CN113151223B

CN113151223B - Method for preparing kelp hydrolysate

Info

Publication number: CN113151223B
Application number: CN202010074535.9A
Authority: CN
Inventors: 王本新; 姜周
Original assignee: Shandong Hongye Marine Technology Co ltd
Current assignee: Shandong Hongye Marine Technology Co ltd
Priority date: 2020-01-22
Filing date: 2020-01-22
Publication date: 2022-08-30
Anticipated expiration: 2040-01-22
Also published as: CN113151223A

Abstract

The invention discloses a method for preparing kelp hydrolysate. The invention provides an artificial enzyme, which is a recombinant protein obtained by connecting Aspergillus niger pectinase and exoglucanase CEL2 combined protein through connecting peptide; the amino acid sequence of the exoglucanase CEL2 is shown as 392-671 th site of SEQ ID No. 1. The artificial enzyme and the matching method provided by the invention are used for preparing kelp hydrolysate, and 1.7g/L reducing sugar can be obtained in 8 hours. Therefore, the method provided by the invention has important application value.

Description

Method for preparing kelp hydrolysate

Technical Field

The invention relates to the fields of kelp processing, industrial enzymes and food industry, in particular to a method for preparing kelp hydrolysate.

Background

The kelp is a large marine brown algae plant growing in low-temperature seawater, and belongs to a seaweed plant. The kelp can be eaten and has wide application value in the fields of medical treatment, health care and the like because the kelp is rich in mineral substances and functional components. In China, the mode of artificial kelp culture is established in the 50 th 20 th century, and with the rapid development of the aquaculture industry and the progress of kelp technology in China, the kelp industry enters a continuous, rapid and healthy development stage. In recent years, the kelp culture scale in China is rapidly developed. The annual output of the Chinese kelp is improved from 398.26 ten thousand tons in 1999 to 539.64 ten thousand tons in 2008, the annual output of the Chinese kelp accounts for about 81-88% of the annual output of the world kelp, and the culture output and the scale of the Chinese kelp are the first in the world seaweed culture.

Kelp contains dozens of nutrient components, mainly including mannitol, algin, etc. Wherein the main functional substances of the kelp are polysaccharide substances. Laminarin has various biological activities and medicinal functions, including enhancing immunity and resisting tumor. The kelp is processed into hydrolysate rich in kelp sugar, and the absorption of functional ingredients of kelp can be promoted. The kelp hydrolysate can be used as food additive and nutrient solution in fields of kelp soy sauce, functional beverage, etc. The traditional kelp hydrolysate is subjected to high-temperature cooking, acid-base treatment and other methods, so that the energy consumption is high, a large amount of sewage is discharged, and the requirements of green and environment-friendly processes cannot be met. Based on cellulase (such as exoglucanase), the technology for processing kelp hydrolysate by using an efficient enzyme method is established by combining protease, pectinase and the like, so that the use amount of chemical reagents such as acid, alkali and the like can be greatly reduced. However, how to improve the catalytic efficiency of the enzyme is the key to reduce the cost. Therefore, the method has important significance for mining novel cellulase resources such as exoglucanase and the like from the metagenome and applying the cellulase resources to the preparation of kelp hydrolysate.

Disclosure of Invention

The invention aims to provide an artificial enzyme and application thereof in preparing kelp hydrolysate.

In a first aspect, the present invention claims a fusion protein.

The fusion protein claimed by the invention is a recombinant protein obtained by connecting aspergillus niger pectinase and exoglucanase CEL2 through a connecting peptide; the amino acid sequence of the exoglucanase CEL2 is shown in the 392-671 th site of SEQ ID No. 1.

Further, the amino acid sequence of the Aspergillus niger pectinase is shown as the 1 st to 335 th sites of SEQ ID No. 1.

Further, the amino acid sequence of the connecting peptide is shown as 336-391 of SEQ ID No. 1.

Still further, the fusion protein may be any of:

(A1) protein with amino acid sequence shown as SEQ ID No. 1;

(A2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in (A1) and having the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In a second aspect, the invention claims nucleic acid molecules encoding the fusion proteins described hereinbefore.

Further, the nucleic acid molecule is a fusion gene encoding the fusion protein.

In the fusion gene, the gene for coding the Aspergillus niger pectinase is shown as 1 st to 1005 th sites of SEQ ID No. 2.

