CN105801675B - A kind of High-activity chitosanase control gene csn and the method using gene production High-activity chitosanase - Google Patents
A kind of High-activity chitosanase control gene csn and the method using gene production High-activity chitosanase Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01132—Chitosanase (3.2.1.132)
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Abstract
The invention belongs to technical field of bioengineering, provide a kind of High-activity chitosanase control gene csn and the method using gene production High-activity chitosanase.High-activity chitosanase control gene csn according to the present invention is in the Bacillus cercus that deposit number is CGMCC No.6129, inventor constructs heterologous expression vector using High-activity chitosanase control gene csn, and it is transferred in host e. coli BL21 body, experiment shows to carry out high density fermentation using the e. coli bl21, it can get active chitosan essence enzyme, enzyme activity 500000U/g or more, and simple production process, controllability are strong, are suitable for industrialized production.
Description
Technical field
The invention belongs to technical field of bioengineering, a kind of High-activity chitosanase control gene csn and utilization are provided
The method of gene production High-activity chitosanase.
Background technique
Chitin is the second largest natural class high-molecular compound that cellulose is only second on the earth, the product after deacetylation
For chitosan.Chitosan contains free amine group, is unique alkaline polysaccharide in natural polysaccharide, but due to chitosan water solubility
Difference, it is difficult to play its due bioactivity, greatly limit the application range of chitosan.The degradation of discovered in recent years chitosan
Product chitosan oligosaccharide has unique physiological function, and chitosan oligosaccharide, by glucosides key connection, has good water solubility, life by 2-20 monosaccharide
The advantages that object activity strong (being ten times of equal quality chitosan or more), it is described as " the sixth-largest biological element " by scientific circles,
Field of medicaments, field of food, agriculture field, filed of daily-use chemical industry, biological veterinary field and feed additive field have very
Extensive potential using value.Therefore, the great attention of the research in relation to chitosan oligosaccharide and production by vast researcher,
Achieve great successes.
The common production method of chitosan oligosaccharide has physical degradation methods and chemical degradation method at present.Physical degradation methods commonly surpass
Sonication prepares chitosan oligosaccharide by ultrasonic degradation chitosan, and reaction process is mild, can carry out at low temperature, and environment is dirty
Dye is smaller, but the water-soluble products yield of low molecular weight is very low in final product, along with the method cost is excessively high, so physics
Edman degradation Edman is difficult to industrial applications.The conventional method of industrialized production chitosan oligosaccharide is chemical degradation method at present, i.e. Acid hydrolysis system
Standby chitosan oligosaccharide, chitosan are unstable under acidic condition, it may occur that the partial hydrolysis of long-chain, i.e. glycosidic bond fracture are formed many
The segment that relative molecular mass does not wait.Acid hydrolysis prepares a kind of method that chitosan oligosaccharide is comparative maturity, is earliest for industry
The method that metaplasia produces chitosan oligosaccharide, the technical requirements of this method are relatively low, and degradation speed is fast, and equipment, cost of material are lower, very
It is suitble to industrialized production；However, the disadvantages of that there are aftertreatment technologies is cumbersome for this method, and environmental pollution is serious, the most serious is systems
Contents of monosaccharides is high in standby chitosan oligosaccharide product, and the above oligosaccharides yield of trimerization is low, and molecular weight distribution is wide, and by-product is more, pacifies product
Full property endures query to the fullest extent.
With the continuous development of biotechnology, chitosan enzyme Hydrolyze method is gradually applied to the preparation of chitosan oligosaccharide.Chitosan
Enzyme is usually prepared by microbial fermentation, is common in the metabolite of bacterium and fungi.Chitosan enzyme category hydrolase, with inscribe
β-Isosorbide-5-Nitrae glycosidic bond in mode of action specificity hydrolyzing chitosan molecule generates the chitosan oligosaccharide that the degree of polymerization is 3-8.With chitosan
Enzyme hydrolysis method prepares chitosan oligosaccharide, and reaction condition is mild, easily controllable, and molecular weight of product is evenly distributed, and the degree of polymerization is the oligosaccharides of 3-8
For yield up to 90% or more, contents of monosaccharides is very low, and for production process it is not necessary that a large amount of chemical reagent are added, environmental pollution is small, and
Without other by-products, Product Safety ensure that, be the chitosan oligosaccharide production method of a kind of " green ".
