CN106399285A - Facultative incision type recombinant alginate lyase rAly-1 as well as coding gene and application thereof - Google Patents
Facultative incision type recombinant alginate lyase rAly-1 as well as coding gene and application thereof Download PDFInfo
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Abstract
The invention relates to facultative incision type recombinant alginate lyase rAly-1 as well as a coding gene and application thereof. An amino acid sequence of the facultative incision type recombinant alginate lyase rAly-1 is as shown by SEQ ID NO. 2; a nucleotide sequence of the coding gene of the facultative incision type recombinant alginate lyase rAly-1 is as shown by SEQ ID NO. 1. According to the facultative incision type recombinant alginate lyase rAly-1 as well as the coding gene and the application thereof, the facultative incision type recombinant alginate lyase rAly-1 is obtained from a genome of flammeovirga yaeyamensis MY04 for the first time; not only can the lyase be used for degrading a guluronic acid segment from alginate, but also can be used for degrading a mannuronic acid segment. The lyase can be used for thoroughly degrading alginate polysaccharide in any M/G ratio, and efficiently preparing alginate oligosaccharide with a lower polymerization degree.
Description
Technical field
The present invention relates to a kind of facultative endo-type restructuring algin catenase rAly-1 and its encoding gene and application, belong to
Gene engineering technology field.
Background technology
Algin is a kind of linear acidic polysaccharide, and its backbone molecule is linear, by mannuronic acid (Mannuronate, M)
And its C-5 difference conformational isomer guluronic acid (Guluronate, G), by glycosidic bond, continuously or alternate links form.Algin
There is homogeneous poly- M section, poly- G section, and MG the or GM section of heterozygosis in intramolecular.Algin mainly originate from the medicines such as Thallus Laminariae (Thallus Eckloniae), Alga Sgrgassi Enervess,
Eat dual-purpose large ocean Eukaryotic Algae, be the main component constituting cell wall and intercellular matrix.Additionally, conditioned pathogen copper
Green pseudomonass and some soil microorganisms such as azotobacter vinelandii also can secrete Algin, and difference is microbe-derived Brown algae
Glue has acetylation modification.At present, the main body that daily-use chemical industry, food and medicine field are applied is the Algin of algal source.Brown
Algin, together with agar, carrageenan, is yield maximum, economic worth highest, three Large Marine Ecosystem polysaccharide being most widely used.For a long time
Since, the high-valued research of Algin is always one of emphasis of field of marine biotechnology.In the recent period, Geng Meiyu etc. is with poly- M section
Algin oligosaccharide is raw material, successfully develops the drug candidate 971 with directed against amyloid-beta protein aggregation activity, is near completion anti-
The III phase of Alzheimer's disease (Alzheimer Disease, AD) is clinical, is expected to become a kind new medicine.Current research also table
Bright, algin oligosaccharide has dynamic regulation antiinflammatory/proinflammatory important with antioxidation/enzymatic oxidation, Inhibiting α-glucosidase activity etc.
Function.Therefore, there is specific M/G ratio and the algin oligosaccharide of degree of polymerization size have significant application value and economic worth,
Realize this kind of oligosaccharide efficient prepare significant.
Algin catenase, is catalyzed the fracture of Algin intramolecular glycosidic bond by β-cancellation mechanism, and in product oligosaccharides
Non reducing end form C4=C5 unsaturated double-bond, form conjugated structure with the C=O (carboxylic carbonyl) of C5- position, thus producing
Product oligosaccharides containing unsaturated ends (Δ).Compared with chemically or physically learning biodegrading process, the enzymic degradation of Algin has
Mild condition, the advantageous characteristic such as easy to control, environmental pollution is little, thus there is the potential quality of popularization and application.Algin catenase is main
From the digestive system of Carnis Rapanae thomasianae, Sargassum degradation bacteria or virus etc..It is limited to yielding poorly of natural algin catenase, transgenic
Although algin catenase yield is high but the impact of the water solublity of most enzyme and the composite factor such as activity is poor, so far, with β-fine jade
The like products such as glue enzyme, cellulase are compared, rare wide variety of commercialization algin catenase.
Also find during the reason analysis causes this situation, though the patent application with regard to algin catenase and scientific documents
So quantity is more, but focuses primarily upon the preliminary study such as the Resource exploitation of bacterium producing multi enzyme preparation and enzyme, and the oligosaccharide with regard to enzyme generates feature
And its explaination of degradation of substrates characteristic is relatively fewer, it is not provided that the enzymatic property information of abundance on the whole, this leads to toolenzyme
Scarcity, and cannot accurately apply.
Because Algin is made up of two kinds of sugar units of M, G, the selectivity to substrate therefore according to enzyme, generally can will be brown
The three major types such as algin lyases are divided into M- specificity, G- specificity and M/G- is facultative.Additionally, the degradation of substrates mould according to enzyme
Formula, is further divided into the two types such as restriction endonuclease, excision enzyme.Restriction endonuclease can at random, efficient degradation polysaccharide, generate the degree of polymerization different
Series unsaturation oligosaccharide；Excision enzyme can be degraded substrate in order, and specificity generates basic sugar unit, absorbs for organism.
Wherein, the efficient preparation of the thorough enzymolysis of Algin to be realized or oligosaccharide, facultative restriction endonuclease is one of highly important instrument.?
In existing Chinese patent application, the application with regard to the facultative or difunctional algin catenase of M/G is less, Jin Jianru：
1st, a kind of Algin cracking disclosed in Chinese patent literature CN105624137A (application number 201410660235.3)
Gene order of enzyme Algb and preparation method and application.2nd, Chinese patent literature CN105154458A (application number
201510672140.8) a kind of method that restructuring algin catenase and its engineering bacteria and hydrolysis disclosed in prepare brown alga oligose.
Application with regard to endo-type algin catenase is relatively less, Jin Jianru：
1st, a kind of restructuring Algin disclosed in Chinese patent literature CN105154458A (application number 201510672140.8)
The method that lyases and its engineering bacteria and hydrolysis prepare brown alga oligose.2nd, Chinese patent literature CN104293754A (application number
201410469861.4) disclosed in a kind of endo-type algin catenase and its encoding gene and application.
