CN109609485A - A kind of chitin deacetylase and its application - Google Patents
A kind of chitin deacetylase and its application Download PDFInfo
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Abstract
The present invention is analyzed and researched by the different genes group sequence to different plant species, screening has obtained several agnoproteins, and further therefrom identify the chitin deacetylase with outstanding deacetylase activity, more specifically, it withers the chitin deacetylase of bacterium (Verticillium longisporum) Vlo the present invention relates to a kind of from rape Huang, with it has been reported that chitin deacetylase compared with, the chitin deacetylase requires working condition lower, biological safety with higher, and there is the characteristic for being more suitable for industrial applications, it shows to be widely applied potentiality.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to one kind is withered bacterium (Verticillium from rape Huang
Longisporum) the chitin deacetylase of VL1 and its application.
Background technique
Chitin also known as chitin are to pass through β-Isosorbide-5-Nitrae glycosidic bond by N- acetylaminohydroxyphenylarsonic acid D-Glucose monomer (D-GlcNAc)
The straight chain polymer glycosaminoglycan being formed by connecting, being primarily present in invertebrate, (especially the shell-fish such as shrimp, crab, insect are dynamic
The shell of object), seaweed (such as green alga), be the important polysaccharide of another major class in addition to cellulose in fungi (such as mould).So
And chitin is not soluble in water, diluted acid, alkali, ethyl alcohol or other organic solvents, so its utility value is greatly limited, and it is de-
Product chitosan after deacetylate depends on deacetylation difference, can be dissolved in acid and neutral aqueous solution.Since chitosan has
There are the good characteristics such as biological functionality and compatibility, blood compatibility, safety, microbic resolvability, therefore in medicine, food
The numerous areas such as product, chemical industry, cosmetics, water process, METAL EXTRACTION and recycling, biochemistry and biomedical engineering have obtained answering extensively
With (Kaur and Dhillon 2014).Currently, concentrated base pyrolysismethod (40% or more the hydrogen-oxygen that production chitosan mainly uses
Change sodium) there are problems, such as the reaction time is long, energy consumption is high, product quality (is primarily referred to as average molecular mass and deacetylated
Degree) it is unstable, and will cause serious problem of environmental pollution.
Chitin deacetylase (Chitin deacetylase, abbreviation CDA, E.C.3.5.1.41) can will be in chitin
Acetyl group removing, so that it be made to be converted into chitosan (Ghormade, Kulkarni et al.2010), can effectively solve tradition
Method prepare chitosan there are the problem of, provide a kind of high-quality, low cost, the novel producer of energy-saving and environment-friendly chitosan
Method.
Currently, several relevant reports of existing CDA research both at home and abroad, generating species includes fungi, bacterium and insect etc.,
Wherein main source is fungi (Zhao, Park et al.2010).1973, Araki etc. (Araki and Ito 1974) was first
CDA is had found in the secondary Zygosaccharomyces rouxii (Mucor rouxii) from Zygomycetes (Zygonycetes).Nineteen eighty-two, Kauss etc.
(Kauss, Jeblick et al.1983) is from phytopathogen Kidney bean anthrax-bacilus (Colletotrichum
Lindemuthianum isolated CDA in), this is the earliest report that the enzyme is found from disengaged bacterium, optimal reaction temperature
Degree is 60 DEG C, and optimal pH is slight alkali environment.Then, more and more fungies, bacterium are reported with the deacetylated enzyme activity of chitin
Property, such as aspergillus nidulans (Aspergillus nidulans) (Alfonso, Nuero et al.1995), Absidia are mould
(Absidia coerulea) (Gao, Katsumoto et al.1995), schizosaccharomyces pombe (Schizosaccharomyces
Pombe) (Matsuo, Tanaka et al.2005), saccharomyces cerevisiae (Saccharomyces cerevisiae) (Martinou,
Koutsioulis et al.2002), needle mushroom (Flammulina velutipes) (Yamada, Kurano et
Al.2008), tang mould (Rhizopus circinans) (Gauthier, Clerisse et al.2008) and gemma bar are rolled up
Bacterium (Bacillus) (He, Xu et al.2014) (Sun, Zhang et al.2016) (Raval, Simsa et al.2017)
With rhodococcus erythropolis (Rhodococcus erythropolis) (Sun, Zhang et al.2014).Wherein, waxy brood cell's bar
The CDA in the source bacterium (Bacillus cereus) shows enzyme activity (Sun, Zhang et similar with wild-type fungal
al.2016)。
The CDA of the originated from fungus found so far is substantially single chain polypeptide glycoprotein, shows good thermostabilization
Property, but the position in the cell the CDA of separate sources, optimum pH, optimum temperature, molecular weight, isoelectric point and to metal from
The reaction etc. of son and acetic acid suffers from biggish difference.In addition, the CDA in reported phytopathogen Kidney bean anthrax-bacilus source
The optimal pH of (hereinafter also referred to CliCDA) is slight alkali environment, is not suitable with the reaction condition (slant acidity) of industrial material.
In conclusion the activity (activity especially under acidic conditions) of the CDA found so far still can not
Sufficiently meet the actual demand of industrialized production chitosan, therefore, the CDA for screening high activity is still to need to solve in industrial applications
Major issue certainly.
