CN109750022A - A kind of algin catenase Alg2A and its preparation method and application - Google Patents
A kind of algin catenase Alg2A and its preparation method and application Download PDFInfo
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- CN109750022A CN109750022A CN201910234540.9A CN201910234540A CN109750022A CN 109750022 A CN109750022 A CN 109750022A CN 201910234540 A CN201910234540 A CN 201910234540A CN 109750022 A CN109750022 A CN 109750022A
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- Prior art keywords
- alg2a
- algin catenase
- algin
- gene
- catenase
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
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Abstract
The invention discloses a kind of algin catenase Alg2A and its preparation method and application.The present invention is optimized the algin catenase encoding gene in Flavobacterium bacterial strain S20 (Flavobacterium sp.S20), the nucleic acid sequence after optimization is as shown in SEQ ID NO.2 according to the Preference of Pichia pastoris codon.Efficient secretory expression further is carried out using algin catenase encoding gene of the pichia yeast expression system to the optimization, obtains algin catenase, amino acid sequence is as shown in SEQ ID NO.1.The algin catenase that the present invention obtains has higher hydrolysing activity to sodium alginate substrate, the crude enzyme liquid that shake flask fermentation generates is the ability with the thorough degradation 1g sodium alginate of 0.1ml (protein content about 0.042mg), has good prospects for commercial application.
Description
Technical field
The invention belongs to algin catenase preparation technical fields, and in particular to a kind of efficient preparation of algin catenase
Method and application.
Background technique
Alginic acid is the different polysaccharide formed by guluronic acid and mannuronic acid by Isosorbide-5-Nitrae glucosides key connection, is brown alga
The main component of cell wall.Alginic acid and its esters, because it forms the performances such as gel with high viscosity and easily, in food, change
The fields such as work and pharmacy obtain wide application.However, higher molecular weight and limited water solubility limit alginic acid and its
The application of salt.In contrast, catabolite alginic acid oligosaccharides, because of its lower molecular weight and better bioactivity, tool
There is broader practice prospect.
Algin catenase can crack alignic glycosidic bond by β cancellation, and generation contains the brown of unsaturated double-bond
Alginic acid oligosaccharides.Currently, the algin catenase for having a large amount of separate sources is cloned, expresses and identifies.Application No. is
201110424529.2 patent discloses the gene of algin catenase Alg2A from Flavobacterium bacterial strain a kind of, and
Further the alginate lyase gene is cloned on coli expression carrier, expression obtains algin catenase Alg2A.
However, finding in further R&D process, there are still have technology to lack for the above-mentioned technical solution for preparing algin catenase Alg2A
It falls into, for example, resistant gene present in the bacteria residue that recombinant bacterial strain industrial fermentation processes generate pollutes the environment；Free matter
Grain carrier unstable expression, it is easy to be lost after repeatedly passing on；The algin catenase Alg2A of Bacillus coli expression in the cell,
Target enzyme could be obtained by having to pass through clasmatosis, increase the difficulty of large scale preparation.
Summary of the invention
It is an object of the present invention to provide the preparation method and applications of algin catenase Alg2A a kind of；It is intended to provide one kind
The specific algin catenase of economical and efficient.For the algin catenase for obtaining highly effective and safe, to deriving from, Flavobacterium is thin
The algin catenase encoding gene of bacterium optimizes, fully synthetic and secreting, expressing is realized in Pichia pastoris.That expresses is brown
Phycocolloid lyases has the application potential for realizing that alginic acid oligosaccharides is industrially prepared.
Present invention technical solution used for the above purpose is as follows:
The present invention is optimized by the codon to algin catenase encoding gene, so that it is in Pichia pastoris
Secreting, expressing, to efficiently quickly obtain the high algin catenase of hydrolysing activity.
A kind of algin catenase Alg2A provided by the invention, amino acid sequence is as shown in SEQ ID NO.1.It is described
The encoding gene of algin catenase Alg2A, nucleotide sequence is as shown in SEQ ID NO.2.
Algin catenase of the present invention Alg2A's the preparation method comprises the following steps: by nucleotide sequence such as SEQ ID NO.2 institute
The alginate lyase gene shown is constructed into expression vector, is then introduced into Pichia pastoris, and Fiber differentiation is simultaneously secreted
The algin catenase of expression.
The specific preparation step of the algin catenase Alg2A include: (1) used according to Pichia pastoris codon it is inclined
Good property carries out codon optimization to the alginate lyase gene original series from Flavobacterium；(2) by the base after optimization
Because carrying out fully synthetic and constructing into expression vector pGBG1；(3) after carrying out linearization for enzyme restriction to the expression vector of building, give up
Segment containing resistant gene recycles the segment containing alginate lyase gene and imports in Pichia pastoris GS115, lures
It leads culture and obtains the algin catenase of secreting, expressing.
