CN106399334A - Thermally stable mutant aromatic sulfatase and its gene and use - Google Patents
Thermally stable mutant aromatic sulfatase and its gene and use Download PDFInfo
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Abstract
The invention discloses thermally stable mutant aromatic sulfatase and its gene and use. Through an error-prone PCR technology, random mutagenesis is introduced, a P. carrageenovora aromatic sulfatase mutant library is constructed and through screening, the mutant aromatic sulfatase with improved thermal stability is obtained. The result shows that compared with WT, H260L thermal stability is obviously improved. Based on potassium p-nitrophenyl sulfate as a substrate, the H260L has the optimal reaction temperature of 55 DEG C and pH of 8.0. The H260L is stable in the pH range of 6.0-9.0. EDTA has a strong inhibitory effect on the activity of the mutant enzyme and it is proved that a metal ion produces an important effect in the catalytic process of the mutant sulfatase. H260L has good tolerance to a detergent and has a gracilaria lemaneiformis crude polysaccharide sulfuric acid group desulfurization rate of 82%. The invention also discloses genetic engineering bacteria containing the mutant aromatic sulfatase gene. The genetic engineering bacteria realize the heterologous expression of the sulfatase and provide a good foundation for the industrial production and application of the sulfatase.
Description
Technical field
The present invention relates to the technical field of genetic engineering and enzyme engineering, more particularly, to a kind of thermally-stabilised mutation aromatic radical sulphuric acid
Esterase, gene and its application.
Background technology
Agar, also known as agar, is a kind of natural polysaccharide extracting from marine red alga, has excellent gelling and thickening
Property, it is widely used in the fields such as food, light industry, medicine and biological engineering.A large amount of sulfur are carried on natural agar molecule in red algae
Acid esters group, is the main cause of impact agar gel intensity, electroendosmosis and protein adsorption ability, and removing sulfate group is fine jade
Necessity and key link that fat produces.At present, commonly use the sulfate group that alkaline process removes in agar in commercial production, not only
Produce serious environmental issue, also result in agar and degrade in a large number loss.Compared with alkali pretreatment, removed using hydrolysis technology
Sulfate group in agar has the characteristics that reaction condition is gentle, specificity is high, and environmental pollution is little, and is not easy to cause
Agar hydrolysis run off, and are the developing direction of new agar production technology.
Agar is typical hot melt macromolecule, requires heat to more than 45 DEG C ability water solubles, and below 43 DEG C just
Solidification may form gel.Additionally, agar solution has larger viscosity, its viscosity gradually rises with the reduction of temperature.These
Characteristic determine agar only after heating for dissolving and be maintained at more than 45 DEG C (so that agar is under dissolved state) could be to it
Carry out ferment treatment.Therefore, the enzyme of hydrolysis agar sulfuric ester not only will have significant agar sulfate group hydrolysing activity, also will
There is good heat stability.
Arylsulfatase (Arylsulfatase) is a kind of catalytic pyrolysiss sulfuric acid ester bond, generates corresponding alcohol and 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 heat stability of enzyme is not high, be to affect this sulfatase to remove agar sulfur
The main property defect of acid esters efficiency.
In view of this, the present inventor establishes P.carrageenovora arylsulfatase clonal expression skill in early stage
On the basis of art, the technology using orthogenesiss is transformed to this enzyme, and the heat stability of evolution enzyme is studied and devised one kind
Mutation arylsulfatase, gene and its application, this case thus produces.
Content of the invention
It is an object of the invention to provide a kind of thermally-stabilised mutation arylsulfatase, gene and its application.
To achieve these goals, the present invention solves its technical problem and is adopted the technical scheme that：
A kind of gene of encoding mutant arylsulfatase, its nucleotide sequence is as shown in SEQ ID NO.1.Gene is big
Little for 987bp.
A kind of mutation arylsulfatase, its aminoacid sequence is as shown in SEQ ID NO.2.This encoding histone 328
Amino acid residue.
A kind of mutation arylsulfatase expression vector, the table containing described arylsulfatase H260L gene
Reach carrier pET-28a-ars.