In the fusion gene, the gene coding the exoglucanase CEL2 is shown as the 1174-2013 site of SEQ ID No. 2.

In the fusion gene, the gene encoding the linker peptide is shown as position 1006-1173 of SEQ ID No. 2.

Further, the fusion gene may be any of:

(B1) a DNA molecule shown as SEQ ID No. 2;

(B2) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the fusion protein;

(B3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the DNA sequence defined in any one of (B1) to (B2) and encoding the fusion protein.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a third aspect, the invention claims recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria comprising the nucleic acid molecules described above.

In a fourth aspect, the present invention claims the use of the fusion protein as described above as an artificial enzyme in the preparation of kelp hydrolysate.

In a fifth aspect, the invention claims a complete set of enzyme preparations for preparing kelp hydrolysate.

The complete set of enzyme preparation for preparing kelp hydrolysate, which is claimed by the invention, consists of artificial enzyme and protease; the artificial enzyme is a fusion protein as described above.

Further, in the enzyme preparation set, the two enzymes can be packaged separately or in a mixed manner.

In a sixth aspect, the invention claims a kit for preparing kelp hydrolysate.

The kit for preparing kelp hydrolysate claimed by the invention contains the enzyme preparation set and kelp powder.

In a seventh aspect, the invention claims the application of the fusion protein or the nucleic acid molecule or the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium or the enzyme kit or the kit in preparing kelp hydrolysate.

In an eighth aspect, the present invention claims a method for preparing kelp hydrolysate.

The method for preparing kelp hydrolysate as claimed in the invention can comprise the following steps: adding artificial enzyme (i.e. the fusion protein described above) and protease into the kelp powder to react; the reaction may be carried out using a reaction buffer having a pH of 4.5 to 5.5 (e.g., pH5.0) and a reaction temperature of 42 to 47 ℃ (e.g., 45 ℃).

In a specific embodiment of the present invention, the reaction buffer is specifically 0.1M acetic acid-sodium acetate buffer at pH 5.0.

In the method, the reaction time may be 8 hours; the rotational speed during the reaction may be 200 rpm.

In the reaction process, the ratio of the artificial enzyme, the protease and the kelp powder can be 5000IU (calculated by the activity of pectinase) and 10000IU:50 g.

In a specific embodiment of the present invention, the final concentration of the artificial enzyme in the reaction system is about 5000IU/L pectinase; the final concentration of the protease in the reaction system is about 10000 IU/L; the final concentration of the kelp powder in the reaction system is 50 g/L.

In a specific embodiment of the invention, the artificial enzyme is 95% pure. The cellulase is a commodity with the product number of C805042, made by Beijing Taize Jia industry science and technology development Co., Ltd; the protease is a commodity with the product number of P0029 of Beijing Yinuoka science and technology Limited.

In each of the above aspects, the artificial enzymes may be prepared according to a method comprising the steps of: introducing the nucleic acid molecule as described in the second aspect above (encoding the fusion protein as described above) into an E.coli recipient cell to obtain a recombinant E.coli; culturing the recombinant escherichia coli to obtain the artificial enzyme.

Wherein the nucleic acid molecule can be introduced into the E.coli recipient cell in the form of a recombinant vector.

In a specific embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing a small fragment between the cleavage sites NotI and BamHI of the pET28a vector with the nucleic acid molecule (SEQ ID No. 2).

Further, the recombinant E.coli was cultured under conditions of 2 hours at 30 ℃ followed by inoculation of IPTG to a final concentration of 0.1mM, followed by culture for 16 hours. And collecting the thallus after culture, and extracting protein by a nickel affinity chromatography after ultrasonic crushing to obtain the artificial enzyme protein solution.

In the invention, the particle size of the kelp powder is less than 40 meshes.

Further, the kelp powder can be obtained by drying fresh kelp at 60 ℃, crushing and sieving with a 40-mesh sieve.

Experiments prove that 1.7g/L of reducing sugar can be obtained in 8 hours by preparing kelp hydrolysate by using the artificial enzyme (namely the recombinant protein obtained by connecting aspergillus niger pectinase and exoglucanase CEL2 through connecting peptide) and the matching method. Therefore, the method provided by the invention has important application value.

