CN110452919A - A kind of truncated algin catenase Aly7B-CDII gene and its application - Google Patents
A kind of truncated algin catenase Aly7B-CDII gene and its application Download PDFInfo
- Publication number
- CN110452919A CN110452919A CN201910854192.5A CN201910854192A CN110452919A CN 110452919 A CN110452919 A CN 110452919A CN 201910854192 A CN201910854192 A CN 201910854192A CN 110452919 A CN110452919 A CN 110452919A
- Authority
- CN
- China
- Prior art keywords
- aly7b
- cdii
- truncated
- algin
- algin catenase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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- 239000000737 potassium alginate Substances 0.000 description 1
- 235000010408 potassium alginate Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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Abstract
The present invention relates to a kind of truncated alginate lyase gene Aly7B-CDII, and nucleotide sequence is as shown in SEQ ID NO.1.By on this alginate lyase gene Aly7B-CDII Cloning Transformation to coli expression carrier, obtain can heterogenous expression E. coli recombinant stain, with the bacterial strain heterogenous expression truncate.Zymologic property characterization result shows that truncate Aly7B-CDII shows activity to algin, poly M and poly G.Truncate Aly7B-CDII shows substrate specificity similar with holoenzyme, but is better than holoenzyme in pH and temperature stability.The present invention, which excavates novel algin catenase to reconstruct, has very big reference value, and algin catenase truncate obtained can be used as the potential tool that production prepares algin oligosaccharide.
Description
Technical field
The invention belongs to technique for gene engineerings and field of protein expression, and in particular to a kind of truncated algin catenase base
Cause and its application.
Background technique
China possesses living marine resources extremely abundant, especially marine algae resource.Brown alga as a macro-algae resource, by
It is rich in this with unique properties and very big Development volue polysaccharide of algin in its cell wall, is favored by developers.
Algin is a kind of straight chain acidic polysaccharose, by beta-D-mannuronic acid (mannuronic, M) and α-L- guluronic acid
The linear anionic polysaccharide that (guluronic, G) is combined by α -1,4- glycosidic bond.Under native state, the master of algin
Wanting existence is alginic acids salt and the water-insoluble such as water-soluble sodium alginate (sodium alginate), potassium alginate
Alginic acid (alginic acid).Sodium alginate (trade name sodium alginate) or other alginates master currently on the market
It is obtained if being extracted from brown alga.Studies have shown that algal polysaccharide is according to abilities such as its high viscosity and gel characteristics, food,
The industries such as pharmacy are widely used.The degradation obtained oligosaccharides of algal polysaccharide, while retaining a variety of unique bioactives,
The problem of also avoiding the poor solubility that polysaccharide itself has has good bioavailability, with wide exploitation benefit
Use prospect.By present, there are many methods availalbes of degradation sodium alginate, including chemical degradation method, physical degradation methods and enzyme
Edman degradation Edman.Wherein, the chemical degradation method based on acid degradation is difficult to control with degradation condition, and operation is more complex, and time-consuming
Disadvantage.Physical degradation methods include radiation method and ultrasonic method etc., and generally as householder method, and it is not easy that oligosaccharides is made.And make
There is the advantages that mild condition, yield is high, and substrate and product specificities are strong with corresponding algin catenase degradation sodium alginate, it is brown
Phycocolloid lyases is therefore more and more for producing the tools of algin oligosaccharide as degradation.In addition, algin catenase is also
Application with some unique physiology and other field, such as can apply to treatment, the seaweed fodder of pulmonary cyst fibrosis
Processing and the research in the fields such as seaweed genetic engineering (Wong TY et al.Annual Review of Microbiology,
2000,54:289-340)。
Algin catenase is a kind of polysaceharide lyase, decomposes algin by β-elimination reaction.According to cracking substrate point
Class can be divided into polyM obligatory type (EC4.2.2.3), polyG obligatory type (EC 4.2.2.11) and bifunctional.According further to
Degradation model can be divided into endo-type and circumscribed-type.It is distributed that (including ocean is thin from marine animal and plant and multiple-microorganism
Bacterium, soil bacteria and fungi).By present research, has been carried out and functionality can be used for produce by algin catenase
The algin oligosaccharide of food.Therefore.In order to cope with industrialization demand, there is the algin of unique specificity and high-efficiency catalytic activity
Lyases right and wrong are often with there is realistic meaning.
