CN110066779A - Agarase gene, recombinant plasmid, recombinant bacterial strain, agarase and its application - Google Patents
Agarase gene, recombinant plasmid, recombinant bacterial strain, agarase and its application Download PDFInfo
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Abstract
The present invention discloses a kind of agarase gene, recombinant plasmid, recombinant bacterial strain, agarase and its application, is related to gene engineering technology field.The amino acid sequence of the agarase is as shown in SEQ ID NO:2；The nucleotide sequence of the agarase gene caAgaA is as shown in SEQ ID NO:1；The recombinant plasmid carries the agarase gene caAgaA；The recombinant bacterial strain includes the agarase gene caAgaA.Agarase provided by the invention has preferable thermal stability and pH stability, can be used for the preparation of new fine jade oligosaccharides and the recycling of the nucleic acid after Ago-Gel.
Description
Technical field
The present invention relates to gene engineering technology field, in particular to a kind of agarase gene, recombinant plasmid, recombinant bacterium
Strain, agarase and its application.
Background technique
Agar is a kind of heteroglycan in red algae cell wall, mainly by by two kinds of polysaccharide groups of agarose and agaropectin
At.Agar or agarose hydrolyze available biologically active oligosaccharides substance through agarase, utilize agarase
This characteristic, agarase in the industrial production with important application value is all had in biological study.
However, often having the following problems in industrial production and biological study: agarase is often to solid bottom
Object activity is very poor, but agarose condensing temperature is higher than 60 DEG C；Graininess enzyme effect more preferably, but enzyme product granulating when need
Undergo 80 DEG C of pyroprocesses；In addition, in industrialized production, pH environment can also change with process conditions, but existing at present
Agarase it is poor to the adaptability of high temperature and acid-base environment, do not adapt to above-mentioned application scenarios, this is greatly
The application and development of agarase are limited, therefore, it is necessary to excavate the agar for providing good thermal stability and pH stability
Carbohydrase.
Summary of the invention
The main object of the present invention be propose a kind of agarase gene, recombinant plasmid, recombinant bacterial strain, agarase and its
Using, it is desirable to provide a kind of agarase, the agarase have preferable thermal stability and pH stability.
To achieve the above object, the present invention proposes a kind of agarase, the amino acid sequence of the agarase such as SEQ
Shown in ID NO:2.
The present invention also proposes a kind of agarase gene caAgaA, for encoding above-mentioned agarase, the agarase
The nucleotide sequence of gene caAgaA is as shown in SEQ ID NO:1.
The invention also provides a kind of recombinant plasmids, carry agarase gene caAgaA as claimed in claim 2.
Preferably, the recombinant plasmid includes expression vector pET-28a.
The invention also provides a kind of recombinant bacterial strains, include above-mentioned agarase gene caAgaA.
Preferably, the recombinant bacterial strain is by the recombinant plasmid transformed large intestine bar comprising above-mentioned agarase gene caAgaA
Bacterium BL21 (Rosetta) is obtained.
In addition, the application the invention also provides above-mentioned agarase in the preparation of new fine jade oligosaccharides.
And answering in the nucleic acid recycling that the invention also provides above-mentioned agarases after agarose gel electrophoresis
With.
In technical solution of the present invention, the recombinant plasmid and again that provides agarase gene caAgaA, carry the gene
Group bacterial strain, agarase gene caAgaA can encode out a kind of agarase, and the agarase is not only with higher
Agar or agarose can be hydrolyzed to the new fine jade oligosaccharides of 4 and 6 residues, effectively so as to be used for bioactivity by catalytic activity
The preparation of new fine jade oligosaccharides, and there is splendid thermal stability and pH stability, so that the application scenarios of agarase have been expanded,
Technology difficulty when its application is reduced, input cost is reduced, therefore, agarase provided by the invention is in the industrial production
With stronger applicability.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the SDS-PAGE test result figure of agarase provided by the invention；
Fig. 2 is the enzyme activity determination result figure of agarase provided by the invention at various ph values；
The stability test result figure of Fig. 3 agarase provided by the invention at various ph values；
Fig. 4 is the enzyme activity determination result figure of agarase provided by the invention at different temperatures；
Fig. 5 is the stability test result figure of agarase provided by the invention at different temperatures；
Fig. 6 is enzyme activity determination result figure of the agarase provided by the invention in different metal ions environment；
Fig. 7 is the thin-layer chromatography test result figure that agarase provided by the invention hydrolyzes agarose；
Fig. 8 is the test result figure of nucleic acid recycling of the agarase provided by the invention after agarose gel electrophoresis.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
In industrial production and biological study, often have the following problems: agarase is often living to solid substrate
Property is very poor, but agarose condensing temperature is higher than 60 DEG C；Graininess enzyme effect more preferably, but enzyme product granulating when need to undergo
80 DEG C of pyroprocesses；In addition, in industrialized production, pH environment can also change with process conditions, but existing fine jade at present
Lipolysaccharide enzyme is poor to the adaptability of high temperature and acid-base environment, does not adapt to above-mentioned application scenarios, this is greatly limited
The application and development of agarase.
In consideration of it, the invention proposes a kind of agarase, the amino acid sequence of the agarase such as SEQ ID NO:2
It is shown.