Traditional chitosan enzyme production method is obtained by nature wild mushroom spontaneous fermentation, different strain fermentation items
Part, fermentation form are different, and wild mushroom fermentation condition is immature, and that there are producing enzyme vigor is low, the producing enzyme period is long, is difficult to purify place
Reason, difficult the disadvantages of realizing commercialization, directly limit the industrial applications that enzyme hydrolysis method prepares chitosan oligosaccharide technique, such as: journey bodyguard
Big grade, which is screened and identified on fresh shrimp surface, produces chitosan enzyme bacterium-clostridium perfringen, produces chitosan enzyme highest enzyme activity and is
15.8U/mL；Wang Yanjun, which is equal in mud sample, to be separated and identifies and produces chitosan enzyme bacterial strain-Penicillium notatum, and to its condition of enzyme production into
Row optimization, fermented and cultured 72h, enzyme activity is up to 18U/mL.With the progress of science and technology, some scholars attempt to utilize gene work
Journey technological means improves chitosan enzyme vigor, such as Yabuki.M constructs recombination using the chitosanase gene of bacillus alcalophilus
Engineering bacteria, chitosan enzyme enzyme activity is up to 1051U/mL after high density fermentation, and enzyme activity level improves several times, but above-mentioned acquisition
Various bacterial strains be not able to satisfy industrial requirement still.Therefore, screening produces high vigor chitosan enzyme bacterial strain, and utilizes advanced biology
Technological means, which is continuously improved, improves chitosan enzyme vigor, is to realize that chitosan enzyme Hydrolyze method prepares the pass of chitosan oligosaccharide process industry
Key.
Summary of the invention
The present invention is existing insufficient in view of the above technology, provides a kind of High-activity chitosanase control gene csn and benefit
With the method for gene production High-activity chitosanase.High-activity chitosanase according to the present invention controls the source gene csn
In the Bacillus cercus that deposit number is CGMCC No.6129, inventor controls gene csn using the High-activity chitosanase
Heterologous expression vector is constructed, and is transferred in host e. coli BL21 body, experiment shows to carry out using the e. coli bl21 high
Density fermentation can get active chitosan essence enzyme, enzyme activity 500000U/g or more, and simple production process, controllability are strong, fit
In industrialized production.
Present invention firstly provides a kind of High-activity chitosanases to control gene csn, the High-activity chitosanase control
Gene csn processed, nucleotide sequence is as shown in Seq ID No:1, and the amino acid sequence of coding is as shown in Seq ID No:2；
The High-activity chitosanase control gene csn derives from bacillus cereus JBSH-003, and the bacterial strain is Chinese common micro-
The deposit number of biological inoculum collection is CGMCC No.6129.
The acquisition methods of the gene are as follows:
(1) the acquisition of target gene:
Design upstream primer F:GCGCCATATGAAAATCAGTATGCAAAAAGC, nucleotide sequence such as Seq ID No:
Shown in 3, downstream primer R:GCGCGAATTCTTATTTGATTACAAAATTACCGT, nucleotide sequence such as Seq ID No:4 institute
Show；Using Bacillus cercus genomic DNA as template, PCR amplification is carried out under the action of Taq archaeal dna polymerase, verifying is correct
Glue is carried out afterwards to recycle to obtain genetic fragment, and the segment and carrier are then subjected to blunt end cloning, construct cloning vector；It will clone
Carrier is transferred in bacillus coli DH 5 alpha body by physical method, and bacterium solution is coated on to the LB solid culture containing Ampicillin
On base, 32 DEG C~37 DEG C, stationary culture 10h, positive transformant is obtained, by positive transformant sequencing, compares and analyzes；As the result is shown
Objective gene sequence contains 834 bases, and nucleotide sequence encodes 277 amino acid as shown in Seq ID No:1, compiles
Code amino acid sequence as shown in Seq ID No:2,
Wherein used cloning vector is selected from pMD-T series or pUC serial carrier；
After obtaining above-mentioned purpose gene, inventor has been transferred to common engineering bacteria using the gene to verify its function:
By target gene csn from being extracted in positive transformant and under 30~37 DEG C of water bath conditions through restriction enzyme
Enzyme NdeI and EcoRI double digestion 30min is recycled the gene csn after digestion using plastic recovery kit, then in T4DNA
It is connected under connection enzyme effect with expression vector, constructs heterologous expression vector；Heterologous expression vector is transferred to host by physical method
In e. coli bl21 body, bacterium solution is coated on the LB solid medium containing Kanamycin, 32 DEG C~37 DEG C, stands training
10h is supported, obtains positive transformant to get the engineered strain of production High-activity chitosanase；
Wherein building heterologous expression vector is used for pET series or pGEM serial carrier；
Other than the host e. coli BL21 of above-mentioned use, can also use other hosts, as bacillus coli DH 5 alpha,
Bacillus subtilis, salmonella, helicobacter pylori.