Wherein, but rarely seen Chinese patent literature CN104293754A (application number 201410469861.4) provide detailed
Data is it was demonstrated that the AlgL-5 enzyme advocated is the degradation model of endo-type.At present, have no with regard to facultative endo-type Algin
The relevant report of lyases.
Content of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of facultative endo-type restructuring algin catenase rAly-1 and its
Encoding gene and application.
Technical scheme is as follows：
A kind of facultative endo-type restructuring algin catenase rAly-1, aminoacid sequence is as shown in SEQ ID NO.2.
Above-mentioned facultative endo-type restructuring algin catenase rAly-1 can degrade few from the guluronic acid of Algin
Bglii fragment, also can drop mannuronic acid oligosaccharide fragment, be facultative algin catenase.Can finally produce after this enzymatic degradation Algin
With unsaturated disaccharide, the unsaturated series of oligosaccharides product as principal product for the trisaccharide, its mol ratio is about 1：1；And in unsaturation disaccharide
It is virtually all Δ G；Unsaturated trisaccharide product contains two kinds of non reducing ends such as Δ G, Δ M, and the two mol ratio is close
5：4.The minimum unsaturation oligosaccharide substrates of this enzyme are tetroses, and minimum unsaturation product oligosaccharides are disaccharide.This enzyme is with internal-cutting way
The unsaturated pentasaccharides of degraded, and produce the unsaturated disaccharide of equimolar amountss and unsaturated trisaccharide.This enzyme is in degraded saturation pentasaccharides substrate
When, saturation disaccharide product results from the non reducing end of substrate, and unsaturated trisaccharide product is created on the reproducibility end of substrate
End.
The encoding gene of above-mentioned facultative endo-type restructuring algin catenase rAly-1, nucleotide sequence such as SEQ ID
Shown in NO.1.
The encoding gene of above-mentioned facultative endo-type restructuring algin catenase rAly-1, the common 1335bp of total length, coded egg
White matter contains 444 aminoacid, and molecular weight is about 50.7kD.
A kind of recombinant expression carrier, inserts above-mentioned facultative endo-type restructuring algin catenase in expression vector
The encoding gene of rAly-1.
A kind of recombinant bacterium, has proceeded to the weight of above-mentioned facultative endo-type restructuring algin catenase rAly-1 in host cell
Group expression vector.
The encoding gene of above-mentioned facultative endo-type restructuring algin catenase rAly-1, recombinant expression carrier, recombinant bacterium exist
Prepare the application that facultative endo-type is recombinated in algin catenase rAly-1.
Above-mentioned facultative endo-type restructuring algin catenase rAly-1 is in degraded Algin, degraded algin oligosaccharide or preparation
Application in unsaturated oligosaccharide.
Beneficial effect
1st, the present invention is first in the genome by heat color Bacillus bacteria (Flammeovirga yaeyamensis) MY04
Obtain facultative endo-type restructuring algin catenase rAly-1, this enzyme can be degraded from the guluronic acid fragment of Algin,
Mannuronic acid fragment also can drop.This enzyme thoroughly degrade the product after Algin be equimolar amountss unsaturated disaccharide with unsaturated
Trisaccharide, wherein unsaturated disaccharide product is essentially all Δ G, and unsaturated trisaccharide product to contain two kinds of Δ G, Δ M etc. non-reduced
Property end, and the two mol ratio is close to 5：4.On the whole, this enzyme can be used for any M/G ratio Brown algae glue polysaccharide of thoroughly degrading,
And efficiently prepare the algin oligosaccharide of low degree.
2nd, when facultative endo-type restructuring algin catenase rAly-1 of the present invention is with Algin for substrate, 50
DEG C, there is highest enzyme activity under the conditions of pH6.0, Rate activity reaches 1261 ± 8.2U/mg, the temperature stability of enzyme and pH stability
Good, have broad application prospects.
Brief description
The BLASTp analysis result that Fig. 1, restructuring algin catenase rAly-1 functional module are constituted；
Polyacrylamide gel electrophoresis figure (the SDS- of Fig. 2, restructuring algin catenase rAly-1 expression and purification situation
PAGE)；
Wherein：M, protein molecular weight standard, size is 116kD, 66.2kD, 45kD, 35kD to band from top to bottom,
25kD, 18.4kD, 14.4kD；Thalline before swimming lane 1, control strain breaking cellular wall, applied sample amount 10 μ L, bacterium before swimming lane 2, recombinant bacterium breaking cellular wall
Body, applied sample amount 10 μ L, supernatant after swimming lane 3, recombinant bacterium breaking cellular wall, applied sample amount 10 μ L, swimming lane 4, the rAly-1 through ni-sepharose purification, loading
Measure 10 μ L；
Fig. 3, the temperature influence curve to restructuring algin catenase rAly-1 activity；
Fig. 4, the pH value influence curve to restructuring algin catenase rAly-1 activity；
Fig. 5, the temperature influence curve to restructuring algin catenase rAly-1 stability；
Fig. 6, the pH value influence curve to restructuring algin catenase rAly-1 stability；
The impact bar diagram to restructuring algin catenase rAly-1 activity of Fig. 7, metal ion and chemical reagent；
Molecular gel chromatograph-the HPLC of product oligosaccharides during Fig. 8, restructuring algin catenase rAly-1 degraded Algin
Analysis chart；
In figure：(1) UDP2, unsaturated disaccharide；(2) UDP3, unsaturated trisaccharide；(3) UDP4, unsaturated tetrose；(4)
UDP5, unsaturated pentasaccharides；
Prepared unsaturated oligose fragment after Fig. 9, Algin of thoroughly being degraded with restructuring algin catenase rAly-1
The HPLC analysis chart of UDP2, UDP3；
Figure 10-1, unsaturated oligose fragment UDP2 prepared by Algin of thoroughly being degraded with restructuring algin catenase rAly-1
MS analysis chart；
Figure 10-2, unsaturated oligose fragment UDP3 prepared by Algin of thoroughly being degraded with restructuring algin catenase rAly-1
MS analysis chart；
Figure 11-1, unsaturated oligose fragment UDP2 prepared by Algin of thoroughly being degraded with restructuring algin catenase rAly-1
's1H-NMR schemes；
Figure 11-2, unsaturated oligose fragment UDP3 prepared by Algin of thoroughly being degraded with restructuring algin catenase rAly-1
's1H-NMR schemes；
Separating obtained UDP2- in Figure 12, the product of Algin of not exclusively degrading from restructuring algin catenase rAlgL-5
UDP6HPLC analysis chart；
Figure 13, with restructuring algin catenase rAly-1 thoroughly degrade with oligosaccharide UDP2-UDP6 prepared by rAlgL-5
HPLC analysis chart；
Figure 14, the HPLC analysis chart (purple of saturation poly- M pentasaccharides (M5) of thoroughly being degraded with restructuring algin catenase rAly-1
Outward)；
Figure 15, the HPLC analysis chart (purple of saturation poly- G pentasaccharides (G5) of thoroughly being degraded with restructuring algin catenase rAly-1
Outward)；
Figure 16, use restructuring algin catenase rAly-1 thorough degradative reduction end are by the poly- M of the saturation of 2-AB labelling five
The HPLC analysis chart (fluorescence) of sugared (M5)；
Figure 17, use restructuring algin catenase rAly-1 thorough degradative reduction end are by the poly- G of the saturation of 2-AB labelling five
The HPLC analysis chart (fluorescence) of sugared (G5).