A kind of preparation method of recombination mutation arylsulfatase, including using described mutation arylsulfatase
Expression vector converts host cell, cultivates transformant, obtains recombination mutation arylsulfatase from culture.
As the optimal way of embodiment, described host cell is escherichia coli.
As the optimal way of embodiment, thin to host including being converted using described arylsulfatase expression vector
Born of the same parents' e. coli bl21 (DE3), through IPTG induction, obtains the recombination mutation arylsulfatase of solubility expression.
As the optimal way of embodiment, the final concentration of 0.05mmol/L of described IPTG, inducing temperature is 25 DEG C.
A kind of recombination mutation arylsulfatase, is turned including using described mutation arylsulfatase expression vector
Change host cell, cultivate transformant, obtain recombinase from culture.
As the optimal way of embodiment, the temperature range of this mutation arylsulfatase catalyzing hydrolysis is 30～80
DEG C, optimum temperature is 55 DEG C；Described hydrolysis pH scope is 5.0～10.0, and optimum pH is 8.0.This mutant enzyme is 45,50,55
Process after 30min with 60 DEG C, remain 87%, 88%, 62% and 18% residual activity respectively.In pH 6.0～9.0 condition
Lower process 1h, pheron still can keep more than 70% relative activity.EDTA can suppress the activity of recombinase, and metal ion is described
The catalytic process of mutant enzyme plays an important role.Mutant enzyme to test use some detergents, including Triton X-100,
Tween 20, Tween 80 and Chaps, have good toleration.The desulfurization degree to asparagus rough polysaccharide sulfate group for the H260L
For 82%.
The present invention passes through the mutant that mutation obtains the raising of arylsulfatase heat stability, can be applicable to the enzyme of agar
Method is extracted.Meanwhile, also clone has obtained to can achieve this mutation aromatic radical sulfuric ester with the engineering bacteria of great expression for this invention
The large-scale production of enzyme, provides good basis for follow-up industrial applications.
Brief description
Fig. 1 is the abduction delivering of arylsulfatase H260L and its SDS-PAGE figure of purification assays；Wherein, M：Point
Son amount standard protein；1：α containing pET-28 (+) negative bacterium, IPTG induction；2：- ars the positive bacteria of α containing pET-28, does not induce；3：Contain
PET-28 α-ars positive bacteria, IPTG induces；4：Recombiant protein after purification；
Fig. 2 is the optimal reactive temperature curve chart of mutation arylsulfatase H260L；
Fig. 3 is the optimal reaction pH curve chart of mutation arylsulfatase H260L；
Fig. 4 is the heat stability curve chart of mutation arylsulfatase H260L；
Fig. 5 is the pH stability curve figure of mutation arylsulfatase H260L.
Specific embodiment
Embodiment 1：Heat stability improves the screening of mutation arylsulfatase
With the restructuring containing wild type Pseudoalteromonas carrageenovora arylsulfatase gene
Plasmid (WT) is template, carries out fallibility PCR, expands arylsulfatase：
Forward primer (SEQ ID NO.3)：5′-CGCGGATCCTTTACGTTTAACGGCAGC-3′；
Downstream primer (SEQ ID NO.4)：5′-CCCAAGCTTGCGTTTTAGTTCGTAAC-3′；
50 μ L amplification systems comprise：5 μ L 10 × buffer, 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 recombiant plasmid template of enzyme gene.PCR reaction condition is：95℃5min；94 DEG C of 1min, 55 DEG C of 45sec, 72 DEG C, 1min 30
Circulation；72℃10min.Method using enzyme cutting clone builds recombinant expression plasmid, that is, use BamHI and HindIII double digestion PCR
Product, the fragment after enzyme action is reclaimed in rubber tapping, with same plasmid pET-28a through BamHI and HindIII double digestion (+) carry out even
Connect.Using CaCl2Conversion method, often pipe E.coli BL21 competent cell addition 2 μ L connection products, after conversion, often pipe adds
0.8mL LB culture medium, 37 DEG C, 50r/min recovery 30min.Add in 3 times of volume LB fluid mediums, add kanamycin,
Its final concentration is made to reach 50 μ g/mL.37 DEG C, 180r/min culture 5-6h.Bacterium solution adds final concentration of 20% glycerol, after mixing
Subpackage, as arylsulfatase libraries of random mutants, are placed in -70 DEG C of preservations.
