CN112979819B

CN112979819B - Method for preparing kelp hydrolysate by using artificial enzyme

Info

Publication number: CN112979819B
Application number: CN201911278062.8A
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: 2019-12-12
Filing date: 2019-12-12
Publication date: 2022-08-30
Anticipated expiration: 2039-12-12
Also published as: CN112979819A

Abstract

The invention discloses an artificial enzyme and application thereof in kelp hydrolysate. The invention provides an artificial enzyme, which is a recombinant protein obtained by connecting aspergillus niger pectinase and trichoderma reesei cellulose binding protein through a connecting peptide. The kelp hydrolysate prepared by the artificial enzyme and the matching method can obtain 3g/L reducing sugar in 6 hours. Therefore, the method provided by the invention has important application value.

Description

Method for preparing kelp hydrolysate by using artificial enzyme

Technical Field

The invention relates to the fields of kelp processing, industrial enzymes and food industry, in particular to an artificial enzyme and application thereof in 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.

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, the technology for processing kelp hydrolysate by an efficient enzyme method is established by combining protease, pectinase and the like, and 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.

Disclosure of Invention

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

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

The fusion protein claimed by the invention is a recombinant protein obtained by connecting aspergillus niger pectinase and trichoderma reesei cellulose binding protein through a connecting peptide.

Furthermore, the amino acid sequence of the Aspergillus niger pectinase is shown as 1 st to 335 th positions of SEQ ID No. 2.

Further, the amino acid sequence of the Trichoderma reesei cellulose binding protein is shown in SEQ ID No.2, position 392-905.

Further, the amino acid sequence of the connecting peptide is shown as 336-391 of SEQ ID No. 2.

Still further, the fusion protein may be any of:

(A1) protein with amino acid sequence shown as SEQ ID No. 2;

(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. 1.

In the fusion gene, the gene encoding the cellulose binding protein of Trichoderma reesei is shown as SEQ ID No.1 at position 1174-2715.

In the fusion gene, the gene encoding the linker peptide is shown as position 1006-1173 of SEQ ID No. 1.

Further, the fusion gene may be any of:

(B1) a DNA molecule shown as SEQ ID No. 1;

(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, cellulase and protease; the artificial enzyme is a fusion protein as described above.

Further, in the enzyme preparation set, the three enzymes can be packaged separately or in a mixed manner. When the three enzymes are mixed and packaged, the three enzymes can be mixed by taking the same amount of enzyme activity units.

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

The kit for preparing kelp hydrolysate, which is 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 (namely the fusion protein mentioned above), cellulase 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., pH 5.0) 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 6 hours; the rotational speed during the reaction may be 200 rpm.

In the reaction process, three enzymes can be used by taking the same amount of enzyme activity units.

Correspondingly, the ratio of the artificial enzyme, the cellulase, the protease and the kelp powder can be 10000IU, 10000IU and 50 g.

In a specific embodiment of the present invention, the final concentration of the artificial enzyme in the reaction system is about 10000IU/L of pectinase; the final concentration of the cellulase in the reaction system is about 10000 IU/L; 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 of Beijing Taize Jia industry science and technology development Limited company; 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 enzyme may be prepared according to a method comprising the steps of: introducing a nucleic acid molecule as described in the second aspect above (encoding a 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 enzyme cutting sites NotI and BamHI of the pET28a vector with the nucleic acid molecule (SEQ ID No. 1).

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 3g/L of reducing sugar can be obtained in 6 hours by preparing kelp hydrolysate by using the artificial enzyme (namely, the recombinant protein obtained by connecting aspergillus niger pectinase and trichoderma reesei cellulose binding protein 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 a hydrolyzed solution of kelp using an 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 strain for producing artificial enzyme

1. Total Gene Synthesis of SEQ ID No.1 (Nanjing Jinzhi Biotech Co., Ltd.) to obtain the artificial enzyme gene gnlcA. The artificial enzyme gene gnlcA contains aspergillus niger pectinase gene, artificial design joint gene and trichoderma reesei cellulose binding protein gene sequence. Wherein, the 1 st-1005 th site of SEQ ID No.1 is Aspergillus niger pectinase gene, the 1006 th-1173 th site is artificial adaptor gene, and the 1174 th-2715 th site is Trichoderma reesei cellulose binding protein gene sequence. SEQ ID No.1 encodes an amino acid sequence shown in SEQ ID No. 2. The 1 st to 335 th sites of SEQ ID No.2 are the amino acid sequence of Aspergillus niger pectinase; position 336-391 is the amino acid sequence of an artificial linker (linker peptide); position 392-905 is the amino acid sequence of the cellulose binding protein of Trichoderma reesei.

