CN110438097B - L-isoleucine hydroxylase mutant with improved heat stability and enzyme activity - Google Patents
L-isoleucine hydroxylase mutant with improved heat stability and enzyme activity Download PDFInfo
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Abstract
The invention discloses an L-isoleucine hydroxylase mutant with improved thermal stability and enzyme activity, in particular to an L-isoleucine hydroxylase mutant with improved thermal stability and enzyme activity and application thereof in production of 4-hydroxyisoleucine, belonging to the technical field of genetic engineering. The method for improving the thermal stability can be used for modifying other hydroxylases and provides theoretical guidance for producing other hydroxylated amino acids with high added values. The specific enzyme activity of the L-isoleucine hydroxylase produced by using the bacillus subtilis engineering bacteria can reach 2.4 +/-0.08U/mg, 5 batches of whole cells are repeated, continuous conversion is carried out for 45 hours, 856.91mM (126.1g/L) 4-HIL can be obtained, and after 5 batches, the conversion capacity of the L-isoleucine hydroxylase is still 84.1% of that of the first conversion.
Description
Technical Field
The invention relates to an L-isoleucine hydroxylase mutant with improved thermal stability and enzyme activity, in particular to an L-isoleucine hydroxylase mutant with improved thermal stability and enzyme activity and application thereof in production of 4-hydroxyisoleucine, belonging to the technical field of genetic engineering.
Background
4-Hydroxyisoleucine (4-hydroxyisoeucine, 4-HIL) is a natural, non-protein amino acid that was first found in fenugreek seeds. It is a promising drug, mainly applied in pharmaceutical industry.
Since 4-HIL has the effects of promoting insulin secretion, improving the resistance of peripheral tissues to insulin, and regulating dyslipidemia, great attention and research have been drawn. Up to now, the synthesis method of 4-HIL mainly comprises plant separation and extraction, chemical enzyme synthesis and microbial transformation. The plant isolation procedure mainly focused on the extraction of 4-HIL from fenugreek seeds, but the yield was low (about 4mg of 4-HIL from 1kg of fenugreek seeds). Chemical and enzymatic synthesis methods have the problems of low conversion efficiency, high cost, serious pollution and the like. Therefore, with the increasing awareness of environmental protection and the further development of green technology, pollution-free and non-toxic biotechnology is inevitably the main direction of industry development.
Currently, the specific catalysis of L-ILe to produce 4-HIL by L-isoleucine hydroxylase (IDO) has been extensively studied. B.thuringiensis 2e2, B.thuringiensis TCCC11826, B.thuringiensis YBT1520 and Bacillus weihenstephanensis-derived ido genes achieve their expression in Escherichia coli or Corynebacterium glutamicum, and whole cell transformation is used for 4-HIL production or direct fermentation is used for 4-HIL production using a strain with self high L-ILe yield. However, due to the poor thermal stability of IDO from the above sources, its enzymatic activity decreases significantly when the temperature exceeds its optimum temperature.
Therefore, the method for improving the thermal stability and the enzyme activity of the L-isoleucine hydroxylase is provided, and has important value for the production and the application of the L-isoleucine hydroxylase.
Disclosure of Invention
The first purpose of the invention is to provide an L-isoleucine hydroxylase mutant with improved heat stability and enzyme activity, which comprises an amino acid sequence shown in SEQ ID NO. 4.
It is a second object of the present invention to provide a gene encoding the mutant.
In one embodiment of the invention, the nucleotide sequence of the gene is shown in SEQ ID NO. 3.
The third purpose of the invention is to provide a vector containing the gene.
It is a fourth object of the present invention to provide a cell expressing the L-isoleucine hydroxylase mutant.
The fifth purpose of the invention is to provide a genetically engineered bacterium, which takes Bacillus subtilis as a host to express an L-isoleucine hydroxylase mutant shown in SEQ ID No. 4.
In one embodiment of the present invention, the genetically engineered bacterium is a bacillus subtilis168 as a host.
In one embodiment of the invention, the genetically engineered bacterium uses pMA5 as a vector.