Chromogenic reaction according to producing arylsulfatase bacterial strain hydrolysis substrate p-nitrophenyl potassium sulfate (p-NPS) is carried out
The primary dcreening operation of mutated library.The bacterial strain flat board coming from library, after 50 DEG C process 2h, is covered with the soft agar containing p-NPS, its
Middle wild type (WT) no chromogenic reaction, the bacterium colony with chromogenic reaction is considered the mutant that heat stability improves.Bacterial strain is overnight
After culture, by volume 1：100 are transferred in 5mL LB fluid medium (kanamycin containing 50mg/mL), 37 DEG C, 180r/min
Cultivate to OD600Reach 0.6 0.8, add final concentration of 0.05mmol/L IPTG, 25 DEG C, 180r/min culture 10h.Centrifugation is received
Collection thalline, is resuspended in 50mmol/L Tris-HCl buffer (pH 7.5), carries out ultrasonic disruption process on ice, obtains
Crude enzyme liquid.After crude enzyme liquid processes 2h at 50 DEG C, with p-NPS as substrate, the remaining vigor of detection enzyme, carry out secondary screening checking.
Through primary dcreening operation and secondary screening, finally give the mutant that one plant of heat stability improves.Gene sequencing analysis display, mutation
Arylsulfatase, compared with wild type arylsulfatase, has a base to be replaced, leads to enzyme in enzyme gene sequence
An amino acid residue is had to change, that is, the histidine (H) of 260 is mutated into leucine (L).This mutation aromatic radical sulfuric ester
Enzyme is named as H260L.
Embodiment 2：It is mutated arylsulfatase using recombinant strains expression and purification of Recombinant
After recombinant strains incubated overnight, by volume 1：100 are transferred to 250mL LB fluid medium (containing 50mg/
ML kanamycin) in, 37 DEG C, 180r/min cultivates to OD600Reach 0.6 0.8, add final concentration of 0.05mmol/L IPTG,
25 DEG C, 180r/min culture 10h.Thalline is collected by centrifugation, is resuspended in Buffer A (50mmol/L NaH2PO4, 300mmol/L chlorine
Change sodium, 15mmol/L imidazoles, pH 8.0) in, carry out ultrasonic disruption process on ice.It is centrifuged at 4 DEG C, collect supernatant, then
Carry out Ni-NTA affinity chromatograph, through lavation buffer solution (50mmol/L NaH2PO4, 300mmol/L sodium chloride, 30mmol/L miaow
Azoles, pH 8.0) after washing, using elution buffer (50mmol/L NaH2PO4, 300mmol/L sodium chloride, 250mmol/L miaow
Azoles, pH 8.0) eluting, collect eluent.Through SDS-PAGE detection, recombination mutation arylsulfatase albumen after purification
Molecular weight is 35.7kDa, and result is as shown in Figure 1.Measure protein concentration with Bradford method, obtain concentration about 1.0mg/mL's
Mutant enzyme H260L.
Embodiment 3：The viability examination of arylsulfatase
20 μ L enzyme liquids (400ng enzyme) are added in 80 μ L 20mmol/L p-NPS substrate solutions.After incubating 10min at 55 DEG C,
Add 25 μ L NaOH (5mol/L) terminating reactions, supply volume to 1mL with distilled water, measure the absorption value of 410nm.Aromatic radical
The vigor of sulfatase is defined as under these conditions, and catalysis per minute generates the enzyme needed for 1 μm of oL paranitrophenol (p-NP)
Amount.
Embodiment 4：The research of mutation arylsulfatase H260L optimum reaction conditionses
Mutation arylsulfatase optimal reactive temperature measures in the range of 30～80 DEG C.Concrete operations：Before taking
System used by literary composition, 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.Result such as Fig. 2 institute
Show.
