CN109652437A - It is a kind of produce chitosan enzyme recombinant bacterium construction method and its application - Google Patents
It is a kind of produce chitosan enzyme recombinant bacterium construction method and its application Download PDFInfo
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Abstract
The present invention discloses construction method and its application of a kind of recombinant bacterium of production chitosan enzyme.The present invention co-expresses bacillus subtilis chitosanase gene and Vitreoscilla hemoglobin gene in Pichia pastoris, and recombinant bacterial strain efficiently prepares chitosan enzyme through high density fermentation strategy.It is up to 50000U/mL or more using the enzyme activity that this method prepares chitosan enzyme, protein content is up to 16.0mg/mL.Recombinase specific enzyme activity power is 4065.7U/mg, and optimal pH 6.0, optimum temperature is 55 DEG C.Chitosan oligosaccharide is prepared using enzyme hydrolysis high concentration (>=10%) chitosan, chitosan oligosaccharide yield is greater than 75%, and primary product is disaccharides, trisaccharide and tetrose.Chitosan enzyme preparation method producing enzyme level of the invention is significantly larger than the report of existing literature and patent, and institute's producing enzyme has excellent hydrolysis properties, in industrial production chitosan enzyme and prepares in chitosan oligosaccharide with good application value.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to it is a kind of produce chitosan enzyme recombinant bacterium construction method and its answer
With.
Background technique
Chitosan is the deacetylated product in chitin part, by D- Glucosamine and 2-acetylamino-2-deoxy-D-glucose with
The straight chain natural polysaccharide that β -1,4- glycosidic bond is formed by connecting at random.Chitosan enzyme (EC3.2.1.132) is single-minded hydrolyzing chitosan
Enzyme, cut β-Isosorbide-5-Nitrae-glycosidic bond at random from chitosan chain, product is chitosan oligosaccharide and Glucosamine.It is same based on its sequence
Source property, chitosan enzyme are divided into glycoside hydrolase (Glycoside Hydrolases, GH) 5,7,8,46,75 and 80 families
(Thadathil et al.Food Chemistry,2014,150:392–399)。
There are many documents and patent report that microorganism produces chitosan enzyme in recent years, but most of producing enzymes are horizontal partially
It is low, it is difficult to be used for industrialized production, only some microorganisms produce the horizontal higher of chitosan enzyme.As patent CN102399727A is disclosed
A kind of bacillus subtilis utilizes the method that konjaku flour is that carbon source produces chitosan enzyme, enzyme activity 11762U/mL.Chen etc. will
Aspergillus fumigatus chitosanase gene heterogenous expression in pichia pastoris yeast, enzyme activity are 25000U/mL (Chen et
Al.Biotechnology Letters, 2012,34:689-694), this is the highest producing enzyme water of chitosan enzyme at present
It is flat.
Pichia pastoris yeast is widely used in the various heterologous proteins of production as a kind of mature, efficient expression system,
High cell density fermentation is the effective ways for obtaining high yield recombinant protein.However, high-cell-density cultivation needs a large amount of oxygen,
Cause dissolved oxygen easy to form limitation during methanol induction, and due to the increase of biomass and defoaming agent, the solubility of oxygen and
Mass-transfer efficiency gradually decreases, and influences expression quantity (Yang the et al., Biotechnology of cellular biomass and destination protein
Advances,2017,36(1):182-195).Vitreoscilla (Vitreoscillastercoraria) hemoglobin can promote
It conveys into oxygen so as to improve cellular respiration and energetic supersession.Existing research co-expresses itself and galactosidase, phosphatidase etc.
Improve dissolved oxygen restricted problem when high density fermentation, cellular biomass and destination protein expression quantity is made to increase substantially (Wu et
al.,Journal of Bioscience&Bioengineering,2012,113:332-337；Wang et al.,Enzyme
and Microbial Technology,2012,50:22-28).Although many chitosanase genes are cloned expression, have no
The pertinent literature and patent report of chitosan enzyme expression are improved using coexpression Vitreoscilla hemoglobin.
Chitosan oligosaccharide is the product of degradation of chitosan, and the degree of polymerization (degree of polymerization, DP) is generally in 2-
Between 10, molecular weight is low, good water solubility, have many physiological activity, as biocidal property, anti-oxidant, antitumor, norcholesterol,
Blood pressure lowering, anti-infection and control arthritis etc., are widely used in the industries such as food, medicine, agricultural and cosmetics (Liaqat
et al.Carbohydrate Polymers,2018:184-243).Chitosan oligosaccharide preparation method mainly has enzyme process, physical method and change
Method.Wherein enzyme process has many advantages, such as that environmental-friendly, reaction condition is mild, product composition is ideal, is to be widely applied and most send out
Open up potentiality chitosan oligosaccharide preparation method (Cabrera et al.Biochemical Engineering Journal, 2005,25:
165-172).Although enzyme process prepares chitosan oligosaccharide with lot of advantages, there is also many deficiencies.Firstly, chitosan enzyme producing enzyme water
Putting down low is enzymatic hydrolysis chitosan using bottleneck；Secondly, the concentration of substrate used in existing enzymatic conversion method relevant report is low, lead
Cause later period concentrated cost high, production efficiency is low.Currently, the concentration of substrate that enzyme hydrolysis method uses is generally 2-5%.Although Chen
The concentration of substrate of equal reports is 10%, but needing is more than hydrolysis for 24 hours, and energy consumption and cost increase considerably (Chen et
al.Biotechnology Letters,2012,34:689-694).Therefore, the producing enzyme level and optimization water of chitosan enzyme are improved
Solution technique is the key that realize chitosan enzyme industrial production chitosan oligosaccharide.
Summary of the invention
It is an object of the present invention to provide a kind of construction methods of recombinant bacterium for producing chitosan enzyme.