Inventor additionally provides the method for High-activity chitosanase control gene csn production High-activity chitosanase, including
Following steps, by taking e. coli bl21 as an example:
(1) kind activation: on aseptic operating platform, the production High-activity chitosanase for the above-mentioned acquisition for taking 1~5 μ l to freeze
Engineering bacteria is in the test tube containing LB liquid medium, and 32 DEG C~37 DEG C, 130~180rpm cultivates 10h；
(2) prepared by seed: on aseptic operating platform, the strain of activation being transferred to the triangular flask containing sterilized liquid culture medium
It is interior, the 200ml culture medium is dispensed in every triangular flask, the test tube strains of an activation are inoculated with a triangular flask, and then 32 DEG C~37
DEG C, 130~180rpm cultivates 10h；
(3) inoculation fermentation tank: fermentation tank, pipeline, air filter are carried out sky and disappeared first, 121 DEG C, sky disappears
30min injects culture medium after temperature reduction, then disappear in fact to culture medium, 121 DEG C, disappear 30min in fact；It is down to temperature
It is inoculated at 32 DEG C~37 DEG C；
(4) fermentation condition: inoculum concentration v/v 5%~10%；35 DEG C~37 DEG C of cultivation temperature；PH:7.0；Initial speed:
200rpm；Fermentor pressure: 0.05Mpa；Ventilatory capacity ratio: 1:1；Feed supplement: concentration is the glycerol aqueous mixtures of 50vt%；Fermentation week
Phase: 45~50h；
(5) induce: as fermentation liquid OD600When=40~60, the inducer IPTG of final concentration of 0.1~0.4mmol/L is added,
Induce target gene csn great expression；
(6), from the inducer beginning is added, every 2h sampling is primary, and detects enzyme activity using DNS method, when the continuous 4h of enzyme activity no longer rises
Stop fermentation when high, and put tank, obtains zymocyte liquid；
(7) broken wall treatment: carrying out thallus broken wall treatment to zymocyte liquid using high pressure homogenizer, and setting pressure 90~
Between 120Mpa, chitosan enzyme a large amount of in somatic cells is discharged into extracellular, acquisition shell by broken wall treatment by flow velocity 60L/h
Dextranase crude enzyme liquid.
Wherein the formula of culture medium described in step (2) and (3) is calculated as: 1.0~1.5 parts of peptones, 2.0~
2.5 parts of yeast extracts, 0.4~0.6 part of glycerol, 0.2~0.3 part of dipotassium hydrogen phosphate, 1.0~1.3 parts of potassium dihydrogen phosphates, 93.8
~95.4 parts of softened waters；
The magnesium sulfate for being also 2% containing g/v in the glycerol aqueous mixtures of 50vt% described in step (4).
The thick enzyme of the chitosan enzyme of above-mentioned acquisition, the final thick enzyme sporoderm-broken rate of chitosan enzyme is up to 90%, through DNS method enzyme activity determination,
Its enzyme activity is 7000~9000U/ml.
In order to further increase the quality of chitosan enzyme, inventor also discloses the purification to above-mentioned chitosan enzyme crude enzyme liquid
Processing, to obtain active chitosan essence enzyme, the specific steps are as follows:
(1) crude enzyme liquid will be obtained by, which being centrifuged using centrifuge, is separated by solid-liquid separation, and centrifugal rotational speed 4000rpm is centrifuged 10min, receives
Collect clear liquid；
(2) micro-filtration processing is carried out by 0.22um microfiltration membranes to the clear liquid after centrifugation, further remove thallus and molecule
Impurity；The ultrafiltration membrane through 6000Dalton is concentrated by ultrafiltration micro-filtrate again, concentration rate 1/5~1/10, collects liquid in ultrafiltration；
(3) 95% ethyl alcohol of liquid product 30%~50% in ultrafiltration is added in liquid into ultrafiltration, carries out alcohol precipitation under room temperature,
It is centrifuged after alcohol precipitation, collects precipitating, obtain wet solid enzyme；
(4) dry: obtained wet solid enzyme is dried in vacuo under the conditions of 50 DEG C to get active chitosan essence enzyme.Through DNS
Method enzyme activity determination, enzyme activity are 500000U/g or more.
By calculating, the comprehensive recovery of entire technical process enzyme activity is 75% or more, and gained purifies active chitosan
Enzyme enzyme activity reaches 500000U/g or more；In addition, gained High-activity chitosanase temperature and pH value wide adaptation range, 40 DEG C~
65 DEG C all have higher bioactivity, have very strong the enzyme activity between pH value 4.5~6.5.
From the above, it can be seen that present invention chitosan enzyme essence enzyme enzyme activity obtained reaches 500000U/g, and Bacillus cercus JBSH-
It is 25000U/g that 003 fermentation, which produces chitosan enzyme essence enzyme enzyme activity level, therefore, the final gained chitosan enzyme essence enzyme enzyme activity water of the present invention
Flat more former starting strain improves 20 times or more；The engineering bacteria host that the present invention uses preferably uses e. coli bl21, is
Type strain, high density fermentation condition maturity, the maximum cell density that ferments is compared with Bacillus cercus JBSH-003 high several times, separately
Outside, the Escherichia coli fermentation period is 45~50h, and Bacillus cercus JBSH-003 fermentation period is 72~96h, therefore is being sent out
It is more much lower than former starting strain in ferment cost.