Drawings

FIG. 1 shows the results of producing kelp hydrolysate by using artificial enzyme. The ordinate g/L refers to the reducing sugar content per liter of fermentation broth.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

Example 1 preparation of Artificial enzyme

Construction of Escherichia coli strains for producing artificial enzymes

1. Total gene synthesis SEQ ID No.2 (Nanjing Jinzhi Biotechnology Co., Ltd.) to obtain the artificial enzyme gene gc 2A. The artificial enzyme gene gc2A contains Aspergillus niger pectinase gene, artificially designed linker gene and exoglucanase cel2 gene sequence. Wherein, the 1 st-1005 th site of the SEQ ID No.2 is the Aspergillus niger pectinase gene, the 1006 th-1173 th site is an artificial adaptor gene, and the 1174 th-2013 th site is an exoglucanase cel2 gene sequence. SEQ ID No.2 encodes the amino acid sequence shown in SEQ ID No. 1. 1-335 of SEQ ID No.1 is the amino acid sequence of Aspergillus niger pectinase; position 336-391 is the amino acid sequence of an artificial linker (linker peptide); the 392-671 th site is the amino acid sequence of exoglucanase CEL 2.

2. The gc2A gene was constructed on pET28a plasmid by Gibson method

(1) PCR amplification of the gc2A gene. A gene fragment GC2A was PCR-amplified with high fidelity TransStart FastPfu DNA polymerase (Beijing Quanji Biotechnology Co., Ltd., Cat. AP221) using a plasmid containing the GC2A gene (SEQ ID No.2) provided by Nanjing Jinzhi Biotech Ltd as a template and GC-F and GC-R as primers.

GC-F：5’-GCCGCGCGGCAGCCAT-ATGGGCAGCTGCACCTTTAAAACCGCGGCG-3’；

GC-R：5’-GTCGACGAGCTCGAATTCG-TTAATCTTTCGGTTTGCCGCCCGGGTTCGGGCA-3’。

(2) Constructing a recombinant expression vector containing the gc2A gene. Carrying out agarose gel electrophoresis on the PCR amplification fragment obtained in the step (1), and recovering a target fragment; the vector pET28a (Wuhan vast Ling Bioceae) was simultaneously digested with NotI and BamHITechnology ltd, cat # VT0331-01), recovering a carrier large fragment ET28 a. The gnlcA fragment was ligated to the ET28a fragment using the Gibson assembly method (Gibson DG, Young L, ET al. enzymatic assembly of DNA molecules up to basic cloned kits. Nat. methods. 2009; 6(5): 343-345). With CaCl ₂ Escherichia coli DH 5. alpha. competent cells (Beijing Quanjin Biotechnology Co., Ltd., cat # CD201) were transformed by the method. This was spread evenly on LB plates containing kanamycin and cultured overnight at 37 ℃. Clones were selected and sequenced, and the resulting positive plasmid was designated pETC 02.

The structure of pETC02 is described as: and (3) replacing a small fragment between the cleavage sites NotI and BamHI of the pET28a vector by the DNA fragment shown in SEQ ID No. 2.

(3) Construction of Escherichia coli Strain GCA-02: the pETC02 plasmid was transformed into Transetta (DE3) competent cells (Beijing Quanjin Biotechnology Co., Ltd., product No. CD801-01) by calcium chloride transformation, cultured overnight at 37 ℃ on an LB plate containing kanamycin, and then cloned to obtain an E.coli strain GCA-02 expressing an artificial enzyme protein.

Second, preparation of Artificial enzyme

After activation of GCA-02 strain, the strain was inoculated in a 5L fermentor (3L of LB medium at 30 ℃ C., and IPTG concentration was added to a final concentration of 0.1mM at 2 hours after the transfer) at a ratio of 1:100 for 16 hours. The cells were centrifuged at 5000rpm and collected. After the cells were sonicated, proteins were extracted by nickel affinity chromatography (Beijing Quanyu gold Biotechnology Co., Ltd., product No. DP101-01) (all methods were as described in the specification). The protein was then analyzed for molecular weight and purity by SDS-PAGE. As a result, it was confirmed that the molecular weight of the purified protein was about 70KD, which substantially coincided with the theoretical molecular weight of the artificial enzyme (SEQ ID No. 2). SDS-PAGE shows that the purified product has no impurity band (purity reaches 95%). Then, the concentration of the artificial enzyme was measured by using the Brandford reagent (Bio-engineering (Shanghai) Co., Ltd., product No. C600641), and the method was performed in accordance with the product instructions.