The construction method of the recombinant bacterium provided by the invention for producing chitosan enzyme includes the following steps: bacillus subtilis shell
Xylanase gene and Vitreoscilla hemoglobin gene import Pichia pastoris, obtain the recombinant bacterium for producing chitosan enzyme.
In the construction method of above-mentioned recombinant bacterium, the bacillus subtilis chitosanase gene be following a1) or a2) or
A3):
A1) DNA molecular shown in sequence 1；
A2) hybridize under strict conditions with the a1) DNA molecular limited and encode the bacillus subtilis chitosan enzyme
DNA molecular；
A3) and a1) or a2) identity of the DNA molecular with 90% or more that limits and the coding bacillus subtilis
The DNA molecular of chitosan enzyme；
The Vitreoscilla hemoglobin gene be following b1) b2) or b3):
B1) DNA molecular shown in sequence 2；
B2) hybridize under strict conditions with the b1) DNA molecular limited and encode the DNA of the Vitreoscilla hemoglobin
Molecule；
B3) and b1) or b2) DNA molecular that limits is blood red with 90% or more identity and the coding Vitreoscilla
The DNA molecular of albumen.
Further, the bacillus subtilis chitosanase gene is led by recombinant expression carrier pPICZA-BsCsn46
Enter in the Pichia pastoris；The recombinant expression carrier pPICZA-BsCsn46 is to replace DNA molecular shown in sequence 1
The carrier obtained after small fragment between I restriction enzyme site of pPICZA carrier EcoR I and Not；
The Vitreoscilla hemoglobin gene imports the Pichia pastoris by recombinant expression carrier pPIC3.5K-VHb
In；The recombinant expression carrier pPIC3.5K-VHb is that DNA molecular shown in sequence 2 is replaced I He of pPIC3.5K carrier EcoR
The carrier obtained after small fragment between I restriction enzyme site of Not.
Further, the bacillus subtilis chitosanase gene passes through recombinant expression carrier pPICZA-BsCsn46
It imports in pichia pastoris yeast GS115, building obtains recombinant bacterium GS115/BsCsn46；The Vitreoscilla hemoglobin base
Because importing in the recombinant bacterium GS115/BsCsn46 by recombinant expression carrier pPIC3.5K-VHb, the production chitosan is obtained
The recombinant bacterium of enzyme.
In the construction method of above-mentioned recombinant bacterium, the Pichia pastoris can be pichia pastoris yeast；The Pasteur is finished red
Yeast concretely pichia pastoris yeast GS115.
Another object of the present invention is to provide following (1)-(4) any described biomaterial:
(1) recombinant bacterium of the production chitosan enzyme constructed using above-mentioned construction method；
(2) above-mentioned bacillus subtilis chitosanase gene；
(3) above-mentioned Vitreoscilla hemoglobin gene；
(4) containing the blood red egg of Vitreoscilla described in bacillus subtilis chitosanase gene and/or (3) described in (2)
Expression cassette, recombinant vector and the recombinant bacterium of white gene.
The new application that the present invention has a purpose to be to provide above-mentioned biomaterial again.
The present invention provides above-mentioned biomaterials to prepare the application in chitosan enzyme.
The present invention also provides application of the above-mentioned biomaterial in hydrolyzing chitosan.
The present invention also provides above-mentioned biomaterials to prepare the application in chitosan oligosaccharide.
It is a still further object of the present invention to provide a kind of preparation methods of chitosan enzyme.
The preparation method of chitosan enzyme provided by the invention includes the following steps: to construct to obtain using above-mentioned construction method
Production chitosan enzyme recombinant bacterium carry out fermented and cultured, obtain tunning, from the tunning be made chitosan enzyme.
In above-mentioned chitosan enzyme preparation method, the fermented and cultured uses fermentation process in high density, the high density fermentation
Method and culture medium specifically can refer to the hair in Pichia Fermentation Process Guideline (Invitrogen)
Fermenting process.
Further, the method for the fermented and cultured includes the following steps:
1) above-mentioned recombinant bacterium is seeded in seed culture medium, culture to OD600When for 8-12, seed liquor is obtained；
2) seed liquor is seeded in the fermentation medium containing glycerol and carries out fermented and cultured, to sweet in cultivating system
Oil consumption starts to flow glycerol adding solution when most；
3) stop stream glycerol adding solution, hungry bacterial strain (20- when thallus weight in wet base in cultivating system reaches (200-250) g/L
40) start stream after min plus methanol solution carries out Fiber differentiation.
Further, it is described 1) in, the seed culture medium can be YPD culture medium, be formulated it is specific as follows: tryptose
Peptone 20g/L, yeast extract 10g/L, glucose 20g/L.
Above-mentioned recombinant bacterium is seeded in the YPD culture medium and is cultivated to OD600When being 10, seed liquor is obtained.
It is described 2) in, the formula of the fermentation medium is specific as follows: solvent is water, and solute and its concentration difference are as follows:
Glycerol 40g/L, CaSO40.93g/L, K2SO418.2g/L MgSO4·7H2O 14.9g/L, KOH 4.13g/L, 85% phosphoric acid
26.7mL/L PTM1 4.35mL/L。
The PTM1Formula it is specific as follows: solvent is water, and solute and its concentration difference are as follows: CuSO4·7H2O 6g/L,
NaI 0.08g/L, MnSO4·H2O 3g/L, NaMoO4·2H2O 0.2g/L, boric acid 0.02g/L, CoCl20.5g/L, ZnCl2
20g/L, FeSO4·7H2O 65g/L, biotin 0.2g/L, concentrated sulfuric acid 5mL/L.
The condition of the fermented and cultured is as follows: the starting pH (4.0-4.5) (28% ammonium hydroxide tune pH can be used) of fermentation system,
Temperature (28-30) DEG C, originates speed of agitator (500-700) rpm, and ventilatory capacity (1.0-2.0) vvm maintains minimum dissolved oxygen to be greater than
20%.Specifically, the starting pH 4.0 of fermentation system, originates speed of agitator 600rpm, ventilatory capacity 1.5vvm by 30 DEG C of temperature.