Summary of the invention
To solve the above subject, present inventor has performed further investigations, pass through the different genes group sequence to different plant species
It analyzes and researches, screening has obtained several agnoproteins, and further therefrom identifies with outstanding deacetylase activity
Chitin deacetylase, so as to complete the present invention.
Therefore, in a first aspect, the present invention provides one kind to wither bacterium (Verticillium from rape Huang
Longisporum) the chitin deacetylase (hereinafter also referred to VloCDA) of VL1, the amino acid of the chitin deacetylase
Sequence is as shown in SEQ ID NO:1:
MYTTTVLSLLALTGTTLTAPTALHLRDSTPPSPTRHRRAPALGQTLYSCVNPGQVALTYDDGPYTFTS
SLLDVLDEEGVTATFFLTGSNFGREMTSDPWSAIVQRTYAAGHQLASHTYTHPDLSALTPAARAAEMAANDDAFRA
ILGFAPRYMRAPFLSCDAACAADMAALGFHIVDASIDTKDFEHNQYGTVYAAEAKFDAELGWDPAVDSAIVLAHDV
HETTVSVLTRHMISTLRARGFRAVTVGECLGDSPDGWYKA (SEQ ID NO:1).
Compared with reported chitin deacetylase, the chitin deacetylase is lower to working condition requirement, favorably
It in reducing production cost, reduces environmental pollution, prepares the process exploitation of chitosan for enzyme process and industrial applications are laid a good foundation,
There is important practice significance to mycelial higher value application.
It is excellent the present invention provides the DNA molecular of the chitin deacetylase of coding as described in relation to the first aspect in second aspect
Selection of land, the nucleotide sequence of the DNA molecular is as shown in SEQ ID NO:2: ATGTACACAACAACAGTGCTGAGCCTGCTT
GCACTGACAGGAACAACACTTACAGCACCGACAGCGCTGCATCTGAGAGATTCAACACCGCCGTCACCGACAAGAC
ATAGAAGAGCGCCGGCACTTGGACAAACACTGTATTCATGCGTTAATCCGGGACAAGTTGCACTGACATATGATGA
TGGACCGTATACATTCACTAGCAGCCTTCTGGATGTGCTTGATGAAGAAGGCGTTACAGCGACATTTTTCCTTACA
GGCAGCAATTTTGGAAGAGAAATGACAAGCGATCCGTGGTCAGCAATTGTGCAGAGAACATATGCAGCGGGACATC
AACTGGCAAGCCATACATATACACATCCGGATCTTTCAGCACTGACACCGGCAGCAAGAGCGGCAGAAATGGCGGC
GAATGATGATGCATTTCGCGCAATTCTGGGCTTTGCGCCGAGATATATGAGAGCACCGTTTCTTAGCTGTGATGCA
GCATGCGCGGCGGATATGGCGGCACTTGGCTTTCATATTGTTGATGCAAGCATTGATACAAAAGATTTTGAACATA
ACCAGTACGGCACAGTTTATGCAGCGGAAGCAAAATTTGATGCAGAACTGGGCTGGGACCCTGCGGTTGATTCAGC
AATTGTCCTGGCACATGATGTTCATGAAACAACAGTTTCAGTGCTGACAAGACATATGATTTCAACACTTAGAGCG
AGAGGATTTCGCGCGGTGACAGTTGGCGAATGCCTTGGAGATAGCCCGGATGGCTG GTATAAAGCA (SEQ ID NO:
2)。
The nucleotide sequence (SEQ ID NO:2) is by the coded sequence of optimization, in Escherichia coli (Escherichia
Coli) and in bacillus subtilis (B.subtilis) can high efficient expression, thus obtained chitin deacetylase has
High biological safety, can be directly used for industrialized production, advantageously reduce production cost.
In the third aspect, the present invention provides the expression vectors comprising the DNA molecular as described in second aspect, it is preferable that
The expression vector is pET24a (+) (general biosystem (Anhui) Co., Ltd) or pHThis (CN104263711A, Lee
It is refreshing, Huang Xiongliang, Wang Jufang;A kind of alkali-resistant xylanase and its encoding gene and recombinant vector, 2015.).
In fourth aspect, the present invention provides the transformed cells for importing the expression vector as described in the third aspect, it is preferable that
The transformed cells are E. coli transformant cells or bacillus subtilis transformed cells;It is highly preferred that the transformed cells are
Escherichia coli Transetta (DE3) cell (Beijing Quanshijin Biotechnology Co., Ltd) or 1012 cell of bacillus subtilis
(MoBiTec company, Germany).
At the 5th aspect, the present invention provides include chitin deacetylase as described in relation to the first aspect;Such as second aspect
The DNA molecular;Expression vector as described in the third aspect;And one of transformed cells as described in fourth aspect or
A variety of kits.
It is described the present invention provides the method for the chitin deacetylase of preparation as described in relation to the first aspect at the 6th aspect
Method includes the following steps: to cultivate the transformed cells as described in fourth aspect in the medium;And it collects in the culture medium
The chitin deacetylase.