During the encoding gene to algin catenase Alg2A optimizes, in order to make algin catenase exist
It is capable of the secreting, expressing of efficient stable in Pichia pastoris, the alginate lyase gene after optimization has lacked 5 ' end signal peptide sequences of coding
22 amino acid of column.It is that the signal peptide carried in pichia vector is utilized to realize secretion table when carrying out gene expression
It reaches, this is a key point so that optimization gene high efficient expression.
Using the algin catenase of inducing expression thick enzyme supernatant sodium alginate is hydrolyzed and is used efficient liquid phase and
Mass spectrometry method analyzes the degree of polymerization and composition of product.Analysis the result shows that: obtained crude enzyme liquid can be realized to brown alga
The complete hydrolysis of sour sodium.
Algin catenase of the present invention can be individually used for alginic acid and the degradation of its esters prepares alginic acid oligosaccharides；
Or the algin catenase is used in mixed way with other algin catenases, Synergistic degradation alginic acid and its esters.
Compared with prior art, the invention has the benefit that
1. alginate lyase gene of the invention derives from Flavobacterium, pichia pastoris yeast expression system point is used
Secrete expression.Pichia pastoris yeast (Pichia pastoris) expression system has been used for the food-grades such as lactase and phospholipase C
The expression of enzyme preparation, its own is also used for the production (GB2760-2014) of zytase.Therefore, using Flavobacterium source
Alginate lyase gene in Pichia pastoris secreting, expressing, it is few that product algin catenase can become food-grade alginic acid
The production enzyme preparation of sugar.
2. the alginate lyase gene in the present invention is according to the codon-bias of Pichia pastoris due to being optimized,
It may be implemented in efficient secretory expression in Pichia pastoris.Through determination of activity and conversion, 0.1mL crude enzyme liquid (about contains 0.042mg enzyme egg
It is white) can complete hydrolysis 1g sodium alginate, prepare the application potential of alginic acid oligosaccharides with scale.
3. compared with existing patented technology: the target gene after a, optimization eliminates ampicillin resistance before conversion,
Avoid resistant gene pollution on the environment present in the bacteria residue of later period recombinant bacterial strain industrial fermentation processes generation.B, lead to
It crosses genetic recombination mode target gene is integrated into Pichia pastoris genome, the plasmid vector expression of specific ionization is more stable,
It is not easy to lose because of repeatedly passing on.C, in Pichia pastoris secreting, expressing enzyme, since secretion is extracellular, there is no need into
The techniques such as row clasmatosis, the crude enzyme liquid that fermentation liquid obtains can be directly used for the preparation of alginic acid oligosaccharides without purifying.
Detailed description of the invention
Fig. 1 is that recombinant expression carrier alg2A-pGBG1 and its Xho I and Not I double digestion in the embodiment of the present invention 2 are produced
Object gel electrophoresis spectrum.
At the beginning of Fig. 2 is the recombinant expression Pichia pastoris GS115 bacterial strain sodium alginate substrate plate enzyme activity in the embodiment of the present invention 3
Sieve figure.
Fig. 3 is the fermented liquid supernatant of the Pichia yeast engineering containing alginate lyase gene in the embodiment of the present invention 3
SDS-PAGE map.
The HPLC that Fig. 4 is enzymolysis product alginic acid oligosaccharides AOS-Alg2A in the embodiment of the present invention 4 analyzes map.
Fig. 5 is the mass spectrogram of enzymolysis product alginic acid oligosaccharides AOS-Alg2A in the embodiment of the present invention 4.
Specific embodiment
Technical solution of the present invention is described in detail below with reference to embodiment.The reagent and biomaterial used below
It if not otherwise specified, is commercially produced product.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer suggests
Condition carry out.
The codon optimization of 1 alginate lyase gene of embodiment and full genome synthesis
Under the premise of not changing amino acid sequence, to from Flavobacterium bacterial strain S20 (Flavobacterium
Sp.S20 algin catenase encoding gene) carries out codon optimization, all Pichia pastoris preferences of the codon after optimization
Codon, particular sequence are shown in SEQ ID NO.2.The original series of gene order and algin catenase Alg2A after optimization are (such as
Shown in sequence SEQ ID NO.3, GenBank accession number:JF412659) consistency (identity) is 77%.
