CN109929860A - A kind of preparation and application of fucoidin enzyme coding gene and enzyme - Google Patents

Abstract

The invention discloses a kind of fucoidin enzyme gene from bacillus cereus (Bacillus cereus) and its preparation methods and application of enzyme, utilize the technical method of genetic engineering, it will be in the gene cloning to coli expression carrier of the fucoidanase, obtain can the heterogenous expression enzyme E. coli recombinant stain, the fucoidanase of bacterial strain heterogenous expression preparation, can efficient degradation fucoidin.Fucoidanase provided by the invention can be widely applied to the fields such as the preparation of agricultural, food, feed addictive, medicine and low weight molecular fucoidan.

Description

A kind of preparation and application of fucoidin enzyme coding gene and enzyme

Technical field

The present invention relates to gene orders of a kind of fucoidanase and its preparation method and application.The present invention provides the rocks The recombinant plasmid and recombination engineered strain of polysaccharides enzyme and its application in terms of polysaccharide degradation.Rock algae provided by the invention Polysaccharase can be widely applied to agricultural, food, feed addition, medicine and oligomeric fucoidin the fields such as preparation.

Background technique

Fucoidin (fucoidan), also known as fucoidan are a kind of heteroglycan of high molecular weight, are usually existed In brown alga and some echinoderms.Fucoidin is mainly formed with L-fucose and sulfate group, and contains other monosaccharide groups Divide the uronic acid with different proportion.Fucoidin is a kind of unique active polysaccharide, is the main attack heat of current marine drug research and development One of point.It has a variety of physiological activity, it has been investigated that a variety of physiological activity of fucoidin and its molecular weight and sulfate radical Content and position have important relationship.Although fucoidin is a kind of water-soluble polysaccharide, its high molecular weight makes it each The application of aspect receives certain limitation.So seeking one kind in order to the multiple biological activities for preferably it being utilized to have It is particularly important that the method that fucoidin is degraded to low molecular weight fucosan can be simple and efficient.

It there is no the report of a large amount of industrial production low weight molecular fucoidans at present, production still rests on the laboratory research phase Between, the method for low weight molecular fucoidan is obtained mainly include the following types: (1) is extracted from natural material (brown alga), but is extracted Complex process, and yield is extremely low；(2) pass through physical method, such as radiation method, microwave method, supercritical ultrasonics technology.Although physical method It operates fairly simple, is easy to control, but since its degradation efficiency is lower and palliating degradation degree is limited, be unfavorable for mass production；(3) sharp It is obtained with the method for sour water solution fucoidin, but the segment of the sour water solution degree of polymerization easy to form not etc., the molecule of catabolite Amount distribution is larger, causes difficulty to isolating and purifying.Meanwhile sour water solution will be completed under the high temperature conditions, reaction condition is violent, therefore It is easy to damage the active structure of polysaccharide, and then influences lytic activity.(4) it is cut using hydrolase or cracking enzyme spcificity Disconnected fucoidin, to realize the reduction of its molecular weight.The great advantage of enzymatic isolation method is while reducing molecular weight to sulfuric acid Root does not have an impact.Meanwhile the reaction efficiency of enzyme is high, method is simple, easy to control, and later period separation is also easy, and enzyme is as a kind of Biological agent, the unfavorable factor that chemical reagent will not be brought to generate.So producing oligomeric rock using the method for enzymatic hydrolysis fucoidin Algae sugar is one relatively easy, is easy the production method of research.

Fucoidanase (EC3.2.1.51) is the class of enzymes of fucoidin of capable of effectively degrading.Both at home and abroad to fucoidin The research of degrading enzyme focuses mostly in microorganism, and wherein bacterium includes Flavobacterium section (Flavobacteriaceae), bacillus (Bacillus) and ocean proteus (Pseudoalteromonas).Fungi include Fusarium, Dendryphiella and Aspergillus.The bacterial strain of bacillus has certain general character of bacillus, can generate the inverse gemma of heat-resistant, have Conducive to the production of enzyme preparation, formulation and in the environment survives, colonizes and breed.Existing many researchs both at home and abroad are to this at present The a variety of bacterial strains of Pseudomonas carried out the research of physiological activity and antibacterial activity, but very to the report of fucoidin degrading enzyme gene It is few, and reported fucoidanase is weaker to the degrading activity of fucoidin, for example, 2003 Nian Wuke from The fucoidin enzymatic activity that Dendryphiella Arenaria TM94 is obtained is 24.3IU/mL；2004 Nian Wangpeng from The fucoidin enzymatic activity that Bacillus sp.H-TP2 is obtained is 1.29IU/mL；Dong Shujun in 2016 from The fucoidin enzymatic activity that Wenyingzhuangiafucanilytica is obtained is 2.9U/mg；It is unsuitable for large-scale application.Cause This, find it is a kind of be capable of efficiently, to stablize the fucoidanase of degradation fucoidin be to reduce low component amount fucoidin to be produced into This beneficial way.And since the content of fucoidanase in vivo is considerably less, a large amount expression is carried out by gene level It is to improve a kind of effective measures of fucoidin production of enzyme with characterization.

