CN106399334B - A kind of thermostabilization mutation arylsulfatase, gene and its application - Google Patents
A kind of thermostabilization mutation arylsulfatase, gene and its application Download PDFInfo
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Abstract
The invention discloses a kind of thermostabilization mutation arylsulfatase, gene and its applications.Random mutagenesis, building are introduced using fallibility round pcrP.carrageenovoraArylsulfatase mutant library.By screening, the mutation arylsulfatase that thermal stability improves is obtained.The result shows that the thermal stability of H260L significantly improves compared with WT.Using p-nitrophenyl potassium sulfate as substrate, the optimal reactive temperature and pH of H260L is respectively 55 DEG C and 8.0.H260L stablizes within the scope of the pH of 6.0-9.0.EDTA has strong inhibition effect to mutation enzyme activity, illustrates that metal ion plays an important role in the catalytic process of mutant enzyme.H260L has good tolerance to detergent.H260L is 82% to the desulfurization degree of asparagus rough polysaccharide sulfate group.The present invention also obtains the genetic engineering bacterium containing above-mentioned mutation arylsulfatase gene simultaneously, realizes the heterogenous expression of enzyme, provides good basis for the industrialized production and application of the enzyme.
Description
Technical field
The present invention relates to the technical fields more particularly to a kind of thermostabilization of genetic engineering and enzyme engineering to be mutated aromatic radical sulfuric acid
Esterase, gene and its application.
Background technique
Agar is also known as agar-agar, is a kind of natural polysaccharide extracted from marine red alga, has excellent gelling and thickening
Property, it is widely used in the fields such as food, light industry, medicine and bioengineering.A large amount of sulphur are had in red algae on natural agar molecule
Acid esters group is the main reason for influencing agar gel intensity, electroendosmosis and protein adsorption ability, and removal sulfate group is fine jade
Necessity and key link of rouge production.Currently, generally using the sulfate group in alkaline process removal agar in industrial production, not only
Serious environmental issue is generated, agar is also resulted in and largely degrades loss.Compared with alkali pretreatment, removed using hydrolysis technology
Sulfate group in agar has the characteristics that reaction condition is mild, specificity is high, and environmental pollution is small, and is not easy to cause
Agar hydrolysis is lost, and is the developing direction of novel agar production technology.
Agar is typical hot melt macromolecule, and it is just water-soluble to require heat to 45 DEG C or more, and just at 43 DEG C or less
It may solidify and form gel.In addition, agar solution has larger viscosity, viscosity gradually rises with the reduction of temperature.These
Characteristic determine agar only after heating for dissolving and be maintained at 45 DEG C or more (being in agar under dissolved state) could be to it
Carry out enzymatic treatment.Therefore, the enzyme for hydrolyzing agar sulfuric ester will not only have significant agar sulfate group hydrolysing activity, also want
There is good thermal stability.
Arylsulfatase (Arylsulfatase) is a kind of catalytic pyrolysis sulfuric acid ester bond, generates corresponding pure and mild nothing
The enzyme of machine sulfate.The arylsulfatase of Pseudoalteromonas carrageenovora has agar sulfuric ester
Hydrolysing activity can remove the sulfate group on agar, but the thermal stability of enzyme is not high, be to influence sulfatase removal agar sulphur
The main property defect of acid esters efficiency.
In view of this, the present inventor establishes P.carrageenovora arylsulfatase clonal expression skill in early period
On the basis of art, the enzyme is transformed using the technology of directed evolution, one kind is studied and devised to the thermal stability of evolution enzyme
Thus mutation arylsulfatase, gene and its application, this case generate.
Summary of the invention
The purpose of the present invention is to provide a kind of thermostabilization mutation arylsulfatase, gene and its applications.
To achieve the goals above, the technical scheme adopted by the invention to solve the technical problem is that:
A kind of gene of encoding mutant arylsulfatase, nucleotide sequence is as shown in SEQ ID NO.1.Gene is big
Small is 987bp.
A kind of mutation arylsulfatase, amino acid sequence is as shown in SEQ ID NO.2.The encoding histone 328
Amino acid residue.
A kind of mutation arylsulfatase expression vector, the table containing the arylsulfatase H260L gene
Up to carrier pET-28a-ars.