2. The gnlcA gene was constructed on the pET28a plasmid using the Gibson method

(1) PCR amplification of the gnlcA gene. The gene fragment gnlcA was amplified by PCR using high fidelity TransStart Fastpfu DNA polymerase (Beijing Quanji Biotech Co., Ltd., product No. AP 221) with the plasmid containing the gnlcA gene (SEQ ID No. 1) provided by Nanjing Jinzhi Biotech Co., Ltd as a template and g-F and g-R as primers.

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

g-R：5’-GTCGACGAGCTCGAATTCGTTA-CAGGCACTGGCTATAATACGGGTTCAGCAC-3’。

(2) A recombinant expression vector containing the gnlcA gene was constructed. 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 # VT 0331-01), and recovering the 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 sectional and cloned nucleic acids. nat. methods 2009; 6(5): 343-345). With CaCl ₂ Escherichia coli DH 5. alpha. competent cells (Beijing Quanjin Biotechnology Co., Ltd., cat # CD 201) were transformed by the method. These were spread evenly on LB plates containing kanamycin and cultured overnight at 37 ℃. Clones were selected and sequenced, and the positive plasmid obtained was designated pETG 01.

The structure of pet 01 is described as: the DNA fragment shown in SEQ ID No.1 was substituted for a small fragment between the cleavage sites NotI and BamHI of the pET28a vector to obtain a recombinant plasmid.

(3) Construction of E.coli strain GC 01: the pETG01 plasmid was transformed into Transetta (DE3) competent cells (Beijing Quanjin Biotechnology Co., Ltd., product No. CD 801-01) by calcium chloride transformation, cultured overnight at 37 ℃ on an LB plate containing kanamycin, and then clones were selected to obtain an E.coli strain GC01 expressing an artificial enzyme protein.

Second, preparation of Artificial enzyme

After activation of the strain GC01, 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. DP 101-01) (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 110KD, 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%). It was demonstrated that purified artificial enzyme had been obtained. 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.

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

The procedure was as in example 1, part one, part two.

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

n-R：5’-GTCGACGAGCTCGAATTCGTTA-GCTCGCCACGCTCGGATAGTTTTTGCACGC-3’。

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

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

Example 2 production of sea tangle hydrolysate by 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 10000IU/L (IU is defined as the enzyme which catalyzes the hydrolysis of pectin into 1 micromole of galacturonic acid per minute under the optimal conditions and is one activity unit);

cellulase (beijing taize jia science and technology development ltd, cat # C805042), final enzyme concentration: about 10000IU/L (IU is defined as the amount of enzyme that catalyzes the hydrolysis of sodium carboxymethylcellulose to 1 micromole of reducing sugars per minute as one activity unit);

protease (beijing einoka technologies 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 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: 6 h;

reaction speed: 200 rpm.

The test group containing all the components is set as a test group T1, the test group T2 is set by replacing the artificial enzyme with the Aspergillus niger pectinase with the same enzyme activity concentration, and the test group C is set by not containing the artificial enzyme component.

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 3g/L of reducing sugar, the experimental group T2 produced about 1.9g/L of reducing sugar, and the control group C produced about 1.8g/L of reducing sugar. The preparation of the kelp hydrolysate by using the artificial enzyme has obvious advantages.