The formula of every liter of glycerite is specific as follows: glycerol 500g, PTM1 12mL。
It is described 3) in, the condition of the Fiber differentiation is as follows: pH (5.0-6.0), temperature (22-28) DEG C (such as 22 DEG C, 25 DEG C
Or between point value described in 28 DEG C or above-mentioned any two in the range of temperature value), revolving speed (800-1000) rpm, maintain it is minimum molten
Oxygen is greater than 15%.Specifically, the condition of the Fiber differentiation is as follows: pH6.0,25 DEG C of temperature, revolving speed 800rpm.
Methanol feeding strategy is used during the Fiber differentiation, specially according to dissolved oxygen feedback mechanism, using four-part form
Methanol feeding strategy.According to the variation of different phase dissolved oxygen, methanol feeding rate is adjusted.That is, 0-12h, flow rate are
3.6mL/h/L preliminary fermentation culture medium (fermentation medium in above-mentioned 2))；13-48h, flow rate are that 5.6mL/h/L is initial
Fermentation medium；49-120h, 8.6mL/h/L preliminary fermentation culture medium；121-168h, flow rate are that 7.2mL/h/L is just originated
Ferment culture medium.
The formula of every liter of methanol solution is specific as follows: 100% methanol solution 1000mL, PTM1 12mL。
The chitosan enzyme that the above method is prepared also belongs to protection scope of the present invention.
It is a still further object of the present invention to provide the new applications for the chitosan enzyme being prepared using the above method.
The present invention provides application of the chitosan enzyme being prepared using the above method in hydrolyzing chitosan.
The present invention also provides the chitosan enzymes being prepared using the above method to prepare the application in chitosan oligosaccharide.
Final object of the present invention be to provide a kind of chitosan method for hydrolysis or a kind of preparation method of chitosan oligosaccharide.
The method for hydrolysis of chitosan provided by the invention or the preparation method of chitosan oligosaccharide include with above-mentioned chitosan enzyme to shell
The step of glycan is hydrolyzed.
In above-mentioned chitosan hydrolyzate method or the preparation method of chitosan oligosaccharide, the mass concentration model of the chitosan in hydrolyzation system
Enclose can for 10-20%, concretely 10%, 12%, 14%, 16%, 18%, 20% or above-mentioned any two described between point value
In the range of concentration value；The enzyme concentration of chitosan enzyme can be 100-300U/g chitosan, concretely 100U/g, 150U/g,
Enzyme concentration in the range of between point value described in 200U/g, 250U/g, 300U/g or above-mentioned any two.
In above-mentioned chitosan hydrolyzate method or the preparation method of chitosan oligosaccharide, the pH value of hydrolyzation system can be 4.5-6.5；Specifically
It can be the pH value described in 4.5,5,5.5,6,6.5 or above-mentioned any two between point value in range.The temperature of the hydrolysis can be
(30-55)℃；Range between point value described in concretely 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C or above-mentioned any two
Interior temperature value；The time of the hydrolysis can be (4-12) h, concretely 4h, 6h, 8h, 10h, 12h or above-mentioned any two
Time value in the range of between the point value.
The present invention realizes chitosan enzyme in Pichia pastoris efficiently using coexpression Vitreoscilla hemoglobin strategy intracellular
Expression, recombinant bacterium are 50370U/mL, protein content 16.0mg/mL through high density fermentation ectoenzyme vigor.Recombinase specific enzyme activity
Power is up to 4065.7U/mg.The enzyme optimal pH is 6.0, keeps stablizing in pH4.5-10.0 range, optimum temperature is 55 DEG C, 45 DEG C
It keeps stablizing below.The chitosan enzyme is used to hydrolyze high concentration chitosan (>=10%), chitosan oligosaccharide yield > 70%.This hair
Bright chitosan enzyme producing enzyme level is significantly larger than the reported values of existing literature and patent, and hydrolysis properties are excellent, in chitosan enzyme
It is had a good application prospect in industrial production and chitosan conversion.
Detailed description of the invention
Fig. 1 is the plasmid map of recombinant expression carrier.A is pPICZA-BsCsn46；B is pPIC3.5k-VHb.
Fig. 2 is the SDS- of recombinant yeast pichia pastoris growth and its course for secreting chitosan enzyme (BsCsn46) and extracellular protein
PAGE analysis.A is recombinant yeast pichia pastoris growth and its course for secreting chitosan enzyme (BsCsn46)；B is the SDS- of extracellular protein
PAGE analysis.
The purification process SDS-PAGE analysis and deglycosylation and enzyme spectrum analysis that Fig. 3 is BsCsn46.
Fig. 4 is BsCsn46 optimal pH, pH stability, optimum temperature, temperature stability and the test curve figure of half-life period.
Wherein, (a) is BsCsn46 optimal pH；It (b) is BsCsn46pH stability；It (c) is BsCsn46 optimum temperature；(d) it is
BsCsn46 temperature stability；It (e) is BsCsn46 half-life period.
Fig. 5 is that TLC the and MALDI-TOF MS of BsCsn46 hydrolyzing chitosan product is analyzed.(a) shell is hydrolyzed for BsCsn46
The TLC of glycan product is analyzed；(b) it is analyzed for the MALDI-TOF MS of BsCsn46 hydrolyzing chitosan product.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Without departing substantially from spirit of that invention
In the case where essence, the method for the present invention, step or condition are modified or replaceed, all belonged to the scope of the present invention.It is following
Experimental method in embodiment is unless otherwise specified conventional method.Test material as used in the following examples, such as nothing
Specified otherwise is to be commercially available from routine biochemistry reagent shop.Quantitative test in following embodiment, is respectively provided with and weighs three times
Multiple experiment, results are averaged.