Verification that the CEL2 protein shown in 392-671 th site of SEQ ID No.1 has exoglucanase activity: according to the procedures of the first and second embodiments of the example 1, the CEL2 gene shown in the 1174-2013 position of SEQ ID No.2 is subjected to prokaryotic expression in Escherichia coli, and the CEL2 protein is obtained. And then determining the activity of the exoglucanase by adopting a pNPG method, and performing according to the literature (bud inclusion, screening, cloning and identification of cellulose degrading enzymes derived from uncultured microorganisms in rumens of Chinese yaks, doctor's academic thesis of university of Compound denier, 2010), wherein the determined and purified CEL1 protein has the activity of the exoglucanase, and the activity of the enzyme can reach 32IU/mg (the unit of the activity of the enzyme is defined as the enzyme amount which catalyzes the pNPG to hydrolyze at the pH of 5 and 45 ℃ per minute to generate 1 micromole of p-nitrophenol and is one unit of the activity).

Third, construction of escherichia coli strain for producing aspergillus niger pectinase and preparation of enzyme

The procedure of example 1 was followed.

Wherein in the process of constructing and producing the Aspergillus niger pectinase Escherichia coli strain, a primer GC-R is replaced by n-R, and the sequence of the n-R is as follows:

n-R：5’-GTCGACGAGCTCGAATTCG-TTAGCTCGCCACGCTCGGATAGTTTTTGCACGC-3’。

the gene fragment gnA was PCR-amplified using GC-F and n-R as primers, and the plasmid pETG-C1 was obtained in the same manner as in examples 1 and 2.

The structure of pETG-C1 is described as: the recombinant plasmid obtained after replacing a small fragment between the cleavage sites NotI and BamHI of the pET28a vector with the DNA fragment shown at positions 1-1005 of SEQ ID No. 2.

pETG-C1 was transformed into Transetta (DE3) competent cells by the same method as in the first and second examples 1, 2, to obtain E.coli strain GC02 expressing the A.niger pectinase protein.

According to the same manner as that described in example 1, Aspergillus niger pectinase was prepared.

Example 2 production of kelp hydrolysate Using Artificial enzyme

Cleaning fresh herba Zosterae Marinae (Weihai, etc.) and oven drying at 60 deg.C overnight, pulverizing in a high-power pulverizer, and sieving with 40 mesh sieve to obtain herba Zosterae Marinae powder.

Reaction in a 5L reaction tank:

the reaction system comprises the following components:

artificial enzyme obtained in example 1, final enzyme concentration: the activity of the pectinase is about 5000IU/L (IU is defined as the amount of enzyme which catalyzes the hydrolysis of pectin to generate 1 micromole of galacturonic acid per minute under the optimal conditions (pH5, 45 ℃) is one activity unit); the corresponding cellulase activity was approximately 10000IU/L (IU is defined as the amount of enzyme that catalyzes the hydrolysis of sodium carboxymethylcellulose to 1. mu. mol reducing sugar per minute at 45 ℃ and pH5 as one activity unit).

Protease (Beijing Yinaoka science and technology Co., Ltd., cat # P0029), final enzyme concentration: about 10000IU/L (IU is defined as the amount of enzyme that catalyzes the hydrolysis of casein to 1 micromole tyrosine per minute at 45 ℃ and pH5 as one activity unit).

50g/L of kelp powder.

Reaction conditions of the reaction tank:

reaction buffer: 0.1M sodium acetate/acetic acid buffer (pH 5).

Reaction temperature: 45 ℃;

the reaction time is as follows: 8 h;

reaction speed: 200 rpm.

The group containing all the components was designated as test group T1, the one obtained by replacing the artificial enzyme with aspergillus niger pectinase (prepared in step three of example 1) having the same enzyme activity concentration was designated as test group T2, and the one containing no artificial enzyme component was designated as control group C.