Summary of the invention

Novel bacillus cereus (Bacillus cereus) is derived from the first purpose of the invention is to provide a kind of Fucoidanase BcFucA and its encoding gene.

A second object of the present invention is to provide a kind of methods for preparing novel fucoidanase BcFucA.

Third object of the present invention is to provide containing the fucoidanase BcFucA DNA recombinant expression plasmid and Recombination engineered strain.

Fourth object of the present invention is to provide a kind of application of novel fucoidanase in fucoidin degradation.

Fucoidanase BcFucA provided by the present invention, the bacillus cereus isolated and purified in rotten seaweed Bacillus cereus, the fucoidin enzyme coding gene BcFucA therefrom amplified have in following nucleotide sequence feature One or two or more kinds:

1) in sequence table SEQ ID NO.1 DNA (DNA) sequence；

2) in polynucleotide SEQ ID NO.2 amino acid sequence DNA (DNA) sequence；

3) homology with SEQ ID NO.1 DNA (DNA) sequence limited reaches 80% or more, and energy DNA (DNA) sequence of the protein of coding degradation fucoidin；

4) one or several nucleotide are carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table to take Coding obtained from generation, missing or addition has the active nucleotide sequence of fucoidanase.

The present invention also provides the amino acid sequences of fucoidanase BcFucA, have one of following feature or two kinds More than:

1) 1-1136 amino acids residue sequence of the SEQ ID NO.2 since aminoterminal in sequence table, wherein 1- 1128 is, with the active amino acid sequence of fucoidanase BcFucA, 1129-1136 are restriction enzyme site and His-Tag Amino acid sequence；

2) by 1-1136 or 1-1128 amino acids residue of the SEQ ID NO.2 since aminoterminal in sequence table into Row one or more amino acid substitutions, deletions, or additions and formed constant with fucoidanase fucoidin enzymatic activity Amino acid sequence.

The amino acid sequence and its nucleotide coding sequence of fucoidanase BcFucA of the invention can also be according to prediction Fucoidanase BcFucA amino acid sequence and its artificial synthesized acquisition of nucleotide coding sequence.

The method of preparation and reorganization enzyme BcFucA is that fucoidanase gene cloning is entered to recombinant expression carrier, imports host Cell obtains the fucoidanase of recombinant expression.

Above-mentioned fucoidin enzyme gene, nucleotide sequence have one of following feature or two kinds or more:

1) DNA (DNA) sequence with SEQ ID NO.1 in sequence table；

2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequence is encoded；

3) one or more nucleosides is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table Coding obtained from acid replaces, misses or adds has the active nucleotide sequence of fucoidanase；

The expression vector of the recombinant expression fucoidanase BcFucA can be coli expression carrier, yeast table Up to carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungi expression Carrier, plant expression vector, insect expression vector or mammalian cell expression vector etc..

For recombinantly expressing the recombinant bacterium or transgenic cell line of fucoidanase BcFucA, escherichia coli host can be Cell (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α), Yeast host cells (such as Saccharomyces cerevisiae, Pichiapastoris, Kluyveromyceslactis Deng), hay bacillus host cell (such as Bacillus subtilis R25, Bacillussubtilis9920), lactic acid bacteria place Chief cell (such as Lactic acid bacteria COCC101), actinomyces host cell (such as Streptomyces spp. Deng), filamentous fungal host cell (such as Trichodermaviride, Trichodermareesei, Aspergillusniger, Aspergillusnidulans etc.), insect cell (such as Bombyxmori, Antharaea eucalypti) or mammal Cell (such as Chinese hamster ovary cell CHO, baby hamster kidney cell BHK, CHL cells CHL).

The gene order of fucoidanase BcFucA of the invention is by round pcr from bacillus cereus Clone obtains in (Bacillus cereus).The long 3411bp in the gene coding region, belongs to glycoside hydrolase glycoside 65 family of hydrolase (GH).

Fucoidanase provided by the invention degradation fucoidin in can apply, including in applying below one kind or Two kinds:

1) in the glycosidic bond of fracture fucoidin, the application in oligomeric fucoidin or oligosaccharides is obtained；

2) it after being mixed with other fucoidanases, is applied in terms of collaboration is broken fucoidin glycosidic bond.

The fucoidanase BcFucA that the present invention is obtained from Recombinant protein expression, can with efficient degradation fucoidin, When using fucoidin as substrate, having best Rate activity under conditions of 50 DEG C, pH4.0 is 3.0U/mg.