Sequence listing

<110> Shandong Hongye ocean science and technology Co., Ltd

<120> a method for preparing kelp hydrolysate by using artificial enzyme

<130> GNCLN192758

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2715

<212> DNA

<213> Artificial sequence

<400> 1

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 gtgagctatc gcaaactggc ggtgattagc 1200

gcgtttctgg cgaccgcgcg cgcgcagagc gcgtgcaccc tgcagagcga aacccatccg 1260

ccgctgacct ggcagaaatg cagcagcggc ggcacctgca cccagcagac cggcagcgtg 1320

gtgattgatg cgaactggcg ctggacccat gcgaccaaca gcagcaccaa ctgctatgat 1380

ggcaacacct ggagcagcac cctgtgcccg gataacgaaa cctgcgcgaa aaactgctgc 1440

ctggatggcg cggcgtatgc gagcacctat ggcgtgacca ccagcggcaa cagcctgagc 1500

attggctttg tgacccagag cgcgcagaaa aacgtgggca gccgcctgta tctgatggcg 1560

aacgatagca cctatcagga atttaccctg agcggcaacg aatttagctt tgatgtggat 1620

gtgagccagc tgccgtgcgg cctgaacggc gcgctgtatt ttgtgagcat ggatgcggat 1680

ggcggccaga gcaaatatcc gggcaacgcg gcgggcgcga aatatggcac cggctattgc 1740

gatagccagt gcccgcgcga tctgaaattt attaacggcc aggcgaacgt ggaaggctgg 1800

gaaccgagca gcaacaacgc gaacaccggc gtgggcggcc atggcagctg ctgcagcgaa 1860

atggatattt gggaagcgaa cagcattagc gaagcgctga ccccgcatcc gtgcgaaacc 1920

gtgggccaga ccatgtgcag cggcgatagc tgcggcggca cctatagcaa cgatcgctat 1980

ggcggcacct gcgatccgga tggctgcgat tggaacccgt atcgcctggg caacaccagc 2040

ttttatggcc cgggcagcag ctttgcgctg gataccacca aaaaactgac cgtggtgacc 2100

cagtttgcga ccgatggcag cattagccgc tattatgtgc agaacggcgt gaaatttcag 2160

cagccgaacg cgcaggtggg cagctatagc ggcaacacca ttaacaccga ttattgcgcg 2220

gcggaacaga ccgcgtttgg cggcaccagc tttaccgata aaggcggcct gacccagttt 2280

aaaaaagcga ccagcggcgg catggtgctg gtgatgagcc tgtgggatga ttattatgcg 2340

aacatgctgt ggctggatag cacctatccg accaacgaaa ccagcagcac cccgggcgcg 2400

gtgcgcggca gctgcagcac cagcagcggc gtgccggcgc aggtggaaag ccagagcccg 2460

aacgcgaaag tgacctttag caacattaaa tttggcccga ttggcagcac cggcaacccg 2520

agcggcggca acccgccggg cggcaacccg ccgggcacca ccaccacccg ccgcccggcg 2580

accaccaccg gcagcagccc gggcccgacc cagagccatt atggccagtg cggcggcatt 2640

ggctatagcg gcccgaccgt gtgcgcgagc ggcaccacct gccaggtgct gaacccgtat 2700

tatagccagt gcctg 2715

<210> 2

<211> 905

<212> PRT

<213> Artificial sequence

<400> 2

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 Ser Tyr Arg Lys Leu Ala Val Ile Ser