Specific embodiment
The elaboration of following examples, is some common technologies how implemented for the comprehensive disclosure present invention, rather than is
Limit the range of application of the present invention.Inventor tried one's best guarantee in embodiment the accuracy of a parameter (for example measure,
Temperature, etc.), but some experimental erroies and deviation also should be paid attention to.Unless otherwise stated, middle-molecular-weihydroxyethyl of the present invention is
Refer to weight average molecular weight, temperature is degree Celsius.
Biological material source
Heat color bacillus (Flammeovirga yaeyamensis) MY04 derives from Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, protects
Hide November 27 2008 date, deposit number CGMCC NO.2777.
Embodiment 1, the extraction of heat color bacillus (Flammeovirga yaeyamensis) MY04 strain gene group DNA
Heat color bacillus (Flammeovirga yaeyamensis) MY04 is seeded in fluid medium YT04,28
DEG C, under conditions of 200rpm, shaken cultivation to 600nm light absorption value (OD600) it is 1.2；Take culture bacterium solution 10mL, 12,000 × g
It is centrifuged 15min, collects thalline precipitates under the conditions of (g, Gravitational coefficient of the Earth)；Bacteriolyze enzyme buffer liquid (10mM Tris- with 10mL
HCl, pH 8.0) suspension thalline, it is centrifuged 15min under the conditions of 12,000rmp, collects thalline precipitates.
Aforesaid liquid culture medium YT04, every liter of component is as follows：
Tryptone 10g, yeast extract 5.0g, sodium chloride 30g, with water dissolution and be settled to 1L, pH 7.2.
To in above-mentioned bacterial sediment, often pipe adds bacteriolyze enzyme buffer liquid (purchased from Shanghai Sheng Gong biological engineering company limited)
6.0mL, obtains the bacterium solution of about 7.0mL, is separately added into each 280 μ L of lysozyme soln that concentration is 20mg/mL, makes lysozyme dense eventually
Spend for 800 μ g/mL；It is placed in 1.0h in ice-water bath, be then transferred in 37 DEG C of water-baths, temperature bath 2h, sticky to reaction system；Add
Concentration is the Proteinase K Solution 30 μ L of sodium cetanesulfonate solution 0.41mL, 100mg/mL of 100mg/mL, in 52 DEG C of temperature baths
1.0h；Add the equilibrated phenol/chloroform/isoamyl alcohol (volume ratio 25 of Tris-：24：1) solution 7.5mL, gently overturns and mixes；?
10,000 × g, it is centrifuged 10min under the conditions of 4 DEG C, collect supernatant, and add the NaAc-HAc (pH 5.2,3.0M) of 1.0mL to buffer
Liquid, and the dehydrated alcohol of 8.5mL, fully mix；Choose thread DNA with pipette tips, be transferred in the centrifuge tube of 1.5mL, with
70% ethanol (stores in -20 DEG C), washs 2 times, discards supernatant after micro- centrifugation；10,000 × g, it is centrifuged 2min under the conditions of 4 DEG C, thorough
Bottom discards supernatant；DNA is deposited in aseptic working platform apoplexy and dries up dry, then use sterile deionized water in 4 DEG C of overnight dissolving DNAs
Sample, prepared genomic DNA.
Embodiment 2, the scanning of heat color bacillus (Flammeovirga yaeyamensis) MY04 strain gene group and its sequence
Row analysis.
The genomic DNA that embodiment 1 is obtained, carries out the scanning sequencing of genome, by upper using pyrosequencing techniques
Hai Meiji biotech firm completes.With NCBI (National Center for Biotechnology Information,
http://www.ncbi.nlm.nih.gov/) online software of website is analyzed to DNA sequencing result.Used NCBI
The analysis software of website is Open Reading Frame Finder (ORF Finder, http://
) and Basic Local Alignment Search Tool www.ncbi.nlm.nih.gov/gorf/gorf.html
(BLAST,http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Shown with the result of above-mentioned biological software analysis, heat color bacillus (Flammeovirga yaeyamensis) MY04
The encoding gene aly-1 of one algin catenase, the coding head of district 1335bp of gene aly-1 are carried on strain gene group DNA,
Nucleotide sequence is as shown in SEQ ID NO.1.Restructuring algin catenase rAly-1 coded by gene aly-1 contains 444 altogether
Individual aminoacid, its aminoacid sequence is as shown in SEQ ID NO.2.