The agarase contains 310 amino acid residues, and theoretical molecular weight is 35496.89 dalton, and isoelectric point is
4.42.Its sequence is as shown in SEQ ID NO:2, with the agarase of the same glycoside hydrolase Families (GH16) in existing report
Amino acid sequence (NCBI accession number are as follows: AAF21821.1, AXY54922.1, ATG22743.1ALO78721.1,
ATI96843.1、AXK59832.1、WP_066965750.1、ABW77762.1ANT83229.1、BAQ95400.1、
AGU13985.1、AIF29515.1、AGW43026.1AFR90184.1、ACM50513.1、ADD60418.1、AAN39119.2、
BAF34350.1ABL06969.1, AAF21820.1, BAD29947.1, BAC99022.1) consistency it is lower, about 20~
63%, that is to say, that this is a kind of completely new agarase.
Inventors have found that the agarase is endo-type: the new fine jade that agarose can be hydrolyzed to 4 and 6 residues is few
Sugar, rather than hydrolyzing becomes disaccharides (i.e. new fine jade disaccharides)；And optimal reactive temperature is 40 DEG C, optimal reaction pH is 7.0.Meanwhile
Its biochemical character is also embodied by: Zn2+With reproducibility reagent (dithiothreitol (DTT), DTT) to agarase under the concentration of 5mM
CaAgaA has apparent activation effect.Specific manifestation are as follows: under conditions of DTT concentration is 5mM, agarase caAgaA performance
Activity out is 2.1 times of enzyme activity under the conditions of no DTT；In Zn2+Under conditions of concentration is 5mM, agarase caAgaA performance
About 165% enzyme activity out.In addition, the agarase be incubated at a temperature of 70 DEG C can still be maintained after 2h about 80% enzyme activity,
It still is able to the enzyme activity for maintaining to be greater than 40% after being incubated for 2h under the conditions of pH is 3~11, i.e., agarase tool provided by the invention
There are fabulous thermal stability and pH stability, that is to say, that requirement of the agarase provided by the invention to temperature and pH condition
Lower, it can be adapted for more application scenarios, therefore, have stronger applicability in the industrial production.
For this purpose, the present invention proposes a kind of agarase gene caAgaA, for encoding above-mentioned agarase, the agarose
The nucleotide sequence of enzyme gene caAgaA is as shown in SEQ ID NO:1.
Inventors have found that deriving from the agarase encoding gene sequence of Deep-Sea Microorganisms Cellulophaga algicola
The protein of the predictable coding of column has fabulous thermal stability and pH stability, is based on this reason, and in order to adapt to have
There is the engineering bacteria of different codon preferences to realize the great expression of agarase, inventor derives from Deep-Sea Microorganisms to this
The agarase coding gene sequence of Cellulophaga algicola is designed, to agarase coding gene sequence into
Go codon optimization, it is final to obtain nucleotide sequence agarase gene caAgaA as shown in SEQ ID NO:1.To the base
Because recombinant expression can obtain agarase as described above, agarase catalytic activity with higher and biology are living
Property, and after Function Identification, it was demonstrated that it is endo-type, has above-mentioned biochemical character and wide with high temperature resistant and pH tolerance range
General advantage.
Specifically, agarase gene caAgaA provided by the invention is searched in GenBank gene database by comparison
Rope (PHI-BLAST, for the free sequence similarity search program of US National biological information research center exploitation, network address are as follows:
Https: //blast.ncbi.nlm.nih.gov/Blast.cgi) it finds a kind of from marine microorganism
The agarase encoding gene of (Cellulophaga algicola) is then obtained its codon and is optimized by artificial synthesized
Nucleotide.1098 bases of the full length gene.54, gene 5 ' end nucleotide may be signal peptide sequence, therefore carry out base
Because removing 54 nucleotide when expression, nucleotides sequence when eventually for construction of recombinant plasmid is classified as shown in SEQ ID NO:1,
Totally 933 bases.
Further, concrete operations when above-mentioned agarase gene is excavated can be accomplished by the following way: firstly, structure
The mode sequences (obtaining by HMMER program) of agarase protein sequence are built, sequence is search sequence in this mode, is used
Blast program searches for the protein sequence in NCBI Protein Data Bank, finds agarase gene proposed by the invention
caAgaA；Then according to the preferences of e. coli codon, which is optimized, is obtained
Agarase gene caAgaA.It should be noted that agarase gene caAgaA is synthesized in Suzhou Jin Weizhi company, synthesis
Agarase gene caAgaA be located on pU57 carrier, the 5 ' ends and 3 ' ends for synthesizing gene are with restriction enzyme site
EcoR1 and Not1.Two restriction enzyme sites will be used for an agarase gene caAgaA and be cloned into Bacillus coli expression
Carrier pET-28a.
Meanwhile the invention discloses a kind of recombinant plasmid, which carries above-mentioned agarase gene caAgaA.
It should be appreciated that in the recombinant vector for inserting above-mentioned agarase gene caAgaA belongs to the scope of protection of the present invention.