Fucoidanase BcFucA of the invention can be widely applied to agricultural, food, feed addition, medicine and oligomeric rock algae The fields such as the preparation of polysaccharide.

Detailed description of the invention

Fig. 1: fucoidin enzyme gene BcFucA agarose gel electrophoresis detection.

The SDS-PAGE figure of Fig. 2: fucoidanase BcFucA expression and purifying.The sample that each swimming lane is added is respectively: swimming Road M: Protein Marker, swimming lane 1-BcFucA do not induce thallus always to precipitate, swimming lane 2-BcFucA bacteria break supernatant, swimming lane 3- BcFucA upper prop flows through liquid three times, and the elution of swimming lane 4-20mM imidazoles flows through liquid, and the elution of swimming lane 5-200mM imidazoles flows through liquid.

Fig. 3: influence curve of the pH value to fucoidanase BcFucA.

Fig. 4: influence curve of the temperature to fucoidanase BcFucA.

Fig. 5: fucoidanase BcFucA to the MALDI-TOF-MS map of fucoidin catabolite.

Specific embodiment

Sequence table

The information of SEQIDNo.1

(a) sequence signature

Length: 3411 nucleotide

Type: nucleotide

Chain: single-stranded

(b) molecule type: DNA

Sequence description: SEQ ID NO.1

ATGAAAGCAACAGCACCTATTACGATTGAAAATCCAAAACCAGAGGGAAAGAAACTATCACTTTGGTAC AATGAGCCAGCAAAAGATTGGGAAAAACAAGCATTGCCAATCGGTAATGGCTATATGGGTGGAATGGTCTTTGGTGG AGTCCAACAAGAACGTATTCAGTTTAATGAAAAAACGCTTTGGACTGGCGGACCAAGCAGTACAAGTGAGTATACGT ACGGGAATCGTGATGGGGCAGCGAGTCACTTAGGTAGCATTAGAGAAAAGTTATCAAAAGGTGATAAATCAGGAGCA GAAAGGGAATCTACGCAATTTCTCACAGGTCTCCAAAAAGGCTTTGGTTCCTATCAAAATTTCGGGGACATATACTT AGATTTTAATATGCCAGATGGGTCTTCATTTTCCAATTATCGCAGGGAATTAAATTTAAATGAGGGAATTTCTACGG TGTCCTATAACTATAAAGGTGTTCAATATAATAGGGAATATTTTGCAAGTTACCCTGATCGCGTTATGGTCATGCGT TTAACTGCTAGTGAATCGAAACAGCTTTCATTAGATGTTAGACCAACGAGTGCACAGGGAGGGCAAGTAACGTCCAA AGATAACAAAATCACGATAAAAGGACAAATAGCGAATAATGGAATGAAATATGAATCGGAATTTAAAGTACTAAATG AAGGCGGTACATTAACAGCAGAGAATGGGAAAATTAAAGTAGCAAATGCAGATAGTTTAACAATTATTATGACTGCA GCTACTGATTATGAAAATAAGTACCCGAGTTATAAGGGGGAAGATCCACATCAAAAGGTTGAAAAAATTATGTCCGC TATTTCTAACAAGAGTTATGAGGTTTTAAAATATACACATATAAAAGACTATTACTCTTTATTTAATCGTGTTTCTT TAAATTTAGGAGGAGAAAAACCATCCGTTCCAACAAATGAATTACTAGCATCTTACAGTAAAGAAAACAGTAAGTAT TTAGAAGAACTATTCTTCCAATATGGAAGATATTTGCTTATTTCTTCTTCAAGACCAGGAACGCTTCCTGCAAATTT