Bacillus subtilis (Bacillus subtilis) WY-34 in following embodiments is recorded in document " Jiang et
al.,High-level production,purification and characterization of a thermostable
beta-mannanase from the newly isolated Bacillus subtilis WY34,Carbohydrate
Polymers, 2006, in 66:88-96 ", the public can obtain from applicant, which only attaches most importance to the correlation of duplicate invention
Used in experiment, it not can be used as other purposes and use.
PPICZA in following embodiments is purchased from U.S. Invitrogen.
PPIC3.5K in following embodiments is purchased from U.S. Invitrogen.
Pichia pastoris yeast GS115 in following embodiments is purchased from U.S. Invitrogen.
Specific enzyme activity power defines in following embodiments: enzyme activity unit possessed by every milligram of albumen (U/mg).
The formula of fermentation medium is as follows in following embodiments: solvent is water, and solute and its concentration difference are as follows: glycerol
40g/L, CaSO40.93g/L, K2SO418.2g/L MgSO4·7H2O 14.9g/L, KOH 4.13g/L, 85% phosphoric acid
26.7mL/L PTM14.35mL/L.Wherein, PTM1Be formulated as follows: solvent is water, and solute and its concentration difference are as follows: CuSO4·
7H2O 6g/L, NaI 0.08g/L, MnSO4·H2O 3g/L, NaMoO4·2H2O 0.2g/L, boric acid 0.02g/L, CoCl2
0.5g/L, ZnCl220g/L, FeSO4·7H2O 65g/L, biotin 0.2g/L, concentrated sulfuric acid 5mL/L.
The preparation of embodiment 1, recombinant bacterium GS115/BsCsn46/VHb
1, using bacillus subtilis WY-34 genome as template, PCR is carried out using primer BsCsn46-F and BsCsn46-R
Amplification, obtains bacillus subtilis chitosanase gene BsCsn46.Primer sequence is as follows:
BsCsn46-F:AACCGGAATTC(underscore is the I digestion position EcoR to ATGGGACTGAATAAAGATCAAAAGC
Point)；
BsCsn46-R:AAGAATGCGGCCGC(underscore is the I digestion position Not to TTATTTGATTACAAAATTACCGTAC
Point).
Codon optimization is carried out to bacillus subtilis chitosanase gene BsCsn46, the BsCsn46 gene sequence after optimization
Column are carried out as shown in sequence 1, and using the sequence after codon optimization as template using primer BsCsn46-F and BsCsn46-R
PCR amplification obtains PCR product.
2, double digestion is carried out to the PCR product and pPICZA of step 1 acquisition respectively using EcoR I and Not I, using T4DNA
It is connected at 16 DEG C of ligase, building obtains recombinant expression carrier pPICZA-BsCsn46 (Figure 1A).
3, PCR amplification using vitreoscilla genome as a template, is carried out using primer VHb-EcoRI and VHb-NotI, is obtained
PCR product is Vitreoscilla hemoglobin gene sequence.Primer sequence is as follows:
VHb-EcoRI:CCGGAATTCATGTTGGATCAACAGACTATC (underscore is I restriction enzyme site of EcoR)；
VHb-NotI:AAATATGCGGCCGCTTATTCAACAGCTTGAGCGT (underscore is I restriction enzyme site of Not).
4, double digestion is carried out to the PCR product and pPIC3.5K of step 3 acquisition respectively using EcoR I and Not I, used
It is connected at 16 DEG C of T4DNA ligase, building obtains recombinant expression carrier pPIC3.5K-VHb (Figure 1B).
5, the recombinant expression carrier pPIC9K-BsCsn46 electrotransformation pichia pastoris yeast GS115 for obtaining step 2 turns
For beggar using bleomycin as selection markers, the screening through high copy transformant obtains an active recombinant bacterium of plant height chitosan enzyme,
It is named as GS115/BsCsn46.
6, the recombinant bacterium GS115/BsCsn46, His for obtaining the pPIC3.5K-VHb electrotransformation step 5 that step 4 obtains+
For transformant using Geneticin as selection markers, the screening through high copy transformant obtains coexpression bacterial strain, is named as
GS115/BsCsn46/VHb。
The secreting, expressing and height of recombinant expression carrier electrotransformation Pichia pastoris, recombinant protein in pichia pastoris yeast are copied
The screening of shellfish transformant is with specific reference to the method in Multi-Copy Pichia Expression Kit (Invitrogen).Through
It crosses the above method and realizes that chitosan enzyme and hemoglobin co-express in pichia pastoris yeast, transformant is sieved by the above method
5 plants of higher coexpression bacterial strains (table 1) of production chitosan enzyme activity are obtained after choosing, selection produces chitosan enzyme activity highest (415U/
ML coexpression bacterial strain) is used for following experimental analyses.
1,5 plant of higher coexpression bacterial strain of production chitosan enzyme activity of table
| Strain number | Gene copy number | Chitosan enzyme enzyme activity (U/mL) |
| 1 | 8 | 342 |
| 2 | 10 | 384 |
| 3 | 12 | 415 |
| 4 | 14 | 370 |
| 5 | 16 | 413 |
Embodiment 2, the preparation and purifying for recombinating chitosan enzyme (BsCsn46)
One, recombinant bacterium high density fermentation preparation and reorganization chitosan enzyme (BsCsn46)
The high density fermentation and culture medium of recombinant bacterium are referring to Pichia Fermentation Process Guideline
(Invitrogen) method in, the specific steps are as follows:
1, the recombinant bacterium for the production chitosan enzyme activity higher (415U/mL) that acquisition is screened in embodiment 1 is seeded to 150mL
YPD culture medium (tryptone 20g/L, yeast extract 10g/L, glucose 20g/L), 30 DEG C, 200rpm is cultivated to OD600About
It is 10, as seed liquor.