Further, in repeatedly considering by the amino acid structure to expression cell and agarase proposed by the present invention
Afterwards, preferably using coli expression carrier pET-28a as the carrier of recombinant plasmid.Its reason is: Bacillus coli expression
Carrier pET-28a itself has restriction enzyme site as described above, is convenient for reorganization operation, and itself has kalamycin resistance base
Cause can be used for vector selection.It should be noted that the preparation method of above-mentioned recombinant plasmid may comprise steps of:
By artificial synthesized acquisition agarase gene caAgaA, synthesized agarase gene caAgaA is located at pU57
On carrier, agarase gene caAgaA is scaled off from pU57 carrier by restriction enzyme site EcoR1 and Not1, digestion obtains
DNA fragmentation be then connected on the coli expression carrier pET-28a through same digestion (EcoR1 and Not1 digestion), connect
Product is transformed into bacillus coli DH 5 alpha, and kit extracts plasmid, is identified with EcoR1 and Not1 double digestion plasmid, digestion
It is correctly recombinant plasmid pET-28a-caAgaA.
In addition, including above-mentioned agarase gene caAgaA the invention also provides a kind of recombinant bacterial strain.
The recombinant bacterial strain can be by the recombinant plasmid transformed Escherichia coli comprising above-mentioned agarase gene caAgaA
BL21 (Rosetta) is obtained.That is, the recombinant bacterial strain can also include above-mentioned recombinant plasmid.Above-mentioned recombinant plasmid is imported and is expressed
It can be obtained recombinant bacterial strain in cell, target gene can be replicated with the breeding of host cell.Usually, the expression
Cell can be Escherichia coli, be also possible to the cells such as yeast or other kinds of cell such as zooblast etc., at this
In embodiment, expression cell is preferably e. coli bl21 (Rosetta).
Based on this, above-mentioned recombinant bacterial strain can be made by following step:
Step S1, agarase gene caAgaA is connected to carrier pET-28a by restriction enzyme site EcoR1 and Not1
On, connection product is transformed into bacillus coli DH 5 alpha, is extracted plasmid with kit, is reflected by EcoR1 and Not1 double digestion
Fixed, digestion is correctly recombinant plasmid pET-28a-caAgaA；
Step S2, recombinant plasmid pET-28a-caAgaA is used into CaCl2Method converts e. coli bl21 (Rosetta),
Obtain the recombination bacillus coli BL21 (Rosetta) containing recombinant plasmid pET-28a-caAgaA, as recombinant bacterial strain.
And the preparation method based on above-mentioned recombinant bacterial strain, it can be deduced that cultivate above-mentioned recombination thallus to form above-mentioned agarose
The preparation method of enzyme, the preparation method can be realized when specific operation by following steps:
S10, agarase gene caAgaA is connected on carrier pET-28a by restriction enzyme site EcoR1 and Not1, even
Object of practicing midwifery is transformed into bacillus coli DH 5 alpha, is extracted plasmid with kit, is identified by EcoR1 and Not1 double digestion, enzyme
Cutting correctly is recombinant plasmid pET-28a-caAgaA；
S20, recombinant plasmid pET-28a-caAgaA is used into CaCl2Method converts e. coli bl21 (Rosetta), obtains
E. coli bl21 (Rosetta) (as recombinant strains) containing recombinant plasmid pET-28a-caAgaA；
S30, the e. coli bl21 (Rosetta) containing recombinant plasmid pET-28a-caAgaA is pressed into 1:90~110
Ratio (preferably 1:100) is seeded in ZYP-5052 culture medium, in 37 DEG C, 180~220rpm (preferably 200rpm) condition
It is lower to cultivate to OD600It is 0.95~1.05, temperature is then reduced to 18~22 DEG C (preferably 20 DEG C), continues 20~26h of culture
After (preferably for 24 hours), thalline were collected by centrifugation, and thallus is suspended in HisTrap A buffer, then by being centrifuged after pressure breaking
Take supernatant (i.e. comprising the recombinant protein formed that fermented by recombinant bacterial strain in supernatant)；
S40, by supernatant by after HisTrap affinity column, using 20%HisTrap B buffer in AKTA albumen
The recombinant protein on instrument on elution chromatography column is purified, then will be weighed using the Millipore concentration tube that molecular cut off is 10kDa
It is 1mg/mL, the agarase purified that histone, which changes liquid and is concentrated into protein concentration into phosphate buffer,.
Wherein, the pH value of the HisTrap A buffer is 7.4, and the HisTrap A buffer includes following mole dense
The component of degree: the imidazoles of NaCl, 20mM of 500mM and the Na of 20mM2HPO4；The pH value of the HisTrap B buffer is 7.4,
The HisTrap B buffer includes the component of following molar concentration: the imidazoles and 20mM of NaCl, 500mM of 500mM
Na2HPO4。
The specific formula of ZYP-5052 culture medium is as follows: 1% peptone, 5% yeast extract, 50mM Na2HPO4, 50mM
KH2PO4, 25mM (NH4)2SO4, 0.5% glycerine, 0.05% glucose, 0.2% lactose, 2mM MgSO4, 100 μ of kanamycins
G/ml, 34 μ g/ml of chloramphenicol.
The phosphate buffer contains the component of following molar concentration: 137mM NaCl, 2.7mM KCl, 10mM
Na2HPO4, 1.8mM KH2PO4。
It should be noted that above-mentioned be related to gene chemical synthesis, recombinant plasmid and recombinant bacterial strain preparation and recombinant bacterial strain hair
Ferment culture obtains in the embodiments such as recombinant protein, specific experiment mode, experiment raw material person is not specified, according to genetic engineering
And the conventional method progress that microbial fermentation culture prepares protein is carried out based on genetic engineering.