ACAAGGAGTATGGAATAATTCGAACACACCACCGTGGGAAAGTGATTATCATTTTAATATTAATTTGCAAATGAATT ATTGGCCTGCAGAAGTGACAAATCTATCTGAAACAGCTGAGCCATTAATGGATTATGTTGATTCATTGAGAGAACCG GGAAGGGTTTCAGCAGAAAAACATTTTGGTGTTACAGGCGGCGGGTGGACCGTTAACACGATGAATAATCCATTTGG ATTTACTGCGCCTGGATGGGGATTAGGCTGGGGATGGGCACCTAGTGCGAATGCCTTTATTGGTCAAAACCTTTGGG AGCATTACAAGTTTACGGATGATAAACAATACTTACAAGAAAAGATTTATCCGATACTGAAAGAAGCGGCAGTGTTT CATAGTAAGTTTTTAGTAGAAGATCAAAATAAGAAGTTAGTTGTTTCTCCGTGCTGGTCTCCAGAGCTTGGTGGCAT TTCTAATGGATGTGCCTTTGACCAACAGTTAGTATATGAATTGTTCTCTAATGTAATTGAAGCGAGTGAAGTTTTAC AGGTTGATAATGTATTTCGGGATGAGTTAAAGGCGAAAAGGGATAAATTATTCCCGCCAATTCAAATTGGTAGATAT GGTCAGGTGCAAGAGTGGAAGGACGATATAGATGACCCTGGCGAAACGCATAGGCATATATCCCAGTTAGTTGCGCT ATATCCGGGAAGTATGATTAATCACAATACACCAGAATGGCTAGAGGCAGCTAAAGTTACTTTAAACCATCGTGGTG ACGAGGGAACAGGCTGGAGTAAAGCGAATAAAATTAACTTATGGGCACGTTTGTTAGATGGTGACCATGCGTATAAA ATCCTGCAAGGGCAACTTACTGGAAGTACGCTCAGCAACTTATTTGATACCCATCCGCCGTTTCAAATTGATGGGAA TTTTGGCGCAACATCAGGAATTGCTGAAATGTTAATTCAAAGCCATACCGACTCCATTCAACTATTGCCTGCACTTC CGAAGGCTTGGAAAGACGGTTCTTATAAAGGATTAAGAGCACGCGGTGCATTTACGATTGATGCGGATTGGAAAAAT GGCACGCCTACAGTTATTCAGGTGACTTCTGATCATGGAAACGATGTAAAGTTAAAAAGTCCGATATTCAATACATC GTTTACTGTGACAAGAGTCGGTACGAATACACCGGTGCCATTTACGAAGGATGGCGATACGATTTCATTTAAAACGG AAGCAGGTAAAAAGTATAAAATTGAGTCTATGCTTAGTTTCGATCTAGAATCTCCCAGCAGTGTAACAGCAGGAAAT ACAGTTAAGGTAAAGGCAAACTTATCAAACTTTGGTACGCTGAAATCTTCATCTGGAGAAGTGGTTGTAAAGGCTCC AGAGAGCTGGAATGTGAAGCCAATTAAAGTAGCTTTTGAAGGCGTTGAACCAGGGGAATCGAAAACAATTGAGGCAG ATTTATCTGTCCCTATTGATGTAGTTGCTAATAAGTATTCCATTGAAGCGGAGGTAACTACAGATAGCGGGACGATT AGTAAATCCAATCAAATAGAAGTAACGCCAGCAGTAAAGCTAATTTCGGCAAATGTTGACCCATATCCAATTAGTAG TGAAGGTGGTTCGACTACATTAAAGGTAAAGGTCCAAAATGAAATAAAGGAAAAAGTTACACCTGGAAAAATTGAAT TACAATTACCAGAAGGCTGGAATGCACAGCCATTAGCGACAGACTTTCAATTATCGGCAGGAGGAGAGGAAACATAT TCGTTTGTTATCACGCCACCATCCCAGTTTAAAGGAGTAAAAGAAGTTGAAGTTTCTGTAAAATTGGGGAATGCGGT GGTAACATCAACGAAAGTGCAAGTTGCTTCCGGCGGCATCTACCTTAGTGACATTTCATGGGTAAAAGCAACTGCTG GCTGGGCAACGGTTCAAAAGGATAAAAGTACCGACAAAAATCCAATTTCATTACTCGGAACGACAGGGCCGATTACG TACAAAAAAGGAATCGGAACGCATTCGAAGTCAGAAATTACGTACGACATATCAAATTCAACTTACAAACGATTTCA TTCATACGTAGGAATCGACCAGGAGCCTGGTGGTAAAGGCGGTTCAGTCGTATTTAAAGTGCTACTTGATGGGGCCG AAGTATTTAATAGCGGTACAATGTACTATAATACGCCAGCAAAATTTGTTGATGTTGACCTTACAGGTAAAAAAGAA TTAAAACTAGTCGTTGATGATGCAGGTAACGGTAACGGAAATGATCATGCGGACTGGGCGGATGCTTGGTTGTGTTT TAAACTCGAGCACCACCACCACCACCACTGA