2, it selects 5L fermentor (working volume 1.5L) to be used as high density fermentation container, the step 1 of 150mL is obtained
Seed liquor, which is added in the fermentor of the fermentation medium containing 1350mL, carries out batch fermentation culture.During fermented and cultured, adopt
With 28% ammonium hydroxide tune pH to 4.0,30 DEG C of temperature, speed of agitator 600rpm, ventilatory capacity 1.5vvm are originated.To glycerol in cultivating system
Start stream plus 50% glycerol (glycerol 500g/L, PTM when exhausting112mL/L) 6-8h is monitored by control flow feeding speed
Dissolved oxygen (Dissolved oxygen, DO) maintains minimum dissolved oxygen to be greater than 20%.Reach 220g/L to thallus weight in wet base in cultivating system
Stop stream glycerol adding afterwards, hungry bacterial strain 30min starts stream plus 100% methanol (containing PTM1Fiber differentiation 12mL/L) is carried out, more
Changing pH, temperature and revolving speed is respectively 6.0,25 DEG C and 800rpm, maintains minimum dissolved oxygen to be greater than 15%, Fiber differentiation 168h is obtained
Fermentation liquid.Fiber differentiation stage methanol feeding recharge rate is as shown in table 2.
Table 2, induction period methanol feeding recharge rate
Fiber differentiation starts rear every 12h and thallus weight in wet base, extracellular chitosan enzyme enzyme activity and extracellular protein content is measured by sampling.
The measuring method of thallus weight in wet base is as follows: it takes 1mL fermentation liquid 12000rpm to be centrifuged 3min, discards supernatant liquid, it is accurate to claim
Take thallus weight.
The standard method of extracellular chitosan enzyme enzyme activity determination is as follows: using 3,5- dinitrosalicylic acid (3,5-
Dinitrosalicylic acid, DNS) method, using Glucosamine as standard.400 μ L, 0.5% (w/v) chitosan (DDA=
75-85%, pH 6.0) the suitably diluted enzyme solution of solution and 100 μ L mixes, 10min kept the temperature at 55 DEG C, 500 μ L DNS are added
Reagent terminates reaction, boiling water bath 10min colour developing.Fermentation liquid 12000rpm is centrifuged 10min, supernatant is taken to survey suction under 540nm
Light value.Enzyme activity calculation formula is as follows: H=Cx × n/ (T × V), wherein H is represented enzyme activity (U/mL), and Cx represents reaction and generates
The amount (μm ol) of the substance of reduced sugar, n represent the extension rate of enzyme solution, and T is represented reaction time (min), and V, which is represented, is added enzyme solution
Volume (mL).Enzyme activity definition: under the above-described reaction conditions, enzyme amount needed for reacting 1 μm of oL reduced sugar of release per minute defines
For an enzyme activity unit (U).
Extracellular protein content is using bovine serum albumin as standard protein, using Lowry method (Lowry et al., The
Journal of Biological Chemistry, 1951,193:265-275) measurement protein content (mg/mL).
Fig. 2 is the SDS- of recombinant yeast pichia pastoris growth and its course for secreting chitosan enzyme (BsCsn46) and extracellular protein
PAGE analyzes result.Fig. 2 (a) intermediate cam form point is thallus weight in wet base (g/L), square dot is fermented supernatant fluid chitosan enzyme enzyme activity
(U/mL), circular dot is the protein concentration (mg/mL) of fermented supernatant fluid.M is Protein Marker, swimming lane 1-8 in Fig. 2 (b)
For Fiber differentiation 0h, for 24 hours, 48h, 72h, 96h, 120h, 144h and 168h when supernatant.The result shows that: what embodiment 1 obtained
After recombinant bacterial strain carries out high density fermentation 144h in 5L fermentor, the chitosan enzyme vigor and protein content of supernatant are respectively
50370.0U/mL and 16.0mg/mL, thallus weight in wet base are 428.6g/L.SDS-PAGE analyzes extracellular protein during methanol induction
Composition is as it can be seen that purpose band is gradually thicker as induction time extends, and recombination chitosan enzyme content gradually increases in fermented supernatant fluid
It is more.
Two, the purifying of chitosan enzyme (BsCsn46) is recombinated
The fermentation liquid 10000rpm that step 1 is obtained is centrifuged 10min, and collecting supernatant is crude enzyme liquid.Then by supernatant
Liquid is in 50mM KH2PO4Dialyse at 4 DEG C 12h in-NaOH (pH8.0) buffer, obtains dialysis sample.It again will dialysis sample warp
QSFF (1.0 × 10cm) strong anion displacement chromatography isolates and purifies, 0.5mL/min flow velocity loading.Chromatographic column is in advance with initial slow
Fliud flushing (50mM KH2PO4- NaOH pH8.0) balance.Use the initial buffer liquid containing (0-200) mM NaCl with 1.0mL/min
Flow velocity linear elution destination protein, the chitosan enzyme solution purified (pure enzyme solution), the shell with SDS-PAGE detection purifying are poly-
Chitosan enzyme purity in carbohydrase solution.
BsCsn46 deglycosylation analysis: using deglycosylation enzyme (endo- β-N-acetylglucosaminidase,
Endo H) processing purifying chitosan enzyme solution, SDS-PAGE detects deglycosylation as a result, enzyme solution to handle without Endo H
As control.
Chitosan enzyme enzyme spectrum analysis: being added the chitosan of 0.1% (w/v) in SDS-PAGE separation gel, will after the completion of electrophoresis
Separation gel is in renaturation buffer (50mM KH2PO4- NaOH pH 7.5,1% (w/v) TritonX-100) in embathe 6-8h；Again will
Separation gel is placed in buffer (10mM acetic acid-sodium acetate, pH 6.0), 45 DEG C of reaction 2h.0.1% congo red staining 15min, 1M
NaCl decolourizes clear to band.