At the 7th aspect, the present invention provides a kind of method of catalysis de-acetyl chitin production chitosan, features
It is, uses chitin deacetylase as described in relation to the first aspect;DNA molecular as described in second aspect;Such as third aspect institute
The expression vector stated;Transformed cells as described in fourth aspect;And/or the kit as described in terms of the 5th.
In eighth aspect, the present invention provides chitin deacetylases as described in relation to the first aspect;As described in second aspect
DNA molecular;Expression vector as described in the third aspect;Transformed cells as described in fourth aspect;And/or such as institute in terms of the 5th
Purposes of the kit stated in catalysis de-acetyl chitin production chitosan.
Beneficial effect
Compared with prior art, the present invention has the advantage that (1) rape Huang of the invention withers, the chitin in bacterium source is de-
Acetyl enzyme gene can be expressed in the host cells endocrine such as such as Bacillus coli cells and B. subtilis cell;(2) this hair
The producing strains (especially bacillus subtilis) of bright chitin deacetylase have biological safety, to working condition require compared with
It is low, without using strong base solution, lay a good foundation for green large-scale production chitosan.Specifically, chitin of the invention
The acquisition of deacetylase (the chitin deacetylase especially produced by bacillus subtilis) is for mycelium higher value application
For have great theoretical and practical significance.
Detailed description of the invention
Fig. 1 is shown according to embodiment 2, the albumen of the VloCDA of engineered strain Escherichia coli Transetta (DE3) expression
Matter electrophorogram.M: albumen marker;1:VloCDA purifying protein.
Fig. 2 shows according to embodiment 4-1, temperature is on the active influence of different CDA.
Fig. 3 is shown according to embodiment 4-2, and pH is on the active influence of VloCDA.
Specific embodiment
It hereafter will be apparent from the present invention.
Chitin deacetylase and its determination of activity one, of the invention
Chitin deacetylase of the invention can be obtained by artificial synthesized;Can also by first synthesize its encoding gene,
It carries out biological expression again and obtains.
The various methods of chitin deacetylase activity are measured as known to those skilled in the art.For example, chitin is deacetylated
3,5- dinitrosalicylic acid can be restored and generate brownish red by the acetylglucosamine (NAG) that enzyme hydrolysis chitin generates
Amino-compound, the amount of reduzate of the depth and generation of brownish red is directly proportional, therefore, utilizes 3,5- dinitrosalicylic acid colorimetric
Method can measure the activity of chitin deacetylase.Alternatively, paranitroacetanilide is a kind of colourless compound, second is sloughed
Apparent yellow is presented in product p-nitroanilide obtained by acyl group, therefore it is de- as chitin that paranitroacetanilide can also be used
The tracking reagent of acetyl enzyme enzyme activity determination.
According to embodiment of the present invention, the work of chitin deacetylase of the invention can be measured by following methods
Property: using certain density paranitroacetanilide solution as substrate, under the conditions of specific pH, keep substrate deacetylated with chitin
Enzyme reacts under water bath with thermostatic control, boils termination reaction after reaction, obtains chitin by measurement reaction system absorbance
The enzyme activity of deacetylase.
Two, encode the DNA molecular of chitin deacetylase of the invention
DNA molecular of the invention is the DNA molecular of coding chitin deacetylase of the invention.According to the present invention one it is excellent
The embodiment of choosing, DNA molecular of the invention can obtain in the following manner: obtain wild type first for example, by using the methods of PCR
Shell element deacetylase gene.
Another preferred embodiment according to the present invention can also obtain DNA molecular of the invention by chemical synthesis.
A particularly preferred embodiment according to the present invention, to improve expression efficiency of the chitin deacetylase in host cell,
The coded sequence of wild type chitin deacetylase can be replaced with the DNA sequence dna being made of host cell preference codon, then
DNA molecular of the invention is prepared by chemically synthesized mode.Some most preferred embodiments according to the present invention, the host
Cell is Bacillus coli cells or B. subtilis cell, correspondingly, by the coded sequence of wild type chitin deacetylase
Replace be capable of in Escherichia coli and bacillus subtilis high efficient expression the coded sequence by optimization it is (such as but unlimited
In SEQ ID NO:2).But the as long as DNA molecular of coding chitin deacetylase of the invention and can be suitable
Chitin deacetylase of the invention is expressed in host cell, then is not limited to the DNA molecular.
Another preferred embodiment according to the present invention is divided as the DNA for encoding chitin deacetylase of the invention
Son is also possible to following DNA moleculars: under strict conditions with the complementary series of the nucleotide sequence as shown in SEQ ID NO:2
Being hybridized and being encoded has desired active chitin deacetylase.Wherein, stringent condition refers to form so-called spy
Condition of the specific hybridization without forming non-specific hybridization.Although the condition is different because of nucleotide sequence or its length, in fact
Example includes with high sequence identity (for example, having the sequence one of the sequence identity not less than 75%, preferably not less than 90%
Cause property, further much more desirably not less than 95% sequence identity, most desirably not less than 98% sequence identity) DNA molecular
Phase mutual cross, and sequence identity is lower than the condition that the DNA molecular of above-mentioned standard does not hybridize;Or for floating in Southern hybridization
The hybridization conditions for the typical conditions washed (for example, 60 DEG C and 1 × SSC, 0.1%SDS, preferably 0.1 × SSC and are equivalent to 0.1%
The salinity of SDS).