A kind of preparation method of recombination mutation arylsulfatase, including using the mutation arylsulfatase
Expression vector converts host cell, cultivates transformant, recombination mutation arylsulfatase is obtained from culture.
As the preferred embodiment of embodiment, the host cell is Escherichia coli.
It is thin to host including being converted using the arylsulfatase expression vector as the preferred embodiment of embodiment
Born of the same parents' e. coli bl21 (DE3), induces through IPTG, obtains the recombination mutation arylsulfatase of solubility expression.
As the preferred embodiment of embodiment, the final concentration of 0.05mmol/L of the IPTG, inducing temperature is 25 DEG C.
A kind of recombination mutation arylsulfatase, including being turned using the mutation arylsulfatase expression vector
Change host cell, cultivates transformant, recombinase is obtained from culture.
As the preferred embodiment of embodiment, the temperature range of the mutation arylsulfatase catalyzing hydrolysis is 30~80
DEG C, optimum temperature is 55 DEG C；The hydrolysis pH range is 5.0~10.0, optimal pH 8.0.The mutant enzyme is 45,50,55
After 60 DEG C of processing 30min, 87%, 88%, 62% and 18% residual activity is remained respectively.In 6.0~9.0 condition of pH
Lower processing 1h, zymoprotein can still keep 70% or more relative activity.EDTA can inhibit the activity of recombinase, illustrate metal ion
It plays an important role in the catalytic process of mutant enzyme.Some detergent that mutant enzyme uses test, including Triton X-100,
Tween 20, Tween 80 and Chaps have good tolerance.Desulfurization degree of the H260L to asparagus rough polysaccharide sulfate group
It is 82%.
The present invention obtains the mutant of arylsulfatase thermal stability raising by mutation, can be applied to the enzyme of agar
Method is extracted.Meanwhile this invention also clones that obtained can be with the engineering bacteria of great expression, it can be achieved that the mutation aromatic radical sulfuric ester
The large-scale production of enzyme provides good basis for subsequent industrial applications.
Detailed description of the invention
The SDS-PAGE figure of inducing expression and its purification assays that Fig. 1 is arylsulfatase H260L；Wherein, M: point
Son amount standard protein；1: α containing pET-28 (+) negative bacterium, IPTG induction；2: α containing pET-28-ars positive bacteria, does not induce；3: containing
PET-28 α-ars positive bacteria, IPTG induction；4: recombinant protein after purification；
Fig. 2 is the optimal reactive temperature curve graph for being mutated arylsulfatase H260L；
Fig. 3 is the optimal reaction pH curve graph for being mutated arylsulfatase H260L；
Fig. 4 is the thermal stability curve graph for being mutated arylsulfatase H260L；
Fig. 5 is the pH stability curve figure for being mutated arylsulfatase H260L.
Specific embodiment
Embodiment 1: thermal stability improves the screening of mutation arylsulfatase
With the recombination containing wild type Pseudoalteromonas carrageenovora arylsulfatase gene
Plasmid (WT) is template, carries out fallibility PCR, expands arylsulfatase:
Upstream primer (SEQ ID NO.3): 5 '-CGCGGATCCTTTACGTTTAACGGCAGC-3′；
Downstream primer (SEQ ID NO.4): 5 '-CCCAAGCTTGCGTTTTAGTTCGTAAC-3′；
50 μ L amplification systems include: 5 μ 10 × buffers of L, 0.2 μm of ol/L primer, 0.5 μm of ol/L dTTP, 0.5 μm of ol/
L dGTP, 0.1 μm of ol/L dATP, 0.1 μm of ol/L dCTP, 1U rTaq polymerase, 7mmol/L MgCl2Contain wild type with 2ng
The recombinant plasmid template of enzyme gene.PCR reaction condition are as follows: 95 DEG C of 5min；94 DEG C of 1min, 55 DEG C of 45sec, 72 DEG C, 1min 30
Circulation；72℃10min.Recombinant expression plasmid is constructed using the method for enzyme cutting clone, that is, uses BamHI and HindIII double digestion PCR
Product, the segment after being tapped and recovered digestion are connected with the plasmid pET-28a (+) equally through BamHI and HindIII double digestion
It connects.Using CaCl22 μ L connection products are added in conversion method, every pipe E.coli BL21 competent cell, and every pipe is added after conversion
0.8mL LB culture medium, 37 DEG C, 50r/min recovery 30min.It is added in 3 times of volume LB liquid mediums, kanamycins is added,
Its final concentration is set to reach 50 μ g/mL.37 DEG C, 180r/min culture 5-6h.Final concentration of 20% glycerol is added in bacterium solution, after mixing
Packing, as arylsulfatase libraries of random mutants, are placed in -70 DEG C of preservations.