385 390 395 400

Ala Phe Leu Ala Thr Ala Arg Ala Gln Ser Ala Cys Thr Leu Gln Ser

405 410 415

Glu Thr His Pro Pro Leu Thr Trp Gln Lys Cys Ser Ser Gly Gly Thr

420 425 430

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

435 440 445

Thr His Ala Thr Asn Ser Ser Thr Asn Cys Tyr Asp Gly Asn Thr Trp

450 455 460

Ser Ser Thr Leu Cys Pro Asp Asn Glu Thr Cys Ala Lys Asn Cys Cys

465 470 475 480

Leu Asp Gly Ala Ala Tyr Ala Ser Thr Tyr Gly Val Thr Thr Ser Gly

485 490 495

Asn Ser Leu Ser Ile Gly Phe Val Thr Gln Ser Ala Gln Lys Asn Val

500 505 510

Gly Ser Arg Leu Tyr Leu Met Ala Asn Asp Ser Thr Tyr Gln Glu Phe

515 520 525

Thr Leu Ser Gly Asn Glu Phe Ser Phe Asp Val Asp Val Ser Gln Leu

530 535 540

Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp

545 550 555 560

Gly Gly Gln Ser Lys Tyr Pro Gly Asn Ala Ala Gly Ala Lys Tyr Gly

565 570 575

Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn

580 585 590

Gly Gln Ala Asn Val Glu Gly Trp Glu Pro Ser Ser Asn Asn Ala Asn

595 600 605

Thr Gly Val Gly Gly His Gly Ser Cys Cys Ser Glu Met Asp Ile Trp

610 615 620

Glu Ala Asn Ser Ile Ser Glu Ala Leu Thr Pro His Pro Cys Glu Thr

625 630 635 640

Val Gly Gln Thr Met Cys Ser Gly Asp Ser Cys Gly Gly Thr Tyr Ser

645 650 655

Asn Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp Trp Asn

660 665 670

Pro Tyr Arg Leu Gly Asn Thr Ser Phe Tyr Gly Pro Gly Ser Ser Phe

675 680 685

Ala Leu Asp Thr Thr Lys Lys Leu Thr Val Val Thr Gln Phe Ala Thr

690 695 700

Asp Gly Ser Ile Ser Arg Tyr Tyr Val Gln Asn Gly Val Lys Phe Gln

705 710 715 720

Gln Pro Asn Ala Gln Val Gly Ser Tyr Ser Gly Asn Thr Ile Asn Thr

725 730 735

Asp Tyr Cys Ala Ala Glu Gln Thr Ala Phe Gly Gly Thr Ser Phe Thr

740 745 750

Asp Lys Gly Gly Leu Thr Gln Phe Lys Lys Ala Thr Ser Gly Gly Met

755 760 765

Val Leu Val Met Ser Leu Trp Asp Asp Tyr Tyr Ala Asn Met Leu Trp

770 775 780

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

785 790 795 800

Val Arg Gly Ser Cys Ser Thr Ser Ser Gly Val Pro Ala Gln Val Glu

805 810 815

Ser Gln Ser Pro Asn Ala Lys Val Thr Phe Ser Asn Ile Lys Phe Gly

820 825 830

Pro Ile Gly Ser Thr Gly Asn Pro Ser Gly Gly Asn Pro Pro Gly Gly

835 840 845

Asn Pro Pro Gly Thr Thr Thr Thr Arg Arg Pro Ala Thr Thr Thr Gly

850 855 860

Ser Ser Pro Gly Pro Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile

865 870 875 880

Gly Tyr Ser Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val

885 890 895

Leu Asn Pro Tyr Tyr Ser Gln Cys Leu

900 905

Claims

1. The fusion protein is a recombinant protein obtained by connecting aspergillus niger pectinase and trichoderma reesei cellulose binding protein through a connecting peptide;

the amino acid sequence of the Aspergillus niger pectinase is shown as the 1 st-335 th site of SEQ ID No. 2;

the amino acid sequence of the Trichoderma reesei cellulose binding protein is shown as 392-905 of SEQ ID No. 2;

the amino acid sequence of the connecting peptide is shown as 336-391 position of SEQ ID No. 2;

the fusion protein is any one of the following:

(A1) protein with amino acid sequence shown in SEQ ID No. 2;

(A2) and (C) 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;

in the fusion gene, the gene for coding the Aspergillus niger pectinase is shown as 1 st-1005 th site of SEQ ID No. 1.

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

in the fusion gene, the gene coding the cellulose binding protein of trichoderma reesei is shown as the 1174-2715 position of SEQ ID No. 1.

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

in the fusion gene, the gene coding the connecting peptide is shown as 1006-1173 of SEQ ID No. 1.

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

the fusion gene is a DNA molecule shown in SEQ ID No. 1.

7. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the nucleic acid molecule of any one of claims 2 to 6.

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

9. A kit for preparing a kelp hydrolysate comprising the enzyme preparation set according to claim 8 and kelp powder.

10. 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, expression cassette, transgenic cell line or recombinant bacterium according to claim 7 or the enzyme kit according to claim 8 or the kit according to claim 9 for the preparation of a kelp hydrolysate.

11. A method for preparing kelp hydrolysate comprises the following steps: adding artificial enzyme, cellulase 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.