Summary of the invention
Goal of the invention: the technical problem to be solved in the present invention is to provide a kind of novel truncated algin catenase Aly7B-
CDII。
The present invention also technical problems to be solved are to provide above-mentioned algin catenase Aly7B-CDII in cracking algin
In application.
By carrying out truncation acquisition to two structural domains of Aly7B overall length enzyme (calling holoenzyme in the following text), according to poor with the property of holoenzyme
Other and corresponding computer simulation experiment, the inherent mechanism that catalytic domain is shown in enzyme analysis.
In order to solve the above technical problems, the present invention adopts the following technical scheme:
A kind of truncated algin catenase Aly7B-CDII, amino acid sequence is as shown in SEQ ID NO.2.It is by complete
Enzyme Aly7B intercepts its second catalysis domain amino acid sequence 190-477 totally 288 amino acid residue, building recombinant expression for
The Aly7B-CDII of catalytic activity.Above-mentioned truncation enzyme Aly7B-CDII, nucleotide sequence are as shown in SEQ ID NO.1
864bp。
Above-mentioned truncation enzyme Aly7B-CDII is expressed in coli expression carrier after isolating and purifying, and obtains albumen
Molecular weight is 32.51kDa.By comparing, the basic zymetology such as temperature and pH in nature, truncate enzyme Aly7B-CDII show with
Optimum temperature similar in holoenzyme, but the enzyme activity of truncation enzyme Aly7B-CDII is without influence after 35 DEG C of incubation 1h, and holoenzyme is only protected
Stay 50%.This shows to truncate enzyme Aly7B-CDII after another truncation enzyme Aly7B-CDI structural domain of knock-off, may be presented one
Protein structure more closely, to guarantee thermal stability more higher than holoenzyme；Also, it truncates enzyme Aly7B-CDII to show more
High optimum pH, and more wide in range pH stability boundary.In metal ion influence, enzyme Aly7B-CDII and complete is truncated
Enzyme difference in general trend is little, but truncates enzyme and show more sensitive function and effect in facilitation.This may be
It is easier to influence with combined outside due to after the inhibition of no Aly7B-CDI structural domain, truncating enzyme Aly7B-CDII.In
Deeper into dynamics research in, holoenzyme is shown for sodium alginate than truncating the Rate activity that doubles of enzyme Aly7B-CDII,
But enzyme Aly7B-CDII is truncated for poly G and shows the Rate activity higher than holoenzyme.In addition, truncating Aly7B-CDII pairs of enzyme
Poly M and the Km value of poly G are lower than holoenzyme, this illustrate the presence of Aly7B-CDI may hinder holoenzyme and poly M with
The affinity of poly G.On artifact schemas, degraded in the form of inscribe sodium alginate, poly M and poly G production of holoenzyme polymerize
The oligosaccharides of 2-5 is spent, and monosaccharide can further be generated on this basis by truncating enzyme Aly7B-CDII.According to docking as a result, truncating
The residue Y257, Q143, H145 and R82 that enzyme Aly7B-CDII is catalyzed in tunnel can be respectively and in sublocus -1 ,+1 ,+2 and+3
Carboxyl forms hydrogen bond.Then, the glycosidic bond between -1 and+1 is cut and discharges monosaccharide.
Expression vector comprising above-mentioned truncated alginate lyase gene Aly7B-CDII is in protection scope of the present invention
Within.
Cell comprising above-mentioned truncated alginate lyase gene Aly7B-CDII is within protection scope of the present invention.
A kind of construction method for the genetic engineering bacterium producing algin catenase, includes the following steps:
(1) truncated alginate lyase gene is cloned into expression plasmid, obtains recombinant expression carrier；
(2) recombinant expression carrier is converted into host strain, obtains the genetic engineering bacterium for producing algin catenase.
In step (1), the plasmid is pET21a (+).
In step (2), the host strain is bacillus coli DH 5 alpha.
Above-mentioned truncated algin catenase Aly7B-CDII prepares algin widow in cracking alginate or algal polysaccharide
Application in sugar is within protection scope of the present invention.
The cell of above-mentioned truncated alginate lyase gene Aly7B-CDII is prepared in algin catenase in fermentation
Using within protection scope of the present invention.