Compared with prior art, the present invention, which utilizes, utilizes High-activity chitosanase control gene csn building production high activity
The engineering bacteria of chitosan enzyme, while providing the producer that high density fermentation production High-activity chitosanase is carried out using the bacterial strain
Method achieves following usefulness: (1) by the building of expression vector, target gene csn being transformed, is recombinated, place is transferred to
In main thallus, great expression is carried out in inducer inductive condition, expression quantity is it in the intracorporal decades of times of former starting strain；(2) select
The engineered strain that e. coli bl21 carries out building production High-activity chitosanase as host strain is selected, e. coli bl21 is mould
Formula bacterial strain, high density fermentation condition maturity, the producing enzyme period is short, is widely used in industrial field of microorganism fermentation；(3) obtained
High-activity chitosanase crude enzyme liquid enzyme activity is high, reaches 8000U/ml through DNS method enzyme activity determination, average level improves number than before
Times；(4) High-activity chitosanase essence enzyme can be obtained after this method production thick enzyme later period purified processing of chitosan, through DNS method
Enzyme activity determination reaches 500000U/g, and used simple process realizes the requirement of merchandized handling；(5) this method obtains high activity
Chitosan enzyme, enzyme activity greatly improve, and can be applied to the industrial applications of enzyme hydrolysis method production chitosan oligosaccharide technique.
Detailed description of the invention
Fig. 1 is the electrophoresis result schematic diagram that active chitosan of the present invention controls gene csn.
Specific embodiment
The specific embodiment of form by the following examples does further specifically above content of the invention
It is bright, but the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention, and it is complete to be all made of conventional prior unless otherwise specified, in following embodiments
At.
The acquisition of 1 target gene of embodiment
Design upstream primer F:GCGCCATATGAAAATCAGTATGCAAAAAGC, nucleotide sequence such as Seq ID No:
Shown in 3, downstream primer R:GCGCGAATTCTTATTTGATTACAAAATTACCGT, nucleotide sequence such as Seq ID No:4 institute
Show；Using bacillus cereus genomic DNA as template, PCR amplification, the wax are carried out under the action of Taq archaeal dna polymerase
Sample subtilis genomic dna derives from bacillus cereus JBSH-003, and the bacterial strain is in China General Microbiological culture presevation
The deposit number at center is CGMCC No.6129；
Using single factor experiment and orthogonal experiment, amount, the genomic DNA of primer is added in PCR amplification system by changing
Amount and PCR amplification condition annealing temperature (TM), it is single, enough to obtain to determine optimal PCR amplification system and condition
Amplified production；Optimized, final amplification system and amplification condition is shown in Tables 1 and 2 respectively:
Table 1.PCR amplification system (50 μ l)
Table 2.PCR amplification condition
Construct cloning vector
Pcr amplification product is verified by Agarose horizontal electrophoresis, verifies correct rear utilization " Ago-Gel reclaim reagent
Box " carries out target gene fragment recycling, and the genetic fragment of acquisition and pMD19-T vector are carried out flat end company at 16 DEG C
It connects (linked system is shown in Table 3), obtains cloning vector.
3. cloning vector linked system of table (10 μ l)
Cloning vector is transferred in bacillus coli DH 5 alpha body, and bacterium solution is coated on the training of the LB solid containing Ampicillin
It supports on base, 37 DEG C of stationary culture 10h, obtains positive transformant.And analyze positive transformant sequencing, comparison, sequencing result is aobvious
Show, the objective gene sequence of acquisition contains 834 bases, and nucleotide sequence encodes 277 ammonia as shown in Seq ID No:1
Base acid, the amino acid sequence of coding is as shown in Seq ID No:2；It is finally csn by the unnamed gene.Electrophoresis detection result is such as
Shown in Fig. 1.
Embodiment 2 produces the engineered strain building of High-activity chitosanase
It selects e. coli bl21 for host strain, selects pET-30b vector to be connected with target gene csn and construct expression
Carrier carries out double digestion first with restriction enzyme NdeI and EcoRI at 37 DEG C, makes pET-30b vector and csn
Identical cohesive end is formed, the two is then connected into (linked system is shown in Table 4) using T4DNA ligase, was connected at 16 DEG C
Night.
4. expression vector linked system of table (10 μ l)
Connection product is transferred in e. coli bl21 body, and bacterium solution is coated on the training of the LB solid containing Kanamycin
It supports on base, 37 DEG C of stationary culture 10h, obtains positive transformant, that is, produce the engineered strain of High-activity chitosanase.
3 high density fermentation of embodiment obtains High-activity chitosanase
The engineered strain of the production High-activity chitosanase obtained using embodiment 2 carries out high density fermentation, hair as strain
Yeast-like fungi liquid is after broken wall treatment, the chitosan crude enzyme liquid enzyme activity through the measurement acquisition of DNS enzyme activity determination method.