It is carried out according to the chromogenic reaction for producing arylsulfatase bacterial strain hydrolysis substrate p-nitrophenyl potassium sulfate (p-NPS)
The primary dcreening operation of mutated library.Bacterial strain flat board from library is covered after 50 DEG C of processing 2h with the soft agar containing p-NPS,
For middle wild type (WT) without chromogenic reaction, the bacterium colony with chromogenic reaction is considered the mutant strain that thermal stability improves.Bacterial strain is stayed overnight
After culture, 1:100 is transferred in 5mL LB liquid medium (kanamycins containing 50mg/mL) by volume, 37 DEG C, 180r/min
It cultivates to OD600Reach 0.6-0.8, final concentration of 0.05mmol/L IPTG, 25 DEG C, 180r/min culture 10h is added.Centrifugation is received
Collect thallus, be resuspended in 50mmol/L Tris-HCl buffer (pH 7.5), carry out ultrasonic disruption processing on ice, obtains
Crude enzyme liquid.Crude enzyme liquid is after 50 DEG C of processing 2h, using p-NPS as substrate, detects the remaining vigor of enzyme, carries out secondary screening verifying.
By primary dcreening operation and secondary screening, the mutant strain of one plant of thermal stability raising is finally obtained.Gene sequencing analysis shows that, mutation
Arylsulfatase has a base to be replaced, leads to enzyme compared with wild type arylsulfatase in enzyme gene sequence
There is an amino acid residue to change, i.e., 260 histidines (H) are mutated into leucine (L).The mutation aromatic radical sulfuric ester
Enzyme is named as H260L.
Embodiment 2: recombination mutation arylsulfatase is expressed and purified using recombinant strains
After recombinant strains are incubated overnight, 1:100 is transferred to 250mL LB liquid medium (containing 50mg/ by volume
ML kanamycins) in, 37 DEG C, 180r/min cultivates to OD600Reach 0.6-0.8, final concentration of 0.05mmol/L IPTG be added,
25 DEG C, 180r/min culture 10h.Thalline were collected by centrifugation, is resuspended in Buffer A (50mmol/L NaH2PO4, 300mmol/L chlorine
Change sodium, 15mmol/L imidazoles, pH 8.0) in, ultrasonic disruption processing is carried out on ice.It is centrifuged at 4 DEG C, collects supernatant, then
Ni-NTA affinity chromatography is carried out, by washing buffer (50mmol/L NaH2PO4, 300mmol/L sodium chloride, 30mmol/L miaow
Azoles, pH 8.0) washing after, utilize elution buffer (50mmol/L NaH2PO4, 300mmol/L sodium chloride, 250mmol/L miaow
Azoles, pH 8.0) elution, collect eluent.It is detected through SDS-PAGE, recombination mutation arylsulfatase albumen after purification
Molecular weight is 35.7kDa, as a result as shown in Figure 1.Protein concentration is measured with Bradford method, obtains concentration about 1.0mg/mL's
Mutant enzyme H260L.
Embodiment 3: the viability examination of arylsulfatase
20 μ L enzyme solutions (400ng enzyme) are added in 80 μ L 20mmol/L p-NPS substrate solutions.After 55 DEG C of incubation 10min,
25 μ L NaOH (5mol/L) are added and terminate reaction, supplies volume to 1mL with distilled water, measures the absorption value of 410nm.Aromatic radical
The vigor of sulfatase is defined as under the above conditions, enzyme needed for catalysis generates 1 μm of oL p-nitrophenol (p-NP) per minute
Amount.
Embodiment 4: the research of mutation arylsulfatase H260L optimum reaction conditions
Mutation arylsulfatase optimal reactive temperature measures in the range of 30~80 DEG C.Concrete operations: before taking
System used in text reacts 10min under the conditions of 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C and 80 DEG C respectively, measures 410nm's
Light absorption value.Measurement result shows: the optimal reactive temperature of mutation arylsulfatase H260L is 55 DEG C.As a result such as Fig. 2 institute
Show.