The reducing sugar detection method comprises the following steps:

taking out a sample, centrifuging at 13000rpm for 5min, taking the supernatant, boiling water bath for 5min, taking 100 μ l of the supernatant, adding 100 μ l of DNS solution (the formula is that sodium hydroxide 21g and DNS 6.3g are fully dissolved in 500mL of distilled water, adding potassium sodium tartrate 182g, phenol 5g and sodium metabisulfite 5g into the solution, stirring until the solution is completely dissolved, fixing the volume to 1000mL, keeping the solution out of the sun), boiling water bath for 5min, centrifuging, taking 100 μ l of the supernatant, adding the supernatant into a 96-well plate, and measuring the content of reducing sugar in an enzyme labeling instrument.

The results are shown in FIG. 1: the experimental group T1 produced about 1.7g/L of reducing sugar, the experimental group T2 produced about 0.6g/L of reducing sugar, and the control group C produced about 0.1g/L of reducing sugar. The preparation of the kelp hydrolysate by using the artificial enzyme has obvious advantages.

<110> Shandong Hongye ocean science and technology Co Ltd

<120> a method for preparing kelp hydrolysate

<130> GNCLN200367

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 671

<212> PRT

<213> Artificial sequence

<400> 1

Met Gly Ser Cys Thr Phe Lys Thr Ala Ala Ala Ala Lys Ala Gly Lys

1 5 10 15

Ala Gly Cys Ser Thr Ile Thr Leu Asp Asn Ile Glu Val Pro Ala Gly

20 25 30

Thr Thr Leu Asp Leu Thr Gly Leu Thr Ser Gly Thr Lys Val Ile Phe

35 40 45

Glu Gly Thr Thr Thr Phe Asp Tyr Glu Glu Trp Ala Gly Pro Leu Ile

50 55 60

Ser Met Ser Gly Lys Asp Ile Thr Val Thr Gly Ala Ser Gly His Leu

65 70 75 80

Ile Asn Cys Asp Gly Ala Arg Trp Trp Asp Gly Lys Gly Thr Ser Gly

85 90 95

Lys Lys Lys Pro Lys Phe Phe Tyr Ala His Gly Leu Asp Ser Ser Ser

100 105 110

Ile Thr Gly Leu Asn Ile Lys Asn Thr Pro Leu Met Ala Phe Ser Val

115 120 125

Gln Ala Asp Asp Ile Thr Leu Thr Asp Ile Thr Ile Asn Asn Ala Asp

130 135 140

Gly Asp Thr Leu Gly Gly His Asn Thr Asp Ala Phe Asp Val Gly Asn

145 150 155 160

Ser Val Gly Val Asn Ile Ile Lys Pro Trp Val His Asn Gln Asp Asp

165 170 175

Cys Leu Ala Ile Asn Ser Gly Glu Asn Ile Trp Phe Thr Ser Gly Thr

180 185 190

Cys Ile Gly Gly His Gly Leu Ser Ile Gly Ser Val Gly Gly Arg Ser

195 200 205

Asn Asn Val Val Lys Asn Val Thr Ile Glu His Ser Thr Val Ser Asn

210 215 220

Ser Glu Asn Ala Val Arg Ile Lys Thr Val Ser Gly Ala Thr Gly Ser

225 230 235 240

Val Ser Glu Ile Thr Tyr Ser Asn Ile Val Met Ser Gly Ile Ser Asp

245 250 255

Tyr Gly Val Val Ile Gln Gln Asp Tyr Glu Asp Gly Lys Pro Thr Gly

260 265 270

Lys Pro Thr Asn Gly Val Thr Ile Thr Asp Val Lys Leu Glu Ser Val

275 280 285

Thr Gly Thr Val Asp Ser Lys Ala Thr Asp Ile Tyr Leu Leu Cys Gly

290 295 300

Ser Gly Ser Cys Ser Asp Trp Thr Trp Asp Asp Val Lys Val Thr Gly

305 310 315 320

Gly Lys Lys Ser Thr Ala Cys Lys Asn Tyr Pro Ser Val Ala Ser Cys

325 330 335

Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Ser Ser Val Glu Gln Leu

340 345 350

Gly Gly Gly Gly Ser Glu Ala Ala Ala Ala Lys Ser Val Glu Gly Ser

355 360 365

Ser Gly Gly Gly Gly Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

370 375 380

Ser Gly Gly Gly Gly Ser Val Met Arg Glu Ser Gly Leu Gly Thr Val

385 390 395 400

Thr Ser Gly Asn Thr Ala Cys Ser Ser Ala Thr Thr Cys Ser Ala Leu

405 410 415

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

420 425 430

Val Thr Ser Ala Pro Pro Ser Gln Gln Thr Asn Val Gly Thr Arg Val

435 440 445

Tyr Met Glu Ser Gly Lys Arg Tyr Gln Met Phe Asp Leu Leu Asn Gln

450 455 460

Glu Leu Thr Phe Asp Val Asp Val Ser Lys Val Pro Cys Gly Gly Thr

465 470 475 480

Asn Gly Ala Leu Tyr Phe Ile Ser Leu Met Glu Asp Gly Gly Met Ser

485 490 495

Lys Phe Ser Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys

500 505 510

Asp Ser Gln Cys Pro Arg Asp Ile Lys Phe Ile Asn Gly Glu Asn Trp

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Glu Ala Asn Asn Leu Asn Pro Phe Arg Met Gly Asn Arg Glu Phe Tyr

530 535 540

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

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Ile Thr Gln Phe Ile Thr Asp Asn Asp Thr Glu Thr Asp Asp Arg Val

565 570 575

Thr Glu Ser Asn Pro Asn Thr Asn Phe Pro Gly Leu Met Gly Thr Asp

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Ser Ile Thr Asp Ala Met Cys Asp Asp Ala Lys Ala Leu Phe Glu Asp

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His Pro Tyr Val Met Gly Gly Leu Ala Gln Leu Ser Ser Leu Ala Lys

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Gly Thr Gly Leu Ala Leu Ser Ile Trp Asn Asp His Thr Ala Asn Met

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Leu Trp Leu Asp Ser Phe Lys Thr Gly Glu Asp Pro Lys Asp Pro Gly

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Ala Leu Arg Cys Thr Cys Pro Asn Pro Gly Gly Lys Pro Lys Asp