The information of SEQIDNo.2

(a) sequence signature

Length: 1136 amino acid

Type: amino acid

Chain: single-stranded

(b) molecule type: albumen

Sequence description: SEQ ID NO.2

MKATAPITIENPKPEGKKLSLWYNEPAKDWEKQALPIGNGYMGGMVFGGVQQERIQFNEKTLWTGGPSS TSEYTYGNRDGAASHLGSIREKLSKGDKSGAERESTQFLTGLQKGFGSYQNFGDIYLDFNMPDGSSFSNYRRELNLN EGISTVSYNYKGVQYNREYFASYPDRVMVMRLTASESKQLSLDVRPTSAQGGQVTSKDNKITIKGQIANNGMKYESE FKVLNEGGTLTAENGKIKVANADSLTIIMTAATDYENKYPSYKGEDPHQKVEKIMSAISNKSYEVLKYTHIKDYYSL FNRVSLNLGGEKPSVPTNELLASYSKENSKYLEELFFQYGRYLLISSSRPGTLPANLQGVWNNSNTPPWESDYHFNI NLQMNYWPAEVTNLSETAEPLMDYVDSLREPGRVSAEKHFGVTGGGWTVNTMNNPFGFTAPGWGLGWGWAPSANAFI GQNLWEHYKFTDDKQYLQEKIYPILKEAAVFHSKFLVEDQNKKLVVSPCWSPELGGISNGCAFDQQLVYELFSNVIE ASEVLQVDNVFRDELKAKRDKLFPPIQIGRYGQVQEWKDDIDDPGETHRHISQLVALYPGSMINHNTPEWLEAAKVT LNHRGDEGTGWSKANKINLWARLLDGDHAYKILQGQLTGSTLSNLFDTHPPFQIDGNFGATSGIAEMLIQSHTDSIQ LLPALPKAWKDGSYKGLRARGAFTIDADWKNGTPTVIQVTSDHGNDVKLKSPIFNTSFTVTRVGTNTPVPFTKDGDT ISFKTEAGKKYKIESMLSFDLESPSSVTAGNTVKVKANLSNFGTLKSSSGEVVVKAPESWNVKPIKVAFEGVEPGES KTIEADLSVPIDVVANKYSIEAEVTTDSGTISKSNQIEVTPAVKLISANVDPYPISSEGGSTTLKVKVQNEIKEKVT PGKIELQLPEGWNAQPLATDFQLSAGGEETYSFVITPPSQFKGVKEVEVSVKLGNAVVTSTKVQVASGGIYLSDISW VKATAGWATVQKDKSTDKNPISLLGTTGPITYKKGIGTHSKSEITYDISNSTYKRFHSYVGIDQEPGGKGGSVVFKV LLDGAEVFNSGTMYYNTPAKFVDVDLTGKKELKLVVDDAGNGNGNDHADWADAWLCFKLEHHHHHH

1 fucoidanase full-length gene of embodiment clone

Reference gene group DNA purification kit (green skies LOT030217170602) operating procedure extracts waxy gemma bar The genomic DNA of bacterium.To The National Center for Biotechnology Information (NCBI) database After middle fucoidanase gene order carries out Multiple Sequence Alignment analysis, specific primer BcFucA-F:5 '-is designed CGGACGCATATGAAAGCAACAGCACCTATTAC-3'；BcFucA-R:5' GACGCGCTCGAGTTTAAAACTCAACCAAGC-3 ', using the genomic DNA of the bacillus cereus of extraction as template, amplification Encode the gene order (not including signal peptide gene) of fucoidanase maturation protein.PCR reaction condition are as follows: 94 DEG C of 3min, 1 Circulation；94 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 3min30s, 30 circulations；72 DEG C of 5min, 1 circulation.PCR product carries out agarose (see Fig. 1) after gel electrophoresis analysis, gel extraction is carried out to target gene, the method through double digestion is connected to prokaryotic expression carrier It is sequenced after pET21a is upper.

2 fucoidanase gene sequencing of embodiment

Sequencing result is using the Basic Local Alignment Search Tool (BLAST) in GenBank database Analysis, DNAMAN software carry out Multiple Sequence Alignment, VectorNTI analytical sequence information.

The fucoidin enzyme gene (being named as BcFucA) of acquisition encodes head of district 3411bp, nucleotide sequence such as SEQ ID Shown in NO 1.BcFucA encodes 1136 amino acid and a terminator codon, amino acid sequence such as 2 institute of SEQ ID NO Show, protein theoretical molecular weight is 127kDa, and prediction isoelectric point is 5.8.The amino acid of BcFucA coding includes 3 carbon hydrates Object binding modules NPCBM (Carbohydrate Binding Module) and a GH65 family structure domain.

Recombinant expression and purifying of the embodiment 3BcFucA gene in Escherichia coli

For the ease of the recombinant expression of gene, NdeI and XhoI digestion position is introduced respectively in the upstream and downstream primer of design Point.PCR cleaning product BcFucA and expression vector pET21a is subjected to double digestion with NdeI and XhoI respectively, digestion products are through clear After clean recycling, T is used₄DNA ligase connects (linked system: (5mLT₄DNALigase 0.5 μ L, 10 × T₄DNALigase 2 μ L of Buffer 0.5mL, pET21a, 2 μ L of PCR product), condition of contact: ambient temperature overnight connection.).5 μ L connection products are taken to convert E.coli TOP10 competent cell is coated on the solid Luria-Bertani culture medium containing 100 μ g/mL ampicillins, 37 DEG C of culture 16-18h.Picking monoclonal carries out bacterium colony PCR verifying using degenerate primer, will expand correct monoclonal access It is cultivated in liquid Luria-Bertani culture medium containing 100 μ g/mL ampicillins, extracts plasmid；Using enzyme NdeI and XhoI carries out double digestion to the plasmid of extraction, and as a result correct recombinant plasmid send Hua Da gene sequencing.Sequencing result shows BcFucA gene shown in SEQ ID NO 1 is inserted between NdeI the and XhoI restriction enzyme site of pET21a, and direction of insertion is just Really, it was demonstrated that the recombinant plasmid is named as pET21a-BcFucA by construction of recombinant plasmid success.