With BLASTp software on-line analyses, result shows, with restructuring algin catenase rAly-1's in ncbi database
Amino acid sequence similarity be more than 30% be all function without identification protein.As shown in figure 1, BLASTp analysis also pushes away
Conservative supposition catalyst structure domain in algin catenase super families 2 is contained at the protein C-end surveying Aly-1.Use biology
Software BioEdit 7.0.5.3 is analyzed, and the theoretical molecular of display a-protein ly-1 is about 50.7kD.Existed with signal peptide
Line forecasting software SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) online point
Analysis, the N- end 1-20 amino acids of this protein are coding sequence of secretory signal peptide.
Embodiment 3, gene aly-1 are recombinant expressed in e. coli bl21 (DE3) bacterial strain
With the prepared genomic DNA of embodiment 1 as template, enter performing PCR amplification.Primer sequence is as follows：
Forward primer Aly1-F：5’-cgcGGATCCAACAATAAAGTAGAGGACGAG-3’(BamH I)；
Reverse primer Aly1-R：5’-ccgCTCGAGTATAAGTTTCTTTTAATTCTATAG-3’(Xho I)；
In forward primer Aly1-F, underscore mark is restricted enzyme BamH I site, under reverse primer Aly1-R
Underlining is restricted enzyme Xho I site.High-fidelity DNA polymerase PrimeSTAR HS DNA used
Polymerase is purchased from DaLian, China treasured biotech firm, and PCR reaction reagent used is carried out according to the description of product that the said firm provides
Operation.
PCR reaction condition：95 DEG C of denaturations 4min；94 DEG C of degeneration 40s, 60 DEG C of annealing 30s, 72 DEG C of extension 75s, 35 are followed
Ring；72 DEG C of extension 10min；Stablize 10min for 4 DEG C.
PCR primer is carried out double digestion with restricted enzyme BamH I and Xho I, is reclaimed by agarose gel electrophoresiies
PCR primer after enzyme action.By be purchased from Invitrogen company of the U.S. product pET-30a (+) plasmid DNA, with BamH I and Xho
I double digestion, the product fragment after entering row agarose gel electrophoresis and reclaiming enzyme action.Restricted enzyme BamH I and Xho I is equal
It is purchased from DaLian, China treasured biotech firm, the system of enzyme-to-substrate reaction used in enzyme action, temperature and time, all according to the said firm
The description of product operation providing.
By the PCR primer through BamH I and Xho I double digestion, with also pass through double digestion pET-30a (+) plasmid carries
Body, is connect under the catalysis of DNA ligase；Connection product converts e.colistraindh5α, coats containing 50 μ g/mL cards
On the Luria-Bertani culture medium solid plate of that mycin, after 37 DEG C of culture 16h, picking monoclonal；Monoclonal is accessed and contains
There is culture in the liquid Luria-Bertani culture medium of 50 μ g/mL kanamycin, extract plasmid；Plasmid amplimer is entered
Performing PCR is verified, result obtains the amplified production that size is 1.3kb, and the recombiant plasmid that preliminary proof builds is correct；Then this is heavy
Group plasmid be sequenced, result shows, pET-30a (+) BamH I and Xho I restriction enzyme site between insert SEQ ID
Gene aly-1 shown in NO.1, and direction of insertion is correct, so proving that the recombiant plasmid building is correct further, this is recombinated
Plasmid is named as pE30a-Aly1.
Recombiant plasmid pE30a-Aly1 conversion coli strain BL21 (DE3) is (public purchased from U.S. Invitrogen
Department), then according to the operating procedure that the said firm provides, carry out restructuring Algin using isopropylthiogalactoside (IPTG) and split
The abduction delivering of solution enzyme rAly-1.8,000 × g, it is centrifuged 15min under the conditions of 4 DEG C, collects thalline, and with the resuspended bacterium of buffer A
Body, ultrasonication in ice water bath environment.15,000 × g, it is centrifuged 30min further under the conditions of 4 DEG C, collect water-soluble component,
And with Ni- agarose gel, purification is carried out to group algin catenase rAly-1.With containing imidazole concentration be 10,50,100,
250th, the buffer A of 500mM carries out gradient elution, and purification condition operates according to the product manual of gel.Coagulated with polyacrylamide
The purification situation of degeneration gel electrophoresis detection restructuring algin catenase rAly-1.Result is as shown in Figure 2：After IPTG abduction delivering
BL21 (DE3) thalline in, restructuring algin catenase rAly-1 yield be not less than 500mg/L thalline culture；After purification
Restructuring algin catenase rAly-1 on running gel be in single band, and position with predict molecular weight match；Will be pure
Restructuring algin catenase rAly-1 sample after change loads the bag filter that smallest molecule interception is 10kD, in 4 DEG C of environment
Buffer A is dialysed.The composition of described buffer A is 50mM Tris, 150mM NaCl, pH 7.9, and restructuring Brown algae is obtained
Glue lyases rAly-1 enzyme liquid.
Embodiment 4, the mensure of restructuring algin catenase rAly-1 optimum temperature
It is the Algin of 0.1-1.2%, polymannuronate fragment (polyM) that deionized water prepares mass-volume concentration
With poly- three kinds of different substrates of guluronic acid fragment (polyG), after heating for dissolving, be placed in 0 DEG C, 10 DEG C, 20 DEG C, 25 DEG C, 30 DEG C,
35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, lower the temperature in 100 DEG C of water bath 1h.To every 900 μ L
Add the diluent 100 μ L of the restructuring algin catenase rAly-1 that embodiment 3 is obtained, restructuring Algin cracking in substrate solution
The diluent concentration of enzyme rAly-1 be 10 μ g/mL, after mixing continue reaction, every when sampling.Lower 3 Duplicate Samples of each temperature conditions
Product, with the restructuring enzyme preparation of boiling water bath inactivation as matched group.