In addition, the feature of endo-type, high temperature resistant and acid-base property based on above-mentioned agarase, the present invention also proposes institute as above
Application of the agarase stated in the new fine jade oligosaccharides preparation of bioactivity or nucleic acid recycling.
Since agarase proposed by the present invention is endo-type, i.e. agar or agarose can be hydrolyzed to by the agarase
Therefore the agarase can be applied in the preparation of the new fine jade oligosaccharides of bioactivity, i.e., by the new fine jade oligosaccharides of 4 and 6 residues
Using agar or agarose as raw material, agarase proposed by the present invention is added and is hydrolyzed, is then obtained by purification procedures
Get Xin Qiong oligosaccharides.Moreover, because the feature that agarase proposed by the present invention is wide in range with high temperature resistant and pH tolerance range, the fine jade
Lipolysaccharide enzyme can be prepared to graininess enzyme, while bring convenience for the design of the technique for applying of agarase, for example, by agarose
Enzyme carries out hydrolysis together with agar or agarose after High-temperature Liquefaction.
Agarose gel electrophoresis detection method is common a kind of detection method in molecular biology, after detection such as
Nucleic acid substances recycling in gel is a ring particularly important in the detection method by what.It can be degraded agar based on agarase
This sugared function, agarase can be applied in the step for nucleic acid recycles, since agarase is often to solid substrate
Activity is very poor, and agarase provided by the invention has characteristic resistant to high temperature therefore in the specific implementation, can be by the fine jade
After High-temperature Liquefaction, reaction is hydrolyzed together with the Ago-Gel after electrophoresis in lipolysaccharide enzyme, is degraded by agarose thus by core
Acid substance releases, and then achievees the purpose that recycle nucleic acid substances.
In conclusion agarase gene caAgaA provided by the invention, after being transferred to Escherichia coli by carrier, is weighed
Group expression bacterial strain, then agarase, institute is made after high cell density fermentation, affinity purification method in recombinant strains
Agarase high temperature resistant obtained and acid-base property are fabulous, and the new fine jade that agarose effectively can be hydrolyzed to 4 and 6 residues is few
Sugar can be used for the preparation of the new fine jade oligosaccharides of bioactivity and the recycling of nucleic acid after nucleic acid electrophoresis.
Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that
Following embodiment is only used to explain the present invention, is not intended to limit the present invention.
The acquisition of 1 agarase gene caAgaA of embodiment
Using the protein sequence of reported agarase as search sequence, with PHI-BLAST program search NCBI albumen
Matter sequence database finds agarase gene caAgaA according to the present invention；Then according to the inclined of e. coli codon
Love property, optimizes agarase gene caAgaA codon, obtains the agarase gene for recombinant expression,
Nucleotide sequence is as shown in SEQ ID NO:1.Agarase gene caAgaA is synthesized in Suzhou Jin Weizhi company, the agar of synthesis
Carbohydrase gene caAgaA is located on pU57 carrier, synthesize gene 5 ' ends and 3 ' ends with restriction enzyme site EcoR1 and
Not1。
The preparation of 2 recombinant plasmid of embodiment
(1) agarase for being obtained embodiment 1 with restriction enzyme EcoR1 and Not1 (being purchased from Takara company)
Gene caAgaA is scaled off from pU57 carrier, is tapped and recovered the caAgaADNA segment cut；
(2) caAgaA DNA fragmentation is connected to pET-28a carrier by T4DNA ligase (being purchased from Takara company)
(it is a kind of for recombinantly expressing the commercial carrier of foreign gene in Escherichia coli, it is purchased from Novagen company, band on the carrier
Have kalamycin resistance gene) on；
(3) connection product is passed through into CaCl2Method is transformed into a kind of bacillus coli DH 5 alpha (clone of biology laboratory routine
Bacterial strain is purchased from Invitrogen company, is stored in Wuhan Polytechnic University laboratory) in competent cell.The cell of conversion is applied
Cloth carries out positive transformant screening on the LB agar plate containing 100 μ g/ml kanamycins (being purchased from Sigma company)；Wherein
The preparation for the bacillus coli DH 5 alpha competent cell being related to and CaCl2Method for transformation is the routine operation of biology laboratory,
Specific method can refer to the chapters and sections in mono- book of Current Protocols in Molecular Biology about DNA conversion
(doi.org/10.1002/0471142727.mb0108s37)；The LB agar plate wherein used is by LB culture medium
The agar that (containing 10g NaCl, 5g yeast extract and 10g peptone in every liter) is added 2.5% is prepared.
(4) picking monoclonal is seeded in the LB culture medium containing 100 μ g/ml kanamycins, is incubated overnight, then with examination
Agent box extracts plasmid, is identified by EcoR1 and Not1 double digestion, and digestion result is correctly recombinant plasmid pET-28a-
caAgaA。
The preparation of 3 recombinant bacterial strain of embodiment
(1) the recombinant plasmid pET-28a-caAgaA for obtaining embodiment 2 uses CaCl2Method converts e. coli bl21
(Rosetta) (it is purchased from Novagen company, is the common gene engineering expression bacterium in laboratory, it is anti-that which comes with chloramphenicol
Property, it is stored in Wuhan Polytechnic University laboratory) competent cell；The wherein preparation of competent E.coli BL21 (Rosetta)
And method for transformation is identical as the method in embodiment 2 (3)；
(2) that the e. coli bl21 (Rosetta) after conversion is coated on (100 μ g/ml) and chlorine containing kanamycin is mould
On the LB agar plate of element (34 μ g/ml are purchased from Sigma company).The bacterium colony grown on this Double plate is recombinant bacterial strain.
