCTTTACTACCGCAAGTTACCGGGCCACAGCAAAGGTTCTGTGTATATCGCCCATGAGCCAAACGGCGGCAGCGACAGCTGGTACGACATGATTGGCAGCCGTTCCAGCAGCGCCTCGGACCCGTCCGACGGTATCGCACTGGATGAAGTCTGGAGCTACGAGGTCAAGGTTGTCGGTAACACCCTCACCGTGACCATCTTCCGTGCTGGTAAAGACGATGTGGTACAGGTTGTGGATATGGGCAACAGCGGTTACGACGTCGCCGACCAGTACCAGTACTTCAAGGCCGGGGTGTACAACCAGAACAACACCGGCAATGCCAGTGACTATGTCCAGGTGACCTTCTACGCCCTGGAGCAGTCGCACGATTAA(SEQ ID NO:6)。

in another aspect, the invention also provides a recombinant expression vector, wherein the nucleic acid segment of the coding gene is inserted into the recombinant expression vector.

The invention also provides a genetic engineering bacterium which is used for recombining and expressing the mutant enzyme.

The mutant enzyme provided by the invention is used for preparing alginate oligosaccharides.

The invention also provides a method for preparing the alginate oligosaccharide, which comprises the following steps:

1) preparing a substrate: mixing the algin raw material with water to prepare an enzymolysis substrate solution with the concentration of 2-8% and the pH of 7.0-8.0;

2) step-by-step enzymolysis: adding alginate lyase, stirring and performing enzymolysis for 1-2h at 50-65 ℃, adding 0.5-2% of the alginate lyase again, stirring and performing enzymolysis for 2-4 h;

3) preparing oligosaccharide: and after the enzymolysis is finished, filtering or centrifuging, concentrating supernate, and freeze-drying to obtain the algal oligosaccharide.

Compared with the existing method for preparing the alginate oligosaccharides by enzymatic hydrolysis, the modified recombinant alginate lyase is used, so that the enzyme adding amount can be reduced, the enzymolysis time can be shortened, and the enzymolysis efficiency can be improved. The enzyme of the invention has high enzyme activity and high thermal stability, and is more suitable for preparing alginate oligosaccharides by industrially hydrolyzing algin. The prepared alginate oligosaccharide has the effects of resisting tumor, resisting inflammation, reducing blood fat, improving immunity and the like, can be used in the fields of food and health care products, and has wide application prospect.

Drawings

FIG. 1: SDS-PAGE electrophorograms of the original enzyme cAlyM and its mutants, in which lane 1 is the mutant enzyme D59C-A267C; lane 2 is the mutant enzyme G60C-T70C;

FIG. 2: the optimal reaction temperature (left figure) and the optimal reaction pH (right figure) of the original enzyme cAlyM and mutants thereof D59C-A267C and G60C-T70C;

FIG. 3: a heat stability diagram of original enzyme cAlyM and mutants D59C-A267C and G60C-T70C at 45 ℃;

FIG. 4: thermal stability profiles of X33-cAlyM and X33-D59C-A267C at 45 deg.C (left panel) 50 deg.C (right panel);

FIG. 5: and (4) mass spectrogram of the enzymolysis product.

Detailed Description

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1: construction of mutants and their expression in E.coli

In order to improve the thermal stability and activity of alginate lyase, the applicant modified the original alginate lyase with the following amino acid sequence:

MKVSCAVVLSACIASANADNNGDGKADSIKENDLNAGYADGTYFYTAADGGMVFRCPIDGYKTSTNTSYTRTELREMLRRGDTSIATQGVNGNNWVFGSAPASAREAAGGVDGVLRATLAVNHVTTTGDSGQVGRVIVGQIHANNDEPLRLYYRKLPGHSKGSVYIAHEPNGGSDSWYDMIGSRSSSASDPSDGIALDEVWSYEVKVVGNTLTVTIFRAGKDDVVQVVDMGNSGYDVADQYQYFKAGVYNQNNTGNASDYVQVTFYALEQSHD(SEQ ID NO:1)

the original algin lyase, the nucleotide sequence of its coding gene is as follows:

ATGAAAGTAAGTTGCGCTGTCGTACTGTCTGCTTGTATTGCCAGTGCCAACGCAGACAACAATGGCGATGGCAAGGCCGACTCCATCAAGGAAAATGACCTGAATGCAGGCTATGCAGATGGCACCTACTTCTATACTGCTGCCGATGGCGGCATGGTGTTCCGCTGCCCGATCGATGGCTATAAAACATCGACCAACACGTCCTATACCCGCACCGAGCTGCGCGAGATGCTACGTCGTGGCGACACCAGCATTGCCACCCAGGGGGTCAATGGAAACAACTGGGTATTCGGCTCCGCACCCGCTTCGGCACGTGAAGCAGCCGGCGGTGTCGACGGTGTTTTACGCGCAACCCTCGCGGTAAACCATGTCACCACTACCGGAGATAGCGGCCAGGTTGGACGGGTGATTGTTGGACAGATTCACGCCAACAACGACGAACCGCTGCGTCTTTACTACCGCAAGTTACCGGGCCACAGCAAAGGTTCTGTGTATATCGCCCATGAGCCAAACGGCGGCAGCGACAGCTGGTACGACATGATTGGCAGCCGTTCCAGCAGCGCCTCGGACCCGTCCGACGGTATCGCACTGGATGAAGTCTGGAGCTACGAGGTCAAGGTTGTCGGTAACACCCTCACCGTGACCATCTTCCGTGCTGGTAAAGACGATGTGGTACAGGTTGTGGATATGGGCAACAGCGGTTACGACGTCGCCGACCAGTACCAGTACTTCAAGGCCGGGGTGTACAACCAGAACAACACCGGCAATGCCAGTGACTATGTCCAGGTGACCTTCTACGCCCTGGAGCAGTCGCACGATTAA(SEQ ID NO:2)。

after further analysis of the original enzyme sites, amino groups 59, 60, 70 and 267 were selected for mutation. On the sequence of the coding gene of SEQ ID NO. 2, 10 to 15 bases were selected as forward primer sequences from the left and right, respectively, centered on the mutated base, and the reverse primer sequences were completely reverse-complementary thereto. DNAMAN 6.0 was used to verify the quality of the primers. The primer design results are as follows:

D59C-F GCTGCCCGATCTGTGGCTATAAAAC、

D59C-R GTTTTATAGCCACAGATCGGGCAGC、

A267C-F CACCGGCAATTGCAGTGACTATGTC、

A267C-R GACATAGTCACTGCAATTGCCGGTG、

G60C-F GCCCGATCGATTGCTATAAAACATCG、

G60C-R CGATGTTTTATAGCAATCGATCGGGC、

T70C-F CACGTCCTATTGCCGCACCGAGCTG、

T70C-R CAGCTCGGTGCGGCAATAGGACGTG；

a bacterial plasmid miniextract kit is used for extracting a plasmid of DH5 alpha-HTa-cAlyM containing a coding gene segment of SEQ ID NO. 2, and the plasmid is placed in an ultra-low temperature refrigerator for long-term storage and used as a template of PCR. The full length of the plasmid is amplified by using a mutation primer,

the amplification system consisted of:

the amplification conditions were as follows:

the amplification result was verified by 1% agarose gel electrophoresis, and the amplification product was digested with Dpn I in the following digestion system for 3 hours.

The Dpn I digestion product is transformed into Escherichia coli competent cell DH5 alpha, cultured overnight at 37 ℃, and 3-5 recombinant bacteria are selected from each mutant and sent to the company for sequencing. The recombinant plasmid with correct sequencing was extracted and transformed into E.coli competent cells BL21(DE3) and cultured overnight at 37 ℃. The positive recombinant bacteria were selected and cultured overnight at 37 ℃ in LB medium containing 100. mu.g/mL AMP, and inoculated as seed liquid to the fermentation medium, and expression was induced. The fermentation broth was centrifuged, the supernatant was collected as a crude enzyme, and the precipitated cells were suspended in 100mM phosphate buffer pH7.0 and disrupted by sonication to obtain an intracellular enzyme.

The crude extracellular enzyme solution was passed through a 0.22 μm filter, purified by Ni-NTA affinity chromatography column (1.6X 5cm), and the elution peaks at each stage were collected, and the degree of purification was checked by 12% SDS-PAGE while verifying the molecular weight. Electrophoresis results show that a single band is obtained after purification, and the molecular weight of the mutant is consistent with that of the original enzyme, and is 35Kda (FIG. 1).

Sodium alginate substrate was prepared in 50mM phosphate buffer pH7.0 and the enzyme activity of the recombinase was measured at different temperatures (40-60 ℃) to determine the optimum reaction temperature for the recombinase.

Dissolving sodium alginate in 50mM, citric acid buffer solution (pH 5.0), phosphate buffer solution (pH 6.0-8.0) and glycine buffer solution (pH 9.0), respectively, and determining the optimum reaction pH of the recombinase by measuring the enzyme activity of the recombinase at the optimum temperature; the purified mutant enzyme was incubated at 45 ℃ for 6h, and samples were taken at 0.5h, 1h, 2h, 3h and 6h, respectively, to determine the thermostability of the enzyme.

