阅读说明：本技术 一种新琼二糖的生产方法 (Production method of neoagarobiose ) 是由 何雄飞 侯艳平 易志伟 产竹华 张春毅 王斐 于 2021-11-09 设计创作，主要内容包括：本发明属于新琼二糖生产领域,具体涉及一种新琼二糖的生产方法,该方法包括将琼胶水溶液在β-琼胶酶的存在下酶解为低聚新琼寡糖液,所述β-琼胶酶的基因序列为SEQ ID NO:1；往低聚新琼寡糖液中加入硫酸进行酸解反应,再往所得酸解产物中加入碳酸钡充分搅拌沉淀,之后将体系中的沉淀通过固液分离去除,所得上清液干燥后得到新琼二糖干粉。本发明提供的方法所得产物中新琼二糖的占比较高。(The invention belongs to the field of production of new agarobiose, and particularly relates to a production method of new agarobiose, which comprises the steps of carrying out enzymolysis on an agar aqueous solution in the presence of beta-agarase to obtain oligomeric new agaro-oligosaccharide solution, wherein the gene sequence of the beta-agarase is SEQ ID NO: 1; and adding sulfuric acid into the oligomeric new agaro-oligosaccharide solution to carry out acidolysis reaction, adding barium carbonate into the obtained acidolysis product, fully stirring and precipitating, removing the precipitate in the system through solid-liquid separation, and drying the obtained supernatant to obtain the new agarobiose dry powder. The neoagarobiose in the product obtained by the method provided by the invention has a high proportion.)

1. A method for producing neoagarobiose, which is characterized by comprising the following steps:

s1, carrying out enzymolysis on the agar aqueous solution in the presence of beta-agarase to obtain a new oligomeric agar oligosaccharide solution, wherein the gene sequence of the beta-agarase is SEQ ID NO: 1;

s2, adding sulfuric acid into the oligomeric new agaro-oligosaccharide solution to carry out acidolysis reaction, adding barium carbonate into the obtained acidolysis product, fully stirring and precipitating, removing the precipitate in the system through solid-liquid separation, and drying the obtained supernatant to obtain the new agaro-disaccharide dry powder.

2. The method for producing neoagarobiose according to claim 1, wherein the conditions for the enzymatic hydrolysis in step S1 are such that the degree of polymerization of the resulting oligo-neoagaroose solution is 20 or less.

3. The method for producing neoagarobiose according to claim 1, wherein the conditions for the enzymatic hydrolysis in step S1 are such that the resulting oligomeric neoagaro-oligosaccharide solution contains neoagarotetraose and/or neoagarohexaose as a main component.

4. The method for producing neoagarobiose according to claim 1, wherein in step S1, the conditions of the enzymatic hydrolysis include a temperature of 40 to 50 ℃ and a time of 3 to 8 hours.

5. The method for producing neoagarobiose according to claim 1, wherein in step S1, the amount of β -agarase is 0.1 to 1mL, based on 100g of the total weight of the aqueous agar solution.

6. The method for producing neoagarobiose according to any one of claims 1 to 5, wherein the acidolysis reaction is performed at a temperature of 20 to 70 ℃ for 2 to 5 hours in step S2.

7. The method for producing neoagarobiose according to any one of claims 1 to 5, wherein in step S2, the amount of sulfuric acid used is such that the concentration of sulfuric acid in the acidolysis reaction system is 0.05 to 0.2 mol/L; the molar ratio of the barium carbonate to the sulfuric acid is (2-3) to 1.

8. The method for producing neoagarobiose according to any one of claims 1 to 5, wherein in step S2, the solid-liquid separation method is centrifugal separation; the drying method is at least one selected from vacuum freeze drying, evaporation drying and crystallization filtration drying.

9. The method for producing neoagarobiose according to any one of claims 1 to 5, further comprising a step S1 of purifying and concentrating the oligomeric neoagaroose solution obtained by the enzymatic hydrolysis.

10. The method for producing neoagarobiose according to any one of claims 1 to 5, wherein the method further comprises a step S2 of subjecting the supernatant to gel column purification to isolate neoagarobiose before drying.

Technical Field

The invention belongs to the field of production of neoagarobiose, and particularly relates to a production method of neoagarobiose.

Background

Agar polysaccharide is a plant polysaccharide separated from marine red algae, and can be prepared from red algae of Gelidium and Gracilaria. Agar polysaccharide is a macromolecular substance in nature, so that a human body cannot directly utilize the component, the possibility of further utilization of the component can be realized only after the agar polysaccharide is converted into ultra-low agarose (DP < 100) or agar oligosaccharide (DP is more than or equal to 2 and less than 20), and the component can be widely used for subsequent molecular modification only after the agar polysaccharide is degraded into a small molecular structure. However, the existing agar modification means can only obtain ultra-low agarose generally, and the ultra-low agarose can not reach the target level.

Among these oligomeric structures, neoagarobiose (C)₁₂H₂₀O₁₀) Is a component monomer of agar polysaccharide, has the molecular weight of only 324.28, and has good biological activity and biological safety. According to the existing literature reports, the neoagarobiose has wide application prospects in the fields of liver injury nursing, cancer treatment, skin repair and the like. Meanwhile, the neoagarobiose also has good drug modification prospect, and the combination of the neoagarobiose with bioactive substances such as protein, lipid and the like also has potential drug-forming possibility.