The optimal reaction pH of mutation arylsulfatase H260L measures in the range of 5.0～10.0.Measure use
Buffer is：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, result is as shown in Figure 3 in the range of pH scope 7.0～9.0.
Embodiment 5：The zymetology stability analyses of mutation arylsulfatase H260L
The heat stability of mutation arylsulfatase is measured at 45,50,55 and 60 DEG C.Concrete operations are：Will
After wild-type enzyme after purification or mutant enzyme (400ng) process 30min at different temperatures, add substrate p-NPS, measure enzyme
Residual activity.Measurement result shows：After mutant enzyme H260L is processed under the conditions of 45,50,55 and 60 DEG C, remain respectively
87%th, 88%, 62% and 18% residual activity.Wild-type enzyme remains 79%, 68%, 21% and under similarity condition respectively
1% residual activity.As can be seen here, the heat stability of H260L significantly improves.Result is as shown in Figure 4.
The pH stability of mutation arylsulfatase is measured in the range of 5.0～10.0.Concrete operations are：By enzyme
It is placed in the buffer of different pH value, after 37 DEG C incubate 1h, add substrate p-NPS, measure the residual activity of enzyme.Measurement result shows
Show：After mutant enzyme H260L is processed under the conditions of pH 6.0～9.0, pheron still can keep more than 70% residual activity.Result
As shown in Figure 5.
Mutation arylsulfatase adds respectively 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 in and place at 37 DEG C
After 1h, measure enzyme residual activity, be not added with metal ion enzyme activity for 100%.Measurement result shows：K+To mutant enzyme
Vigor has no significant effect, and Mg2+、Cu2+、Zn2+、Cd2+The activity of meeting inhibitory enzyme, the higher suppression of concentration is more obvious, or even Zn2+
Cu with high concentration2+Enzyme complete deactivation can be led to.Na+And Ba2+Certain promotion is had to make mutation arylsulfatase vigor
With.Ca2+And Mn2+At low concentrations enzyme activity is not affected, they have facilitation to enzyme activity in higher concentrations.Co2+
Have facilitation in low concentration to enzymatic activity, when concentration increases to 10mmol/L can faint inhibitory enzyme vigor.Result is such as
Shown in table 1.
The impact to mutation arylsulfatase activity for table 1 metal ion
NA represents does not have vigor.
The inhibitor adding final concentration of 1mmol/L or 10mmol/L in mutation arylsulfatase respectively (includes
EDTA, β-ME, DTT and PMSF) and 0.1% (w/v or v/v) or 1% (w/v or v/v) detergent (include Triton X-
100th, Tween 20, Tween 80, Chaps and SDS).It is placed in the residual activity measuring enzyme after placing 1h at 37 DEG C, not add
The enzyme activity of inhibiting or detergent is 100%.Measurement result shows：Inhibitor EDTA, β-ME and DTT has aobvious to enzyme activity
The inhibitory action writing, most obvious of which is EDTA so that mutation arylsulfatase loses the enzyme activity more than 70%, says
Bright metal ion plays an important role in the catalytic process of mutant enzyme.PMSF does not have strong inhibitory action to enzymatic activity, dense
When spending for 10mmol/L, mutant enzyme still can retain more than 80% enzyme activity.Mutation arylsulfatase is to Triton X-
100th, Tween 20, Tween 80 and Chaps have good toleration.SDS has inhibitory action to enzyme activity, when SDS concentration
During for 1%, mutation arylsulfatase can retain 23.9% enzyme activity.Result is as shown in table 2.