Measure the concentration (OD of newly-generated reducing sugar in each reaction system with DNS- reducing sugar method540), and calculate meansigma methodss,
Carry out variance analyses.The corresponding reaction temperature of obtained the maximum absorption is the optimum temperature of recombinase, and enzyme activity (RA) relatively is defined as：Respectively
Absorption value and the percentage ratio of obtained the maximum absorption.Result is as shown in Figure 3：When measuring enzyme activity with three kinds of substrates as described above, restructuring is brown
Algin lyases rAly-1 reaches maximum vigor in 50 DEG C of reactions, and this shows the most suitable of algin catenase rAly-1 of recombinating
Reaction temperature is 50 DEG C.When meanwhile, with Algin for substrate, restructuring algin catenase rAly-1 illustrates 73% at 0 DEG C
Enzyme activity relatively, this shows that restructuring algin catenase rAly-1 has suitable cold zymologic property；Also show at 80-100 DEG C
More than 45% relative enzyme activity, this shows that restructuring algin catenase rAly-1 has certain suitable hot zymologic property.
In addition, as shown in figure 4, restructuring algin catenase rAly-1 is to Algin, poly- guluronic acid fragment (polyG)
Degraded with polymannuronate fragment (polyM) is respectively provided with preferable degrading activity, tentatively shows algin catenase of recombinating
RAly-1 is a facultative algin catenase.
Embodiment 5, the mensure of restructuring algin catenase rAly-1 optimum pH
The NaAc-HAC buffer being 50mM with concentration respectively, the NaH of 50mM2PO4-Na2HPO4Buffer, 50mM
Tris-HCl buffer, prepares the Algin substrate that mass-volume concentration (g/mL) is 0.1～1.2%, institute respectively with Algin
Corresponding pH value is respectively 5,6,6,7,8,7,8,9,10 3 sections, and each pH value is set up all under optimum temperature.Substrate is molten
Xie Hou, is placed in incubation 1h in optimum temperature, then adds the restructuring Algin cracking that embodiment 3 is obtained in every 900 μ L substrates
The diluent 100 μ L of enzyme rAly-1, start after mixing react, every when sampling.3 parallel sample under the conditions of each pH, with boiling water
The restructuring enzyme preparation of bath inactivation is matched group.Measure newly-generated concentration of reduced sugar in each reaction system with DNS- reducing sugar method
(OD540), and calculate meansigma methodss and deviation.Enzyme (RA) work relatively is defined as：Each group mean absorbance and the percentage of obtained the maximum absorption
Than.The corresponding pH of obtained the maximum absorption is the optimum pH of recombinase.Result is as shown in Figure 4：Restructuring algin catenase rAly-1's
Optimal reaction pH is 6.0.In the range of pH 5～10, restructuring algin catenase rAly-1, shows relative to enzyme activity more than 78%
Show that this enzyme has extensive pH reactivity.
Embodiment 6, the temperature stability analysis of restructuring algin catenase rAly-1
By embodiment 3 prepared restructuring algin catenase rAly-1 enzyme liquid heat treatment under different temperatures (0～70 DEG C)
After 0.5h, 1h, 2h, the Algin being 0.1～1.2% with the mass-volume concentration (g/mL) being configured with distilled water respectively, by 1：9
The ratio mixing of (volume ratio), then measures remnant enzyme activity under optimum temperature, with the enzyme liquid enzyme activity definition without Overheating Treatment
For 100% relative activity.Result is as shown in Figure 5：Restructuring algin catenase rAly-1 pretreatment at a temperature of less than 50 DEG C
After 2h, still have>90% residual activity, shows that this enzyme has certain heat stability.
Embodiment 7, the pH stability analyses of restructuring algin catenase rAly-1
The enzyme liquid of the restructuring algin catenase rAly-1 that embodiment 3 is obtained, in optimum temperature (50 DEG C), different pH
After difference preincubate 2h in (pH5～10) environment, molten with the Algin substrate that mass-volume concentration (g/mL) is 0.1～1.2%
Liquid presses 1：The ratio mixing of 9 (volume ratios), then measures remnant enzyme activity, with the enzyme liquid enzyme without pretreatment under optimum temperature
Work is defined as 100% relative activity.Result as shown in fig. 6, in the range of pH5～10 pretreatment 2h, rAly-1 enzyme activity still protects
Hold more than 70%.This shows that restructuring algin catenase rAly-1 is wider to pH value tolerance range.
The impact to restructuring algin catenase rAly-1 activity of embodiment 8, metal ion
The restructuring that the mass concentration that deionized water is configured is 0.1～1.2% Algin substrate, embodiment 3 is obtained is brown
Algin lyases rAly-1 enzyme liquid and water press 5：1：After the ratio mixing of 4 (volume ratios), then add not in reaction system
Same metal ion, final concentration of 1mM or 10mM of ion of interpolation, then reacts 4h at 50 DEG C, by aforesaid DNS- reducing sugar
Method surveys the vigor of enzyme.Matched group is the activity (being set as 100%) of rAly-1 when being not added with any metal ion.Shown in result Fig. 7,
Under 1mM or 10mM concentration：(1)Na+、K+、Li+Deng three kinds of monovalent metal reagent in 10mM to the activities present of rAly-1 it is
Facilitation, but Ag+There is the effect of significantly inhibiting；(2)Co2+、Fe2+、Mg2+、Ni2+To enzyme activity, there is rush Deng divalent metal ions
Enter effect, and remaining bivalence, trivalent metal ion are inhibited to enzyme activity；(3) glycerol, beta -mercaptoethanol and DTT exist
During 10mM, activity is obviously promoted to the activity of rAly-1, enzyme activity can be made respectively to improve to 125.8%, 130.4% and
116.4%.