The degradation method of the common agar polysaccharide mainly comprises a chemical method, a physical method and an enzymatic hydrolysis method. Among them, the fenton method and the acid hydrolysis method are common chemical methods, and the radiation method is common physical methods. However, since chemical and physical methods are very random at the degradation site of agaropectin, the degree of degradation of the product cannot be precisely controlled, and such methods usually have very strict requirements on the process and damage the environment to a certain extent, so the industrial realization value is not high. The enzymolysis method is widely researched by students due to high environmental friendliness and easily controlled reaction conditions.

Because agar polysaccharide has stable physicochemical properties and few microorganisms capable of being efficiently degraded in a land environment, most of the currently obtained agarase has unsatisfactory activity, the main degradation products are mostly concentrated on agar oligosaccharide with the polymerization degree of 4-20, and a product with a high neoagarobiose ratio is difficult to obtain. A small amount of research reports that microorganisms or agarase which can degrade to obtain neoagarobiose are found, but the activities of the microorganisms or agarase are poor, most of the finished products are polysaccharide which is not completely degraded, and the proportion of the neoagarobiose is low.

Disclosure of Invention

The invention aims to overcome the defect that the neoagarobiose obtained by the existing method is low in proportion, and provides a novel production method of the neoagarobiose.

After intensive research, the inventor of the invention finds that the main reason that the new agarobiose cannot be accurately degraded by the existing enzymatic hydrolysis method is that the existing agarase carries out enzymatic hydrolysis on agarase polysaccharide in a random endo-or exo-cleavage mode, and after a micromolecule product is obtained by degradation, the active site of the agarase is difficult to further effectively combine with the micromolecule to continue degradation, so that the new agarobiose cannot be obtained or the occupation ratio of the new agarobiose is low. After intensive research, the inventor of the invention also finds that the ratio of neoagarobiose in the obtained product is higher by carrying out enzymolysis on the agar by adopting beta-agarase with the gene sequence of SEQ ID NO:1 and introducing an acidolysis process at the end point of the enzymolysis reaction. Based on this, the present invention has been completed.

Specifically, the invention provides a production method of neoagarobiose, which comprises the following steps:

s1, carrying out enzymolysis on the agar aqueous solution in the presence of beta-agarase to obtain a new oligomeric agar oligosaccharide solution, wherein the gene sequence of the beta-agarase is SEQ ID NO: 1;

s2, adding sulfuric acid into the oligomeric new agaro-oligosaccharide solution to carry out acidolysis reaction, adding barium carbonate into the obtained acidolysis product, fully stirring and precipitating, removing the precipitate in the system through solid-liquid separation, and drying the obtained supernatant to obtain the new agaro-disaccharide dry powder.

According to a specific embodiment of the invention, the beta-agarase obtains an enzyme gene from the genus or the strain by cloning or a chemical synthesis method, the enzyme gene is introduced into a prokaryotic expression vector to obtain a recombinant vector, then the recombinant vector is transformed into escherichia coli to obtain a recombinant strain, and the recombinant strain is fermented, separated and purified to obtain the beta-agarase. The gene sequence of the beta-agarase is SEQ ID NO. 1, and the corresponding amino acid sequence is SEQ ID NO. 2. The methods of obtaining the enzyme gene by cloning or chemical synthesis, introducing the enzyme gene into a prokaryotic expression vector to obtain a recombinant vector, and transforming the recombinant vector into escherichia coli to obtain a recombinant strain are well known to those skilled in the art, and are not described herein. The prokaryotic expression vector may be, for example, a PET-series expression vector (e.g., PET-14, PET-21, PET-22, PET-25, PET-28), a PGEX-series expression vector (e.g., PGEX-4T-2, PGEX-6T-1), or the like. The Escherichia coli may be, for example, Escherichia coli BL21(DE 3).