The impact to mutation arylsulfatase activity of table 2 inhibitor and detergent
Embodiment 6：Mutation arylsulfatase H260L removes the research of asparagus rough polysaccharide sulfate group
Weigh in the buffer that 0.1g asparagus rough polysaccharide is dissolved in 20mL 50mmol/L Tris-HCl (pH 7.5).Point
Not Jia Ru 30,60 and 90U wild type or mutant enzyme, 45 DEG C process 4h.Mixed solution after processing is placed on nylon cloth,
After fully being washed with ultra-pure water, dries pulverizing.Weigh etc. weight after ferment treatment with untreated asparagus rough polysaccharide, high temperature
After carbonization, it is placed in 550 DEG C of ashing 4h in Muffle furnace, gained ash is all dissolved in ultra-pure water, is settled to 25mL.Take 1mL molten
Liquid with after 0.22 μm of membrane filtration on chromatography of ions determination sample sulfate group content.Measurement result shows：At 45 DEG C
Reason 4h, increasing with enzyme concentration, the sulfate group that asparagus rough polysaccharide is removed is more.When enzyme concentration is for 90U, mutation
Enzyme H260L can remove 82.1% sulfate group.Result is as shown in table 3.
Table 3 is mutated the research that arylsulfatase removes asparagus rough polysaccharide sulfate group
In sum, the pheron of mutation arylsulfatase H260L gene code has excellent enzymatic property, can
It is applied to the Enzymatic Extraction of agar.Meanwhile, this invention also obtain and can achieve this aromatic radical with the engineering bacteria of great expression
The large-scale production of sulfatase, provides good basis for follow-up industrial applications.
All deformation that those of ordinary skill in the art can directly derive or associate from the disclosure of invention, all should
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 aminoacid sequence is as shown in SEQ ID NO.2.
3. a kind of arylsulfatase expression vector it is characterised in that：Containing the 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 described in claim 3
Mutation arylsulfatase expression vector conversion host cell, cultivates transformant, obtains recombination mutation fragrance from culture
Base sulfatase.
5. a kind of recombination mutation arylsulfatase according to claim 4 preparation method it is characterised in that：Described
Host cell be escherichia coli.
6. a kind of recombination mutation arylsulfatase according to claim 5 preparation method it is characterised in that：Including
Converted to host cell e. coli bl21 using described mutation arylsulfatase expression vector（DE3）, lure through IPTG
Lead, obtain the recombination mutation arylsulfatase of solubility expression.
7. a kind of recombination mutation arylsulfatase according to claim 6 preparation method 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 recombination mutation arylsulfatase according to claim 4 preparation method it is characterised in that：Obtained
The recombination mutation arylsulfatase obtaining has following heat stability：After processing 30min at 45,50,55 and 60 DEG C, protect respectively
Stay 87%, 88%, 62% and 18% residual activity.
9. a kind of recombination mutation arylsulfatase according to claim 4 preparation method it is characterised in that：Obtained
The recombination mutation arylsulfatase obtaining has following characteristics：The temperature of this recombination mutation arylsulfatase catalyzing hydrolysis
Scope is 30 ~ 80 DEG C, and pH scope is 5.0 ~ 10.0.
10. a kind of recombination mutation arylsulfatase according to claim 9 preparation method it is characterised in that：Institute
The recombination mutation arylsulfatase obtaining has following characteristics：This recombination mutation arylsulfatase catalyzing hydrolysis is
Thermophilic degree is 55 DEG C, and optimum pH is 8.0.
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| CN110564791A (en) * | 2019-08-20 | 2019-12-13 | 集美大学 | Modified agar powder |
| CN115896138A (en) * | 2022-10-12 | 2023-04-04 | 吉林大学 | Anaerobic sulfatase maturase gene, anaerobic sulfatase maturase, and preparation method and application thereof |
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| CN110241096B (en) * | 2019-06-28 | 2021-04-20 | 集美大学 | Sulfatase Sulf1694 capable of being used for removing agar sulfate groups and application thereof |
| CN110564791A (en) * | 2019-08-20 | 2019-12-13 | 集美大学 | Modified agar powder |
| CN115896138A (en) * | 2022-10-12 | 2023-04-04 | 吉林大学 | Anaerobic sulfatase maturase gene, anaerobic sulfatase maturase, and preparation method and application thereof |
| CN115896138B (en) * | 2022-10-12 | 2023-08-08 | 吉林大学 | Anaerobic sulfatase mature enzyme gene, anaerobic sulfatase mature enzyme, and preparation methods and application thereof |
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