Embodiment 9, DNS- reducing sugar method measure the enzyme activity of restructuring algin catenase rAly-1
The restructuring Brown algae that the mass concentration that deionized water is configured is the Algin of 0.1-1.2%, concentration is 10 μ g/mL
Glue lyases rAly-1 enzyme liquid, HAc-NaAc (pH 6.0) buffer of 150mmol/L and water press 1：1：The ratio of 1 (volume ratio)
After example mixing, at 50 DEG C, react 4h.Product is heated 10min in boiling water bath, proceeds to 5min in ice-water bath, 12,000
× g, it is centrifuged 15min under the conditions of 4 DEG C, collect supernatant；By the supernatant of certain volume, (3,5- is to nitro diformazan with isopyknic DNS
Benzene) mixing of-reactant liquor, heat 10min in boiling water bath, be down to room temperature, measure absorption value in 540nm.With analyzing pure glucose
Standard substance made by aldehydic acid sodium lactone, and same method operates, and draw molar concentration and the OD of Glucuronic acid lactone540Between dose-effect
Relation curve.With being purchased from the quantification of protein kit measurement restructuring algin catenase of Shanghai Sheng Gong biological engineering company limited
Protein content in rAly-1 enzyme liquid.Define the unit of activity calculating enzyme, that is, at the standard conditions, every point according to international standard
Producing the enzyme amount needed for 1 μm of ol product in clock is 1 IU.Result shows：Restructuring rAly-1 can be digested simultaneously with Algin as substrate
Produce reducing sugar product, enzyme activity is 1261 ± 8.2U/mg.
Embodiment 10, efficient liquid phase (HPLC) analysis of the product of restructuring algin catenase rAly-1 degraded Algin
Deionized water prepares the Algin substrate that mass-volume concentration (g/mL) is 0.1～1.2%, after heating for dissolving,
It is placed in cooling 1h in 50 DEG C of water bath.Add the diluent of the recombinase rAly-1 that embodiment 3 is obtained in every 100 μ L substrates
10-100 μ L, during less than 200 μ L volume, is supplied with sterile deionized water；After mixing continue reaction, every when sampling.Reaction is produced
Thing heats 10min in boiling water bath, proceeds to 5min in ice-water bath.12,000 × g, it is centrifuged 15min, in collection under the conditions of 4 DEG C
Clearly.
The NH being 0.20mol/L with concentration4HCO3Solution, balance Superdex Peptide 10/300GL (GE company) point
Sub- gel chromatographic columnses, flow velocity 0.40mL/min, at least 2 post beds.By the sample of the different enzymolysis times of above-mentioned Algin, with automatic
Injector loads 100 μ g/ samples, and other conditions are constant, and 232nm detects.Operate software with HPLC, analyze the long-pending of each oligosaccharide compositions
Facet is amassed, and calculates relative molar concentration.
As shown in figure 8, under these conditions, after Algin substrate is degraded, with the increase of enzyme digestion reaction time, have
The content of the product oligosaccharides of 235nm characteristic absorption is gradually increased, and relative amount finally tends towards stability.This tentatively shows to recombinate brown
Algin lyases rAly-1 is algin catenase.The final principal product of enzyme digestion reaction is that in HPLC analysis, appearance time is
Two product oligosaccharides of 40.5' and 43.2', area ratio stabilizes to 1:1, show that the two mol ratio is about 1:1.
Embodiment 11, restructuring algin catenase rAly-1 thoroughly degrade Algin oligosaccharide principal product molecular weight identification
According to described in embodiment 10, common 200mg Algin restructuring algin catenase rAly-1 is thoroughly digested, sample
Loading, excessively Superdex Peptide 10/300GL molecular gel chromatographic column (GE company) in batches, and according to appearance time respectively
Collect two oligosaccharide sample that appearance time is 40.5' and 43.2'.Repeatedly collect two oligosaccharide sample are concentrated respectively
Afterwards, repeatedly lyophilization with desalination.Dissolve gained oligosaccharide sample with sterile deionized water, carry out first mass spectrometric (MS) analysis, really
The relative molecular weight of fixed each oligosaccharide.Also use heavy water (D2O) dissolve the oligosaccharide sample of gained, lyophilization repeatedly, to complete hydrogen deuterium
Displacement, and carry out1H-NMR analyzes, and finally determines chemical constitution and the feature of each oligosaccharide.
As shown in figure 9, thoroughly being degraded prepared unsaturated oligosaccharide after Algin with restructuring algin catenase rAly-1
Piece degree UDP2, UDP3 carry out HPLC analysis, and result display product has characteristic absorption in 235nm, and appearance time is respectively 40.5'
With 43.2, purity is all higher than 99%, meets further experiment requirement.
The result (Figure 10) of the first mass spectrometric analysis carrying out under ion mode shows, this two oligosaccharide principal products point
Son amount is 352,528 respectively.This shows, above-mentioned two oligosaccharide principal product is that restructuring algin catenase rAly-1 thoroughly drops respectively
Produced unsaturation disaccharide (UDP2, Figure 10-1) and unsaturated trisaccharide (UDP3, Figure 10-2) after solution Algin.
The result of comprehensive Fig. 8,9,10, unsaturated disaccharide is poly- after restructuring algin catenase rAly-1 degraded Algin
Right minimum oligosaccharide principal product.
Pass through further1The architectural feature of H-NMR data analysiss above two oligosaccharide：(1) as shown in Figure 11-1,
Characteristic chemical shifts value at 5.75ppm shows, the principal product after restructuring algin catenase rAly-1 degraded Algin is not
Saturation disaccharide UDP2 almost all is Δ G.(2) chemical displacement value at 5.70,5.60ppm two as shown in Figure 11-2, shows to lead
The non reducing end of product unsaturation trisaccharide UDP3 has two types：Δ G and Δ M；And at two the area integral at peak ratio
For 100:83, show that the mol ratio of this two classes unsaturation trisaccharide is about 5：4.
Embodiment 12, restructuring algin catenase rAlgL-5 not exclusively degrade Algin product UDP2-UDP6 preparation
According to patent application (application number 201410469861.4, a kind of endo-type algin catenase and its encoding gene
With application) in described in embodiment 10, common 200mg Algin is not thoroughly digested with recombinase rAlgL-5, product is gone up in batches
Sample, excessively molecule gel column Superdex Peptide 10/300GL (GE company), and collect single widow respectively according to appearance time
Bglii fragment.These oligosaccharide sample are repeatedly collected, respectively concentrate after, lyophilization is with desalination repeatedly.