The preparation of 4 agarase of embodiment
(1) (add 100 μ g/ml kanamycins and 34 μ in culture medium after being incubated overnight recombinant bacterial strain prepared by embodiment 2
The chloramphenicol of g/ml), be seeded in ZYP-5052 culture medium in the ratio of 1:100, cultivated under the conditions of 37 DEG C, 200rpm to
OD600(absorbance at 600nm) is 0.95~1.05, and temperature is then reduced to 20 DEG C, after continuing culture for 24 hours, 5000r/
Thalline were collected by centrifugation by min, and thallus is suspended in HisTrap A buffer, then (is contained by centrifuging and taking supernatant after pressure breaking
There is the recombinant protein for being fermented and being formed by recombinant strains)；Wherein, the pH value of HisTrap A buffer is 7.4, including following
The component of molar concentration: the imidazoles of NaCl, 20mM of 500mM and the Na of 20mM2HPO4；The specific formula of ZYP-5052 culture medium
It is as follows: 1% peptone, 5% yeast extract, 50mM Na2HPO4, 50mM KH2PO4, 25mM (NH4)2SO4, 0.5% the third three
Alcohol, 0.05% glucose, 0.2% lactose, 2mM MgSO4, 100 μ g/ml of kanamycins, 34 μ g/ml of chloramphenicol；
(2) it after taking supernatant made from step (1) to pass through HisTrap affinity column (purchased from General Corporation), uses
Then recombinant protein of the HisTrap B buffer on AKTA in elution supernatant will recombinate egg using Millipore concentration tube
It is white change liquid be concentrated into phosphate buffer protein concentration be 1mg/mL, the agarase purified；Wherein, HisTrap
The pH value of B buffer is 7.4, the component including following molar concentration: the imidazoles of NaCl, 500mM of 500mM and 20mM's
Na2HPO4；Phosphate buffer contains the component of following molar concentration: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4,
1.8mM KH2PO4。
The protein expression situation of agarase prepared by embodiment 4, enzyme activity, optimum reaction conditions, heat resistance and acidproof
Alkalinity analysis measurement (agarase caAgaA as described below represent embodiment 4 preparation agarase), measuring method and
As a result as follows:
1, the protein expression analysis of agarase caAgaA
To the agarase in supernatant made from clasmatosis in 4 step of embodiment (1) and step (2) after purification
CaAgaA carries out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) test, as a result as shown in Figure 1 respectively
(in Fig. 1: swimming lane 1 is full cell pyrolysis liquid, and swimming lane 2 is the supernatant after full clasmatosis, and swimming lane 3 is after full clasmatosis
Precipitating, swimming lane 3 to 7 are the collecting pipe sample that supernatant passes through that chromatographic column gradient elution obtains).
As shown in Figure 1, agarase caAgaA after purification has obtained efficient expression and purifying in Escherichia coli.
2, the enzyme activity of agarase caAgaA analyzes (reducing sugar method namely DNS method)
The reaction system of preparation standard is 2mL, comprising: diluted agarase caAgaA, 1mL reaction of 0.5mL about 1:100
Buffer and 0.5mL agarose (being purchased from Thermo Scientific company) solution (0.25%)；By the reaction system 40
DNS reagent (3,5- dinitrosalicylic acid) is added after being incubated for 20min in DEG C water-bath, boiling water water-bath 5min is then cooled to room temperature；
The reaction system is settled to 25mL again, measures absorbance in 520nm.The quantitative of reduced sugar is cooked standard using gala saccharide
Curve, the activity definition of 1 unit (U) agarose enzyme activity are agarase milli needed for capable of discharging 1 μm of ol reduced sugar per minute
Grams.
It is measured through DNS method, the enzyme activity of agarase caAgaA after purification is about 36U/mg.
3, agarase caAgaA optimal reaction pH temperature analysis
It is respectively 3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0 and that agarase caAgaA, which is dispersed in pH value,
(buffer of pH 3~8 is prepared 11.0 buffer by McIlvaine buffer, and the buffer of pH 9~11 is by glycine-hydrogen
The preparation of sodium oxide molybdena (50mM) buffer) in, at a temperature of 40 DEG C, enzyme activity is measured by above-mentioned DNS method respectively, with the highest measured
Enzyme activity value is set as 100%, calculates the opposite enzyme activity of other each pH reaction intervals.
Wherein, the buffer of pH 3~8 is as follows by method prepared by McIlvaine buffer: being 200mM with molar concentration
Na2HPO4Solution and molar concentration are that the citric acid solution of 100mM is hooked to being formulated into required pH value, specific ratio
It is as shown in table 1 below:
Table 1 is prepared the ratio of corresponding pH value buffer by McIlvaine buffer
| pH | Na2HPO4Volume (mL) | Citric acid volume (mL) |
| 3.0 | 04.11 | 15.89 |
| 4.0 | 07.71 | 12.29 |
| 5.0 | 10.30 | 09.70 |
| 6.0 | 12.63 | 07.37 |
| 7.0 | 16.47 | 03.53 |
| 8.0 | 19.45 | 00.55 |
The buffer of pH 9~11 is as follows by method prepared by Glycine-NaOH (50mM) buffer: by glycine
It is dissolved in water, with sodium hydroxide titration to the pH (9.0,10.0,11.0) needed.