The utility model has the advantages that
Truncation enzyme Aly7B-CDII of the invention is transformed in the Aly7B in the vibrio marinopraesens Vibrio sp.W13 of source
(AIY22661.1), it is expressed by intercepting the 190-477 amino acid residue recombination to construct of the second catalytic domain of Aly7B.Theoretical molecular
Amount is 32.64kDa.In nature, it truncates enzyme Aly7B-CDII and shows the thermal stability for being better than holoenzyme and corresponding pH stability,
And it is easier to generate facilitation by metal ion.Affinity more higher than holoenzyme is shown to poly M and poly G, and can be incited somebody to action
Tri- kinds of sodium alginate, poly M and poly G degradation of substrates generate monosaccharide.Can be used as and accordingly prepare the tool of oligosaccharides, agricultural,
Food and medicine and other fields are applied.
Detailed description of the invention
The polyacrylamide gel electrophoresis figure (SDS-PAGE) of Fig. 1: the Aly7B and Aly7B-CDII of purifying, wherein swimming lane
M is Marker, swimming lane 1 is (Aly7B), swimming lane 2 is (Aly7B-CDII).
Fig. 2: temperature, pH for Aly7B and Aly7B-CDII Activity and stabill influence curve (A. optimum temperature, B.
Temperature stability, C. optimal pH, D.pH stability.)
The thermal denaturation curve of Fig. 3: Aly7B (A) and Aly7B-CDII (B).
The thin layer of Fig. 4: algin catenase Aly7B and Aly7B-CDII degradation algin, poly M and poly G product
Chromatography (TLC) analysis chart.(A, C, E are respectively the product of Aly7B degradation algin, equal polymannuronate, guluronic acid；
B, D, F are respectively the product of Aly7B-CDII degradation algin, equal polymannuronate, guluronic acid)
The ESI-MS analysis chart of Fig. 5: Aly7B-CDII enzymatic hydrolysis algin 48h product.
The ESI-MS analysis chart of Fig. 6: Aly7B-CDII enzymatic hydrolysis poly M 48h product.
The ESI-MS analysis chart of Fig. 7: Aly7B-CDII enzymatic hydrolysis poly G 48h product.
Fig. 8: Aly7B overall structure.
Fig. 9: Aly7B-CDII and AlyA sequence alignment.
Figure 10: Aly7B-CDII computer molecular simulation figure, alginic acid tetrose (GGMG) and Aly7B-CDII tunnel-like
The stereo-picture that active site combines,
Figure 11: Aly7B-CDII computer molecular simulation figure is responsible for the catalytic residue figure for combining and being catalyzed.
Figure 12: influence figure of the different metal ions to Aly7B and Aly7B-CDII.
Specific embodiment
According to following embodiments, the present invention may be better understood.Content described in embodiment is merely to illustrate this hair
It is bright.
Embodiment 1: the culture of bacterial strain Vibrio sp.W13 and genome extract
Used bacterial strain Vibrio sp.W13 is stored in this laboratory.The formula of the culture medium used are as follows:
LB culture medium: tryptone 10g, yeast extract 5g, NaCl 10g, pH7.0.Solid medium addition 1.5%
Agar.High pressure steam sterilization.
Beef extract 5g, glucose 15g, yeast extract 1.0g, NaCl 5.0g, MgSO4·7H2O 0.5g、CaCl2 0.2g、
KH2PO4 1.0g、FeSO4·7H2O 0.02g, pH value 7.0.The agar of solid medium addition 1.5%.
Preservation of bacteria strain is applied in solid selection culture, activation bacterium colony is cultivated in liquid medium.4mL is taken to train
The bacterium solution supported is placed in 2mL tube pipe, and 5000g, 10min is sufficiently centrifuged.It discards supernatant, with 180 μ L digestive juices
(Digestion Solution) thallus is resuspended, and adds 20 μ L Proteinase Ks, after mixing well, 56 DEG C of incubation 30min.Add
Enter 20 μ L RnaseA, after mixing well, is placed at room temperature for 10min.200 μ L lysates (Lysis Solution) are added, it is quickly mixed
Even, process does not exceed 15s.400 μ L 50% (v/v) ethanol solutions are added, mix well.Above-mentioned solution is transferred to absorption
In column, 1min is stood, is centrifuged 6000g, 1min, waste liquid is abandoned, adsorption column is transferred in a new 2mL tube pipe.It is added
500 μ L wash buffer I, 8000g, 1min centrifugations, abandon waste liquid.500 μ L wash buffer II, 8000g, 1min are added
Waste liquid is abandoned in centrifugation.It is repeated once.Suction attached column 12000g, 3min are centrifuged, residual liquid in column is sufficiently dried.It is adsorbing
Base for post plasma membrane middle position is added 200 μ L eluents (Elution Buffer) and stands 1min, and 8000g, 1min are centrifuged to get base
Because of group.