As shown in figure 12, oligose fragment UDP2-UDP6 of preparation tests and analyzes through HPLC, and result display product is in 235nm
There is characteristic absorption, purity is all higher than 99%, meet further experiment requirement
Embodiment 13, the analysis of restructuring algin catenase rAly-1 minimum unsaturation oligosaccharide substrates
Oligose fragment prepared by deionized water and embodiment 12, including：Unsaturated seven sugared (UDP7), unsaturated six sugar
(UDP6), unsaturated pentasaccharides (UDP5), unsaturated tetrose (UDP4), unsaturated trisaccharide (UDP3) and unsaturated disaccharide (UDP2).
These oligosaccharide sample are respectively configured substrate solution, the restructuring algin catenase rAly-1 enzyme liquid being 10 μ g/mL with concentration,
HAc-NaAc (pH 6.0) buffer of 150mmol/L and water press 1：1：After the ratio mixing of 1 (volume ratio), 50 DEG C of reactions
24h.According to operation condition of chromatogram described in embodiment 10, carry out HPLC analysis.As shown in figure 13, herein described restructuring is brown for result
Algin lyases rAly-1：
(1) unsaturated six sugared (UDP6) that degrade produce unsaturated tetrose, unsaturated trisaccharide and unsaturated disaccharide afterwards；
(2) unsaturated pentasaccharides (UDP5) of degrading produces the unsaturated trisaccharide of equivalent and unsaturated disaccharide afterwards；
(3) unsaturated tetrose (UDP4) of degrading produces the unsaturated disaccharide of twice afterwards；
(4) no significant changes before and after reacting with unsaturated trisaccharide or unsaturated disaccharide.
This fully shows, unsaturated four bglii fragments are the minimum unsaturation oligosaccharide bottoms of restructuring algin catenase rAly-1
Thing, and the algin catenase rAly-1 that recombinates degrades oligosaccharide substrates in restriction endonuclease mode.
Embodiment 14, high performance liquid chromatography (HPLC) analysis of restructuring algin catenase rAly-1 enzyme cleavage patterns
Take the solution containing about the poly- M pentasaccharides (M5) of 30 μ g saturations, poly- G pentasaccharides (G5), the HAc-NaAc (pH of 150mmol/L
6.0) diluent of the restructuring algin catenase rAly-1 that buffer, embodiment 3 are obtained, according to volume ratio 1:1:1 mixing, puts
12h is reacted in 50 DEG C of water-baths.Reaction system is put 10min in boiling water bath, goes to ice-water bath 5min, 12,000 × g, 4 DEG C
Under the conditions of be centrifuged at least 15min.Collect supernatant, as the oligosaccharide catabolite of restructuring algin catenase rAly-1.With in advance
The restructuring algin catenase rAly-1 enzyme liquid of inactivation in boiling water bath, does negative control reaction.
According to the chromatographic condition described in embodiment 10, the algin catenase rAly-1 that will recombinate digests saturation poly- M pentasaccharides
(M5), the sample of poly- G pentasaccharides (G5), loads 20 μ g/ samples with automatic sampler, and other conditions are constant, and 232nm detects.With
HPLC operates software, analyzes the integral area of each oligosaccharide compositions, calculates relative molar concentration.With reference to molecular weight standard thing, determine
The relative molecular weight of each oligosaccharide.
As Figure 14, after algin catenase rAly-1 degraded saturation poly- M pentasaccharides (M5) that recombinates, produce unsaturated trisaccharide
UM3.As shown in figure 15, produce unsaturated trisaccharide after restructuring algin catenase rAly-1 degraded saturation poly- G pentasaccharides (G5)
UG3.This shows, restructuring algin catenase rAly-1 is facultative endo-type algin catenase, and in degraded saturation type pentasaccharides
During substrate, its saturation type disaccharide product generates the non reducing end from substrate, and unsaturated trisaccharide product is created on substrate
Reducing end under neutral.
Embodiment 15, fluorescence-high performance liquid chromatography (HPLC) analysis of restructuring algin catenase rAly-1 enzyme cleavage patterns
Take the solution containing about the poly- M pentasaccharides (M5) of 10 μ g saturations, poly- G pentasaccharides (G5), rotation is evaporated.Add containing excessive neighbour's amino
Benzoylamide (2-AB), dimethyl sulfoxide (DMSO) solution of boron nitrilation sodium, mix and incubate 2h in rearmounted 60 DEG C of water-baths.Rotation is steamed
Dry, add 500 μ L deionized water dissolving samples, sample is swung with 200 μ L chloroform resonance, centrifugation, collect supernatant.Continue to use chloroform
Repeatedly extract, no less than 7 times, obtain saturation poly- M pentasaccharides (2AB-M5) that reducing end under neutral has been fluorescently labeled, fluorescently-labeled
Saturation poly- G pentasaccharides (2AB-G5).The molecular weight standard thing of same method labelling saturation oligosaccharide.
Above-mentioned 2AB-M5,2AB-G5 sample, HAc-NaAc (pH 6.0) buffer of 150mmol/L, embodiment 3 is taken to be obtained
Restructuring algin catenase rAly-1 diluent, according to volume ratio 1:1:1 mixing, is placed in reaction 12h in 40 DEG C of water-baths.Will
Reaction system puts 10min in boiling water bath, goes to ice-water bath 5min, 12,000 × g, is centrifuged at least 15min under the conditions of 4 DEG C.Receive
Collection supernatant, as the oligosaccharide catabolite of restructuring algin catenase rAly-1.To heat the weight of 10min in advance in boiling water bath
Group algin catenase rAly-1 enzyme liquid, does negative control reaction.
According to the chromatographic condition described in embodiment 10, by the labelling sample of 2AB-M5,2AB-G5 and its enzymatic hydrolysate, with certainly
Dynamic injector loads 50-200ng/ sample, and other conditions are constant, and 330nm excites, and 420nm detects.Operate software with HPLC, point
Analyse the integral area of each oligosaccharide compositions, calculate relative molar concentration.With reference to molecular weight standard thing, determine the average molecular of each oligosaccharide
Amount.