It is specific as follows:
(1) actication of culture: on aseptic operating platform, the engineered strain kind of the production High-activity chitosanase for taking 5 μ l to freeze in
In test tube containing LB liquid medium, 37 DEG C, 150rpm cultivates 10h.
(2) prepared by seed: on aseptic operating platform, the strain of activation being transferred to the triangular flask containing sterilized liquid culture medium
It is interior, culture medium prescription are as follows: 1.2 parts of peptones, 2.4 parts of yeast extracts, 0.4 part of glycerol, 0.23 part of dipotassium hydrogen phosphate, 1.25 parts
Potassium dihydrogen phosphate, 94.52 parts of softened waters, every triangular flask is interior to dispense the 200ml culture medium, the test tube strains inoculation one of an activation
A triangular flask, then 37 DEG C, 150rpm cultivates 10h.
(3) inoculation fermentation tank: fermentation tank, pipeline, air filter are carried out sky and disappeared first, 121 DEG C, sky disappears
30min injects culture medium, culture medium prescription after temperature reduction are as follows: 1.2 parts of peptones, 2.4 parts of yeast extracts, and 0.4 part
Glycerol, 0.23 part of dipotassium hydrogen phosphate, 1.25 parts of potassium dihydrogen phosphates, 94.52 parts of softened waters.Then culture medium disappear in fact, 121
DEG C, disappear 30min in fact.It is inoculated with when temperature is down to 37 DEG C.
(4) fermentation condition: inoculum concentration 5vt%；37 DEG C of cultivation temperature；PH:7.0；Initial speed: 200rpm；Fermentor pressure:
0.05Mpa；Ventilatory capacity ratio: 1:1；Feed supplement: the glycerol aqueous mixtures (magnesium sulfate containing 2vt%) of 50vt%；
(5) induce: as fermentation liquid OD600When=60 or so, the inducer IPTG of final concentration of 0.2mmol/L is added, and will
Cultivation temperature is controlled at 30 DEG C.
(6) put tank: from be added inducer begin, every 2 hours sample detection enzyme activity, when enzyme activity continue 4 it is small when no longer increase, this
When fermentation time be 45h, terminate ferment simultaneously puts tank.
(7) broken wall treatment is carried out to zymocyte liquid using high pressure homogenizer, obtain chitosan crude enzyme liquid, surveyed through DNS method enzyme activity
It is fixed, enzyme activity 8100U/ml.
4 high density fermentation of embodiment obtains High-activity chitosanase
The engineered strain of the production High-activity chitosanase obtained using embodiment 2 carries out high density fermentation, hair as strain
Yeast-like fungi liquid is after broken wall treatment, the chitosan crude enzyme liquid enzyme activity through the measurement acquisition of DNS enzyme activity determination method.
It is specific as follows:
(1) actication of culture: on aseptic operating platform, the engineered strain kind of the production High-activity chitosanase for taking 5 μ l to freeze in
In test tube containing LB liquid medium, 35 DEG C, 150rpm cultivates 10h.
(2) prepared by seed: on aseptic operating platform, the strain of activation being transferred to the triangular flask containing sterilized liquid culture medium
It is interior, 1.4 parts of peptones, 2.0 parts of yeast extracts, 0.5 part of glycerol, 0.25 part of dipotassium hydrogen phosphate, 1.3 parts of potassium dihydrogen phosphates,
94.55 parts of softened waters, every triangular flask is interior to dispense the 200ml culture medium.The test tube strains of one activation are inoculated with a triangular flask, so
35 DEG C afterwards, 150rpm cultivates 10h.
(3) inoculation fermentation tank: fermentation tank, pipeline, air filter are carried out sky and disappeared first, 121 DEG C, sky disappears
30min injects culture medium, culture medium prescription after temperature reduction are as follows: 1.4 parts of peptones, 2.0 parts of yeast extracts, and 0.5 part
Then glycerol, 0.25 part of dipotassium hydrogen phosphate, 1.3 parts of potassium dihydrogen phosphates, 94.55 parts of softened waters disappear in fact to culture medium, and 121
DEG C, disappear 30min in fact, is inoculated with when temperature is down to 37 DEG C.
(4) fermentation condition: inoculum concentration 5.5vt%；35 DEG C of cultivation temperature；PH:7.0；Initial speed: 200rpm；Fermentor
Pressure: 0.05Mpa；Ventilatory capacity ratio: 1:1；Feed supplement: the glycerol aqueous mixtures (magnesium sulfate containing 2vt%) of 50vt%；
(5) induce: as fermentation liquid OD600When=50 or so, the inducer IPTG of final concentration of 0.35mmol/L is added, and will
Cultivation temperature is controlled at 28 DEG C.
(6) put tank: from be added inducer begin, every 2 hours sample detection enzyme activity, when enzyme activity continue 4 it is small when no longer increase, this
When fermentation time be 48h, terminate ferment simultaneously puts tank.