Algin cracking meanwhile in order to make algin catenase be capable of the secreting, expressing of efficient stable in Pichia pastoris, after optimization
Enzyme gene has lacked 22 amino acid of 5 ' end signal peptide sequences of coding.Gene order after optimization entrusts Beijing to hold up the industrial biology of section
Technology Co., Ltd.'s progress is fully synthetic, and the gene order for synthesizing acquisition is denoted as alg2A.
The expression vector establishment of 2 alginate lyase gene alg2A of embodiment
First using restriction enzyme Xho I and Not I to the cloning vector containing alginate lyase gene alg2A
Double digestion is carried out, target gene fragment is obtained, while double digestion is carried out to expression vector pGBG1 using identical restriction endonuclease, returns
Receive large fragment.Two recovery products are attached, and are obtained recombinant vector, are named as alg2A-pGBG1.To determine target algin
Lyase gene has been built up into carrier, carries out double digestion to recombinant vector using Xho I and Not I, and carry out to product
Agarose gel electrophoresis, as a result as shown in Figure 1.As can be seen from FIG. 1: after double digestion, occurring between 750bp and 1000bp
Target gene fragment is consistent with the fragment length 834bp of alg2A.
The screening of 3 algin catenase Pichia yeast engineering of embodiment and algin catenase preparation
By the recombinant plasmid alg2A-pGBG1 of acquisition after restriction enzyme BglII linearisation, gel electrophoresis is separated simultaneously
The nucleic acid large fragment containing target gene is cut, electric shock imports in Pichia pastoris GS115, passes through histidine auxotrophy MD plate
Upper screening obtains recombination bacterium colony.Picking 8 bacterium colonies therein are crossed to the BMMY agar plate containing 0.2% sodium alginate,
After 30 DEG C of culture 48h, the progress of 10% cetylpyridinium chloride(CPC) (Cetylpyridinium chloride, CPC) aqueous solution is poured into
Colour developing finds that 2,5,6 and No. 7 clone strains therein show algin lytic activity, referring to fig. 2, finishes for recombinant expression red
Yeast GS115 bacterial strain sodium alginate substrate plate enzyme activity primary dcreening operation figure.Picking No. 6 bacterial strain single colonies therein, are inoculated in 50ml
In BMGY culture medium, 48h is cultivated under 30 DEG C and 250rpm, supernatant is abandoned in centrifugation, and the BMMY that equivalent is added carries out inducing expression.24h
After add methanol to its final concentration of 1%, it is later primary every adding for 24 hours, be centrifuged after total induction 72h, supernatant is to contain
There is the crude enzyme liquid of algin catenase Alg2A.Using SDS-PAGE detect protein expression situation, result as shown in figure 3,
Occur two bands between 25-35kDa, is estimated as the algin catenase (predicted molecular weight 31.5kDa) and its sugar of expression
Base product.It is 0.42mg/ml that Bradford method, which measures the protein concentration in crude enzyme liquid,；DNS method (draws mark using glucose
Directrix curve, 1U are defined as enzyme amount needed for 1 μm of ol reduced sugar of hydrolysis generation in 1min), its specific enzyme activity is measured at 40 DEG C is
21.20U/ml.Above-mentioned MD agar plate, BMMY agar plate, BMGY culture medium, BMMY culture medium are that yeast expression system is normal
Culture medium can directly be bought or prepare according to existing literature technology.
Embodiment 4: algin catenase Alg2A enzymatic hydrolysis prepares alginic acid oligosaccharides
20g sodium alginate is weighed, is added in 200ml deionized water, the algin cracking prepared in above-described embodiment 3 is added
Enzyme Alg2A crude enzyme liquid 2ml.It is stirred to react 72h at 40 DEG C, adjusts the temperature to 90 DEG C of maintenance 1h, inactivates enzyme.Centrifugation removal is not allowed
Object, supernatant is freeze-dried to obtain finished product alginic acid oligosaccharides, is denoted as AOS-Alg2A.The alginic acid oligosaccharides for weighing a certain amount of preparation freezes
Dry-eye disease is configured to the acetonitrile solution (acetonitrile: water, 1:1, v/v) that alginic acid oligosaccharide concentration is 5mg/mL, for height after filtering
Effect liquid phase chromatogram analysis.High performance liquid chromatograph connects the signal detection that evaporative light scattering detector is used for oligosaccharides, uses
XAmide chromatographic column (Hua Puxinchuan Science and Technology Ltd.) separates alginic acid oligosaccharides, and acetonitrile concentration successively decreases (70%-
50%) mode elutes, and column temperature is 30 DEG C, detector air pressure 23psi, flow velocity: 1ml/min.Mobile phase is 0.1M ammonium formate
(pH3.2), acetonitrile and water.Elution time: 40min.As a result as shown in figure 4, from fig. 4, it can be seen that having obtained a variety of potential
Brown alga oligose component.Its component is analyzed using mass spectrum.Mass Spectrometry Conditions are using negative ion mode, ion source voltage:
3kV；Sheath gas: 40arb；Capillary temperature: 270 DEG C；Scanning range: 300-2000.Testing result is as shown in figure 5, from Fig. 5
It can be seen that the alginic acid oligosaccharides of different polymerization degree can be detected.