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<210> 2

<211> 2013

<212> DNA

<213> Artificial sequence

<400> 2

atgggcagct gcacctttaa aaccgcggcg gcggcgaaag cgggcaaagc gggctgcagc 60

accattaccc tggataacat tgaagtgccg gcgggcacca ccctggatct gaccggcctg 120

accagcggca ccaaagtgat ttttgaaggc accaccacct ttgattatga agaatgggcg 180

ggcccgctga ttagcatgag cggcaaagat attaccgtga ccggcgcgag cggccatctg 240

attaactgcg atggcgcgcg ctggtgggat ggcaaaggca ccagcggcaa aaaaaaaccg 300

aaattttttt atgcgcatgg cctggatagc agcagcatta ccggcctgaa cattaaaaac 360

accccgctga tggcgtttag cgtgcaggcg gatgatatta ccctgaccga tattaccatt 420

aacaacgcgg atggcgatac cctgggcggc cataacaccg atgcgtttga tgtgggcaac 480

agcgtgggcg tgaacattat taaaccgtgg gtgcataacc aggatgattg cctggcgatt 540

aacagcggcg aaaacatttg gtttaccagc ggcacctgca ttggcggcca tggcctgagc 600

attggcagcg tgggcggccg cagcaacaac gtggtgaaaa acgtgaccat tgaacatagc 660

accgtgagca acagcgaaaa cgcggtgcgc attaaaaccg tgagcggcgc gaccggcagc 720

gtgagcgaaa ttacctatag caacattgtg atgagcggca ttagcgatta tggcgtggtg 780

attcagcagg attatgaaga tggcaaaccg accggcaaac cgaccaacgg cgtgaccatt 840

accgatgtga aactggaaag cgtgaccggc accgtggata gcaaagcgac cgatatttat 900

ctgctgtgcg gcagcggcag ctgcagcgat tggacctggg atgatgtgaa agtgaccggc 960

ggcaaaaaaa gcaccgcgtg caaaaactat ccgagcgtgg cgagctgcgg cggcggcggc 1020

agcgaagcgg cggcgaaaag cagcgtggaa cagctgggcg gcggcggcag cgaagcggcg 1080

gcggcgaaaa gcgtggaagg cagcagcggc ggcggcggca gcgaagcggc ggcgaaagaa 1140

gcggcggcga aaagcggcgg cggcggcagc gtgatgcgcg aaagcggcct gggcaccgtg 1200

accagcggca acaccgcgtg cagcagcgcg accacctgca gcgcgctgga aggcgcggat 1260

tatgaaggcc tgcatgcgac caccgcgggc ggcagcgtga ccagcgcgcc gccgagccag 1320

cagaccaacg tgggcacccg cgtgtatatg gaaagcggca aacgctatca gatgtttgat 1380

ctgctgaacc aggaactgac ctttgatgtg gatgtgagca aagtgccgtg cggcggcacc 1440

aacggcgcgc tgtattttat tagcctgatg gaagatggcg gcatgagcaa atttagcggc 1500

aacaaagcgg gcgcgaaata tggcaccggc tattgcgata gccagtgccc gcgcgatatt 1560

aaatttatta acggcgaaaa ctgggaagcg aacaacctga acccgtttcg catgggcaac 1620

cgcgaatttt atggcccggg caaaagctat gatattgata ccaaccgcaa atttagcgtg 1680

attacccagt ttattaccga taacgatacc gaaaccgatg atcgcgtgac cgaaagcaac 1740

ccgaacacca actttccggg cctgatgggc accgatagca ttaccgatgc gatgtgcgat 1800

gatgcgaaag cgctgtttga agatcatccg tatgtgatgg gcggcctggc gcagctgagc 1860

agcctggcga aaggcaccgg cctggcgctg agcatttgga acgatcatac cgcgaacatg 1920

ctgtggctgg atagctttaa aaccggcgaa gatccgaaag atccgggcgc gctgcgctgc 1980

acctgcccga acccgggcgg caaaccgaaa gat 2013

Claims

1. The fusion protein is a recombinant protein obtained by connecting aspergillus niger pectinase and exoglucanase CEL2 through a connecting peptide;