(7) broken wall treatment is carried out to zymocyte liquid using high pressure homogenizer, obtain chitosan crude enzyme liquid, surveyed through DNS method enzyme activity
It is fixed, enzyme activity 8350U/ml；
In order to further increase the quality of chitosan enzyme, inventor has carried out at purification above-mentioned chitosan enzyme crude enzyme liquid
Reason, the specific steps are as follows:
(1) crude enzyme liquid will be obtained by, which being centrifuged using centrifuge, is separated by solid-liquid separation, and centrifugal rotational speed 4000rpm is centrifuged 10min, receives
Collect clear liquid；
(2) micro-filtration processing is carried out by 0.22um microfiltration membranes to the clear liquid after centrifugation, further remove thallus and molecule
Impurity；The ultrafiltration membrane through 6000Dalton is concentrated by ultrafiltration micro-filtrate again, concentration rate 1/5-1/10, collects liquid in ultrafiltration；
(3) 95% ethyl alcohol of liquid product 30%~50% in ultrafiltration is added in liquid into ultrafiltration, carries out alcohol precipitation under room temperature,
It is centrifuged after alcohol precipitation, collects precipitating, obtain wet solid enzyme；
(4) dry: obtained wet solid enzyme is dried in vacuo under the conditions of 50 DEG C to get active chitosan essence enzyme.Through DNS
Method enzyme activity determination, enzyme activity are 500000U/g or more.
5. high density fermentation of embodiment obtains High-activity chitosanase
The engineered strain of the production High-activity chitosanase obtained using embodiment 2 carries out high density fermentation, hair as strain
Yeast-like fungi liquid is after broken wall treatment, the chitosan crude enzyme liquid enzyme activity through the measurement acquisition of DNS enzyme activity determination method.
It is specific as follows:
(1) actication of culture: on aseptic operating platform, the engineered strain kind of the production High-activity chitosanase for taking 5 μ l to freeze in
In test tube containing LB liquid medium, 32 DEG C, 150rpm cultivates 10h.
(2) prepared by seed: on aseptic operating platform, the strain of activation being transferred to the triangular flask containing sterilized liquid culture medium
It is interior, 1.5 parts of peptones, 2.5 parts of yeast extracts, 0.6 part of glycerol, 0.3 part of dipotassium hydrogen phosphate, 1.3 parts of potassium dihydrogen phosphates, 93.8
Part softened water dispenses the 200ml culture medium in every triangular flask, and the test tube strains of an activation are inoculated with a triangular flask, and then 32
DEG C, 150rpm cultivates 10h.
(3) inoculation fermentation tank: fermentation tank, pipeline, air filter are carried out sky and disappeared first, 121 DEG C, sky disappears
30min injects culture medium, culture medium prescription after temperature reduction are as follows: 1.5 parts of peptones, 2.5 parts of yeast extracts, and 0.6 part
Then glycerol, 0.3 part of dipotassium hydrogen phosphate, 1.3 parts of potassium dihydrogen phosphates, 93.8 parts of softened waters disappear in fact to culture medium, and 121 DEG C,
Disappear 30min in fact, is inoculated with when temperature is down to 32 DEG C.
(4) fermentation condition: inoculum concentration 6vt%；32 DEG C of cultivation temperature；PH:7.0；Initial speed: 200rpm；Fermentor pressure:
0.05Mpa；Ventilatory capacity ratio: 1:1；Feed supplement: the glycerol aqueous mixtures (magnesium sulfate containing 2vt%) of 50vt%；
(5) induce: as fermentation liquid OD600When=45 or so, the inducer IPTG of final concentration of 0.4mmol/L is added, and will
Cultivation temperature is controlled at 28 DEG C.
(6) put tank: from be added inducer make, every 2 hours sample detection enzyme activity, when enzyme activity continue 4 it is small when no longer increase, this
When fermentation time be 50h, terminate ferment simultaneously puts tank.
(7) broken wall treatment is carried out to zymocyte liquid using high pressure homogenizer, obtain chitosan crude enzyme liquid, surveyed through DNS method enzyme activity
It is fixed, enzyme activity 7950U/ml.
Comparative example 1. produces the engineered strain of High-activity chitosanase and former starting strain produces chitosan enzyme enzyme activity and fermentation
Time compares
With in embodiment 3 culture medium prescription and fermentation condition to it is provided by the invention production High-activity chitosanase work
Journey bacterial strain and former starting strain-Bacillus cercus JBSH-003 carry out fermenting experiment, compare the produced chitosan enzyme enzyme activity of the two
And best fermentation period.
It is specific as follows:
(1) actication of culture: on aseptic operating platform, the engineered strain for the production High-activity chitosanase for taking 5 μ l to freeze respectively
Kind and Bacillus cercus JBSH-003 are in two test tubes containing LB liquid medium, and 37 DEG C, 150rpm cultivates 10h.