It should be noted that we attempt to optimize and synthesize during optimizing to alginate lyase gene
8 alginate lyase genes, but the gene that result only has two of them to optimize is realized in pichia yeast expression system
Efficient secreting, expressing, genes of the two optimizations are respectively derived from thermophilic sugar Pseudomonas bacterial strain 2-40 and described herein
Flavobacterium bacterial strain S20.The algin catenase that the gene of other optimizations is expressed in pichia yeast expression system does not have
Activity or activity it is lower, optimization gene be then can not in Bichi yeast system smooth secreting, expressing.
It above are only part preferred embodiment of the invention, the present invention is not limited in the content of embodiment.For ability
For the technical staff in domain, can there are various change and change in the conception range of technical solution of the present invention, it is made any
Variation and change, within that scope of the present invention.
Sequence table
<110>(Suzhou) Biotechnology Co., Ltd is believed in section's honor in
<120>a kind of algin catenase Alg2A and its preparation method and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 274
<212> PRT
<213>Flavobacterium (Flavobacterium sp. 141-8)
<400> 1
Leu Glu Lys Arg Glu Ala Glu Ala Gln Asp Lys Lys Ser Lys Ser Lys
1 5 10 15
Thr Ala Lys Ile Asp Trp Ser His Trp Thr Val Thr Val Pro Glu Glu
20 25 30
Asn Pro Asp Lys Pro Gly Lys Pro Tyr Ser Leu Gly Tyr Pro Glu Ile
35 40 45
Leu Asn Tyr Ala Glu Asp Lys Ile Ala Ser Lys Tyr Met Tyr Asp Asp
50 55 60
Pro Lys Asp Lys Ser Val Val Phe Tyr Ala Phe Pro Ser Gly Val Thr
65 70 75 80
Thr Ala Asn Thr His Tyr Ser Arg Ser Glu Leu Arg Glu Thr Met Glu
85 90 95
Thr Gly Ser Asn Lys Val Asn Trp Thr Phe Ala Lys Gly Gly Lys Met
100 105 110
Arg Gly Thr Tyr Ala Ile Asp Asp Ile Ser Lys Glu Pro Asp Gly Lys
115 120 125
Tyr Ser Arg Val Ile Ile Ala Gln Ile His Gly Val Leu Thr Asp Glu
130 135 140
Gln Arg Asp Leu Ile Gly Gln Lys Asp Asn Asn Ala Pro Pro Ile Leu
145 150 155 160
Lys Val Tyr Trp Asp Lys Gly Lys Ile Arg Val Lys Thr Lys Val Leu
165 170 175
Lys Asp Leu Asn Ala Pro Tyr Lys Glu Met Leu Leu Glu His Ala Trp
180 185 190
Gly Asp Asp Glu Gly Arg Asn Phe Lys Glu Lys Ile Asp Leu Asn Thr
195 200 205
Arg Phe Thr Leu Glu Val Lys Val Ser Asp Gly Arg Met Glu Val Ile
210 215 220
Leu Asn Asp Thr Glu Ser Leu Val Tyr Asp Asp Ile His Met Lys Lys
225 230 235 240
Trp Gly Ile Phe Glu Asn Tyr Phe Lys Ala Gly Asn Tyr Phe Gln Ser
245 250 255
Lys Thr Pro Gly Thr Phe Ala Lys Val Lys Ile Tyr Ser Leu Gln Val
260 265 270
Thr His
<210> 2
<211> 834
<212> DNA
<213>Flavobacterium (Flavobacterium sp. 141-8)
<400> 2
ctcgagaaga gagaggctga ggctcaagac aagaagtcca agtccaagac tgctaagatt 60
gactggtccc actggactgt tactgttcca gaggagaacc cagacaagcc aggtaagcca 120
tactccttgg gttacccaga gattttgaac tacgctgagg acaagattgc ttccaagtac 180
atgtacgacg acccaaagga caagtccgtt gttttctacg ctttcccatc cggtgttact 240
actgctaaca ctcactactc cagatccgag ttgagagaga ctatggagac tggttccaac 300
aaggttaact ggactttcgc taagggtggt aagatgagag gtacttacgc tattgacgac 360
atttccaagg agccagacgg taagtactcc agagttatta ttgctcaaat tcacggtgtt 420
ttgactgacg agcaaagaga cttgattggt caaaaggaca acaacgctcc accaattttg 480
aaggtttact gggacaaggg taagattaga gttaagacta aggttttgaa ggacttgaac 540
gctccataca aggagatgtt gttggagcac gcttggggtg acgacgaggg tagaaacttc 600
aaggagaaga ttgacttgaa cactagattc actttggagg ttaaggtttc cgacggtaga 660