SEQ ID NO:1：

ATGCATCATCATCATCATCATCGACCTAAGTTCATAAACTTTCACGCAAAGTCCCACGATAAAGACGTGACCGCTTTCTACGAAGAATATGACGTATACCTTGGCCGAGGCTTCTGGGGAATGATTAAAACAGTGCTGCGCAAGGGATTTACAAAACGCTCCACGGTGGGTATACACGTTGACACAGCAGACGTCGGCTCGACTTATCCGAATTCTACTATAATTGATAAATCAACGGACGTCAAGGAGGTCTCTAATCTCATAGGAACAGAACACGAGAAACCGTTTTGGCCGGATGCTGGAGAAATGTCGCTGATGGACGCATCTACCTCGGGGGACTTTATGGGTCAGTTTTTTAACGAGTTTGTCGATGCAGGTGCTACTAACTCGGATGATCGGCCAAAATACGTCGAAATCATCAATGAACCCTTTTGGCATGCTCATGACTTTTATGAGATTACTGCAAAGGAGATGGCGGAACTCTTTGGTACTATCGCAGCGCAAGTTCACGATACTCCGTCGCTCGGAAAGATGAAGGTTGGTGGCTATTGTACCGGTTTCCCAGATTTTGAAATCAATAACTTTGCACATTGGGAAGATAATATGAAAATGTTCATGGATGTGGCGGGAGATGATATGGACTTTTGGTCTATACACCTATACGATTTTCCTTCGGGAATAACCCAGAACAATAATCGCTCGGGATCCAACATGGAAGCGGTTCTAGATCTTATAGAGACGTATTCGATGTGGAAGGGAGGCAAGGTCAAACCTCATGCGATCACACAATACGGGGTTATCACCCATGGTTTCGACAATTACACACCGTATCGGGATTGGCTTCACATCAAATCAACAAATTCTATGCTGATGCAGTTTATGGAGCGCACGGACAATATATGCTATGCAATGCCGTTTGCCATGGATAAATCCACGTGGCACCTAACGGAGAATAATGGGCAGCCTGGTGCACTCTTCATACCTACCAATATCGGTGAAAAGGAAGTAGAGCAGTGGGTGTTCACGGAAATGATCAAATTCTATCAACTTTGGAAGACCGGAGTTTCTGTCAATCCGGCGTCAACGTCCGTAGCAGTTGCGGCTACGCAGAAATTCACAGCAGGCATAACGATGGCTGATGCTGGCGTGGTATGGTCCGTTGCTAATGAATCGGTGGCACTAGTCTCCGCGACTGGGAAGGTAACAGCTGTTAAGAAGGGCAAGCTGACCATCACAGCAACCACTACGGACGAATTTGCGGCAGTACTTATCCAAAAGATAGAAGCGGAAGATTTCGGGGCTTATCTAGATCGCATCAACGAGGGCGTTAATATCGATTCTACAACAGCTGGGCAGGAGACGGGCGAATGGACCTCATACGCAGGTACGGATGTTAATATACCCGAATCAGCAATCTATACGATCTCGGTCAACACATCAACTTCGACAGAGTCGTCCCTTGATCTTATAGAGGATAACAAAATCTGTGAAACTATCGAAGTAAATAACAATGCCCTTACGGAGGTAAAAGTGGAGCTAGCCGGTTCACATGTGCTCGGACACGGGGATGTTAAGATAAATTGGCTTTTCAGTTAA。

SEQ ID NO:2：

MHHHHHHRPKFINFHAKSHDKDVTAFYEEYDVYLGRGFWGMIKTVLRKGFTKRSTVGIHVDTADVGSTYPNSTIIDKSTDVKEVSNLIGTEHEKPFWPDAGEMSLMDASTSGDFMGQFFNEFVDAGATNSDDRPKYVEIINEPFWHAHDFYEITAKEMAELFGTIAAQVHDTPSLGKMKVGGYCTGFPDFEINNFAHWEDNMKMFMDVAGDDMDFWSIHLYDFPSGITQNNNRSGSNMEAVLDLIETYSMWKGGKVKPHAITQYGVITHGFDNYTPYRDWLHIKSTNSMLMQFMERTDNICYAMPFAMDKSTWHLTENNGQPGALFIPTNIGEKEVEQWVFTEMIKFYQLWKTGVSVNPASTSVAVAATQKFTAGITMADAGVVWSVANESVALVSATGKVTAVKKGKLTITATTTDEFAAVLIQKIEAEDFGAYLDRINEGVNIDSTTAGQETGEWTSYAGTDVNIPESAIYTISVNTSTSTESSLDLIEDNKICETIEVNNNALTEVKVELAGSHVLGHGDVKINWLFS

In step S1, the conditions of the enzymatic hydrolysis are such that the degree of polymerization of the obtained oligo-new agaro-oligosaccharide solution is 20 or less, preferably 4 to 20.

In step S1, the enzymolysis conditions are such that the resulting oligo-neoagaro-oligosaccharide solution is at least one of neoagarotetraose, neoagarohexaose, and neoagarooctaose, and it is preferable that neoagarotetraose and/or neoagarohexaose.

Further, in the step S1, the enzymolysis condition includes that the temperature is 40-50 ℃ and the time is 3-8 h.

In step S1, the amount of β -agarase is 0.1 to 1mL, and specifically may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0mL, based on 100g of the total weight of the agar aqueous solution.

Further, in step S2, the acidolysis reaction conditions include a temperature of 20 to 70 ℃, specifically 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and the like; the time is 2-5 h, specifically 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h and the like.

Further, in step S2, the sulfuric acid is used in an amount such that the concentration of the sulfuric acid in the acidolysis reaction system is 0.05-0.2 mol/L.

In step S2, the molar ratio of barium carbonate to sulfuric acid is (2-3): 1.

Further, in step S2, the solid-liquid separation method is centrifugal separation.

Further, in step S2, the drying method is at least one selected from vacuum freeze drying, evaporation drying and crystallization filtration drying.

Further, the method for producing neoagarobiose provided by the invention further comprises the step of purifying and concentrating the oligomeric neoagarobiose liquid obtained by enzymolysis in step S1. Wherein the purification method may be specifically selected from at least one of filter paper filtration, plate and frame filtration, membrane filtration, centrifugation, and resin purification. The method of concentration may be specifically selected from at least one of concentration under reduced pressure, concentration under vacuum, and concentration by evaporation.