(2) prepared by seed: on aseptic operating platform, the strain of activation being transferred to the triangular flask containing sterilized liquid culture medium
It is interior, culture medium prescription are as follows: 1.2 parts of peptones, 2.4 parts of yeast extracts, 0.4 part of glycerol, 0.23 part of dipotassium hydrogen phosphate, 1.25 parts
Potassium dihydrogen phosphate, 94.52 parts of softened waters, every triangular flask is interior to dispense the 200ml culture medium, the test tube strains inoculation one of an activation
A triangular flask, then 37 DEG C, 150rpm cultivates 10h.
(3) inoculation fermentation tank: fermentation tank, pipeline, air filter are carried out sky and disappeared first, 121 DEG C, sky disappears
30min injects culture medium, culture medium prescription after temperature reduction are as follows: 1.2 parts of peptones, 2.4 parts of yeast extracts, and 0.4 part
Then glycerol, 0.23 part of dipotassium hydrogen phosphate, 1.25 parts of potassium dihydrogen phosphates, 94.52 parts of softened waters disappear in fact to culture medium, and 121
DEG C, disappear 30min in fact, is inoculated with when temperature is down to 37 DEG C.
(4) fermentation condition: inoculum concentration 5%；37 DEG C of cultivation temperature；PH:7.0；Initial speed: 200rpm；Fermentor pressure:
0.05Mpa；Ventilatory capacity ratio: 1:1；The glycerol aqueous mixtures (magnesium sulfate containing 2vt%) of 50vt%；
(5) induce: as the engineered strain fermentation liquid OD of production High-activity chitosanase600When=60 or so, final concentration is added
For the inducer IPTG of 0.2mmol/L, and cultivation temperature is controlled at 30 DEG C.
(6) put tank: from the 42h that ferments, every 2 hours sample detection enzyme activity, when enzyme activity continue 4 it is small when no longer increase, terminate hair
Ferment simultaneously puts tank.Every sub-sampling measures the thick enzyme enzyme activity of sample through high pressure homogenizer broken wall treatment, and through DNS method.
Testing result is as follows:
From upper table it can be found that the engineered strain of production High-activity chitosanase provided by the invention is when fermenting 46h, institute
The thick enzyme enzyme activity of chitosan enzyme reaches maximum 8125U/mL, and Bacillus cercus JBSH-003 is when fermenting 72h, gained chitosan
The thick enzyme enzyme activity of enzyme reaches maximum 405U/mL, it is possible to conclude that a kind of High-activity chitosanase gene provided by the invention
The engineered strain of the production High-activity chitosanase of csn building, chitosan enzyme enzyme activity produced is horizontal high, and fermentation period is short,
And simple production process, controllability are strong, are suitable for industrialized production.
Claims (5)
1. a kind of High-activity chitosanase controls gene csn, it is characterised in that: the High-activity chitosanase controls gene
Csn, nucleotide sequence is as shown in Seq ID No:1, and the amino acid sequence of coding is as shown in Seq ID No:2.
2. High-activity chitosanase according to claim 1 controls gene csn, it is characterised in that: the high activity shell
Dextranase controls gene csn and derives from bacillus cereus (Bacillus cereus) JBSH-003, and the bacterial strain is Chinese common
The deposit number of Culture Collection is CGMCC No.6129.
3. obtaining the engineering of production High-activity chitosanase using the control of High-activity chitosanase described in claim 1 gene csn
The method of bacterium, it is characterised in that: specific step is as follows:
(1) the acquisition of target gene:
Design upstream primer F:GCGCCATATGAAAATCAGTATGCAAAAAGC, nucleotide sequence such as Seq ID No:3 institute
Show, downstream primer R:GCGCGAATTCTTATTTGATTACAAAATTACCGT, nucleotide sequence is as shown in Seq ID No:4；
Using Bacillus cercus genomic DNA as template, PCR amplification is carried out under the action of Taq archaeal dna polymerase, verifying is correct laggard
Row glue recycles to obtain genetic fragment, and the segment and carrier are then carried out blunt end cloning, constructs cloning vector；By cloning vector
It is transferred in bacillus coli DH 5 alpha body by physical method, and bacterium solution is coated on to the LB solid medium containing Ampicillin
On, 32 DEG C~37 DEG C, stationary culture 10h, positive transformant is obtained, by positive transformant sequencing, compares and analyzes；Mesh as the result is shown
Gene order contain 834 bases, nucleotide sequence encodes 277 amino acid as shown in Seq ID No:1, coding
Amino acid sequence as shown in Seq ID No:2,
Wherein used cloning vector is selected from pMD-T series or pUC serial carrier；
(2) produce the engineered strain building of High-activity chitosanase: target gene csn being connected into carrier, building heterogenous expression carries
Body obtains the engineered strain of production High-activity chitosanase:
By target gene csn from being extracted in positive transformant and under 30~37 DEG C of water bath conditions through restriction enzyme
Gene csn after digestion is recycled using plastic recovery kit, is then connected in T4DNA by NdeI and EcoRI double digestion 30min
It connects and is connected under enzyme effect with expression vector, construct heterologous expression vector；Heterologous expression vector is transferred to host by physical method
It is interior, bacterium solution is coated on the LB solid medium containing Kanamycin, 32 DEG C~37 DEG C, stationary culture 10h, is obtained positive
Transformant is to get the engineered strain for producing High-activity chitosanase；
Wherein building heterologous expression vector is used for pET series or pGEM serial carrier；
The host is selected from e. coli bl21 or bacillus coli DH 5 alpha or bacillus subtilis or salmonella or pylorus spiral shell
Bacillus.