Another preferred embodiment according to the present invention is divided as the DNA for encoding chitin deacetylase of the invention
Son is also possible to (preferably have with nucleotide sequence represented by SEQ ID NO:2 with 90% or more sequence identity
95% or more sequence identity, more preferably with 98% or more sequence identity, even more preferably with 99% or more
Sequence identity) and coding have desired active chitin deacetylase.
Expression vector three, of the invention
Expression vector of the invention is the expression vector for expressing chitin deacetylase of the invention.According to the present invention
One preferred embodiment, the expression vector can have following structure: it is deacetylated that control encodes chitin of the invention
The promoter sequence of the DNA molecular expression of enzyme is connected to the upstream of the DNA molecular.Further, it is also possible to which terminator is connected to
The downstream of the DNA molecular.Other routine operation elements also may be included in expression vector.
As expression vector, any general type known in the art can be used.Come from the angle of copy number and stability
It sees, pET24a (+) expression vector that can preferably play a role in Bacillus coli cells.For example, can be by conventional gene engineering
Means obtain expression of the invention and DNA molecular of the invention is inserted between the multiple cloning sites of pET24a (+) carrier
Carrier.Alternatively, may be based on playing in B. subtilis cell and make for the high efficient expression in B. subtilis cell
PHThis carrier obtains expression vector of the invention.
According to a preferred embodiment of the present invention, for selecting the selected marker of expressed chitin deacetylase
Gene also may be included in expression vector of the invention for detecting the reporter gene that quiding gene is expressed.Selectable marker gene
Example include but is not limited to hygromycin gene, kalamycin resistance gene and ampicillin resistance gene.Report base
The example of cause includes but is not limited to beta-Glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, luciferase
(LUC) gene and green fluorescent protein (GFP) gene.
Another preferred embodiment according to the present invention, for secreting, expressing chitin deacetylase of the invention or
For the ease of purifying expressed chitin deacetylase, appended sequence can further include in expression vector of the invention.?
In this case, chitin deacetylase of the invention is with fusion protein (merging with the albumen or peptide encoded by appended sequence)
Form expression.The example of the appended sequence includes but is not limited to the nucleotide sequence of encoded signal peptide or propetide;And coding
The nucleotide sequence of His label or GST label.
Transformed cells four, of the invention
Transformed cells of the invention are the cells for importing expression vector of the invention, which can express/produce the present invention
Chitin deacetylase.The transformed cells can be prokaryotic cell, be also possible to eukaryocyte.Come from conveniently angle
It sees, preferably prokaryotic cell.
According to a preferred embodiment of the present invention, the prokaryotic cell is Bacillus coli cells.Specifically, due to
There is very in-depth study to the condition of Bacillus coli cells production foreign protein at present, and Bacillus coli cells are made in production
Cost is relatively low during standby, can satisfy the demand of technical need and product marketization, thus Bacillus coli cells are considered as
More preferred transformed cells in the production of chitin deacetylase.On the other hand, it is contemplated that present invention discover that in bacillus subtilis
Chitin deacetylase obtained from expressing in bacterium has high biological safety, can be directly used for industrialized production, favorably
In reducing production cost, it is thin that B. subtilis cell is also considered as more preferred conversion in the production of chitin deacetylase
Born of the same parents.However, if expression vector of the invention can be imported and produce chitin deacetylase of the invention, the present invention is not limited to
This.
It can suitably be selected to import expression vector of the invention into transformed cells according to the type of transformed cells and carry out albumen
The method of expression.These methods are all known to the skilled in the art.
According to a preferred embodiment of the present invention, E. coli transformant cells or withered grass can be prepared by the following
Bacillus transformed cells: using the method for chemical conversion, it is thin that expression vector of the invention is transformed into E. coli competent
In born of the same parents or bacillus subtilis bacterium competence cell;Then thallus suspension is coated on plate, and is cultivated until there is single bacterium
It falls.
Kit five, of the invention
Kit of the invention is the chitin deacetylase of the invention comprising chitin deacetylase of the invention, coding
One of DNA molecular, expression vector of the invention and transformed cells of the invention or a variety of kits.
The kit may include container and on the container or label associated with the container or package insert.It closes
Suitable container includes such as bottle, bottle, syringe.The container can be formed of a variety of materials, such as glass or plastics.The label
The application method and purposes of the kit are indicated with package insert.Optionally, kit of the invention can also additionally comprise
One or more components, the component selected from test tube, reaction buffer, PCR primer, dNTP, Taq polymerase, reverse transcriptase,
DNA enzymatic, RNase inhibitor, DEPC water and sterile water, but always it is not limited to this.
The preparation method of chitin deacetylase six, of the invention
By cultivating transformed cells of the invention, chitin deacetylase of the invention can be produced.By melting with signal peptide
The form of the fusion protein of conjunction expresses chitin deacetylase of the invention with for secreting, chitin deacetylase of the invention
It can accumulate in the medium.Alternatively, when chitin deacetylase of the invention is present in transformed cells, it can be broken by ultrasound
Transformed cells are cracked and obtain chitin deacetylase of the invention by modes such as centrifugations by the modes such as broken.