Embodiment 2: the extraction of enzyme Aly7B-CDII is truncated
Following amplimer: forward primer is designed according to the second catalytic domain gene order in Vibrio sp.W13 genome
(5'-TGGAATATTGACGATTGG-3') and reverse primer (5'-ATGAAGAGTGCTCAAAGCAC-3').With the bacterial strain of extraction
Genome is template, carries out PCR amplification and obtains truncate Aly7B-CDII full length gene sequence.PCR condition are as follows: 94 DEG C of initial denaturations
3min then carries out 30 circulations with 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, finally in 72 DEG C of extension 10min.Agarose is solidifying
Gel electrophoresis, which is shown in close at 1.0kb, a specific band, it is cut from Ago-Gel, is returned using DNA gel
Kit is received to be purified.
The DNA fragmentation of purifying is connected on cloning vector pEASY-Blunt Zero Cloning vector, conversion is big
In enterobacteria DH5 α competent cell, in LB solid medium (containing ampicillin) after culture, picking white colony makes
PCR verifying is carried out with amplimer.PCR condition is 94 DEG C of initial denaturation 3min, then with 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min
30 circulations are carried out, finally in 72 DEG C of extension 10min.Recombinant bacterial strain mass propgation corresponding to specific band to occur, makes
Plasmid extraction is carried out with plasmid extraction kit, carries out sequencing analysis.The result shows that the full nucleotide of alginate lyase gene
Sequence 864bp, nucleotide sequence is as shown in SEQ ID NO.1；Encode 288 amino acid, amino acid sequence such as SEQ ID
Shown in NO.2, albumen theoretical molecular weight is 32.64kDa.With the homologous Modeling Server of PHYRE2 to algin catenase Aly7B with
The protein three-dimensional structure of Aly7B-CDII carries out homologous modeling and it is compared.The egg of finally obtained Aly7B-CDII
White matter 3 d structure model is as shown in Fig. 7~10.SEQ ID NO.1:Aly7B-CDII encoding gene open reading frame (ORF) is complete
Long 864bp does not contain signal peptide sequence.SEQ ID NO.2: truncate Aly7B-CDII is the second catalytic domain of Aly7B.
Embodiment 3: the building of algin catenase Aly7B-CDII recombinant expression carrier and corresponding expression and purification.
Forward primer (5'-TGGAATATTGACGATTGG-3') and reverse primer are designed according to truncate Aly7B-CDII
(5'-ATGAAGAGTGCTCAAAGCAC-3').It carries out PCR amplification and obtains truncate Aly7B-CDII full length gene sequence.PCR
Condition are as follows: 94 DEG C of initial denaturation 3min then carry out 30 circulations with 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, finally prolong at 72 DEG C
Stretch 10min.The transformant with amicillin resistance is screened, extracts positive transformant plasmid with plasmid extraction kit, it will
This recombinant plasmid is named as pET21a-Aly7B-CDII.
Recombinant expression plasmid pET21a-Aly7B-CDII is converted into coli strain BL21 (DE3) and (is purchased from the U.S.
Novagen company), then truncate Aly7B-CDII inducing expression and purifying are carried out according to the operating procedure that the said firm provides.
With the purifying situation of polyacrylamide gel electrophoresis detection truncate Aly7B-CDII, as a result as shown in Figure 1, after purification
Single band is presented in Aly7B-CDII on running gel, and position matches with the molecular weight of prediction.
Embodiment 5: the characterization analysis of algin catenase Aly7B-CDII.