The calculated result of the opposite enzyme activity of each pH reaction interval is as shown in Figure 2.As shown in Figure 2, fine jade manufactured in the present embodiment
Lipolysaccharide enzyme caAgaA is that 5~8 sections are active (> 60%) all with higher in pH value, optimal pH 7.0.
4, the pH stability analysis of agarase caAgaA
Agarase caAgaA is dispersed in the buffer solution system of different pH (pH value is 3~11), is incubated at a temperature of 4 DEG C
After educating 2h, under conditions of 40 DEG C, pH value are 7.0, enzyme activity is measured by above-mentioned DNS method, with the highest enzyme activity value setting measured
It is 100%, calculates the opposite enzyme activity of other each pH reaction intervals, as a result as shown in Figure 3.
From the figure 3, it may be seen that agarase caAgaA manufactured in the present embodiment can after under the conditions of pH is 3~11, being incubated for 1h
Maintain about 40% enzyme activity.
Comprehensive 3,4 two as described above investigations are as a result, it is found that agarase proposed by the present invention has wide in range pH
Tolerance range is suitable for the application scenarios that strong acid-base environment or pH are changed greatly.
5, agarase caAgaA optimal reactive temperature is analyzed
Agarase caAgaA is dispersed in the buffer solution system that pH value is 7.0, respectively at 20 DEG C, 30 DEG C, 40 DEG C, 50
DEG C, 60 DEG C, 70 DEG C, at a temperature of 80 DEG C and 90 DEG C, enzyme activity is measured by above-mentioned DNS method, is set as with the highest enzyme activity value measured
100%, the opposite enzyme activity in other each thermotonus sections is calculated, as a result as shown in Figure 4.
As shown in Figure 4, agarase caAgaA manufactured in the present embodiment is with higher in 30~50 DEG C of temperature ranges
Active (> 75%), optimum temperature are 40 DEG C.
6, the temperature stability analysis of agarase caAgaA
By agarase caAgaA be placed at pH value be 7.0 buffer solution system in, be respectively placed in 30 DEG C, 40 DEG C, 50 DEG C,
After being incubated for 2h, 4h and 8h at a temperature of 60 DEG C, 70 DEG C, 80 DEG C and 90 DEG C, under conditions of 40 DEG C, pH value are 7.0, by above-mentioned
DNS method measures enzyme activity, is set as 100% with the highest enzyme activity value measured, calculates the opposite enzyme activity of other each pH reaction intervals, ties
Fruit is as shown in Figure 5.
As shown in Figure 5, agarase caAgaA manufactured in the present embodiment is poor in 50 DEG C of -60 DEG C of stability, but 70
There is fabulous stability at a temperature of DEG C -80 DEG C, it is good to illustrate that agarase proposed by the present invention has hot environment
Tolerance is suitable for the application scenarios of hot environment.
7, the function analysis of metal ion and chemical reagent to agarase caAgaA
10 parts of standard reaction system are prepared according to the method in the analysis of above-mentioned enzyme activity, is separately added into standard reaction system
The FeSO of final concentration of 5mM4Solution, MnSO4Solution, CaCl2Solution, ZnCl2Solution, CoCl2Solution and MgSO4Solution and
Concentration is respectively the EDTA (disodium ethylene diamine tetraacetate) and DTT (dithiothreitol (DTT)) solution of 5mM, with without containing metal ion or
The standard reaction system of DTT/EDTA measures enzyme activity as a control group, by above-mentioned DNS method, with the highest enzyme activity value setting measured
It is 100%, calculates the opposite enzyme activity of other each pH reaction intervals, as a result if Fig. 6, Fig. 6 are different metal ions and EDTA/DTT
On the active influence of agarase caAgaA.
It will be appreciated from fig. 6 that above-mentioned common metal ion (Fe2+、Mn2+、Ca2+、Co2+And Mg2+) under the concentration of 5mM to fine jade
Lipolysaccharide enzyme caAgaA has different degrees of activation effect, wherein Mn2+To the larger promotion effect of activity of agarase caAgaA
(160%).EDTA shows inhibitory effect to agarase caAgaA.DTT shows greatly to swash to agarase caAgaA
Active fruit, under the concentration of 5mM, agarase caAgaA gives expression to 210% enzyme activity.
The aptitude tests of 5 agarase caAgaA of embodiment hydrolysis agarose
Using agarose as substrate, in standard reaction system (the standard reaction system provided in above-mentioned enzyme activity analysis) and 40
DEG C, react different time (0min is to for 24 hours) under the reaction condition of pH 7.0 after, progress thin layer chromatography analysis (tlc silica gel
Plate is purchased from Merck company).Solvent used in thin-layer chromatography is n-butanol: acetic acid: water=10:5:1 (volume ratio), and band is aobvious
Shadow agent is the concentrated sulfuric acid: lamellae is placed in 115 DEG C, 10min after spray developing agent by methanol=5:95 (volume ratio).
Result is analyzed as shown in fig. 7, in Fig. 7: swimming lane M is new fine jade oligosaccharide standards (new fine jade disaccharides NA2 and Xin Qiong tetrose
NA4), other swimming lanes are the product that agarase caAgaA hydrolyzes agarose in different time.