The sixth purpose of the invention is to provide a method for improving the stability of L-isoleucine hydroxylase, wherein serine 181 of L-isoleucine hydroxylase shown in SEQ ID NO.2 is mutated into cysteine.
The seventh object of the present invention is to provide a method for producing the L-isoleucine hydroxylase mutant, comprising the steps of:
(2) the genetically engineered bacterium B.subtilis168/pMA5-idoT181CRespectively inoculating the single colonies into 10mL LB liquid culture medium, and culturing for 8-12h at 35-39 ℃ and 120-180rpm to obtain seed liquid;
(2) inoculating the seed liquid into LB liquid culture medium with 1-5% of inoculation amount, culturing at 25-37 deg.C and 120-180rpm for 12-24h to obtain genetically engineered bacterium B.subtilis168/pMA5-idoT181CThe culture solution of (4);
(3) the culture solution is crushed by a cell crusher and then centrifuged for 5-10min under the conditions of 8000-10000rpm and 4 ℃ to obtain the cultured recombinant bacterium B.subtilis168/pMA5-idoT181CThe crude enzyme solution obtained.
The invention also provides the application of the L-isoleucine hydroxylase mutant and the genetic engineering bacterium in preparing products containing 4-hydroxyisoleucine.
The invention obtains the L-isoleucine hydroxylase mutant T181C with improved thermal stability and enzyme activity by constructing disulfide bonds, and compared with the wild type L-isoleucine hydroxylase IDO (the amino acid sequence is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO. 1), the mutant T181C is obtained by mutating serine at position 181 into cysteine. The half-life of the mutant T181C at 50 ℃ is 4.03h, which is 10.27 times of that of the wild type 0.39 h; the specific enzyme activity of the mutant T181C is 2.4 +/-0.08U/mg, which is 3.5 times of that of the wild type 0.68 +/-0.06U/mg; mutant T181C was replicated in 5 lots, and the cumulative yield of 4-HIL was 856.91mM (126.1g/L), which was 2.19-fold greater than that of wild type 390.5mM (57.5 g/L). Therefore, the L-isoleucine hydroxylase mutant T181C obtained by the invention has good thermal stability, and lays a foundation for industrial application thereof.
Detailed Description
The invention is further illustrated below with reference to specific examples.
Bacillus subtilis168, escherichia coli e.coli JM109, referred to in the examples below, was purchased from north america; the expression vector pMA5 referred to in the examples below was purchased from Youbao; HEPES buffer, Ellman's reagent (DTNB), cysteine, triton, L-isoleucine, alpha-ketoglutarate, FeSO, referred to in the examples below4·7H2O and ascorbic acid were purchased from Shanghai Allantin Biotechnology Ltd.
The media and the required solutions referred to in the following examples are as follows:
LB liquid medium: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract.
LB solid medium: 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract and 2% (m/v) of agar powder.
TY medium: 8g/L of yeast extract, 12g/L of tryptone, 4g/L of tripotassium phosphate, 3g/L of sodium chloride, 2.1g/L of citric acid monohydrate, 0.3g/L of ferric ammonium citrate, 10g/L of glycerol, 2.5g/L of ammonium sulfate and 0.24g/L of magnesium sulfate.
TB culture medium: peptone 12g/L, yeast extract 24g/L, glycerin 4g/L, KH2PO4 2.31g/L、K2HPO42.54g/L。
Cysteine Standard solution (1 mmol/L): 0.017563g of L-cysteine were weighed out accurately, dissolved in 1mL of formic acid, and made up to 100mL with ultrapure water.
DTNB standard solution (10 mmol/L): 0.198175g of DTNB were weighed out accurately, and prepared into 50mL of a 50mmol/L disodium hydrogenphosphate solution having a pH of 7.0, and stored in a brown bottle and stored at a low temperature in the dark for further use.
DTNB assay solution (0.1 mmol/L): the mixture is prepared by mixing 10mmol/L DTNB standard solution and 0.25mmol/L Tris-HCI buffer solution with the pH value of 7.0 according to the volume ratio of 1: 99, the preparation is prepared in situ.