The results showed that the optimum reaction temperature for mutant D59C-A267C (amino acid sequence SEQ ID NO:3, encoding gene sequence SEQ ID NO:4) and mutant G60C-T70C (amino acid sequence SEQ ID NO:5, encoding gene sequence SEQ ID NO:6) was 55 ℃, which was identical to the original enzyme, and the optimum reaction pH was 8.0, which was higher than the original enzyme 7.0 (FIG. 2).

Under the same reaction conditions, the intracellular enzyme activity of the mutant D59C-A267C is 3.1 times that of the original enzyme, and the total enzyme activity is 2.6 times that of the original enzyme.

Incubation of the original enzyme, mutant D59C-A267C and mutant G60C-T70C enzymes in a 45 ℃ water bath revealed a half-life of 1.90h at 45 ℃ (T1/2, 45 ℃), T1/2 for mutant D59C-A267C and G60C-T70C enzymes, 4.15 and 3.06h at 45 ℃ (FIG. 3).

Example 2: expression of recombinase in pichia pastoris

The plasmids HTa-cAlyM and HTa-D59C-A267C are used as templates, and primers are designed to amplify genes of original enzyme and modified enzyme, wherein the primers are as follows:

EcoR Ⅰ-F:5’-agagaggctgaagctgaattcATGAAAGTAAGTTGCGCTGTC-3’

Not Ⅰ-R:5’-tgttctagaaagctggcggccgcTTAATCGTGCGACTGCTCCAG-3’

the amplification system was as follows:

the amplification conditions were as follows:

after the PCR amplification is finished, agarose gel electrophoresis with the concentration of 1% is used for verifying, whether the size length of the PCR product strip is consistent with the target length or not is judged, and then the PCR product is purified by a PCR product purification kit. The empty plasmid pPICZ alpha A preserved in the laboratory is subjected to double enzyme digestion by using EcoRI and Not I, and is subjected to heat preservation in a water bath at 37 ℃ for 1-3h and inactivation at 80 ℃ for 20 min. The enzyme digestion system is as follows:

by usingII One Step Cloning Kit, the target gene and the linearized vector were recombined and ligated, reacted at 37 ℃ for 30min, and after completion, the recombinant product was immediately cooled on ice.

The 20. mu.L recombination reaction was as follows:

add 20. mu.L of recombinant plasmid to 100. mu.L of E.coli DH 5. alpha. competent cells, mix gently and place on ice for 30 min. After the heat shock in 42 ℃ water bath for 45-60s, the mixture is immediately placed on ice for cooling for 3 min. Adding 900 μ L of non-resistant LLB culture medium, and shake culturing at 37 deg.C and 180r/min for 45 min. After completion of the recovery, the plates were plated on LLB plates (containing 25. mu.g/mL Zeocin) and incubated at 37 ℃ for 16 hours. And (3) selecting a single colony for colony PCR verification, carrying out sample sequencing on a PCR product of the positive colony, selecting a colony with a correct sequencing result, and extracting recombinant plasmids pPICZ alpha A-cAlyM and pPICZ alpha A-D59C-A267C by using a plasmid extraction kit.

The recombinant vector is linearized with a restriction enzyme Sac I, and a 50-L linearization system is as follows:

and (3) preserving the heat of the water bath at 37 ℃ for 1-3h, and purifying the enzyme digestion product by using a PCR product purification kit.

Adding 15 mu L of the linearized recombinant expression vector into 100 mu L of P.pastoris X33 competent cells, gently mixing the cells uniformly, standing the cells on ice for 10min, and transferring the cells to a precooled electric rotating cup for electric conversion under the conditions of 2.00KV and 1 pulse. After the electrotransformation is finished, 1mL of precooled 1mol/L sorbitol is immediately added, the mixture is evenly blown and transferred to a sterilized 1.5mL EP tube, and the incubation is carried out for 1h at the constant temperature of 30 ℃. After completion of the recovery, YPDS plates (containing 100. mu.g/mL bleomycin) were plated and incubated at 30 ℃ for 2-3 days. Randomly selecting a Pichia pastoris single colony from the plate, inoculating the Pichia pastoris single colony to 20mL YPD liquid culture medium, and carrying out shaking culture at 30 ℃ at 200r/min for 24 h. Taking 1mL of bacterial liquid to 20mL of BMGY liquid medium, carrying out shaking culture at 30 ℃ at 200r/min, adding 1% methanol every 24h for induction, and carrying out induction for 3 times in total. After induction is finished, taking a bacterial liquid, centrifuging for 10min at the temperature of 4 ℃ and at the speed of 5000r/min, taking a supernatant as a crude enzyme liquid to measure the enzyme activity, and selecting strains with higher enzyme activity and respectively naming the strains as X33-cAlyM and X33-D59C-A267C.