As shown in Figure 7, over time, gradually start new fine jade oligosaccharides occur in the hydrolysate of agarose, and dense
Spend it is higher and higher, illustrate the embodiment of the present invention preparation agarase caAgaA agarose effectively can be hydrolyzed to 4 and 6
The new fine jade oligosaccharides of residue.
Test of the 6 agarase caAgaA of embodiment for the nucleic acid recycling in Ago-Gel
The DNA fragmentation that agarose gel electrophoresis length with 1% is 1400bp.Buffer used in electrophoresis is TAE (40mM
Tris HCl, 20mM acetic acid, 1mM EDTA), voltage 120V, time 30min.After electrophoresis, in ethidium bromide solution
10min is dyed in (0.5 μ g/ml).The purpose band under ultraviolet light incision, is put into the EP pipe of 1.5ml.1 μ is added in pipe
The RNase A enzyme (the silent winged generation that science and technology of match, article No.: EN0531) of l agarase caAgaA (about 0.5 μ g) and 1U, is placed in 70 DEG C
About 5min in water-bath.Dissolved gel is placed in 40 DEG C of water-baths and digests 15min.EP pipe is placed on 4 DEG C of refrigerators again later
In, after a small amount of remaining agarose solidification, at 4 DEG C, 10min is centrifuged with the revolving speed of 13000rpm.Centrifuged supernatant is taken, is added
The dehydrated alcohol for entering 2 times of volumes places 15min at -20 DEG C.At 4 DEG C, 10min is centrifuged with the revolving speed of 13000rpm, is abandoned
Supernatant is dissolved with 20 μ l desinfection chambers after taking precipitating aeration-drying, can be obtained the DNA fragmentation of recycling.
Meanwhile the gel reclaims kit (article No.: D2501-022) of Omega company is used as control while carrying out nucleic acid
Recovery test (the starting DNA sample of same amount), specific experiment process refer to kit specification.
It is returned respectively using agarase caAgaA and gel reclaims kit with 1% agarose gel electrophoresis analysis is above
Obtained DNA sample is received, records result as shown in figure 8, in figure, No. 1 band is the nucleic acid recycled by agarase caAgaA；
No. 2 bands are the nucleic acid of kit recycling；No. 3 bands are nucleic acid molecular weight Marker.As shown in Figure 8, pass through agarase
CaAgaA can effectively recycle the nucleic acid in Ago-Gel.
In conclusion agarase gene caAgaA provided by the invention, after being transferred to Escherichia coli by carrier, is weighed
Group expression bacterial strain, then the agar of high-purity is made after high cell density fermentation, affinity purification method in recombinant strains
Carbohydrase, obtained agarase have preferable activity, agarose can be effectively hydrolyzed to the new fine jade of 4 and 6 residues
Oligosaccharides can be used for the preparation of the new fine jade oligosaccharides of bioactivity and the recycling of nucleic acid substances.Meanwhile agarase has fabulous heat steady
Qualitative and resistance to acid and alkali.
The above is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc. should all be included within the scope of the present invention.
SEQUENCE LISTING
<110>Wuhan Polytechnic University, Chinese ocean mineral resources research and development association (Chinese ocean transaction management office)
<120>agarase gene, recombinant plasmid, recombinant bacterial strain, agarase and its application
<130> 2019/1/17
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 936
<212> DNA
<213>artificial synthesized
<400> 1
atgagtaaaa gcagcactga tgaaaaacca accagtgaag aagataacca gggcgaagtg 60
attacgccgg aagaggaagt tgaagaagag gtggtgaaag actggaaaac cattccggtt 120
ccggcagacg ccggggcggg taacgtctgg gaattccagg agttaagcga cgatttcgat 180
tacgacgcgc cggccgatgc gaaaggtgca aaatttgata aaaaatggac ggatttttat 240
cacaacctct gggcgggccc gggtctgacc gaatggcgcc gtgatcacag cctggtggtc 300
gatggcaacc tgcagatgat cgccaaccgc gctgaaggca gcaataaaat caatctgggc 360
tgtattacca gcaaagaaca ggtggtttat ccggtgtata ttgaagcgaa cgttaaaatt 420
gccaatacca ccctggccag cgatgtgtgg ctgctgagca gtgatgatac ccaggaaatc 480
gatatcgtgg aagcctatgg cgcgagctac agcgaactgg ctgacagcga tcaaacctgg 540
tacgcagaac gtattcatat cagccatcat atgtttatcc gcgacccgtt tcaggattat 600
cagccgaccg atgccggtag ctggtatcgc gatggtacgc tgtggcgtga agactatcat 660
accgttggcg tttactggaa agatccgttc catctggagt attacatcga cggcaaactg 720
gcgcgtacca cgagcggcac ggaaatgatt gatccgaaca actttgccga aggtaaaggc 780