1, the influence of pH and temperature to enzymatic activity
It is the 50mM phosphorus of 1% sodium alginate substrate, the Aly7B-CDII enzyme solution of purifying and different pH value by mass concentration
Hydrochlorate-citrate (pH 4.0-5.0), 50mM NaH2PO4-Na2HPO4 (pH 6.0-8.0), 50mM Tris-HCl (pH
After 7.0-9.0) mixing with glycine-NaOH (pH 9.0-10.0) in the ratio of 5:2:13 (volume ratio), 30 points are reacted at 35 DEG C
Clock, ultraviolet absorption method survey enzyme activity.Aly7B-CDII reaches maximum vigor in pH 9.0 as the result is shown, is higher than holoenzyme Aly7B.
Mass concentration is anti-in different temperatures (25 DEG C~55 DEG C) respectively for 1% sodium alginate substrate and the Aly7B-CDII enzyme solution of purifying
It answers 30 minutes, surveys enzyme activity by ultraviolet absorption method.Aly7B-CDII reaches maximum vigor at 35 DEG C as the result is shown, shows
The optimal reactive temperature of Aly7B-CDII is 35 DEG C (such as Fig. 2-A, B).
2, the influence of pH and temperature to enzyme stability
Aly7B-CDII enzyme solution and mass concentration after 30min will be heat-treated under different temperatures (25 DEG C -50 DEG C) are 1%
Sodium alginate substrate solution is mixed in the ratio of 1:9 (volume ratio), and remaining enzyme activity is then measured under optimum temperature and optimal pH,
100% relative activity (relative activity) is defined as with the highest enzyme solution enzyme activity of vigor, the results showed that Aly7B-CDII
Lower than 40 DEG C at a temperature of have preferable thermal stability；Will at 4 DEG C, different pH (pH4-10) preincubate for 24 hours after
Aly7B-CDII enzyme solution and mass concentration are that 1% sodium alginate substrate solution is mixed in the ratio of 1:9 (volume ratio), then most
Remaining enzyme activity is measured under thermophilic degree and optimal pH, and 100% relative activity (relative is defined as with the enzyme solution enzyme activity of maximum vigor
Activity), as the result is shown in the range of pH6~10, Aly7B-CDII enzyme activity still keeps 70% or more, shows Aly7B-
CDII is relatively wide (such as Fig. 2-C, D) to pH value tolerance range.
Aly7B-CDII enzyme solution is incubated in 30-45 DEG C of progress 0-1h different time gradient, it is then anti-under optimum condition
30min is answered, using the correspondence enzyme activity for the enzyme solution not being incubated for as 100%.According to Fig. 3 as a result, showing Aly7B-CDII at 35 DEG C
It is incubated for and keeps stablizing in 1h, show thermal stability more higher than holoenzyme.
3, the enzymatic activity and dynamics of Aly7B-CDII
By the Aly7B-CDII of purifying respectively with mass concentration be 0.1% sodium alginate, polymannuronate segment
(polyM) substrates different with poly- guluronic acid segment three kinds of (polyG) are mixed in the ratio of 1:9 (volume ratio), then 35
DEG C, it is reacted under the conditions of pH7.0.It takes the product at differential responses time point to survey its 235nm ultraviolet absorption value, finds with enzymolysis time
Extend, 235nm absorption value gradually increases, and principle (the Qing-Da An et of algin catenase is surveyed according to ultraviolet method
Al.Process Biochemistry, 2008,43:842-847), it was demonstrated that Aly7B-CDII retains cracking enzyme viability.In addition,
According to Fig. 5~8, degradation of the Aly7B-CDII to poly- guluronic acid segment, sodium alginate and polymannuronate segment
Preferable degrading activity is all had, shows that Aly7B-CDII is a difunctional algin catenase.Meanwhile the enzyme is for three kinds
Different substrates all have preferable substrate affinity.
The substrate specificity of 1. truncate Aly7B-CDII of table
2. truncate of table be Aly7B-CDII's and enzyme kinetics
Embodiment 6: metal ion is on the active influence of Aly7B-CDII
It is the Aly7B-CDII enzyme solution mixing of 1% sodium alginate substrate, purifying by mass concentration, while into reaction system
Different metal ions is added, then the final concentration of 1mM of the ion of addition reacts 10 minutes at 30 DEG C, by ultraviolet suction above-mentioned
Receipts method surveys enzyme activity.The activity (being set as 100%) that control group is Aly7B-CDII when any metal ion is not added, as a result as schemed
12.Experimental result shows that the activity of Aly7B and Aly7B-CDII can be by Mg2+, Ca2+And K+Activation, and can also be some
Divalent ion such as Mn2+, Cu2+And Zn2+Inhibit.However, identical ion pair Aly7B and Aly7B-CDII is shown in various degree
Activation, such as Na+, K+, Ca2+And Mg2+。
Embodiment 7:Aly7B-CDII and Aly7B analyzes the TLC and ESI-MS of the catabolite of three kinds of substrates.