When using inducible promoter, preferably induced in the training period.Although cultivating the side of the transformed cells
Method is different with the type of cell, but conventional method can be used.
When chitin deacetylase (when being secreted into culture medium) of the invention is in the shape being present in fermented liquid supernatant
When state, it can be used;The chitin deacetylase can also be used by the way that the fermented liquid supernatant is concentrated.It can purify or part is pure
Change the chitin deacetylase (when being secreted into culture medium).Using the conventional method of protein purification, it can be achieved that purifying or portion
Divide purifying.It is, for example, possible to use include chromatography (such as ion exchange or gel filtration), ammonium sulfate precipitation or organic solvent precipitation
Technology.Enzyme after can also being concentrated and purified by freeze-drying, ultrafiltration membrane and organic solvent precipitation etc..
According to a preferred embodiment of the present invention, six of the C-terminal addition in chitin deacetylase of the present invention are utilized
Polyhistidyl tags carry out purification and recovery to it.
Seven, produce the method and purposes of chitosan using chitin deacetylase catalysis de-acetyl chitin of the invention
Since chitosan can be obtained by de-acetyl chitin, chitin deacetylase of the invention (such as big
The chitin deacetylase produced in enterobacteria or bacillus subtilis, particularly bacillus subtilis) it is enzyme process green high-efficient
Large-scale production chitosan is laid a good foundation.
Some preferred embodiments according to the present invention are 50 DEG C -70 DEG C (preferably 55 DEG C -65 DEG C, more preferable 58 in temperature
DEG C -62 DEG C, most preferably 60 DEG C) and pH be 3.0-8.0 (preferably 3.5-7.5, more preferable 4.0-7.2, further preferred 4.5-
6.5, particularly preferred 5.0-6.0, most preferably 5.5) under conditions of using chitin deacetylase of the invention to chitin carry out
Processing is to produce chitosan.
Embodiment
It is better understood the present invention by means of following embodiments, these embodiments are only used for illustrating the present invention, no
It should be interpreted limitation of the present invention.
In the examples below, unless otherwise instructed, all genetic manipulations can be according to Molecular Cloning
The introduction of (Cold Spring Harbor Laboratory Press (1989)) carries out.In addition, unless otherwise instructed, all
Reagent is from Fisher Scientific.Particularly, the preparation of culture medium and buffer refers to that " Molecular Cloning: A Laboratory refers to
South " volume two (third edition, Science Press, 2002) annex 2 culture medium and buffer section.
The design and screening of 1 chitin deacetylase of embodiment
Potential thermostabilization sugar isomerase is scanned in ncbi database, and is come through sequence alignment analysis and bacterial strain
Source investigation, find it is a kind of wither the agnoprotein of bacterium Vlo from rape Huang, amino acid sequence is pushed away as shown in SEQ ID NO:1
It is surveyed with deacetylase activity.
In turn, codon optimization is carried out to the coded sequence of the amino acid sequence, obtains the core as shown in SEQ ID NO:2
Nucleotide sequence.
2 chitin deacetylase of embodiment is in the intracorporal expression of Escherichia coli
By general biosystem (Anhui), Co., Ltd synthesizes nucleotide sequence shown in SEQ ID NO:2, at its 5 ' end
BamH I restriction enzyme site is added, while adding the coded sequence of hexahistine label before terminator codon, and is added at 3 ' ends
Add Pst I and Not I restriction enzyme site.By BamH I and Not I (New England Biolabs, NEB) to synthesized piece
Duan Jinhang double digestion, and the segment after double digestion is connected to equally through BamH I and Not with T4DNA ligase (Takara)
PET24a (+) carrier (general biosystem (Anhui) Co., Ltd) of I progress double digestion.Connection product is converted to large intestine bar
It is enterprising in the LB solid plate with kanamycins in bacterium DH5 α competent cell (Beijing Quanshijin Biotechnology Co., Ltd)
Row culture and screening positive clone.Picking single colonie carries out bacterium colony PCR verifying;And positive clone carries out to bacterium colony PCR display
Sequence verification.
Show that the correct positive colony of sequence carries out plasmid extraction to through sequence verification using commercial kit, obtains weight
Group plasmid CDA-pET24a (+) is converted to Escherichia coli Transetta (DE3) competent cell (full formula gold biology in Beijing
Technology Co., Ltd.) in, it is cultivated on the LB plate with kanamycins.Picking single colonie carries out bacterium colony PCR verifying, will
The positive clone of bacterium colony PCR display expresses bacterial strain as chitin deacetylase (CDA).
By CDA expression strain inoculated in LB culture medium of the 5mL containing kanamycins, 37 DEG C, 200rpm is incubated overnight.With
1% (v/v) inoculum concentration access 500mL LB culture medium in, 37 DEG C, 200rpm continue cultivate 2-3h, to culture OD600=
When 0.6-0.8, the IPTG of final concentration of 0.5mmol/L is added.It is subsequently placed at 16 DEG C, is incubated overnight under the conditions of 150rpm.