The specific activities of the enzyme activity measured by a DNS method, X33-cAlyM and X33-D59C-A267C crude enzyme liquid were 268.7 and 349.6U/mg respectively.

Incubating the enzyme at 45 ℃ and 50 ℃ to detect the thermal stability of the enzyme, wherein the half-lives of X33-cAlyM and X33-D59C-A267C treated by water bath at 45 ℃ are 2.0h and 5.2h respectively, and the half-life of the mutant is 2.6 times of that of the original enzyme; the half-life of the water bath treatment at 50 ℃ of X33-cAlyM and X33-D59C-A267C is 0.3h and 3.5h respectively, and the half-life of the mutant is 11.7 times of that of the original enzyme.

Example 3: preparation of algin oligosaccharide

Dissolving algin in water with pH value of 7.5 adjusted by NaOH to prepare 200mL of 6% algin solution, adding 0.6mL or 0.8mL of recombinant algin lyase D59C-A267C or cAlyM, stirring and performing enzymolysis for 1.5h at 50 ℃, adding 0.2mL or 0.6mL of recombinant algin lyase D59C-A267C or cAlyM, and continuing stirring and performing enzymolysis for 1h at 50 ℃. Centrifuging the enzymolysis solution at 8000rpm for 10min, removing residue, and collecting supernatant as enzymolysis product.

Carrying out 4 times of alcohol precipitation on the alginate lyase D59C-A267C enzymolysis product to obtain a target product, and carrying out rotary evaporation and freeze drying on the target product to obtain an alginate oligosaccharide crude product, wherein the yield is 84.3%; ESI-MS is used for measuring the polymerization degree of the oligosaccharide, and an ESI-MS spectrum shows that the enzymolysis final products mainly comprise disaccharide, trisaccharide and tetrasaccharide. The yield of the crude alginate-derived oligosaccharide of the original enzyme cAlyM is 69.5%. The result shows that the mutant enzyme has higher enzymolysis efficiency compared with the original enzyme, and the dosage of the enzyme is greatly reduced.

Sequence listing

<110> institute of yellow sea aquatic products of China aquatic science institute of China ocean university

<120> an alginate lyase having better thermal stability and high enzyme activity

<160> 6

<170> SIPOSequenceListing 1.0

<210> 5

<211> 273

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Lys Val Ser Cys Ala Val Val Leu Ser Ala Cys Ile Ala Ser Ala

1 5 10 15

Asn Ala Asp Asn Asn Gly Asp Gly Lys Ala Asp Ser Ile Lys Glu Asn

20 25 30

Asp Leu Asn Ala Gly Tyr Ala Asp Gly Thr Tyr Phe Tyr Thr Ala Ala

35 40 45

Asp Gly Gly Met Val Phe Arg Cys Pro Ile Asp Gly Tyr Lys Thr Ser

50 55 60

Thr Asn Thr Ser Tyr Thr Arg Thr Glu Leu Arg Glu Met Leu Arg Arg

65 70 75 80

Gly Asp Thr Ser Ile Ala Thr Gln Gly Val Asn Gly Asn Asn Trp Val

85 90 95

Phe Gly Ser Ala Pro Ala Ser Ala Arg Glu Ala Ala Gly Gly Val Asp

100 105 110

Gly Val Leu Arg Ala Thr Leu Ala Val Asn His Val Thr Thr Thr Gly

115 120 125

Asp Ser Gly Gln Val Gly Arg Val Ile Val Gly Gln Ile His Ala Asn

130 135 140

Asn Asp Glu Pro Leu Arg Leu Tyr Tyr Arg Lys Leu Pro Gly His Ser

145 150 155 160

Lys Gly Ser Val Tyr Ile Ala His Glu Pro Asn Gly Gly Ser Asp Ser

165 170 175

Trp Tyr Asp Met Ile Gly Ser Arg Ser Ser Ser Ala Ser Asp Pro Ser

180 185 190

Asp Gly Ile Ala Leu Asp Glu Val Trp Ser Tyr Glu Val Lys Val Val

195 200 205

Gly Asn Thr Leu Thr Val Thr Ile Phe Arg Ala Gly Lys Asp Asp Val

210 215 220

Val Gln Val Val Asp Met Gly Asn Ser Gly Tyr Asp Val Ala Asp Gln

225 230 235 240

Tyr Gln Tyr Phe Lys Ala Gly Val Tyr Asn Gln Asn Asn Thr Gly Asn

245 250 255

Ala Ser Asp Tyr Val Gln Val Thr Phe Tyr Ala Leu Glu Gln Ser His

260 265 270

Asp

<210> 6

<211> 822

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaaagtaa gttgcgctgt cgtactgtct gcttgtattg ccagtgccaa cgcagacaac 60