the amino acid sequence of the exoglucanase CEL2 is shown as 392-671 th site of SEQ ID No. 1;

the amino acid sequence of the Aspergillus niger pectinase is shown as the 1 st-335 th site of SEQ ID No. 1; the amino acid sequence of the connecting peptide is shown as 336-391 position of SEQ ID No. 1;

the fusion protein is any one of the following:

(A1) protein with amino acid sequence shown in SEQ ID No. 1;

(A2) and (b) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in (A1).

2. A nucleic acid molecule encoding the fusion protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a fusion gene encoding the fusion protein;

4. The nucleic acid molecule of claim 2, wherein: in the fusion gene, the gene coding the exoglucanase CEL2 is shown as the 1174-2013 site of SEQ ID No. 2.

5. The nucleic acid molecule of claim 2, wherein: in the fusion gene, the gene coding the connecting peptide is shown as the 1006-1173 position of SEQ ID No. 2.

6. The nucleic acid molecule of claim 2, wherein: the fusion gene is a DNA molecule shown in SEQ ID No. 2.

7. A recombinant vector comprising the nucleic acid molecule of any one of claims 2-6.

8. An expression cassette comprising the nucleic acid molecule of any one of claims 2-6.

9. A transgenic cell line comprising the nucleic acid molecule of any one of claims 2-6; the transgenic cell line is a non-animal or plant variety.

10. A recombinant bacterium comprising the nucleic acid molecule of any one of claims 2 to 6.

11. The use of the fusion protein of claim 1 as an artificial enzyme in the preparation of a kelp hydrolysate.

12. A complete set of enzyme preparation for preparing sea tangle hydrolysate comprises artificial enzyme and protease; the artificial enzyme is the fusion protein of claim 1.

13. A kit for preparing a kelp hydrolysate comprising the enzyme preparation set according to claim 12 and kelp powder.

14. Use of the fusion protein according to claim 1 or the nucleic acid molecule according to any one of claims 2 to 6 or the recombinant vector according to claim 7 or the expression cassette according to claim 8 or the transgenic cell line according to claim 9 or the recombinant bacterium according to claim 10 or the enzyme kit according to claim 12 or the kit according to claim 13 for the preparation of kelp hydrolysate.

15. A method for preparing kelp hydrolysate comprises the following steps: adding artificial enzyme and protease into the kelp powder for reaction;

the pH value of the reaction buffer solution adopted for the reaction is 4.5-5.5, and the reaction temperature is 42-47 ℃;

the artificial enzyme is the fusion protein of claim 1.