ctgtataaac cgatggatat tattatcaac gcggaagatc agacctggcg gtctgataat 840
aatgtgacgc catctgacaa agagctggaa aacaaagaaa ataacacctt taaagtggac 900
tggattcgca ttttcaaacc tgtgccggcg aattaa 936
<210> 2
<211> 310
<212> PRT
<213>artificial synthesized
<400> 2
Ser Lys Ser Ser Thr Asp Glu Lys Pro Thr Ser Glu Glu Asp Asn Gln
1 5 10 15
Gly Glu Val Ile Thr Pro Glu Glu Glu Val Glu Glu Glu Val Val Lys
20 25 30
Asp Trp Lys Thr Ile Pro Val Pro Ala Asp Ala Gly Ala Gly Asn Val
35 40 45
Trp Glu Phe Gln Glu Leu Ser Asp Asp Phe Asp Tyr Asp Ala Pro Ala
50 55 60
Asp Ala Lys Gly Ala Lys Phe Asp Lys Lys Trp Thr Asp Phe Tyr His
65 70 75 80
Asn Leu Trp Ala Gly Pro Gly Leu Thr Glu Trp Arg Arg Asp His Ser
85 90 95
Leu Val Val Asp Gly Asn Leu Gln Met Ile Ala Asn Arg Ala Glu Gly
100 105 110
Ser Asn Lys Ile Asn Leu Gly Cys Ile Thr Ser Lys Glu Gln Val Val
115 120 125
Tyr Pro Val Tyr Ile Glu Ala Asn Val Lys Ile Ala Asn Thr Thr Leu
130 135 140
Ala Ser Asp Val Trp Leu Leu Ser Ser Asp Asp Thr Gln Glu Ile Asp
145 150 155 160
Ile Val Glu Ala Tyr Gly Ala Ser Tyr Ser Glu Leu Ala Asp Ser Asp
165 170 175
Gln Thr Trp Tyr Ala Glu Arg Ile His Ile Ser His His Met Phe Ile
180 185 190
Arg Asp Pro Phe Gln Asp Tyr Gln Pro Thr Asp Ala Gly Ser Trp Tyr
195 200 205
Arg Asp Gly Thr Leu Trp Arg Glu Asp Tyr His Thr Val Gly Val Tyr
210 215 220
Trp Lys Asp Pro Phe His Leu Glu Tyr Tyr Ile Asp Gly Lys Leu Ala
225 230 235 240
Arg Thr Thr Ser Gly Thr Glu Met Ile Asp Pro Asn Asn Phe Ala Glu
245 250 255
Gly Lys Gly Leu Tyr Lys Pro Met Asp Ile Ile Ile Asn Ala Glu Asp
260 265 270
Gln Thr Trp Arg Ser Asp Asn Asn Val Thr Pro Ser Asp Lys Glu Leu
275 280 285
Glu Asn Lys Glu Asn Asn Thr Phe Lys Val Asp Trp Ile Arg Ile Phe
290 295 300
Lys Pro Val Pro Ala Asn
305 310
Claims (8)
1. a kind of agarase, which is characterized in that the amino acid sequence of the agarase is as shown in SEQ ID NO:2.
2. a kind of agarase gene caAgaA, for encoding agarase described in claim 1, which is characterized in that described
The nucleotide sequence of agarase gene caAgaA is as shown in SEQ ID NO:1.
3. a kind of recombinant plasmid, which is characterized in that carry agarase gene caAgaA as claimed in claim 2.
4. recombinant plasmid as claimed in claim 3, which is characterized in that include expression vector pET-28a.
5. a kind of recombinant bacterial strain, which is characterized in that include agarase gene caAgaA as claimed in claim 2.
6. recombinant bacterial strain as claimed in claim 5, which is characterized in that by including agarase base as claimed in claim 2
Because the recombinant plasmid transformed e. coli bl21 (Rosetta) of caAgaA obtains.
7. application of the agarase described in claim 1 in the preparation of new fine jade oligosaccharides.
8. application of the agarase described in claim 1 in the nucleic acid recycling after agarose gel electrophoresis.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6511838B1 (en) * | 1998-06-12 | 2003-01-28 | Laboratoires Goemar S.A. | Genes coding for β-agarases and their use for producing agar biodegradation enzymes |
| CN1531595A (en) * | 2001-02-27 | 2004-09-22 | �����﹤����ʽ���� | Agarase and gene thereof |
| CN106544333A (en) * | 2016-11-07 | 2017-03-29 | 新乡医学院 | A kind of β agarases and its encoding gene and application |
| CN108410890A (en) * | 2018-05-16 | 2018-08-17 | 武汉轻工大学 | Xylanase gene, recombinant expression plasmid, recombinant strains, zytase and its preparation method and application |
-
2019
- 2019-04-17 CN CN201910308056.6A patent/CN110066779A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6511838B1 (en) * | 1998-06-12 | 2003-01-28 | Laboratoires Goemar S.A. | Genes coding for β-agarases and their use for producing agar biodegradation enzymes |
| CN1531595A (en) * | 2001-02-27 | 2004-09-22 | �����﹤����ʽ���� | Agarase and gene thereof |
| CN106544333A (en) * | 2016-11-07 | 2017-03-29 | 新乡医学院 | A kind of β agarases and its encoding gene and application |
| CN108410890A (en) * | 2018-05-16 | 2018-08-17 | 武汉轻工大学 | Xylanase gene, recombinant expression plasmid, recombinant strains, zytase and its preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| ABT,B. ET AL.: "Beta-agarase [Cellulophaga algicola DSM 14237]", 《GENBANK: ADV49752.1》 * |
| NONE: "beta-agarase [Cellulophaga algicola]", 《NCBI REFERENCE SEQUENCE: WP_013551224.1》 * |
| 张向阳: "《医学分子生物学》", 28 February 2018 * |
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