By pET21a-BcFucA Transformed E .coli BL21 (DE3), inducing expression and purifying are carried out to it.Use polyacrylamide The expression of amine detected through gel electrophoresis fucoidanase BcFucA and purifying situation, as a result as shown in Fig. 2, fucoidin after purification Enzyme BcFucA is in single band on running gel, and position matches with the molecular weight of prediction.

The determination of activity of 4 fucoidanase BcFucA of embodiment and characterization analysis

(1) vitality test of fucoidanase BcFucA

Using the 4- nitrobenzene-alpha-L- fucopyranoside of 50 μ L0.3mM as substrate, 20mL recombinase is added BcFucA reacts 20min, and the Na of 70 μ L 0.5M is added₂CO₃Reaction is terminated, its absorbance at 410nm is measured.Enzyme activity Unit definition are as follows: enzyme amount needed for 1 μ g p-nitrophenyl of release is an enzyme activity unit (U) per minute.Protein concentration uses green Skies BCA determination of protein concentration kit is measured.

(2) influence of the pH to recombinase BcFucA

Under conditions of 50 DEG C, respectively with 50mL 0.3mM, pH3.0-10.0 (pH3.0Gly-HCl, pH4.0-5.0HAc- NaAc, pH6.0-7.0Na₂HPO₄-NaH₂PO₄, pH8.0Tris-HCl, pH9.0-10.0Gly-NaOH) pNP- fucoidin For substrate, 20mL recombinase BcFucA is added, reacts 20min, the Na of 70 μ L 0.5M is added₂CO₃Terminate reaction, measure its Absorbance at 410nm.Using the enzyme of inactivation as control, with the highest value of activity for 100%, measurement enzyme at each reaction pH Relative activity.Curve is drawn according to relative activity of the enzyme at different pH, determines the optimal reaction pH of enzyme.As a result such as Fig. 3 institute Show, the optimal reaction pH of BcFucA is 4.0, and with being raised and lowered for pH, the activity of BcFucA is reduced.

(3) influence of the temperature to recombinase BcFucA

Under conditions of pH4.0, using the pNP- fucoidin of 50 μ L 0.3mM as substrate, 20mL recombinase is added BcFucA reacts 20min, and the Na of 70mL 0.5M is added₂CO₃Reaction is terminated, its absorbance at 410nm is measured.With inactivation Enzyme be control, be 100% to calculate opposite enzyme activity to react highest enzyme activity, drawn according to the relative activity of enzyme at different temperatures Koji-making line.As a result as shown in figure 4, the optimal reactive temperature of BcFucA is 50 DEG C.

5 recombinase BcFucA of embodiment degradation fucoidin product analysis

After 0.5% (w/v) fucoidin and recombinase BcFucA are mixed in the ratio of 9:1 (volume ratio), in 50 DEG C of items 2h is reacted under part, after Sevage method removing protein, Matrix-Assisted Laser Desorption Ionization Time of Flight is carried out to its product (MALDI-TOF-MS) it analyzes.As shown in figure 5, the oligomeric of a variety of degree of polymerization can be generated to the degradation of fucoidin in BcFucA Sugar, DP=1-5.Therefore, BcFucA can be used for the preparation of oligomeric fucose and the research with fucoidin degradation related fields, Including agricultural, food, feed addition, medicine and low molecular weight fucose the fields such as preparation.

Sequence table

<110>Dalian Inst of Chemicophysics, Chinese Academy of Sciences

<120>preparation and application of a kind of fucoidin enzyme coding gene and enzyme

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3411

<212> DNA

<213>Bacillus cercus (Bacillus cereus)