The detection methods referred to in the following examples are as follows:
the detection method of the enzyme activity of the L-isoleucine hydroxylase comprises the following steps: get1mL of the crude enzyme solution was added with a mixture containing 30mM L-isoleucine, 30mM alpha-ketoglutaric acid, and 1mM FeSO4·7H2O,5mM ascorbic acid and 50mM HEPES buffer pH7.0 in a 9mL reaction system; reacting at 30 ℃ for 1h, and carrying out boiling water bath for 5-10min to terminate the reaction; centrifuging, taking supernatant, performing derivatization treatment, and determining the content of 4-HIL by an HPLC 2, 4-dinitrofluorobenzene pre-column derivatization method;
the sample derivatization treatment method comprises the following steps: add 100. mu.L of the reaction to a 1.5mL centrifuge tube, followed by addition of the derivatization buffer: 0.5M NaHCO pH 9.03And a derivatizing agent: 1% 2, 4-dinitrofluorobenzonitrile solution (1% FDBN) 100 uL each, after mixing, in 60 ℃ dark treatment for 30min, cooling in cold water bath, finally adding 1mL constant volume buffer: 0.01M KH pH7.02PO4；
HPLC analysis: 2, 4-dinitrofluorobenzene pre-column derivatization method, which is determined by C18 column reversed phase high performance liquid chromatography; needle washing: 10% methanol; automatic sample introduction is carried out after needle washing, and the amino acid washing and removing method comprises the following steps: mobile phase a (50mM NaAC, containing 1% N, N-dimethylformamide, adjusted pH to 6.4 with HAC) was gradient eluted with mobile phase B (50% aqueous acetonitrile) for 0-13min, a 70% -38%; 13-21min, A38% -32%; 21-23min, A32% -0%; 23-25min, A0% -0%; 25-26min, A0% -70%; 26-30min, A70% -70%; t is 33 ℃; λ is 360 nm; injecting 20 mu L of sample; time is 30 min; v is1 mL/min;
definition of L-isoleucine hydroxylase enzyme Activity: the amount of enzyme required to catalyze the L-Ile reaction to produce 1. mu. mol of product 4-HIL per minute at 30 ℃ and pH7.0 is one activity unit (U).
The detection method of the specific enzyme activity of the L-isoleucine hydroxylase comprises the following steps: 0.1mg of pure enzyme was added to the mixture containing 30mM L-isoleucine, 30mM alpha-ketoglutaric acid, and 1mM FeSO4·7H2O,5mM ascorbic acid and 50mM HEPES buffer (pH7.0); reacting at 30 ℃ for 1h, and carrying out boiling water bath for 5-10min to terminate the reaction; centrifuging, taking supernatant, performing derivatization treatment, and determining the content of 4-HIL by an HPLC 2, 4-dinitrofluorobenzene pre-column derivatization method;
wherein, the definition of specific enzyme activity of L-isoleucine hydroxylase is as follows: the enzyme activity per mg of enzyme protein is U/mg.
The number detection method of disulfide bonds of wild enzyme IDO and mutant enzyme T181C comprises the following steps: diluting cysteine standard solution with Tris-HCI buffer solution at 25 deg.C to obtain various gradient dilutions 5mL, with concentrations of 0mmol/L, 0.025mmol/L, 0.05mmol/L, 0.1mmol/L, 0.15mmol/L and 0.2mmol/L respectively; and (3) respectively adding 1mL of the gradient diluent, the wild enzyme IDO and the mutant enzyme T181C thereof into 5mL of DTNB analysis solution which is placed in a constant-temperature water tank at 25 ℃ in advance, uniformly mixing, accurately standing for 10min, and immediately measuring the absorbance at the wavelength of 412 nm. The corresponding free cysteine concentration was determined according to the standard curve for cysteine.