aatggcgatg gcaaggccga ctccatcaag gaaaatgacc tgaatgcagg ctatgcagat 120

ggcacctact tctatactgc tgccgatggc ggcatggtgt tccgctgccc gatcgatggc 180

tataaaacat cgaccaacac gtcctatacc cgcaccgagc tgcgcgagat gctacgtcgt 240

ggcgacacca gcattgccac ccagggggtc aatggaaaca actgggtatt cggctccgca 300

cccgcttcgg cacgtgaagc agccggcggt gtcgacggtg ttttacgcgc aaccctcgcg 360

gtaaaccatg tcaccactac cggagatagc ggccaggttg gacgggtgat tgttggacag 420

attcacgcca acaacgacga accgctgcgt ctttactacc gcaagttacc gggccacagc 480

aaaggttctg tgtatatcgc ccatgagcca aacggcggca gcgacagctg gtacgacatg 540

attggcagcc gttccagcag cgcctcggac ccgtccgacg gtatcgcact ggatgaagtc 600

tggagctacg aggtcaaggt tgtcggtaac accctcaccg tgaccatctt ccgtgctggt 660

aaagacgatg tggtacaggt tgtggatatg ggcaacagcg gttacgacgt cgccgaccag 720

taccagtact tcaaggccgg ggtgtacaac cagaacaaca ccggcaatgc cagtgactat 780

gtccaggtga ccttctacgc cctggagcag tcgcacgatt aa 822

<210> 1

<211> 273

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Lys Val Ser Cys Ala Val Val Leu Ser Ala Cys Ile Ala Ser Ala

1 5 10 15

Asn Ala Asp Asn Asn Gly Asp Gly Lys Ala Asp Ser Ile Lys Glu Asn

20 25 30

Asp Leu Asn Ala Gly Tyr Ala Asp Gly Thr Tyr Phe Tyr Thr Ala Ala

35 40 45

Asp Gly Gly Met Val Phe Arg Cys Pro Ile Cys Gly Tyr Lys Thr Ser

50 55 60

Thr Asn Thr Ser Tyr Thr Arg Thr Glu Leu Arg Glu Met Leu Arg Arg

65 70 75 80

Gly Asp Thr Ser Ile Ala Thr Gln Gly Val Asn Gly Asn Asn Trp Val

85 90 95

Phe Gly Ser Ala Pro Ala Ser Ala Arg Glu Ala Ala Gly Gly Val Asp

100 105 110

Gly Val Leu Arg Ala Thr Leu Ala Val Asn His Val Thr Thr Thr Gly

115 120 125

Asp Ser Gly Gln Val Gly Arg Val Ile Val Gly Gln Ile His Ala Asn

130 135 140

Asn Asp Glu Pro Leu Arg Leu Tyr Tyr Arg Lys Leu Pro Gly His Ser

145 150 155 160

Lys Gly Ser Val Tyr Ile Ala His Glu Pro Asn Gly Gly Ser Asp Ser

165 170 175

Trp Tyr Asp Met Ile Gly Ser Arg Ser Ser Ser Ala Ser Asp Pro Ser

180 185 190

Asp Gly Ile Ala Leu Asp Glu Val Trp Ser Tyr Glu Val Lys Val Val

195 200 205

Gly Asn Thr Leu Thr Val Thr Ile Phe Arg Ala Gly Lys Asp Asp Val

210 215 220

Val Gln Val Val Asp Met Gly Asn Ser Gly Tyr Asp Val Ala Asp Gln

225 230 235 240

Tyr Gln Tyr Phe Lys Ala Gly Val Tyr Asn Gln Asn Asn Thr Gly Asn

245 250 255

Ala Ser Asp Tyr Val Gln Val Thr Phe Tyr Cys Leu Glu Gln Ser His

260 265 270

Asp

<210> 2

<211> 822

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaaagtaa gttgcgctgt cgtactgtct gcttgtattg ccagtgccaa cgcagacaac 60

aatggcgatg gcaaggccga ctccatcaag gaaaatgacc tgaatgcagg ctatgcagat 120

ggcacctact tctatactgc tgccgatggc ggcatggtgt tccgctgccc gatctgtggc 180