The purification process SDS-PAGE analysis and deglycosylation and enzyme spectrum analysis that Fig. 3 is BsCsn46.Swimming lane M is albumen point
Sub- amount standard, swimming lane 1 are crude enzyme liquid, and swimming lane 2 be the chitosan enzyme solution of purifying, and swimming lane 3 is Endo H treated enzyme solution, are swum
Road 4 is Endo H enzyme solution, and swimming lane 5 is the zymogram of BsCsn46.
Crude enzyme liquid purifies to obtain the pure enzyme of electrophoresis grade (Fig. 3) through strong anion (QSFF) displacement chromatography.Enzyme activity in purification process
The rate of recovery is 88.3%, purification 1.2, and the specific enzyme activity power of pure enzyme is 4065.7U/mg (table 3).Thick enzyme and pure enzyme have two
Obvious band, zymogram, which shows two bands, has chitosan enzyme active (Fig. 3)；Pure enzyme is shown as after Endo H goes glycosyl to handle
One band (Fig. 2) illustrates that recombinating chitosan enzyme has occurred partial glycosylation phenomenon.
The purifying table of table 3, chitosan enzyme BsCsn46
| Purification step | Total enzyme activity/U | Total protein/mg | Specific enzyme activity/U mg-1 | Purificationa | The enzyme activity rate of recoveryb/ % |
| Crude enzyme liquid | 176315.7 | 52.9 | 3331.2 | 1.0 | 100.0 |
| Pure enzyme solution | 155673.6 | 38.3 | 4065.7 | 1.2 | 88.3 |
A: purification refers to the ratio of the specific enzyme activity of pure enzyme and the specific enzyme activity of thick enzyme；B: the rate of recovery refers to pure enzyme
Total enzyme activity power accounts for the percentage composition of crude enzyme liquid total enzyme activity power.
Embodiment 3, the zymologic property measurement for recombinating chitosan enzyme BsCsn46
1, the measurement of optimal pH and pH stability
Optimal pH measurement: the chitosan enzyme solution of the purifying obtained using in embodiment 2 is as enzyme solution to be measured, in pH3.5-
Optimal pH is measured in 7.5 ranges.Enzyme activity is measured according to the standard method in embodiment 2.With enzyme activity peak for 100%,
Calculate separately the enzyme activity under condition of different pH.
PH Stability Determination: enzyme solution to be measured is diluted using the different buffers within the scope of pH3.0-11.0, is protected at 45 DEG C
Warm 30min, the cooling 30min of ice-water bath, then measures residual enzyme activity under optimum condition according to the standard method immediately.With
Without above-mentioned processing (above-mentioned processing refer to using pH range 3.0-11.0 different buffers dilute chitosan enzyme solution, 45
DEG C heat preservation 30min, then be immediately placed in ice water cooling 30min) enzyme solution as control, calculate residual enzyme activity and account for blank control
The percentage composition of enzyme activity.
Buffer to be measured and its corresponding data point identification are as follows: citric acid-trisodium citrate pH 3.0-6.0 (△)；
Bistris-HCl pH 5.5-7.5(◆)；Acetic acid-sodium acetate pH 3.5-6.0 (■)；MES pH 5-6(◇)；Biphosphate
Sodium-disodium hydrogen phosphate pH 6.0-8.0 (▲)；Tris-HCl pH 7.0-9.0(○)；CHES pH 8.0-10.0(●)；CAPS
pH 10.0-11.0(□)。
Fig. 4 is the measurement result of BsCsn46 optimal pH and pH stability.BsCsn46 optimal reaction pH is 6.0,
It keeps stablizing within the scope of pH4.5-10.0.
2, optimum temperature, temperature stability and the measurement of half-life period
Optimum temperature measurement: substrate is configured with the buffer of optimal pH, enzyme reaction most thermophilic is measured within the scope of 20-75 DEG C
Degree measures enzyme activity according to the standard method, with enzyme activity peak for 100%, calculates separately under condition of different temperatures
Enzyme activity.
The measurement of temperature stability: enzyme solution is placed under different temperatures (20-60 DEG C) and keeps the temperature 30min, ice-water bath is cold immediately
But 30min, it is then described to measure residual enzyme activity under optimum condition according to standard method.Without above-mentioned processing (above-mentioned place
Reason refers to being placed in enzyme solution at 20-60 DEG C and keeps the temperature 30min, immediately ice-water bath 30min) enzyme solution as control, calculate remaining
Enzyme activity accounts for the percentage composition of blank control enzyme activity.
The measurement of half-life period: enzyme solution is respectively placed at 45 DEG C, 50 DEG C and 55 DEG C and handles different time, ice-water bath is cold immediately
But 30min measures residual enzyme activity under optimum condition according to the standard method.With without above-mentioned processing, (above-mentioned processing refers to
Be to handle different time at 45 DEG C, 50 DEG C and 55 DEG C, ice-water bath 30min immediately) enzyme solution as control, calculate residual enzyme
Vigor accounts for the percentage composition of blank control enzyme activity, calculate enzyme at different temperatures enzyme activity decay to 50% time.
Fig. 4 is optimal reactive temperature, temperature stability and the measurement result of half-life period of BsCsn46.Wherein in Fig. 4 (e)
Temperature mark it is as follows: 45 DEG C (●), 50 DEG C (■) and 55 DEG C (▲).Measurement result shows that BsCsn46 optimal reactive temperature is
55 DEG C, stablize at 45 DEG C or less, 45 DEG C of half-life period is 3468min, and 50 DEG C of half-life period is 91.9min.