Thalline were collected by centrifugation, is washed three times with Tris-HCl buffer (pH 7.5), is then resuspended with the 40mL buffer
Thallus.At 4 DEG C using ultrasonic cell disruption instrument carry out clasmatosis, condition be power 20%, ultrasonic 10min, ultrasonic 3s,
It is spaced 2s.10000g is centrifuged 10min at 4 DEG C, collects supernatant, i.e. CDA crude separation protein product.
Using 100 albumin layer analyzer of AKTA purifier, flow velocity is kept into 1.0mL/min, with 0.2M nickel sulfate solution
Pillar is washed, pillar is made to combine upper nickel ion;With Tris-HCl buffer (pH 7.5) pre-equilibrate Ni column (GE, HisTrap FF,
1mL), at least two column volume is balanced;Flow velocity is down to 0.5mL/min when loading;Respectively with the Tris-HCl containing 20mM imidazoles
Buffer (pH7.5) and Tris-HCl buffer (pH 7.5) containing 50mM imidazoles wash away the weak foreign protein of binding force, then
Pillar is rinsed with the Tris-HCl buffer (pH 7.5) containing 250mM imidazoles, what is eluted is the strong purpose egg of binding force
CDA that is white, as purifying.
For electrophoresis result as shown in Figure 1, arrow marks the CDA of purifying, display obtains correct CDA.
Enzyme activity determination shows that the CDA expressed in Escherichia coli body has deacetylase activity.3 chitin of embodiment
Deacetylase is in the endobacillary expression of bacillus subtilis
Nucleotide sequence shown in SEQ ID NO:2 is synthesized by general biosystem (Anhui) Co., Ltd, and its 5 '
End addition BamH I restriction enzyme site, while in 3 ' end addition Pst I restriction enzyme sites.Pass through BamH I and Pst I (New
England Biolabs, NEB) double digestion carried out to synthesized segment, and with T4DNA ligase (Takara) by double digestion
Segment afterwards is respectively connected to equally carry out pHThis carrier (CN104263711A, the Lee of double digestion by BamH I and Pst I
It is refreshing, Huang Xiongliang, a kind of alkali-resistant xylanase of Wang Jufang and its encoding gene and recombinant vector, 2015.).By connection product
Conversion is into bacillus coli DH 5 alpha competent cell (Beijing Quanshijin Biotechnology Co., Ltd), anti-with ampicillin
Property LB solid plate on cultivate and screening positive clone.Picking single colonie carries out bacterium colony PCR verifying;And to bacterium colony PCR
The positive clone of display carries out sequence verification.
Show that the correct positive colony of sequence carries out plasmid extraction to through sequence verification using commercial kit, obtains weight
Group plasmid CDA-pHThis.
The preparation of bacillus subtilis bacterium competence cell and its method for transformation are as follows:
1 required buffer liquid of table and culture medium preparation method
Bacillus subtilis 1012 (MoBiTec company, Germany) is activated on HS solid medium, is connected to 3mL HS
Fluid nutrient medium, 37 DEG C, 250rpm overnight incubation;Bacterium solution will be incubated overnight to be connected in the HS culture medium of Fresh by 1:100,
Make initial OD600About 0.05,37 DEG C, 250rpm culture 5~5.5 hours;250 μ L bacterium solutions (1:20) are taken to be connected to 5mL Fresh
LS culture medium in, cultivate 2h;Culture terminates, and takes 1mL bacterium solution into 1.5mL sterile EP tube, and 10 μ L0.1M EGTA, room is added
Warm bath 5min;The recombinant plasmid CDA-pHThis, 37 DEG C, 180rpm culture 2h as above obtained in right amount is added;Bacterium solution 13,400g
Centrifugation 30 seconds, removes 800 μ L, is gently resuspended, and 200 μ L of residue is all coated with to the LB solid plate of chlorampenicol resistant, 37 DEG C of trainings
Support case overnight incubation.Picking single colonie carries out bacterium colony PCR verifying, and the positive clone of bacterium colony PCR display is taken off second as chitin
Acyl enzyme (CDA) expresses bacterial strain.
By CDA expression strain inoculated in 5mL 2*YT fluid nutrient medium, 37 DEG C, 220rpm overnight incubation;By 1% ratio
Example switching shaking flask (fluid nutrient medium of 2*YT containing 30mL), 37 DEG C, 220rpm culture 2 hours or so to mid-log phase, add IPTG extremely
Final concentration 1mM, 37 DEG C, 180rpm continues to cultivate;Collect bacterium solution after the completion of Fiber differentiation, 4 DEG C, be centrifuged under the conditions of 5000g
10min collects supernatant bacterial sediment respectively;Precipitating using Tris-HCl buffer (pH 7.5) suspend, then using 4 DEG C,
5000g is centrifuged 10min, is repeated twice;After thallus is resuspended using 4mL Tris-HCl buffer, addition lysozyme to 1mg/mL,
5min is placed under the conditions of 37 DEG C;Using ultrasonic cell disruption instrument carry out clasmatosis, condition be power 20%, ultrasonic 10min,
Ultrasonic 3s is spaced 2s;Collect the research that treated sample carries out zymetology performance.