The method for determining the thermal stability of the wild enzyme IDO and the mutant enzyme T181C comprises the following steps: heat treating wild enzyme IDO and mutant enzyme T181C at 10-70 deg.C for 20min, and adding the same amount of wild enzyme IDO and mutant enzyme T181C into a solution containing 30mM L-ILe,30mM alpha-KG, 1mM FeSO4·7H2O,5mM ascorbic acid and HEPES buffer (50mM, pH 7.0) at 30 ℃ for 1 h. To further verify whether T181C was improved in heat stability, the wild enzyme IDO and the mutant enzyme T181C were heat-treated at 50 ℃ for various periods of time, after which the same amount of wild enzyme IDO and mutant enzyme T181C were added to a solution containing 30mM L-ILe,30mM α -KG,1mM FeSO4·7H2O,5mM ascorbic acid and HEPES buffer (50mM, pH 7.0) at 30 ℃ for 1 h.
Example 1: construction of L-isoleucine hydroxylase expression vector
Taking a Bacillus cereus genome as a template, amplifying to obtain an L-isoleucine hydroxylase gene shown as SEQ ID NO.1, and connecting the L-isoleucine hydroxylase gene with a pMA5 expression plasmid subjected to enzyme digestion by restriction enzymes Nde I and Mlu I to obtain a recombinant plasmid pMA 5-ido;
example 2: construction of disulfide bond-containing L-isoleucine hydroxylase mutant T181C
The amino acid sequence of the L-isoleucine hydroxylase is submitted to a Phere 2 online website (http:// www.sbg.bio.ic.ac.uk/Phere 2/html/page. cgi. Uploading pdb files of predicted structures to a Disulfide by Design online server (http:// cptgweb. cpt. wayne. edu/DbD2/index. php) to perform prediction Design of Disulfide bonds, selecting sites which can be paired with free cysteines (C61, C112 and C226) existing in the IDO protein per se to form Disulfide bonds for mutation, and constructing mutants K152C and T181C (C112 has no sites which can be paired to form Disulfide bonds). T181C (mutant K152C was obtained by mutating lysine 152 to cysteine of L-isoleucine hydroxylase shown in SEQ ID NO. 2).
Using the gene of example 1 as a template, site-directed mutagenesis was performed by overlap extension to obtain a mutated gene idoK152C、idoT81CThen the plasmid is connected with a pMA5 expression plasmid which is cut by restriction enzymes Nde I and Mlu I and then the recombinant plasmid pMA5-ido is obtainedK152CAnd pMA5-idoT181C。
Example 3: subtilis168/pMA5-ido, B.subtilis168/pMA5-idoK152CSubtiliss 168/pMA5-idoT181CConstruction and expression of recombinant bacteria
Coli JM109 was transformed with the recombinant plasmids obtained in examples 1 and 2 to obtain recombinant bacteria E.coli JM109/pMA5-ido, E.coli JM109// pMA5-idoK152CColi JM109// pMA5-idoT181C。
plasmid pMA5 and recombinant bacteria E.coli JM109/pMA5-ido, E.coli JM109// pMA5-idoK152CColi JM109// pMA5-idoT181CExtracting the obtained plasmid and transforming the plasmid into Bacillus subtilis168 to express to obtain recombinant strains B.subtilis168/pMA5, B.subtilis168/pMA5-ido and B.subtilis168/pMA5-idoK152CSubtilis168/pMA5-idoT181C。
Recombinant strains B.subtilis168/pMA5, B.subtilis168/pMA5-ido, B.subtilis168/pMA5-idoK152CSubtilis168/pMA5-idoT181CThe single colonies are respectively inoculated into 10mL LB liquid culture medium and cultured for 8-12h under the conditions of the temperature of 37 ℃ and the rotation speed of 120-180rpm to obtain seed liquid.
Inoculating the seed liquid into 600mL LB liquid culture medium with the inoculation amount of 1%, culturing for 12-24h under the conditions of the temperature of 25-37 ℃ and the rotation speed of 120-180rpm, and respectively obtaining the recombinant bacteria B.sub tilis 168/pMA5、B.subtilis 168/pMA5-ido、B.subtilis 168/pMA5-idoK152CSubtilis168/pMA5-idoT181CThe culture solution of (4).