Embodiment 4, chitosan enzyme hydrolyzing chitosan prepare chitosan oligosaccharide
In the aqueous solution of 0.2L chitosan-containing enzyme, (crude enzyme liquid in embodiment 2, chitosan enzyme is in hydrolysis reaction system
Final concentration of 100U/g) in put into 20g Chitosan powder, and 5mL acetic acid is added, concentration of substrate is made to reach 10%, hydrolyze 4h；So
10g Chitosan powder and 2mL acetic acid are added afterwards, so that final concentration of substrate is 15%, continues to hydrolyze 4h, obtains hydrolyzate.It will
Hydrolyzate boiling water bath 5min enzyme deactivation, is added appropriate 5M NaOH tune pH > 9, and 12000rpm is centrifuged 10min, supernatant and precipitating difference
Freeze-drying weighing.Percent hydrolysis and chitosan oligosaccharide yield calculation formula difference are as follows:
Percent hydrolysis (%)=(1-W3/W1) × 100%；Chitosan oligosaccharide yield (%)=(W2-W4-W5)/W1× 100%.
In formula: W1For the quality of chitosan；W2For the quality after supernatant freeze-drying；W3For the matter that pellet frozen is dry
Amount；W4For the quality of acetic acid；W5For the quality of NaOH.
Using Matrix-assisted laser desorption ionization (Matrix-Assisted Laser Desorption
Ionization Time of Flight Mass Spectrometry, MALDI-TOF MS) analysis hydrolyzate oligosaccharides group
At.Hydrolyzate send hundred survey technology Services Co., Ltd, BeiJing ZhongKe to be analyzed by mass spectrometry.Using 4000 QTRAP system of AB SCIEX
System, cation mode, matrix is 2,5-dihydroxybenzoic acid.
Fig. 5 is TLC the and MALDI-TOF MS analysis chart of BsCsn46 hydrolyzing chitosan product, and wherein M is chitosan oligosaccharide standard
Product mixture, G1 are Glucosamine, and G2-G6 is six sugar of chitobiose-shell, and 1 is the composition of hydrolyzate chitosan oligosaccharide.
Chitosan is after 8h is hydrolyzed, and chitosan oligosaccharide yield is 78.0%, percent hydrolysis 80.2%.TLC is analysis shows that hydrolysis
Product is mainly disaccharides, trisaccharide and tetrose, there is a small amount of pentasaccharides and six sugar.Hydrolyzate verifies water through MALDI-TOFMS analysis again
The oligosaccharides that product is DP 2-6 is solved, wherein disaccharides, trisaccharide and tetrose are deacetylated chitosan oligosaccharide, i.e., (GlcN)2、(GlcN)3With
(GlcN)4, pentasaccharides and six sugar be one acetyl group of band chitosan oligosaccharides, i.e., (GlcN)4- GlcNAc and (GlcN)5-GlcNAc。
Embodiment 6, pilot-scale prepare chitosan oligosaccharide
19L water is added into 20L enzymatic vessel, first puts into 1kg chitosan, 200mL acetic acid is added, chitosan enzyme solution is added
(crude enzyme liquid in embodiment 2) hydrolyzes 2h；Then 1kg chitosan is put into again, is added 200mL acetic acid, is added chitosan enzyme
Liquid (crude enzyme liquid in embodiment 2), so that final pH is 5.8, final concentration of substrate is 10% (w/v), and final enzyme concentration is
200U/g, 6h is hydrolyzed at 45 DEG C, and 4000rpm is centrifuged 15min, removes unreacted chitosan, obtain hydrolyzate.
Using solid concentration in refractometer measurement hydrolyzate.The calculation formula of solid content and chitosan oligosaccharide yield is such as
Under: solid content (g)=C × V；Chitosan oligosaccharide yield (%)=(M2/M1) × 100%.In formula: C is solid content in hydrolyzate
Concentration (g/L)；V is hydrolyzate total volume (L)；M1For solid content (g) in solution before hydrolysis, M2After hydrolysis
Solid content (g).
Using the content of chitosan oligosaccharide in HPLC method measurement each sample.HPLC determination condition are as follows: ShodexAsahipak
NH2P-50 4E chromatographic column (250 × 4.6mm), acetonitrile: water=70:30,30 DEG C of column temperature, flow velocity 0.8mL/min, 16min show difference
(RID) detector.
The analysis of chitosan oligosaccharide product oligosaccharide ingredient is as shown in Figure 5.Product composition is mainly chitobiose and chitotriose, is had a small amount of
Shell tetrose, wherein the ratio of disaccharides, trisaccharide and tetrose is respectively 15.0%, 30.4% and 8.5%.Pilot scale chitosan oligosaccharide preparation process
In each step yield be shown in Table 4.Yield after being digested, being centrifuged is 75.2%, and chitosan oligosaccharide yield is 64.6% after spray drying.