<400> 1

atgaaagcaa cagcacctat tacgattgaa aatccaaaac cagagggaaa gaaactatca 60

ctttggtaca atgagccagc aaaagattgg gaaaaacaag cattgccaat cggtaatggc 120

tatatgggtg gaatggtctt tggtggagtc caacaagaac gtattcagtt taatgaaaaa 180

acgctttgga ctggcggacc aagcagtaca agtgagtata cgtacgggaa tcgtgatggg 240

gcagcgagtc acttaggtag cattagagaa aagttatcaa aaggtgataa atcaggagca 300

gaaagggaat ctacgcaatt tctcacaggt ctccaaaaag gctttggttc ctatcaaaat 360

ttcggggaca tatacttaga ttttaatatg ccagatgggt cttcattttc caattatcgc 420

agggaattaa atttaaatga gggaatttct acggtgtcct ataactataa aggtgttcaa 480

tataataggg aatattttgc aagttaccct gatcgcgtta tggtcatgcg tttaactgct 540

agtgaatcga aacagctttc attagatgtt agaccaacga gtgcacaggg agggcaagta 600

acgtccaaag ataacaaaat cacgataaaa ggacaaatag cgaataatgg aatgaaatat 660

gaatcggaat ttaaagtact aaatgaaggc ggtacattaa cagcagagaa tgggaaaatt 720

aaagtagcaa atgcagatag tttaacaatt attatgactg cagctactga ttatgaaaat 780

aagtacccga gttataaggg ggaagatcca catcaaaagg ttgaaaaaat tatgtccgct 840

atttctaaca agagttatga ggttttaaaa tatacacata taaaagacta ttactcttta 900

tttaatcgtg tttctttaaa tttaggagga gaaaaaccat ccgttccaac aaatgaatta 960

ctagcatctt acagtaaaga aaacagtaag tatttagaag aactattctt ccaatatgga 1020

agatatttgc ttatttcttc ttcaagacca ggaacgcttc ctgcaaattt acaaggagta 1080

tggaataatt cgaacacacc accgtgggaa agtgattatc attttaatat taatttgcaa 1140

atgaattatt ggcctgcaga agtgacaaat ctatctgaaa cagctgagcc attaatggat 1200

tatgttgatt cattgagaga accgggaagg gtttcagcag aaaaacattt tggtgttaca 1260

ggcggcgggt ggaccgttaa cacgatgaat aatccatttg gatttactgc gcctggatgg 1320

ggattaggct ggggatgggc acctagtgcg aatgccttta ttggtcaaaa cctttgggag 1380

cattacaagt ttacggatga taaacaatac ttacaagaaa agatttatcc gatactgaaa 1440

gaagcggcag tgtttcatag taagttttta gtagaagatc aaaataagaa gttagttgtt 1500

tctccgtgct ggtctccaga gcttggtggc atttctaatg gatgtgcctt tgaccaacag 1560

ttagtatatg aattgttctc taatgtaatt gaagcgagtg aagttttaca ggttgataat 1620

gtatttcggg atgagttaaa ggcgaaaagg gataaattat tcccgccaat tcaaattggt 1680

agatatggtc aggtgcaaga gtggaaggac gatatagatg accctggcga aacgcatagg 1740

catatatccc agttagttgc gctatatccg ggaagtatga ttaatcacaa tacaccagaa 1800

tggctagagg cagctaaagt tactttaaac catcgtggtg acgagggaac aggctggagt 1860

aaagcgaata aaattaactt atgggcacgt ttgttagatg gtgaccatgc gtataaaatc 1920

ctgcaagggc aacttactgg aagtacgctc agcaacttat ttgataccca tccgccgttt 1980

caaattgatg ggaattttgg cgcaacatca ggaattgctg aaatgttaat tcaaagccat 2040

accgactcca ttcaactatt gcctgcactt ccgaaggctt ggaaagacgg ttcttataaa 2100

ggattaagag cacgcggtgc atttacgatt gatgcggatt ggaaaaatgg cacgcctaca 2160

gttattcagg tgacttctga tcatggaaac gatgtaaagt taaaaagtcc gatattcaat 2220

acatcgttta ctgtgacaag agtcggtacg aatacaccgg tgccatttac gaaggatggc 2280

gatacgattt catttaaaac ggaagcaggt aaaaagtata aaattgagtc tatgcttagt 2340

ttcgatctag aatctcccag cagtgtaaca gcaggaaata cagttaaggt aaaggcaaac 2400

ttatcaaact ttggtacgct gaaatcttca tctggagaag tggttgtaaa ggctccagag 2460

agctggaatg tgaagccaat taaagtagct tttgaaggcg ttgaaccagg ggaatcgaaa 2520

acaattgagg cagatttatc tgtccctatt gatgtagttg ctaataagta ttccattgaa 2580

gcggaggtaa ctacagatag cgggacgatt agtaaatcca atcaaataga agtaacgcca 2640

gcagtaaagc taatttcggc aaatgttgac ccatatccaa ttagtagtga aggtggttcg 2700

actacattaa aggtaaaggt ccaaaatgaa ataaaggaaa aagttacacc tggaaaaatt 2760

gaattacaat taccagaagg ctggaatgca cagccattag cgacagactt tcaattatcg 2820

gcaggaggag aggaaacata ttcgtttgtt atcacgccac catcccagtt taaaggagta 2880

aaagaagttg aagtttctgt aaaattgggg aatgcggtgg taacatcaac gaaagtgcaa 2940

gttgcttccg gcggcatcta ccttagtgac atttcatggg taaaagcaac tgctggctgg 3000

gcaacggttc aaaaggataa aagtaccgac aaaaatccaa tttcattact cggaacgaca 3060

gggccgatta cgtacaaaaa aggaatcgga acgcattcga agtcagaaat tacgtacgac 3120

atatcaaatt caacttacaa acgatttcat tcatacgtag gaatcgacca ggagcctggt 3180

ggtaaaggcg gttcagtcgt atttaaagtg ctacttgatg gggccgaagt atttaatagc 3240

ggtacaatgt actataatac gccagcaaaa tttgttgatg ttgaccttac aggtaaaaaa 3300