The culture solution is crushed by a cell crusher and then centrifuged for 5-10min under the conditions of 8000-10000rpm and 4 ℃ to respectively obtain cultured recombinant bacteria B.subttilis 168/pMA5, B.subttilis 168/pMA5-ido, B.subttilis 168/pMA5-idoK152CSubtilis168/pMA5-idoT181CThe obtained crude enzyme solution roughly detects the enzyme activities of IDO, K152C and T181C.
The detection result shows that IDO, K152C and T181C all have enzyme activity. Therefore, subsequent enzymatic property studies were carried out on the wild enzyme IDO and the mutant enzymes K152C, T181C.
Example 4: enzymological properties of wild enzyme IDO and mutant enzymes K152C and T181C
The cultured recombinant bacteria B.subtilis168/pMA5-ido and B.subtilis168/pMA5-ido obtained in example 3 were culturedK152CSubtilis168/pMA5-idoT181CThe obtained crude enzyme solution is respectively purified by an affinity chromatography column to obtain pure enzymes IDO, K152C and T181C.
1. The optimum reaction temperature of pure enzymes IDO and T181C was determined
1mL of the reaction system was added to a reaction system containing 30mM L-isoleucine, 30mM α -KG, and 1mM FeSO4·7H2O and 5mM ascorbic acid in 50mM HEPES buffer solution with pH of 7.0 are placed in water bath pots with different temperatures, the temperature ranges from 20 ℃ to 60 ℃, then 0.1mg IDO, K152C and T181C are respectively added, after reaction for 1h, the reaction is stopped in boiling water bath for 5min, the supernatant is obtained by centrifugation, and the enzyme activity in the supernatant is measured by HPLC.
The detection result is as follows: the enzyme activities of IDO, K152C and T181C are highest under the condition of 30 ℃, and the optimal reaction temperatures of IDO, K152C and T181C are all 30 ℃.
2. Detection of the optimum pH of the pure enzymes IDO and T181C
Adopting a 1mL reaction system, keeping the reaction temperature at 30 ℃, respectively replacing the HEPES buffer solution for reaction with citric acid-sodium citrate buffer solution (pH range is 3.0-5.0), PBS buffer solution (pH range is 6.0-8.0) and Tris-HCl buffer solution (pH 9.0) with different pH values, with the gradient interval of 1, then respectively adding IDO, K152C and T181C for reaction for 1h, stopping the reaction in a boiling water bath for 5min, centrifuging to take supernatant, and measuring the enzyme activity in the supernatant by HPLC.
The detection result is as follows: when the pH of IDO, K152C and T181C is 7.0, the enzyme activity is highest; as can be seen: the optimum pH of IDO, K152C and T181C was 7.0.
3. Testing the thermal stability of the wild enzyme IDO and the mutant enzymes K152C and T181C
Subjecting the wild enzyme IDO and mutant enzymes K152C and T181C to heat treatment at 10-70 deg.C for 20min, and adding 0.1mg of the wild enzyme and the mutant enzyme to a solution containing 30mM L-isoleucine, 30mM alpha-KG and 1mM FeSO4·7H2O,5mM ascorbic acid in 0.99mL HEPES buffer (pH7.0,50mM) at 30 ℃ for 1h, and residual enzyme activity was detected by HPLC.
And (3) detection results: after the mutant enzyme T181C is subjected to heat treatment at 70 ℃ for 20min, 69.4% of residual enzyme activity is still maintained; while the wild enzyme IDO and the mutant enzyme K152C almost lose the enzyme activity.
To further verify whether or not the thermostability was improved after the mutation, the wild enzyme IDO and the mutant enzymes K152C and T181C were heat-treated at 50 ℃ for various times (0h, 0.5h, 1h, 1.5h and 2h), after which 0.1mg of the wild enzyme and the mutant enzyme were added to a medium containing 30mM L-isoleucine, 30mM α -KG and 1mM FeSO, respectively4·7H2O,5mM ascorbic acid in 0.99mL HEPES buffer (pH7.0,50mM) at 30 ℃ for 1h, and residual enzyme activity was detected by HPLC.
And (3) detection results: T181C mutant enzyme at 50 ℃ T1/2A value of 4.03h, wild type (t)1/20.392) to 10.27 times; t of K152C mutant enzyme at 50 DEG C1/2The value is 0.40h, and no obvious difference is caused with the wild type.