Each step yield in table 4, chitosan oligosaccharide pilot scale preparation process
| Step | Liquid glucose volume (L) | Solid content (g) | Yield (%) |
| Enzymatic hydrolysis | 20 | 2400 | - |
| Centrifugation | 19 | 1805 | 75.2 |
Sequence table
<110>China Agricultural University
<120>a kind of construction method of recombinant bacterium for producing chitosan enzyme and its application
<160>2
<170>PatentIn version 3.5
<210>1
<211>729
<212>DNA
<213>bacillus subtilis (Bacillus subtilis) WY-34
<400>1
atgggtctga acaaggatca aaaaagacgt gcagagcagt tgacttctat ttttgaaaac 60
ggtactacag agatccaata cggttacgtt gaacaactgg atgacggtag aggttacact 120
tgcggaagag ctggctttac tactgctact ggtgatgctt tggaagttgt tgaagtttac 180
actaaggctg ttccaaacaa caagcttaag aagtatttgc cagaattgag aagactagct 240
aaagaagaat ccgatgacac atccaacttg aaaggttttg cttctgcttg gaaatctctg 300
gctaacgata aggaatttag agctgctcaa gataaggtta acgatcattt gtactaccaa 360
cctgcaatga agagatctga taatgctgga ttgaagactg cccttgctag ggctgttatg 420
tacgatactg ttatacaaca tggtgacggt gatgatccag attctttcta cgctttgatt 480
aagagaacta acaagaaggc tggtggttct ccaaaggaag gtatcgatga aaaaaagtgg 540
ttaaacaagt tcttggacgt tagatacgat gatttgatga acccagctaa ccatgatact 600
agagatgaat ggagagagtc tgttgctaga gttgatgttt tgagatctat tgctaaggag 660
aacaattaca atttgaacgg tccaattcat gttagaagta atgaatacgg aaactttgtt 720
attaagtga 729
<210>2
<211>441
<212>DNA
<213>Vitreoscilla (Vitreoscilla stercoraria)
<400>2
atgttagacc agcaaaccat taacatcatc aaagccactg ttcctgtatt gaaggagcat 60
ggcgttacca ttaccacgac tttttataaa aacttgtttg ccaaacaccc tgaagtacgt 120
cctttgtttg atatgggtcg ccaagaatct ttggagcagc ctaaggcttt ggcgatgacg 180
gtattggcgg cagcgcaaaa cattgaaaat ttgccagcta ttttgcctgc ggtcaaaaaa 240
attgcagtca aacattgtca agcaggcgtg gcagcagcgc attatccgat tgtcggtcaa 300
gaattgttgg gtgcgattaa agaagtattg ggcgatgccg caaccgatga cattttggac 360
gcgtggggca aggcttatgg cgtgattgca gatgtgttta ttcaagtgga agcagatttg 420
tacgctcaag cggttgaata a 441
Claims (10)
1. a kind of construction method for the recombinant bacterium for producing chitosan enzyme, includes the following steps: bacillus subtilis chitosan enzyme base
Cause and Vitreoscilla hemoglobin gene import Pichia pastoris, obtain the recombinant bacterium for producing chitosan enzyme.
2. construction method according to claim 1, it is characterised in that:
The bacillus subtilis chitosanase gene be following a1) a2) or a3):
A1) DNA molecular shown in sequence 1；
A2) hybridize under strict conditions with the a1) DNA molecular limited and encode the DNA of the bacillus subtilis chitosan enzyme
Molecule；
A3) and a1) or a2) DNA molecular that limits is poly- with 90% or more identity and the coding bacillus subtilis shell
The DNA molecular of carbohydrase；
The Vitreoscilla hemoglobin gene be following b1) b2) or b3):
B1) DNA molecular shown in sequence 2；
B2) hybridize under strict conditions with the b1) DNA molecular limited and encode the DNA molecular of the Vitreoscilla hemoglobin；
B3) and b1) or b2) identity and the coding Vitreoscilla hemoglobin of the DNA molecular with 90% or more that limit
DNA molecular.
3. construction method according to claim 1 or 2, it is characterised in that: the bacillus subtilis chitosanase gene
It is imported in the Pichia pastoris by recombinant expression carrier pPICZA-BsCsn46；
The Vitreoscilla hemoglobin gene is imported in the Pichia pastoris by recombinant expression carrier pPIC3.5K-VHb；
The recombinant expression carrier pPICZA-BsCsn46 is that DNA molecular shown in sequence 1 is replaced I He of pPICZA carrier EcoR
The carrier obtained after small fragment between I restriction enzyme site of Not；
The recombinant expression carrier pPIC3.5K-VHb is that DNA molecular shown in sequence 2 is replaced I He of pPIC3.5K carrier EcoR
The carrier obtained after small fragment between I restriction enzyme site of Not.
4. construction method according to claim 1 to 3, it is characterised in that: the Pichia pastoris is that Pasteur finishes red ferment
It is female；The pichia pastoris yeast is specially pichia pastoris yeast GS115.
Following 5. (1)-(4) any described biomaterial:
(1) recombinant bacterium of the production chitosan enzyme constructed using any construction method of claim 1-4；
(2) bacillus subtilis chitosanase gene as stated in claim 2；
(3) Vitreoscilla hemoglobin gene as stated in claim 2；
(4) containing Vitreoscilla hemoglobin base described in bacillus subtilis chitosanase gene and/or (3) described in (2)
Expression cassette, recombinant vector and the recombinant bacterium of cause.
6. biomaterial described in claim 5 is preparing the application in chitosan enzyme；
Or, application of the biomaterial described in claim 5 in hydrolyzing chitosan；
Or, biomaterial described in claim 5 is preparing the application in chitosan oligosaccharide.
7. a kind of preparation method of chitosan enzyme, including the following steps: will be using any construction method of claim 1-4
The recombinant bacterium for constructing obtained production chitosan enzyme carries out fermented and cultured, obtains tunning, shell is made from the tunning
Dextranase.
8. according to the method described in claim 7, it is characterized by: the method for the fermented and cultured includes the following steps:
1) recombinant bacterium for the production chitosan enzyme that any construction method of claim 1-4 constructs is seeded to seed training
It supports in base, culture to OD600When for 8-12, seed liquor is obtained；
2) seed liquor is seeded in the fermentation medium containing glycerol and carries out fermented and cultured, consumed to glycerol in cultivating system
Start to flow glycerol adding solution when to the greatest extent；
3) stop stream glycerol adding solution, hungry bacterial strain (20-40) when thallus weight in wet base in cultivating system reaches (200-250) g/L
Start stream after min plus methanol solution carries out Fiber differentiation.
9. the chitosan enzyme being prepared using method described in claim 7 or 8；
Or, application of the chitosan enzyme being prepared using method described in claim 7 or 8 in hydrolyzing chitosan；
Or, the chitosan enzyme being prepared using method described in claim 7 or 8 is preparing the application in chitosan oligosaccharide.
10. a kind of method for hydrolysis of chitosan, including the step that chitosan is hydrolyzed with chitosan enzyme as claimed in claim 9
Suddenly；
Or, a kind of preparation method of chitosan oligosaccharide, including the step that chitosan is hydrolyzed with chitosan enzyme as claimed in claim 9
Suddenly.
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