Further, the method for producing neoagarobiose further comprises step S2, before drying, subjecting the supernatant to gel column purification to separate neoagarobiose, wherein the crude product can be further separated and purified to obtain a high-purity product, and the application range is wider. In addition, the specific operation and conditions of the gel column purification and separation are known to those skilled in the art and will not be described herein.

The invention creatively adopts beta-agarase with a specific gene sequence and introduces an acidolysis process at the end of the enzymolysis reaction, the ratio of neoagarobiose in the obtained product can reach more than 34 percent, and the rest components are agaropectin, agarotriose and neoagarotetraose, so that the invention has no irritation and mutagenicity to cells. In addition, the method takes sulfuric acid as an acidolysis medium, and because a certain amount of sulfate radicals exist in the agar substrate, the sulfuric acid is selected to not introduce new impurity ions, and after acidolysis, the impurity sulfate ions can be precipitated by combining barium carbonate and sulfate radicals, and then all salt impurities can be removed by a simple centrifugal process, so that the difficulty of industrial application is reduced.

Detailed Description

The present invention will be described in detail below by way of examples.

In the following preparation examples, β -agarase was obtained by the following method: according to the method, an enzyme gene with a gene sequence of SEQ ID NO:1 is obtained from corresponding bacteria or strains by a chemical synthesis method, the enzyme gene is introduced into a PET-14 expression vector to obtain a recombinant vector, then the recombinant vector is transformed into escherichia coli BL21(DE3) to obtain a recombinant strain, and the beta-agarase is obtained by fermenting, separating and purifying the recombinant strain.

The manipulations involved in the β -agarase may be performed according to the means conventional in the art without specific mention, for example, as disclosed in "molecular cloning instructions (fourth edition) j. sambrook, m.r. green".

In the following examples and comparative examples, the enzyme activity units are defined as: the amount of enzyme (mL) required to release 1. mu. mol reducing sugar (mmol, U) per minute under optimal conditions.

Example 1

S1, adding 0.1mL of beta-agarase (the gene sequence is SEQ ID NO:1 and the amino acid sequence is SEQ ID NO:2, and the enzyme activity is 200U/mL) into 100g of agar aqueous solution (the agar concentration is 1 wt%), performing enzymolysis for 8 hours at 40 ℃, centrifuging the obtained enzymolysis product through a high-speed refrigerated centrifuge to remove undegraded colloid, and then performing reduced pressure concentration on the supernatant to obtain a new oligosaccharide solution which is mainly a mixture of neoagarotetraose and neoagarohexaose;

s2, adding sulfuric acid into the oligomeric new agaro-oligosaccharide solution to enable the concentration of the sulfuric acid in the system to be 0.05mol/L, carrying out acidolysis for 2 hours at 70 ℃, adding barium carbonate into the acidolysis product, fully stirring and precipitating, wherein the molar ratio of the barium carbonate to the sulfuric acid is 3:1, removing the precipitate in the system through centrifugation, and carrying out vacuum freeze drying on the obtained supernatant to obtain the new agaro-disaccharide dry powder. Through detection, the content of the neoagarobiose in the neoagarobiose dry powder is 41.3%.

Example 2

S1, adding 0.5mL of beta-agarase (the gene sequence is SEQ ID NO:1, the amino acid sequence is SEQ ID NO:2, and the enzyme activity is 200U/mL) into 100g of agar aqueous solution (the agar concentration is 1.5 wt%), carrying out enzymolysis for 5 hours at 45 ℃, centrifuging the obtained enzymolysis product through a high-speed refrigerated centrifuge to remove undegraded colloid, and then carrying out reduced pressure concentration on the supernatant to obtain oligomeric neoagaro-oligosaccharide liquid which is mainly a mixture of neoagarotetraose and neoagarohexaose;

s2, adding sulfuric acid into the oligomeric new agaro-oligosaccharide solution to enable the concentration of the sulfuric acid in the system to be 0.1mol/L, then carrying out acidolysis for 3h at 50 ℃, adding barium carbonate into the acidolysis product, fully stirring and precipitating, wherein the molar ratio of the barium carbonate to the sulfuric acid is 3:1, then removing the precipitate in the system through centrifugation, and carrying out vacuum freeze drying on the obtained supernatant to obtain the new agaro-disaccharide dry powder. Through detection, the content of the neoagarobiose in the neoagarobiose dry powder is 38.7%.