The optimal reaction pH of mutation arylsulfatase H260L is measured in the range of 5.0~10.0.What measurement used
Buffer are as follows: 50mmol/L citric acid-sodium citrate buffer solution (pH 5.0-7.0), 50mmol/L Tris-HCl buffer (pH
7.0-9.0), 50mmol/L Glycine-NaOH (pH 9.0-10.0).Measurement result shows: mutation arylsulfatase
The optimal reaction pH of H260L is 8.0, has greater activity in 7.0~9.0 range of pH range, as a result as shown in Figure 3.
Embodiment 5: the zymetology stability analysis of mutation arylsulfatase H260L
The thermal stability of mutation arylsulfatase is measured at 45,50,55 and 60 DEG C.Concrete operations are as follows: will
After wild-type enzyme or mutant enzyme (400ng) after purification handles 30min at different temperatures, substrate p-NPS is added, measures enzyme
Residual activity.Measurement result is shown: after mutant enzyme H260L is handled under the conditions of 45,50,55 and 60 DEG C, being remained respectively
87%, 88%, 62% and 18% residual activity.Wild-type enzyme remains 79%, 68%, 21% and respectively under similarity condition
1% residual activity.It can be seen that the thermal stability of H260L significantly improves.As a result as shown in Figure 4.
The pH stability of mutation arylsulfatase is measured in 5.0~10.0 ranges.Concrete operations are as follows: by enzyme
It is placed in the buffer of different pH value, after 37 DEG C of incubation 1h, substrate p-NPS is added, measures the residual activity of enzyme.Measurement result is aobvious
Show: after mutant enzyme H260L is handled under the conditions of 6.0~9.0 pH, zymoprotein can still keep 70% or more residual activity.As a result
As shown in Figure 5.
Added respectively in mutation arylsulfatase the different metal salt of final concentration of 1mmol/L or 10mmol/L from
Son, including NaCl, KCl, CaCl2, MgCl2, BaCl2, MnCl2, CuCl2, ZnCl2, CoCl2And CdCl2.It is placed at 37 DEG C and places
After 1h, the residual activity of enzyme is measured, to be not added with the enzyme activity of metal ion as 100%.Measurement result is shown: K+To mutant enzyme
Vigor has no significant effect, and Mg2+、Cu2+、Zn2+、Cd2+It can inhibit the activity of enzyme, the higher inhibition of concentration is more obvious or even Zn2+
With the Cu of high concentration2+It will lead to enzyme complete deactivation.Na+And Ba2+There is certain promotion to make mutation arylsulfatase vigor
With.Ca2+And Mn2+Enzyme activity is not influenced at low concentrations, they have facilitation to enzyme activity in higher concentrations.Co2+
There is facilitation to enzymatic activity in low concentration, it can the faint vigor for inhibiting enzyme when concentration increases to 10mmol/L.As a result such as
Shown in table 1.
1 metal ion of table is on the mutation active influence of arylsulfatase
NA indicates no vigor.
Mutation arylsulfatase in add respectively final concentration of 1mmol/L or 10mmol/L inhibitor (including
EDTA, β-ME, DTT and PMSF) and 0.1% (w/v or v/v) or 1% (w/v or v/v) detergent (including Triton X-
100, Tween 20, Tween 80, Chaps and SDS).It is placed at 37 DEG C after placing 1h, the residual activity of enzyme is measured, not add
The enzyme activity of inhibiting or detergent is 100%.Measurement result is shown: inhibitor EDTA, β-ME and DTT has enzyme activity aobvious
The inhibiting effect of work, most obvious one are EDTA, so that mutation arylsulfatase loses the enzyme activity more than 70%, are said
Bright metal ion plays an important role in the catalytic process of mutant enzyme.Inhibiting effect PMSF not strong to enzymatic activity, dense
When degree is 10mmol/L, mutant enzyme can still retain 80% or more enzyme activity.Arylsulfatase is mutated to Triton X-
100, Tween 20, Tween 80 and Chaps have good tolerance.SDS has inhibiting effect to enzyme activity, when SDS concentration
When being 1%, mutation arylsulfatase can retain 23.9% enzyme activity.The results are shown in Table 2.
