CN109609485A - A kind of chitin deacetylase and its application - Google Patents

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

A kind of chitin deacetylase and its application

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.

Bibliography

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Ghormade,V.,S.Kulkarni,N.Doiphode,P.R.Rajamohanan and M.V.Deshpande (2010)."Chitin deacetylase:A comprehensive account on its role in nature and its biotechnological applications."

He,Y.,J.Xu,S.Wang,G.Zhou and J.Liu(2014)."Optimization of medium components for production of chitin deacetylase by Bacillus amyloliquefaciens Z7,using response surface methodology."Biotechnol Biotechnol Equip 28(2):242- 247.

Jeraj,N.,B.H.Lenasi and K.Breskvar(2006)."Purification and molecular characterization of chitin deacetylase from Rhizopus nigricans." Enzyme&Microbial Technology 39(6):1294-1299.

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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.