Example 3

S1, adding 1mL of beta-agarase (the gene sequence is SEQ ID NO:1, the amino acid sequence is SEQ ID NO:2, and the enzyme activity is 200U/mL) into 100g of agar aqueous solution (the agar concentration is 2 wt%), carrying out enzymolysis for 3h at 50 ℃, centrifuging the obtained enzymolysis product through a high-speed refrigerated centrifuge to remove undegraded colloid, and then carrying out reduced pressure concentration on the supernatant to obtain oligomeric neoagaro-oligosaccharide liquid which is mainly a mixture of neoagarotetraose and neoagarohexaose;

s2, adding sulfuric acid into the oligomeric new agaro-oligosaccharide solution to enable the concentration of the sulfuric acid in the system to be 0.2mol/L, then carrying out acidolysis for 5 hours at 20 ℃, then adding barium carbonate into the acidolysis product, fully stirring and precipitating, wherein the molar ratio of the barium carbonate to the sulfuric acid is 3:1, then removing the precipitate in the system through centrifugation, separating and purifying the obtained supernatant through a gel column, and then carrying out vacuum freeze drying to obtain the new agaro-disaccharide dry powder. Through detection, the content of the neoagarobiose in the neoagarobiose dry powder is 34.2%.

Comparative example 1

A neoagarobiose dry powder was prepared according to the method of example 1, except that the β -agarase having the gene sequence of SEQ ID NO:1 was replaced with the β -agarase disclosed in CN110438182A (the enzyme activity was 200U/mL), and the amino acid sequence of the β -agarase was set to SEQ ID NO: 3:

MTFTKSKIATVLSLSLLGIYGCASTTPQNEQAAAGEQVVEDMGGALPDFESDKFFSKLKAEHAKASAVTDTGVTAGSQALKIDFDSVNEANKFKFWPNVKLHPDTGNWNWNAKGSLTLDVTNPTDSTANIILKIADNVGVMGAGDNQLNYALSVPAGETVPVEMIFNGSKRKLDGYWGGEKINLRKLVEFQIFVQGPIDQQSVIVDNFALVDATGDFVEASGAEEVVTGPVPTVLAITDFEKGQDSFISAERSVATTISPVKTDDGAAIDVLFSASNSYPNITFRPDVPWDWSGQGDFNVAFDMVNKSDEPLQLFVRVDDDEHEAFGGTANGVQNSWSGYVTIAPNDEGTYYLSLMPAGDQMVSGMRGEPPKKSYKAQAISYGWGDNNLDLSNIYSMQLYLQNPTADQKLQISSVRLIPNLESDTSRYEGLLDEFGQYTGQDWAQKVKSLEDLQAAGAAELDSLEHPTQLPDRSKFGGWADGPKLEATGFFRAEKVDGKWALVDPEGYLFFVTGLDNIRMDDTVTITGVDFSNKETREGREVASELRNSMFTWLPEYDDVLAESYDYADWIHTGALKKGEVFSFYSANLQRKYQTSREEALKIWKDVTLNRMQDWGFTTLGNWADPKFYDNQQIAYAANGWIFGDHARISTGNDYWGPIHDPFDPEFAVSTRKMAEKVASEVSKDDPWLMGIFVDNEISWGNTKNEANHYGLVVNALSYDIKESPAKAAFTKHLQDKYSSIDALNQSWGTKVTSWADFEVSFDHRSRLSSSMKKDYSEMLQMLSEKYFSTVQAELKKVLPNHMYLGARFADWGVTPEIARGAAPYVDVMSYNLYAEDLNSKGDWSLLPELDKPSIIGEFHFGATDTGLFHGGIVSASNQADRAKKYTHYMQSIVDNPYFVGAHWFQYLDSPTTGRAWDGENYNVGFVSITDTPYQELIDAAKQFNRDLYNLRYKK, the remaining conditions were the same as in example 1, to obtain a dry powder of reference neoagarobiose. According to detection, the content of the neoagarobiose in the reference neoagarobiose dry powder is 17.5%.

Comparative example 2

A dry neoagarobiose powder was prepared according to the method of example 1, except that the sequence of enzymatic hydrolysis and acid hydrolysis was replaced, and the specific steps were as follows:

s1, adding sulfuric acid into 100g of agar aqueous solution (the concentration of agar is 1 wt%) to enable the concentration of the sulfuric acid in the system to be 0.05mol/L, then carrying out acidolysis for 2h at 70 ℃, then adding barium carbonate into the system, fully stirring and precipitating, wherein the molar ratio of the barium carbonate to the sulfuric acid is 3:1, and then carrying out high-speed freezing centrifugation to remove the precipitate to obtain supernatant, namely an acidolysis product;

s2, adding 0.1mL of beta-agarase (the gene sequence is SEQ ID NO:1 and the amino acid sequence is SEQ ID NO:2, and the enzyme activity is 200U/mL) into the acidolysis product, carrying out enzymolysis for 8h at 40 ℃, carrying out high-speed freezing centrifugation on the obtained enzymolysis product to remove undegraded colloid, and then carrying out reduced pressure concentration on the supernatant to obtain reference neoagarobiose dry powder. According to detection, the content of the neoagarobiose in the reference neoagarobiose dry powder is 3.7%.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