The characterization of 4 CDA zymetology performance of embodiment
CDA enzyme activity determination mode: 200mg/L paranitroacetanilide solution, 0.05mol/L phosphate buffer are prepared;
0.5mL 200mg/L paranitroacetanilide solution, 1.5mL 0.05mol/L phosphate buffer are added in 10mL test tube
Obtained CDA enzyme solution in 0.5mL embodiment 3,60 DEG C of water-bath 15min, boiling is added in (pH 7.2), 60 DEG C of water-bath 3min
Water-bath terminates enzymatic reaction, and water is added to be settled to 10mL.If there is turbid phenomenon, it is centrifuged 10min under the conditions of 3000r/min,
Absorbance value is measured at 400nm.It generates enzyme amount required for 1 μ g paranitroanilinum per hour under the reaction conditions and is defined as 1
A enzyme activity unit (U/mL).Meanwhile by CDA enzyme solution obtained in embodiment 3 in 95 DEG C of water-baths inactivation treatment
10min, then by with operate above it is identical in a manner of, the CDA enzyme solution 0.5mL of the inactivation is added, is carried out as blank control anti-
Ying Hou measures reaction system absorbance value.
<influence of the embodiment 4-1>temperature to enzymatic activity
Other than being reacted under the conditions of 50 DEG C, 60 DEG C, 70 DEG C, according to above-mentioned CDA enzyme activity determination mode
Identical mode, CDA of the invention obtained in the CDA (CliCDA) and embodiment 3 to Kidney bean anthrax-bacilus source
(VloCDA) enzyme activity determination is carried out.Wherein, amino of the CliCDA according to the same manner as in Example 3 based on wild type CliCDA
Acid sequence obtains.
(Fig. 2) as the result is shown, at neutral pH (pH 7.2), VloCDA all shows acceptable under the conditions of each temperature
Deacetylase activity;In addition, VloCDA temperature be 60 DEG C under conditions of active highest similar with CliCDA, and make us
Surprisingly, activity of the VloCDA under this condition than CliCDA improves up to 55.2%.
<influence of the embodiment 4-2>pH to enzymatic activity
Other than being reacted respectively using the phosphate buffer of pH 3, pH 5.5, pH 7.2 and pH 8.11, press
According to mode identical with above-mentioned CDA enzyme activity determination mode, enzyme is carried out to CDA (VloCDA) of the invention obtained in embodiment 3
Measurement living.
(Fig. 3) as the result is shown, on the one hand, the meta-alkali that CliCDA etc. of the VloCDA in Kidney bean anthrax-bacilus source is had a liking for
Good deacetylase activity is shown under the condition (pH 8.11) of property;On the other hand, it is surprising that VloCDA exists
CliCDA is difficult to show more outstanding deacetylase activity under the condition (pH 3.0-5.5) of the slant acidity to play a role, special
It is not the deacetylase activity highest when pH is 5.5.In view of the reaction condition of industrial material is the condition of slant acidity,
CDA of the invention shows the potential for being more suitable for industrial applications.
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(2010)."Chitin deacetylase:A comprehensive account on its role in nature and
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Kafetzopoulos,D.,G.Thireos,J.N.Vournakis and V.Bouriotis(1993)."The
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It will be understood to those of skill in the art that will easily can retouched above to realize purpose same as the present invention
Concept and specific embodiment disclosed in stating are used as basis to modify or design other embodiment.Those skilled in the art
Member will be further understood that such equivalent embodiments without departing from claimed spirit of the invention in the following claims
And range.
Sequence table
<110>Jilin COFCO Biochemical Co., Ltd.
<120>a kind of chitin deacetylase and its application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 260
<212> PRT
<213>rape Huang withers bacterium (Verticillium longisporum)
<400> 1
Met Tyr Thr Thr Thr Val Leu Ser Leu Leu Ala Leu Thr Gly Thr Thr
1 5 10 15
Leu Thr Ala Pro Thr Ala Leu His Leu Arg Asp Ser Thr Pro Pro Ser
20 25 30
Pro Thr Arg His Arg Arg Ala Pro Ala Leu Gly Gln Thr Leu Tyr Ser
35 40 45
Cys Val Asn Pro Gly Gln Val Ala Leu Thr Tyr Asp Asp Gly Pro Tyr
50 55 60
Thr Phe Thr Ser Ser Leu Leu Asp Val Leu Asp Glu Glu Gly Val Thr
65 70 75 80
Ala Thr Phe Phe Leu Thr Gly Ser Asn Phe Gly Arg Glu Met Thr Ser
85 90 95
Asp Pro Trp Ser Ala Ile Val Gln Arg Thr Tyr Ala Ala Gly His Gln
100 105 110
Leu Ala Ser His Thr Tyr Thr His Pro Asp Leu Ser Ala Leu Thr Pro
115 120 125
Ala Ala Arg Ala Ala Glu Met Ala Ala Asn Asp Asp Ala Phe Arg Ala
130 135 140