gaattaaaac tagtcgttga tgatgcaggt aacggtaacg gaaatgatca tgcggactgg 3360

gcggatgctt ggttgtgttt taaactcgag caccaccacc accaccactg a 3411

Claims (8)

1. a kind of fucoidin enzyme gene, nucleotide sequence has one of following feature or two kinds or more:

1) DNA (DNA) sequence with SEQ ID NO.1 in sequence table；

2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequence is encoded；

3) one or more nucleotide is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table to take Coding obtained from generation, missing or addition has the active nucleotide sequence of fucoidanase；

4) homology with SEQ ID NO.1 DNA (DNA) sequence limited reaches 80% or more, and can encode DNA (DNA) sequence of the protein of degradation fucoidin.

2. a kind of fucoidanase of fucoidin enzyme gene coding described in claim 1, it is characterised in that: its amino acid sequence Column have one of following feature or two kinds:

1) 1-1137 amino acids residue sequence of the SEQ ID NO.2 since aminoterminal in sequence table；

2) one or more amino acid substitution, missing are carried out to amino acid sequence shown in SEQ ID NO.2 in sequence table Or addition and formed have the active amino acid sequence of fucoidanase.

3. a kind of preparation method of fucoidanase as claimed in claim 2, it is characterised in that: by fucoidanase gene cloning Enter recombinant expression carrier, imports host cell, obtain the fucoidanase of recombinant expression；

Above-mentioned fucoidin enzyme gene, nucleotide sequence have one of following feature or two kinds or more:

1) DNA (DNA) sequence with SEQ ID NO.1 in sequence table；

2) DNA (DNA) sequence of SEQ ID NO.2 amino acid sequence is encoded；

3) one or more nucleotide is carried out to DNA (DNA) sequence of SEQ ID NO.1 in sequence table to take Coding obtained from generation, missing or addition has the active nucleotide sequence of fucoidanase；

4) homology with SEQ ID NO.1 DNA (DNA) sequence limited reaches 80% or more, and can encode DNA (DNA) sequence of the protein of degradation fucoidin.

4. according to the method for claim 3, it is characterised in that: the expression vector of the recombinant expression fucoidanase, Refer to coli expression carrier, Yeast expression carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomycete expression Carrier, phage vector, filamentous fungi expression vector, plant expression vector, insect expression vector or mammalian cell expression One of carrier or two kinds or more.

5. according to the method for claim 3, it is characterised in that: the host cell, i.e., for recombinantly expressing fucoidin The recombinant bacterium or transgenic cell line of enzyme, refer to e. coli host cell (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichiapastoris, Kluyveromyceslactis etc.), hay bacillus host it is thin Born of the same parents (such as Bacillus subtilis R25, Bacillus subtilis9920), lactic acid bacteria host cell (such as Lactic Acidbacteria COCC101 etc.), actinomyces host cell (such as Streptomyces spp.), filamentous fungi host it is thin Born of the same parents (such as Trichodermaviride, Trichodermareesei, Aspergillusniger, Aspergillusnidulans Deng), insect cell (such as Bombyxmori, Antharaea eucalypti), mammalian cell (such as Chinese hamster ovary One of cell CHO, baby hamster kidney cell BHK, CHL cells CHL etc.).

6. a kind of application of fucoidanase as claimed in claim 2 in degradation fucoidin.

7. applying according to claim 6, it is characterised in that: including in applying below one kind or two kinds:

1) in the glycosidic bond of fracture fucoidin, the application in the fucoidin or oligosaccharides of low molecular weight is obtained；

2) application after being mixed with other fucoidin degrading enzymes, in terms of collaboration is broken fucoidin glycosidic bond.

8. a kind of fucoidanase as claimed in claim 2 is in degradation 4- nitrobenzene-alpha-L- fucopyranoside (pNP- rock Polysaccharides) in application.