SEQUENCE LISTING

<110> blue brain technology (Xiamen) Co Ltd

<120> agarase freeze-drying protective agent and agarase preservation method

<130> NNKE-21002-NUI

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1596

<212> DNA

<213> beta-agarase coding gene

<400> 1

atgcatcatc atcatcatca tcgacctaag ttcataaact ttcacgcaaa gtcccacgat 60

aaagacgtga ccgctttcta cgaagaatat gacgtatacc ttggccgagg cttctgggga 120

atgattaaaa cagtgctgcg caagggattt acaaaacgct ccacggtggg tatacacgtt 180

gacacagcag acgtcggctc gacttatccg aattctacta taattgataa atcaacggac 240

gtcaaggagg tctctaatct cataggaaca gaacacgaga aaccgttttg gccggatgct 300

ggagaaatgt cgctgatgga cgcatctacc tcgggggact ttatgggtca gttttttaac 360

gagtttgtcg atgcaggtgc tactaactcg gatgatcggc caaaatacgt cgaaatcatc 420

aatgaaccct tttggcatgc tcatgacttt tatgagatta ctgcaaagga gatggcggaa 480

ctctttggta ctatcgcagc gcaagttcac gatactccgt cgctcggaaa gatgaaggtt 540

ggtggctatt gtaccggttt cccagatttt gaaatcaata actttgcaca ttgggaagat 600

aatatgaaaa tgttcatgga tgtggcggga gatgatatgg acttttggtc tatacaccta 660

tacgattttc cttcgggaat aacccagaac aataatcgct cgggatccaa catggaagcg 720

gttctagatc ttatagagac gtattcgatg tggaagggag gcaaggtcaa acctcatgcg 780

atcacacaat acggggttat cacccatggt ttcgacaatt acacaccgta tcgggattgg 840

cttcacatca aatcaacaaa ttctatgctg atgcagttta tggagcgcac ggacaatata 900

tgctatgcaa tgccgtttgc catggataaa tccacgtggc acctaacgga gaataatggg 960

cagcctggtg cactcttcat acctaccaat atcggtgaaa aggaagtaga gcagtgggtg 1020

ttcacggaaa tgatcaaatt ctatcaactt tggaagaccg gagtttctgt caatccggcg 1080

tcaacgtccg tagcagttgc ggctacgcag aaattcacag caggcataac gatggctgat 1140

gctggcgtgg tatggtccgt tgctaatgaa tcggtggcac tagtctccgc gactgggaag 1200

gtaacagctg ttaagaaggg caagctgacc atcacagcaa ccactacgga cgaatttgcg 1260

gcagtactta tccaaaagat agaagcggaa gatttcgggg cttatctaga tcgcatcaac 1320

gagggcgtta atatcgattc tacaacagct gggcaggaga cgggcgaatg gacctcatac 1380

gcaggtacgg atgttaatat acccgaatca gcaatctata cgatctcggt caacacatca 1440

acttcgacag agtcgtccct tgatcttata gaggataaca aaatctgtga aactatcgaa 1500

gtaaataaca atgcccttac ggaggtaaaa gtggagctag ccggttcaca tgtgctcgga 1560

cacggggatg ttaagataaa ttggcttttc agttaa 1596

<210> 2

<211> 531

<212> PRT

<213> beta-agarase amino acid sequence

<400> 2

Met His His His His His His Arg Pro Lys Phe Ile Asn Phe His Ala

1 5 10 15

Lys Ser His Asp Lys Asp Val Thr Ala Phe Tyr Glu Glu Tyr Asp Val

20 25 30

Tyr Leu Gly Arg Gly Phe Trp Gly Met Ile Lys Thr Val Leu Arg Lys

35 40 45

Gly Phe Thr Lys Arg Ser Thr Val Gly Ile His Val Asp Thr Ala Asp

50 55 60

Val Gly Ser Thr Tyr Pro Asn Ser Thr Ile Ile Asp Lys Ser Thr Asp

65 70 75 80

Val Lys Glu Val Ser Asn Leu Ile Gly Thr Glu His Glu Lys Pro Phe

85 90 95

Trp Pro Asp Ala Gly Glu Met Ser Leu Met Asp Ala Ser Thr Ser Gly

100 105 110

Asp Phe Met Gly Gln Phe Phe Asn Glu Phe Val Asp Ala Gly Ala Thr

115 120 125

Asn Ser Asp Asp Arg Pro Lys Tyr Val Glu Ile Ile Asn Glu Pro Phe

130 135 140

Trp His Ala His Asp Phe Tyr Glu Ile Thr Ala Lys Glu Met Ala Glu

145 150 155 160

Leu Phe Gly Thr Ile Ala Ala Gln Val His Asp Thr Pro Ser Leu Gly

165 170 175

Lys Met Lys Val Gly Gly Tyr Cys Thr Gly Phe Pro Asp Phe Glu Ile

180 185 190