Ile Leu Gly Phe Ala Pro Arg Tyr Met Arg Ala Pro Phe Leu Ser Cys
145 150 155 160
Asp Ala Ala Cys Ala Ala Asp Met Ala Ala Leu Gly Phe His Ile Val
165 170 175
Asp Ala Ser Ile Asp Thr Lys Asp Phe Glu His Asn Gln Tyr Gly Thr
180 185 190
Val Tyr Ala Ala Glu Ala Lys Phe Asp Ala Glu Leu Gly Trp Asp Pro
195 200 205
Ala Val Asp Ser Ala Ile Val Leu Ala His Asp Val His Glu Thr Thr
210 215 220
Val Ser Val Leu Thr Arg His Met Ile Ser Thr Leu Arg Ala Arg Gly
225 230 235 240
Phe Arg Ala Val Thr Val Gly Glu Cys Leu Gly Asp Ser Pro Asp Gly
245 250 255
Trp Tyr Lys Ala
260
<210> 2
<211> 780
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgtacacaa caacagtgct gagcctgctt gcactgacag gaacaacact tacagcaccg 60
acagcgctgc atctgagaga ttcaacaccg ccgtcaccga caagacatag aagagcgccg 120
gcacttggac aaacactgta ttcatgcgtt aatccgggac aagttgcact gacatatgat 180
gatggaccgt atacattcac tagcagcctt ctggatgtgc ttgatgaaga aggcgttaca 240
gcgacatttt tccttacagg cagcaatttt ggaagagaaa tgacaagcga tccgtggtca 300
gcaattgtgc agagaacata tgcagcggga catcaactgg caagccatac atatacacat 360
ccggatcttt cagcactgac accggcagca agagcggcag aaatggcggc gaatgatgat 420
gcatttcgcg caattctggg ctttgcgccg agatatatga gagcaccgtt tcttagctgt 480
gatgcagcat gcgcggcgga tatggcggca cttggctttc atattgttga tgcaagcatt 540
gatacaaaag attttgaaca taaccagtac ggcacagttt atgcagcgga agcaaaattt 600
gatgcagaac tgggctggga ccctgcggtt gattcagcaa ttgtcctggc acatgatgtt 660
catgaaacaa cagtttcagt gctgacaaga catatgattt caacacttag agcgagagga 720
tttcgcgcgg tgacagttgg cgaatgcctt ggagatagcc cggatggctg gtataaagca 780
Claims (9)
1. one kind is withered the chitin deacetylase of bacterium (Verticillium longisporum) VL1 from rape Huang, described
The amino acid sequence of chitin deacetylase is as shown in SEQ ID NO:1.
2. the DNA molecular of chitin deacetylase as described in claim 1 is encoded,
Preferably, the nucleotide sequence of the DNA molecular is as shown in SEQ ID NO:2.
3. DNA molecular as claimed in claim 2, which is characterized in that the DNA molecular is further connected with coding at its 3 ' end
The nucleotide sequence of hexahistine label.
4. it include the expression vector of DNA molecular as claimed in claim 2 or claim 3,
Preferably, the expression vector is pET24a (+) or pHThis.
5. the transformed cells of expression vector as claimed in claim 4 are imported,
Preferably, the transformed cells are E. coli transformant cells or bacillus subtilis transformed cells;
It is highly preferred that the transformed cells are 1012 cell of Escherichia coli Transetta (DE3) cell or bacillus subtilis.
6. comprising selected from one of following substance or a variety of kits:
Chitin deacetylase as described in claim 1;
DNA molecular as claimed in claim 2 or claim 3;
Expression vector as claimed in claim 4;And
Transformed cells as claimed in claim 5.
7. the method for preparing chitin deacetylase as described in claim 1, described method includes following steps: in culture medium
Middle culture transformed cells as claimed in claim 5;And the chitin deacetylase in the collection culture medium.
8. a kind of method of catalysis de-acetyl chitin production chitosan, which is characterized in that using as described in claim 1
Chitin deacetylase;DNA molecular as claimed in claim 2 or claim 3;Expression vector as claimed in claim 4;As right is wanted
Transformed cells described in asking 5;And/or kit as claimed in claim 6,
Preferably, catalysis de-acetyl chitin production chitosan temperature be 50 DEG C -70 DEG C (preferably 55 DEG C -65 DEG C, more
It is preferred that 58 DEG C -62 DEG C, most preferably 60 DEG C) and pH be 3.0-8.0 (preferably 3.5-7.5, more preferable 4.0-7.2, further preferably
4.5-6.5, particularly preferred 5.0-6.0, most preferably 5.5) under conditions of carry out.
9. chitin deacetylase as described in claim 1;DNA molecular as claimed in claim 2 or claim 3;Such as claim 4
The expression vector;Transformed cells as claimed in claim 5;And/or kit as claimed in claim 6 is in catalysis first
Purposes in the deacetylated production chitosan of shell element,
Preferably, catalysis de-acetyl chitin production chitosan temperature be 50 DEG C -70 DEG C (preferably 55 DEG C -65 DEG C, more
It is preferred that 58 DEG C -62 DEG C, most preferably 60 DEG C) and pH be 3.0-8.0 (preferably 3.5-7.5, more preferable 4.0-7.2, further preferably
4.5-6.5, particularly preferred 5.0-6.0, most preferably 5.5) under conditions of carry out.
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CN114438063A (en) * | 2022-03-07 | 2022-05-06 | 河北农业大学 | Method for efficiently extracting rhodococcus 11-3 intracellular chitin deacetylase |
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