atggaggtta ttttgaacga cactgagtcc ttggtttacg acgacattca catgaagaag 720
tggggtattt tcgagaacta cttcaaggct ggtaactact tccaatccaa gactccaggt 780
actttcgcta aggttaagat ttactccttg caagttactc actaagcggc cgcg 834
<210> 3
<211> 867
<212> DNA
<213>Flavobacterium (Flavobacterium sp. 141-8)
<400> 3
atgagcatac aattttcaaa aatcttatta ctaacggttc tagcaactgc tacaattagt 60
aatgcacagg ataaaaaatc aaaaagcaaa actgctaaaa ttgattggtc tcattggacg 120
gttactgtgc ctgaggagaa tccagataaa ccaggtaagc cgtactcttt agggtatcct 180
gaaatactaa attatgctga ggataaaatc gcatccaagt acatgtacga tgacccaaaa 240
gacaagtctg ttgtttttta tgcctttcct tcgggagtga ccacggctaa tacgcattat 300
tctcgttctg agctaagaga gacaatggaa actggtagca ataaggtcaa ctggacattt 360
gcaaaaggcg gtaaaatgag aggtacgtat gctattgacg acatttcaaa agagccagat 420
ggcaaataca gccgcgttat tattgcgcaa attcacggtg tattaacgga tgaacaacgt 480
gatttaattg gtcaaaaaga caacaatgca ccgcctattt tgaaagtgta ttgggataaa 540
ggaaaaattc gtgtgaaaac aaaagtactt aaagatttga acgcgcctta taaagaaatg 600
cttttagaac atgcttgggg tgatgatgaa ggtcgaaatt ttaaagagaa aatcgattta 660
aacacaaggt ttactctaga agtgaaagtt tcggatgggc gaatggaagt gattttaaat 720
gatacagaat cattggttta cgatgatatt cacatgaaaa aatgggggat attcgaaaat 780
tattttaaag cagggaatta ttttcaatct aaaacaccag gtacctttgc aaaggtaaaa 840
atatattctt tacaagttac tcattag 867
Claims (8)
1. a kind of algin catenase Alg2A, it is characterised in that: the amino acid sequence such as SEQ of the algin catenase Alg2A
Shown in ID NO.1.
2. the encoding gene of algin catenase Alg2A described in claim 1, it is characterised in that: the nucleotides sequence of the gene
Column are as shown in SEQ ID NO.2.
3. the preparation method of algin catenase Alg2A described in claim 1, this method are as follows: by nucleotide sequence such as SEQ
Alginate lyase gene shown in ID NO.2 is constructed into expression vector, is then introduced into Pichia pastoris, Fiber differentiation,
Obtain the algin catenase of secreting, expressing.
4. the preparation method of algin catenase Alg2A as claimed in claim 3, it is characterised in that: the expression vector is
pGBG1。
5. the preparation method of algin catenase Alg2A as claimed in claim 3, it is characterised in that: the Pichia pastoris is
GS115。
6. the preparation method of algin catenase Alg2A as claimed in claim 3, which is characterized in that the method includes such as
Lower step:
(1) nucleotide sequence alginate lyase gene as shown in SEQ ID NO.2 is carried out fully synthetic and constructed to expression
In carrier pGBG1；
(2) after carrying out linearization for enzyme restriction to the expression vector of aforementioned building, give up the segment containing resistant gene, recycling is containing brown
The segment of phycocolloid lyase gene；
(3) expression vector containing alginate lyase gene of aforementioned acquisition is imported in Pichia pastoris GS115, induction
Culture, obtains the algin catenase of secreting, expressing.
7. application of the algin catenase Alg2A described in claim 1 in alginic acid and its esters degradation.
8. application of the algin catenase Alg2A as claimed in claim 6 in alginic acid and its esters degradation, feature exist
In: the algin catenase Alg2A is individually used for alginic acid and its esters degradation prepares alginic acid oligosaccharides；Or it is described
Algin catenase Alg2A is used in mixed way with other algin catenases, Synergistic degradation alginic acid and its esters.
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