Asn Asn Phe Ala His Trp Glu Asp Asn Met Lys Met Phe Met Asp Val

195 200 205

Ala Gly Asp Asp Met Asp Phe Trp Ser Ile His Leu Tyr Asp Phe Pro

210 215 220

Ser Gly Ile Thr Gln Asn Asn Asn Arg Ser Gly Ser Asn Met Glu Ala

225 230 235 240

Val Leu Asp Leu Ile Glu Thr Tyr Ser Met Trp Lys Gly Gly Lys Val

245 250 255

Lys Pro His Ala Ile Thr Gln Tyr Gly Val Ile Thr His Gly Phe Asp

260 265 270

Asn Tyr Thr Pro Tyr Arg Asp Trp Leu His Ile Lys Ser Thr Asn Ser

275 280 285

Met Leu Met Gln Phe Met Glu Arg Thr Asp Asn Ile Cys Tyr Ala Met

290 295 300

Pro Phe Ala Met Asp Lys Ser Thr Trp His Leu Thr Glu Asn Asn Gly

305 310 315 320

Gln Pro Gly Ala Leu Phe Ile Pro Thr Asn Ile Gly Glu Lys Glu Val

325 330 335

Glu Gln Trp Val Phe Thr Glu Met Ile Lys Phe Tyr Gln Leu Trp Lys

340 345 350

Thr Gly Val Ser Val Asn Pro Ala Ser Thr Ser Val Ala Val Ala Ala

355 360 365

Thr Gln Lys Phe Thr Ala Gly Ile Thr Met Ala Asp Ala Gly Val Val

370 375 380

Trp Ser Val Ala Asn Glu Ser Val Ala Leu Val Ser Ala Thr Gly Lys

385 390 395 400

Val Thr Ala Val Lys Lys Gly Lys Leu Thr Ile Thr Ala Thr Thr Thr

405 410 415

Asp Glu Phe Ala Ala Val Leu Ile Gln Lys Ile Glu Ala Glu Asp Phe

420 425 430

Gly Ala Tyr Leu Asp Arg Ile Asn Glu Gly Val Asn Ile Asp Ser Thr

435 440 445

Thr Ala Gly Gln Glu Thr Gly Glu Trp Thr Ser Tyr Ala Gly Thr Asp

450 455 460

Val Asn Ile Pro Glu Ser Ala Ile Tyr Thr Ile Ser Val Asn Thr Ser

465 470 475 480

Thr Ser Thr Glu Ser Ser Leu Asp Leu Ile Glu Asp Asn Lys Ile Cys

485 490 495

Glu Thr Ile Glu Val Asn Asn Asn Ala Leu Thr Glu Val Lys Val Glu

500 505 510

Leu Ala Gly Ser His Val Leu Gly His Gly Asp Val Lys Ile Asn Trp

515 520 525

Leu Phe Ser

530

<210> 3

<211> 955

<212> PRT

<213> beta-agarase

<400> 3

Met Thr Phe Thr Lys Ser Lys Ile Ala Thr Val Leu Ser Leu Ser Leu

1 5 10 15

Leu Gly Ile Tyr Gly Cys Ala Ser Thr Thr Pro Gln Asn Glu Gln Ala

20 25 30

Ala Ala Gly Glu Gln Val Val Glu Asp Met Gly Gly Ala Leu Pro Asp

35 40 45

Phe Glu Ser Asp Lys Phe Phe Ser Lys Leu Lys Ala Glu His Ala Lys

50 55 60

Ala Ser Ala Val Thr Asp Thr Gly Val Thr Ala Gly Ser Gln Ala Leu

65 70 75 80

Lys Ile Asp Phe Asp Ser Val Asn Glu Ala Asn Lys Phe Lys Phe Trp

85 90 95

Pro Asn Val Lys Leu His Pro Asp Thr Gly Asn Trp Asn Trp Asn Ala

100 105 110

Lys Gly Ser Leu Thr Leu Asp Val Thr Asn Pro Thr Asp Ser Thr Ala

115 120 125

Asn Ile Ile Leu Lys Ile Ala Asp Asn Val Gly Val Met Gly Ala Gly

130 135 140

Asp Asn Gln Leu Asn Tyr Ala Leu Ser Val Pro Ala Gly Glu Thr Val

145 150 155 160

Pro Val Glu Met Ile Phe Asn Gly Ser Lys Arg Lys Leu Asp Gly Tyr

165 170 175

Trp Gly Gly Glu Lys Ile Asn Leu Arg Lys Leu Val Glu Phe Gln Ile

180 185 190

Phe Val Gln Gly Pro Ile Asp Gln Gln Ser Val Ile Val Asp Asn Phe

195 200 205

Ala Leu Val Asp Ala Thr Gly Asp Phe Val Glu Ala Ser Gly Ala Glu

210 215 220

Glu Val Val Thr Gly Pro Val Pro Thr Val Leu Ala Ile Thr Asp Phe

225 230 235 240

Glu Lys Gly Gln Asp Ser Phe Ile Ser Ala Glu Arg Ser Val Ala Thr

245 250 255

Thr Ile Ser Pro Val Lys Thr Asp Asp Gly Ala Ala Ile Asp Val Leu

260 265 270

Phe Ser Ala Ser Asn Ser Tyr Pro Asn Ile Thr Phe Arg Pro Asp Val

275 280 285

Pro Trp Asp Trp Ser Gly Gln Gly Asp Phe Asn Val Ala Phe Asp Met

290 295 300