It is the Aly7B-CDII enzyme solution 9:1 of 1% substrate (sodium alginate, poly M and poly G), purifying by mass concentration
System is reacted, and different time samples in 0-48h, tentatively divides processing sample progress thin-layer chromatography after inactivating removing protein
Analysis.As a result as shown in Figure 4.
After product of three kinds of substrates after 48h carries out pretreatment removing protein and metal ion, Electrospray Ionization Mass Spectrometry is carried out.
Electrospray ionization mass spectrum condition is using negative ion mode, ion source voltage: 4.5kV；Sheath gas: 30arb；Capillary temperature: 275
~300 DEG C；Pipe mirror voltage: 250V；Scanning range: 150-2000.As shown in Fig. 5~7.
The computer molecular simulation of embodiment 8:Aly7B-CDII is analyzed
According to the albumen of the algin catenase AlyA (PDB ID:4OZX) of Klebsiella pneumoniae subsp.
3D model structure uses the homologous modeler model of PHYRE2 building Aly7B-CDII.As a result inspection is passed through with 100% confidence level.
Enzyme is carried out molecular simulation with tetrose substrate (GGMG) and docked, and simulates truncation enzyme with this by the model with reference to obtained by homologous modeling
Aly7B-CDII is catalyzed the hydrogen bond of carboxyl in the residue Y257, Q143, H145 and R82 and sublocus -1 ,+1 ,+2 and+3 in tunnel
Connection figure.As a result such as Fig. 8~11.
Sequence table
<110>Nanjing University of Technology
<120>a kind of truncated algin catenase Aly7B-CDII gene and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 864
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tggaatattg acgattggaa attaaccatt cctgcaagca aaaatgattg gtatggtttt 60
ggcggtgaca gcgcggctga attagagcct gagcgttgta attcaagtaa agatcttctg 120
tctaacgaag agagcgttta ccaacgcgag attgatttat catacttcaa tgttattgac 180
ggtagcatgc atttccgcgc cgatatgggt tacggcacgt caacggccaa ctccagttac 240
atccgttcag aattgcgaga gctctacatc agtactaact ccccggattg cagcaccagc 300
gatgaagaga caagttggta tattgaagat agtcgtaccg gtgcaacttc gcatacgcta 360
aacgcaacac taagaattaa cgaataccct aaaatcgacg gtcaattacc aaaagtcgtg 420
gtaggccaga tacatggttg gaaaatcagc caagcactcg tgaagctact ttgggaaggt 480
gacaataagc cagtcagagt tattctgaac gataactaca aacttgataa caacaaagac 540
tgtactgatt gcaacgcatt cagcgttaag cttggtacct acgcggtaaa cgaagactgg 600
caatatacga tccgtgccga taaggaagga ctgtttttag cctcctacga tgcagatggc 660
agtaatatgg tctcgcacac actgaagtgg ggagaagcat actcagacac cgctaacaac 720
aagtcctata ctctgactga aagatgggcg tcgcctgata ttgcgtttta cttcaaagcc 780
ggaatttacc ctcagtttaa acctgataat gcatatcgag gagaaatctt tgatgtgagc 840
tttagtgctt tgagcactct tcat 864
<210> 4
<211> 288
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Trp Asn Ile Asp Asp Trp Lys Leu Thr Ile Pro Ala Ser Lys Asn Asp
1 5 10 15
Trp Tyr Gly Phe Gly Gly Asp Ser Ala Ala Glu Leu Glu Pro Glu Arg
20 25 30
Cys Asn Ser Ser Lys Asp Leu Leu Ser Asn Glu Glu Ser Val Tyr Gln
35 40 45
Arg Glu Ile Asp Leu Ser Tyr Phe Asn Val Ile Asp Gly Ser Met His
50 55 60
Phe Arg Ala Asp Met Gly Tyr Gly Thr Ser Thr Ala Asn Ser Ser Tyr
65 70 75 80
Ile Arg Ser Glu Leu Arg Glu Leu Tyr Ile Ser Thr Asn Ser Pro Asp
85 90 95
Cys Ser Thr Ser Asp Glu Glu Thr Ser Trp Tyr Ile Glu Asp Ser Arg
100 105 110
Thr Gly Ala Thr Ser His Thr Leu Asn Ala Thr Leu Arg Ile Asn Glu
115 120 125
Tyr Pro Lys Ile Asp Gly Gln Leu Pro Lys Val Val Val Gly Gln Ile