2 inhibitor of table and detergent are on the mutation active influence of arylsulfatase
Embodiment 6: the research of mutation arylsulfatase H260L removal asparagus rough polysaccharide sulfate group
0.1g asparagus rough polysaccharide is weighed to be dissolved in the buffer of 20mL 50mmol/L Tris-HCl (pH 7.5).Point
Not Jia Ru 30,60 and 90U wild type or mutant enzyme, 45 DEG C of processing 4h.By treated, mixed solution is placed on nylon cloth,
After sufficiently being washed with ultrapure water, dries pulverizing.Weigh etc. weight after enzymatic treatment with untreated asparagus rough polysaccharide, high temperature
After carbonization, 550 DEG C of ashing 4h in Muffle furnace are placed in, gained ash content is all dissolved in ultrapure water, 25mL is settled to.Take 1mL molten
The sulfate group content of sample is measured after 0.22 μm of film filtering of liquid in ion chromatography.Measurement result is shown: at 45 DEG C
4h is managed, with increasing for enzyme concentration, the sulfate group that asparagus rough polysaccharide is removed is more.When enzyme concentration is 90U, mutation
Enzyme H260L can remove 82.1% sulfate group.The results are shown in Table 3.
Table 3 is mutated the research of arylsulfatase removing asparagus rough polysaccharide sulfate group
In conclusion the zymoprotein of mutation arylsulfatase H260L gene coding has excellent enzymatic property, it can
Enzymatic Extraction applied to agar.Meanwhile also obtain can be with the engineering bacteria of great expression, it can be achieved that the aromatic radical for this invention
The large-scale production of sulfatase provides good basis for subsequent industrial applications.
All deformations that those skilled in the art directly can export or associate from the disclosure of invention, should all
It is considered protection scope of the present invention.
Claims (10)
1. a kind of gene of encoding mutant arylsulfatase H260L, it is characterised in that: its nucleotide sequence such as SEQ ID
Shown in NO.1.
2. a kind of mutation arylsulfatase H260L, it is characterised in that: its amino acid sequence is as shown in SEQ ID NO.2.
3. a kind of arylsulfatase expression vector, it is characterised in that: contain encoding mutant aromatic radical described in claim 1
The expression vector pET-28a-ars of the gene of sulfatase H260L.
4. a kind of preparation method of recombination mutation arylsulfatase, it is characterised in that: including using as claimed in claim 3
It is mutated arylsulfatase expression vector and converts host cell, cultivate transformant, recombination mutation fragrance is obtained from culture
Base sulfatase.
5. a kind of preparation method of recombination mutation arylsulfatase according to claim 4, it is characterised in that: described
Host cell be Escherichia coli.
6. a kind of preparation method of recombination mutation arylsulfatase according to claim 5, it is characterised in that: including
It is converted using the mutation arylsulfatase expression vector to host cell e. coli bl21 (DE3), is lured through IPTG
It leads, obtains the recombination mutation arylsulfatase of solubility expression.
7. a kind of preparation method of recombination mutation arylsulfatase according to claim 6, it is characterised in that: described
Final concentration of 0.05 mmol/L of IPTG, inducing temperature be 25 DEG C.
8. a kind of preparation method of recombination mutation arylsulfatase according to claim 4, it is characterised in that: obtained
The recombination mutation arylsulfatase obtained has following thermal stability: after 45,50,55 and 60 DEG C of processing 30min, protecting respectively
87%, 88%, 62% and 18% residual activity is stayed.
9. a kind of preparation method of recombination mutation arylsulfatase according to claim 4, it is characterised in that: obtained
The recombination mutation arylsulfatase obtained has the feature that the temperature of the recombination mutation arylsulfatase catalyzing hydrolysis
Range is 30 ~ 80 DEG C, and pH range is 5.0 ~ 10.0.
10. a kind of preparation method of recombination mutation arylsulfatase according to claim 9, it is characterised in that: institute
The recombination mutation arylsulfatase of acquisition has the feature that the recombination mutation arylsulfatase catalyzing hydrolysis most
Thermophilic degree is 55 DEG C, optimal pH 8.0.
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