Val Asn Lys Ser Asp Glu Pro Leu Gln Leu Phe Val Arg Val Asp Asp

305 310 315 320

Asp Glu His Glu Ala Phe Gly Gly Thr Ala Asn Gly Val Gln Asn Ser

325 330 335

Trp Ser Gly Tyr Val Thr Ile Ala Pro Asn Asp Glu Gly Thr Tyr Tyr

340 345 350

Leu Ser Leu Met Pro Ala Gly Asp Gln Met Val Ser Gly Met Arg Gly

355 360 365

Glu Pro Pro Lys Lys Ser Tyr Lys Ala Gln Ala Ile Ser Tyr Gly Trp

370 375 380

Gly Asp Asn Asn Leu Asp Leu Ser Asn Ile Tyr Ser Met Gln Leu Tyr

385 390 395 400

Leu Gln Asn Pro Thr Ala Asp Gln Lys Leu Gln Ile Ser Ser Val Arg

405 410 415

Leu Ile Pro Asn Leu Glu Ser Asp Thr Ser Arg Tyr Glu Gly Leu Leu

420 425 430

Asp Glu Phe Gly Gln Tyr Thr Gly Gln Asp Trp Ala Gln Lys Val Lys

435 440 445

Ser Leu Glu Asp Leu Gln Ala Ala Gly Ala Ala Glu Leu Asp Ser Leu

450 455 460

Glu His Pro Thr Gln Leu Pro Asp Arg Ser Lys Phe Gly Gly Trp Ala

465 470 475 480

Asp Gly Pro Lys Leu Glu Ala Thr Gly Phe Phe Arg Ala Glu Lys Val

485 490 495

Asp Gly Lys Trp Ala Leu Val Asp Pro Glu Gly Tyr Leu Phe Phe Val

500 505 510

Thr Gly Leu Asp Asn Ile Arg Met Asp Asp Thr Val Thr Ile Thr Gly

515 520 525

Val Asp Phe Ser Asn Lys Glu Thr Arg Glu Gly Arg Glu Val Ala Ser

530 535 540

Glu Leu Arg Asn Ser Met Phe Thr Trp Leu Pro Glu Tyr Asp Asp Val

545 550 555 560

Leu Ala Glu Ser Tyr Asp Tyr Ala Asp Trp Ile His Thr Gly Ala Leu

565 570 575

Lys Lys Gly Glu Val Phe Ser Phe Tyr Ser Ala Asn Leu Gln Arg Lys

580 585 590

Tyr Gln Thr Ser Arg Glu Glu Ala Leu Lys Ile Trp Lys Asp Val Thr

595 600 605

Leu Asn Arg Met Gln Asp Trp Gly Phe Thr Thr Leu Gly Asn Trp Ala

610 615 620

Asp Pro Lys Phe Tyr Asp Asn Gln Gln Ile Ala Tyr Ala Ala Asn Gly

625 630 635 640

Trp Ile Phe Gly Asp His Ala Arg Ile Ser Thr Gly Asn Asp Tyr Trp

645 650 655

Gly Pro Ile His Asp Pro Phe Asp Pro Glu Phe Ala Val Ser Thr Arg

660 665 670

Lys Met Ala Glu Lys Val Ala Ser Glu Val Ser Lys Asp Asp Pro Trp

675 680 685

Leu Met Gly Ile Phe Val Asp Asn Glu Ile Ser Trp Gly Asn Thr Lys

690 695 700

Asn Glu Ala Asn His Tyr Gly Leu Val Val Asn Ala Leu Ser Tyr Asp

705 710 715 720

Ile Lys Glu Ser Pro Ala Lys Ala Ala Phe Thr Lys His Leu Gln Asp

725 730 735

Lys Tyr Ser Ser Ile Asp Ala Leu Asn Gln Ser Trp Gly Thr Lys Val

740 745 750

Thr Ser Trp Ala Asp Phe Glu Val Ser Phe Asp His Arg Ser Arg Leu

755 760 765

Ser Ser Ser Met Lys Lys Asp Tyr Ser Glu Met Leu Gln Met Leu Ser

770 775 780

Glu Lys Tyr Phe Ser Thr Val Gln Ala Glu Leu Lys Lys Val Leu Pro

785 790 795 800

Asn His Met Tyr Leu Gly Ala Arg Phe Ala Asp Trp Gly Val Thr Pro

805 810 815

Glu Ile Ala Arg Gly Ala Ala Pro Tyr Val Asp Val Met Ser Tyr Asn

820 825 830

Leu Tyr Ala Glu Asp Leu Asn Ser Lys Gly Asp Trp Ser Leu Leu Pro

835 840 845

Glu Leu Asp Lys Pro Ser Ile Ile Gly Glu Phe His Phe Gly Ala Thr

850 855 860

Asp Thr Gly Leu Phe His Gly Gly Ile Val Ser Ala Ser Asn Gln Ala

865 870 875 880

Asp Arg Ala Lys Lys Tyr Thr His Tyr Met Gln Ser Ile Val Asp Asn

885 890 895

Pro Tyr Phe Val Gly Ala His Trp Phe Gln Tyr Leu Asp Ser Pro Thr

900 905 910

Thr Gly Arg Ala Trp Asp Gly Glu Asn Tyr Asn Val Gly Phe Val Ser

915 920 925

Ile Thr Asp Thr Pro Tyr Gln Glu Leu Ile Asp Ala Ala Lys Gln Phe

930 935 940

Asn Arg Asp Leu Tyr Asn Leu Arg Tyr Lys Lys

945 950 955