130 135 140
His Gly Trp Lys Ile Ser Gln Ala Leu Val Lys Leu Leu Trp Glu Gly
145 150 155 160
Asp Asn Lys Pro Val Arg Val Ile Leu Asn Asp Asn Tyr Lys Leu Asp
165 170 175
Asn Asn Lys Asp Cys Thr Asp Cys Asn Ala Phe Ser Val Lys Leu Gly
180 185 190
Thr Tyr Ala Val Asn Glu Asp Trp Gln Tyr Thr Ile Arg Ala Asp Lys
195 200 205
Glu Gly Leu Phe Leu Ala Ser Tyr Asp Ala Asp Gly Ser Asn Met Val
210 215 220
Ser His Thr Leu Lys Trp Gly Glu Ala Tyr Ser Asp Thr Ala Asn Asn
225 230 235 240
Lys Ser Tyr Thr Leu Thr Glu Arg Trp Ala Ser Pro Asp Ile Ala Phe
245 250 255
Tyr Phe Lys Ala Gly Ile Tyr Pro Gln Phe Lys Pro Asp Asn Ala Tyr
260 265 270
Arg Gly Glu Ile Phe Asp Val Ser Phe Ser Ala Leu Ser Thr Leu His
275 280 285
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tggaatattg acgattgg 18
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atgaagagtg ctcaaagcac 20
Claims (9)
1. a kind of truncated alginate lyase gene Aly7B-CDII, nucleotide sequence is as shown in SEQ ID NO.1.
2. a kind of truncated algin catenase Aly7B-CDII, amino acid sequence is as shown in SEQ ID NO.2.
3. the expression vector comprising truncated alginate lyase gene Aly7B-CDII described in claim 1.
4. the cell comprising truncated alginate lyase gene Aly7B-CDII described in claim 1.
5. a kind of construction method for the genetic engineering bacterium for producing algin catenase, which comprises the steps of:
(1) truncated alginate lyase gene is cloned into expression plasmid, obtains recombinant expression carrier；
(2) recombinant expression carrier is converted into host strain, obtains the genetic engineering bacterium for producing algin catenase.
6. producing the construction method of the genetic engineering bacterium of algin catenase according to claim 5, which is characterized in that step
(1) in, the plasmid is pET21a (+).
7. producing the construction method of the genetic engineering bacterium of algin catenase according to claim 5, which is characterized in that step
(2) in, the host strain is bacillus coli DH 5 alpha.
8. truncated algin catenase Aly7B-CDII described in claim 2 prepares brown in cracking alginate or algal polysaccharide
Application in phycocolloid oligosaccharides.
9. cell described in claim 4 prepares the application in algin catenase in fermentation.
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| CN113308455A (en) * | 2021-06-22 | 2021-08-27 | 南京工业大学 | Alginate lyase AlyPL17, truncation and application thereof |
| CN114574472A (en) * | 2020-12-02 | 2022-06-03 | 中国科学院大连化学物理研究所 | Recombinant polysaccharide degrading enzyme and coding gene and application thereof |
| CN115786318A (en) * | 2022-12-20 | 2023-03-14 | 中国海洋大学 | Alginate lyase truncation Algt1 and application thereof |
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| CN114574472B (en) * | 2020-12-02 | 2023-08-18 | 中国科学院大连化学物理研究所 | Recombinant polysaccharide degrading enzyme and encoding gene and application thereof |
| CN113308455A (en) * | 2021-06-22 | 2021-08-27 | 南京工业大学 | Alginate lyase AlyPL17, truncation and application thereof |
| CN115786318A (en) * | 2022-12-20 | 2023-03-14 | 中国海洋大学 | Alginate lyase truncation Algt1 and application thereof |
| CN115786318B (en) * | 2022-12-20 | 2024-05-07 | 中国海洋大学 | Algin lyase truncated Algt1 and application thereof |
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