As shown in Figure 16,17, after being reacted with 2AB-M5 with excessive restructuring algin catenase rAly-1, only dropped on a small quantity
Solution, and produce the 2AB-M3 of equimolar amountss.But, this enzyme can degradable 2AB-G5, and generate the 2AB-G3 of equimolar amountss.This
Show, restructuring algin catenase rAly-1 is facultative endo-type algin catenase, and carrying out during enzyme digestion reaction is from substrate
Non reducing end discharges minimum product, including saturation type disaccharide or unsaturated disaccharide.
Further, 14 and 16, and 15 and 17, explanation are contrasted:(1) 2-AB to saturation pentasaccharides end labelling it is suppressed that
The degrading activity to oligosaccharide for the enzyme rAly-1；(2) rAly-1 is as facultative endo-type algin catenase, the Preference to polyG
Slightly larger than polyM.
Claims (6)
1. a kind of facultative endo-type restructuring algin catenase rAly-1, aminoacid sequence is as shown in SEQ ID NO.2.
2. the encoding gene of facultative endo-type restructuring algin catenase rAly-1 described in claim 1, nucleotide sequence such as SEQ
Shown in ID NO.1.
3. a kind of recombinant expression carrier is it is characterised in that insert the facultative endo-type described in claim 2 in expression vector
The encoding gene of restructuring algin catenase rAly-1.
4. a kind of recombinant bacterium is it is characterised in that proceeded to the recombinant expression carrier described in claim 3 in host cell.
5. the encoding gene of facultative endo-type restructuring algin catenase rAly-1, weight described in claim 3 described in claim 2
Group expression vector, recombinant bacterium described in claim 4 are preparing the application that facultative endo-type is recombinated in algin catenase rAly-1.
6. described in claim 1, facultative endo-type restructuring algin catenase rAly-1 is few in degraded Algin, degraded Algin
Application in sugar or the unsaturated oligosaccharide of preparation.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811454A (en) * | 2017-02-28 | 2017-06-09 | 滕州市悟通香料有限责任公司 | A kind of restructuring algin catenase rAly1 T185N of truncation and its encoding gene and application |
| CN107574196A (en) * | 2017-08-02 | 2018-01-12 | 济南悟通生物科技有限公司 | Application of the algin catenase in series of oligosaccharides product is prepared |
| CN108048435A (en) * | 2017-12-14 | 2018-05-18 | 山东大学 | A kind of difunctional algin catenase Aly2 of endo-type for generating a variety of monosaccharide products and its encoding gene and application |
| CN110387367A (en) * | 2019-09-03 | 2019-10-29 | 中国海洋大学 | A kind of novel algin catenase |
| CN110423787A (en) * | 2019-08-09 | 2019-11-08 | 中国海洋大学 | A kind of preparation method of homogeneity brown alga trisaccharide |
| CN110687154A (en) * | 2019-10-22 | 2020-01-14 | 山东大学 | Alginate oligosaccharide sequencing method based on alginate lyase and application thereof |
| CN115109768A (en) * | 2021-04-06 | 2022-09-27 | 济南爱科替维生物科技有限公司 | Broad-spectrum polysaccharide degrading enzyme rAly16-1 from streptomyces as well as coding gene and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293754A (en) * | 2014-09-12 | 2015-01-21 | 山东大学 | Incision-type sodium alginate lyase as well as encoding gene and application thereof |
| CN105713889A (en) * | 2014-12-02 | 2016-06-29 | 中国科学院大连化学物理研究所 | Alginate lyase Alga and coding gene and application thereof |
| CN105821063A (en) * | 2015-01-05 | 2016-08-03 | 中国科学院大连化学物理研究所 | Incision alginate lyase Alg2B and coding gene, preparation and application thereof |
-
2016
- 2016-09-21 CN CN201610838337.9A patent/CN106399285A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293754A (en) * | 2014-09-12 | 2015-01-21 | 山东大学 | Incision-type sodium alginate lyase as well as encoding gene and application thereof |
| CN105713889A (en) * | 2014-12-02 | 2016-06-29 | 中国科学院大连化学物理研究所 | Alginate lyase Alga and coding gene and application thereof |
| CN105821063A (en) * | 2015-01-05 | 2016-08-03 | 中国科学院大连化学物理研究所 | Incision alginate lyase Alg2B and coding gene, preparation and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "CP003560.1", 《NCBI》 * |
| GENBANK: "WP_066210133.1", 《NCBI》 * |
| 赵琳等: "海藻酸裂解酶异源表达研究进展 ", 《生命科学研究》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN106811454B (en) * | 2017-02-28 | 2021-03-23 | 滕州市悟通香料有限责任公司 | Truncated recombinant alginate lyase rAly1-T185N and coding gene and application thereof |
| CN107574196B (en) * | 2017-08-02 | 2021-05-04 | 济南悟通生物科技有限公司 | Application of alginate lyase in preparation of series oligosaccharide products |
| CN107574196A (en) * | 2017-08-02 | 2018-01-12 | 济南悟通生物科技有限公司 | Application of the algin catenase in series of oligosaccharides product is prepared |
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| CN108048435B (en) * | 2017-12-14 | 2021-05-25 | 山东大学 | Incision type bifunctional alginate lyase Aly2 for generating various monosaccharide products, and coding gene and application thereof |
| CN110423787A (en) * | 2019-08-09 | 2019-11-08 | 中国海洋大学 | A kind of preparation method of homogeneity brown alga trisaccharide |
| CN110423787B (en) * | 2019-08-09 | 2022-03-04 | 中国海洋大学 | Preparation method of uniform brown algae trisaccharide |
| CN110387367A (en) * | 2019-09-03 | 2019-10-29 | 中国海洋大学 | A kind of novel algin catenase |
| CN110387367B (en) * | 2019-09-03 | 2020-05-01 | 中国海洋大学 | Algin lyase |
| CN110687154A (en) * | 2019-10-22 | 2020-01-14 | 山东大学 | Alginate oligosaccharide sequencing method based on alginate lyase and application thereof |
| CN110687154B (en) * | 2019-10-22 | 2023-08-01 | 山东大学 | Algin oligosaccharide sequencing method based on algin lyase and application thereof |
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