4. special using the method for the control gene csn production High-activity chitosanase of High-activity chitosanase described in claim 1
Sign is: the following steps are included:
(1) actication of culture: on aseptic operating platform, production that the vegetation according to the method in claim 3 for taking 1~5 μ l to freeze obtains
The engineering bacteria of High-activity chitosanase is in the test tube containing LB liquid medium, and 32 DEG C~37 DEG C, 130~180rpm is cultivated
10h；
(2) prepared by seed: on aseptic operating platform, the strain of activation being transferred in the triangular flask containing sterilized liquid culture medium, often
Packing 200ml culture medium in triangular flask, test tube strains one triangular flask of inoculation of an activation, then 32 DEG C~37 DEG C, 130
~180rpm cultivates 10h；
(3) inoculation fermentation tank: carrying out fermentation tank, pipeline, air filter sky and disappear first, and 121 DEG C, sky disappears 30min, to
After temperature reduces, culture medium is injected, then culture medium disappear in fact, 121 DEG C, disappear 30min in fact；32 DEG C~37 are down to temperature
DEG C when be inoculated with；
(4) fermentation condition: inoculum concentration v/v 5%~10%；35 DEG C~37 DEG C of cultivation temperature；PH:7.0；Initial speed: 200rpm；
Fermentor pressure: 0.05Mpa；Ventilatory capacity ratio: 1:1；Feed supplement: concentration is the glycerol aqueous mixtures of 50vt%；Fermentation period: 45~
50h；
(5) induce: as fermentation liquid OD600When=40~60, the inducer IPTG of final concentration of 0.1~0.4mmol/L, induction is added
Target gene csn great expression；
(6), from the inducer beginning is added, every 2h sampling is primary, and detects enzyme activity using DNS method, when the continuous 4h of enzyme activity is no longer increased
Stop fermentation, and put tank, obtains zymocyte liquid；
(7) broken wall treatment: carrying out thallus broken wall treatment to zymocyte liquid using high pressure homogenizer, setting 90~120Mpa of pressure it
Between, chitosan enzyme a large amount of in somatic cells is discharged into extracellular by broken wall treatment by flow velocity 60L/h, and it is thick to obtain chitosan enzyme
Enzyme solution；
Wherein the formula of culture medium described in step (2) and (3) is calculated as: 1.0~1.5 parts of peptones, and 2.0~2.5
Part yeast extract, 0.4~0.6 part of glycerol, 0.2~0.3 part of dipotassium hydrogen phosphate, 1.0~1.3 parts of potassium dihydrogen phosphates, 93.8~
95.4 part softened water；
The magnesium sulfate for being also 2% containing g/v in the glycerol aqueous mixtures of 50vt% described in step (4).
5. method according to claim 4, it is characterised in that: the fine purification treatment process of the chitosan enzyme crude enzyme liquid, specifically
Steps are as follows:
(1) crude enzyme liquid will be obtained by, which being centrifuged using centrifuge, is separated by solid-liquid separation, and centrifugal rotational speed 4000rpm is centrifuged 10min, collects clear
Liquid；
(2) micro-filtration processing is carried out by 0.22um microfiltration membranes to the clear liquid after centrifugation, further remove thallus and molecule is miscellaneous
Matter；The ultrafiltration membrane through 6000Dalton is concentrated by ultrafiltration micro-filtrate again, concentration rate 1/5~1/10, collects liquid in ultrafiltration；
(3) 95% ethyl alcohol of liquid product 30%~50% in ultrafiltration is added in liquid into ultrafiltration, carries out alcohol precipitation, alcohol precipitation under room temperature
After be centrifuged, collect precipitating, obtain wet solid enzyme；
(4) dry: obtained wet solid enzyme is dried in vacuo under the conditions of 50 DEG C to get active chitosan essence enzyme.
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| CN102851239A (en) * | 2012-08-22 | 2013-01-02 | 黄河三角洲京博化工研究院有限公司 | Chitosanase producing strain and chitosan production method by using the same |
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