4. Detecting the specific enzyme activities of wild enzyme IDO and mutant enzymes K152C and T181C
In the presence of 30mM L-isoleucine, 30mM alpha-KG, 1mM FeSO4·7H2O,5mM ascorbic acid in 0.99mL HEPES buffer (pH7.0,50mM) were added 100. mu.L of 100. mu.g/mL wild enzyme IDO and mutant enzymes K152C, T181C, respectively, reacted at 30 ℃ for 1 hour, and the yield of 4-HIL was checked by HPLC.
And (3) detection results: the specific enzyme activity of the mutant enzyme T181C is (2.4 +/-0.08) U/mg, which is 3.5 times of wild type IDO [ (0.68 +/-0.06) U/mg ]; the specific enzyme activity of the mutant enzyme K152C is [0.38 +/-0.05U/mg ], and is reduced relative to the wild type.
Example 6: detection of number of disulfide bonds in wild enzyme IDO and mutant enzymes K152C and T181C
Diluting cysteine standard solution with Tris-HCl buffer solution with pH 8.3 to prepare 5mL of various gradient dilutions with concentrations of 0mM, 0.025mM, 0.5mM, 0.1mM, 0.15mM and 0.2mM respectively; and (3) taking 1mL of the gradient diluent, the wild enzyme IDO, the mutant enzyme K152C and the T181C, respectively adding 5mL of DTNB analysis solution which is placed in a constant-temperature water tank at 25 ℃ in advance, uniformly mixing, accurately standing for 10min, and immediately measuring the absorbance at the wavelength of 412 nm.
The corresponding free cysteine concentration was determined from the standard curve for cysteine. The number of free cysteines contained in each molecule of wild enzyme IDO and mutant enzyme K152C, T181C can be calculated according to a proportion formula. X, MrCYS/MrIDO≈CCYS·MrCYS·10-3Wherein X: the number of free cysteines per protein molecule; mrCYS: relative molecular mass of cysteine; mrIDOIs the relative molecular mass of the wild enzyme IDO or the mutant enzyme T181C; cCYS: number of free cysteines per mg protein sample.
And (3) detection results: the number of free cysteine of wild enzyme IDO is 3, and no disulfide bond is formed; the number of free cysteine of the mutant enzyme T181C is 2, and 1 disulfide bond is successfully introduced; the mutant enzyme K152C has the number of free cysteine of 4 and no disulfide bond.
Example 7: recombinant bacterium B.subtilis168/pMA5-idoT181CApplication of
The recombinant bacteria B.subtilis168/pMA5-ido and B.subtilis168/pMA5-ido obtained in example 3 were addedT181CThe culture solution is centrifuged for 5-10min at 8000-10000rpm and 4 ℃ and then the thalli are collected; re-suspending the thalli by using HEPES buffer solution with the concentration of 50mmol/L, pH of 7.0 until the wet cell weight of the thalli in the buffer solution is 10g/L to obtain a reaction system; after a cell wall penetrating agent triton is added into a reaction system by 1 per mill of addition amount, the following scheme is adoptedAdding a substrate and a cofactor into a reaction system, and converting at the temperature of 30 ℃ and the pH value of 7.0 to obtain a reaction solution.
The specific scheme is as follows: and (4) centrifuging and collecting thalli every 9h of conversion, adding the thalli into the newly-prepared substrate conversion solution again, continuing to convert, and continuously converting for 5 batches.
Detecting the yield of the 4-hydroxyisoleucine in the reaction solution, wherein the detection result is as follows: subtilis168/pMA5-idoT181CThe mutant was transformed for 45h by repeating 5 batches to obtain 856.91mM (126.1g/L) of 4-HIL, and the conversion rate after transforming 5 batches was 84.1% of that of the first batch. While the wild strain B.subtilis168/pMA5-ido is repeated for 5 batches and is continuously transformed for 45 hours, 4-HIL of 390.5mM (57.5g/L) can be obtained, and the transformation rate of the transformed wild strain B.subtilis168/pMA is 72.4% of that of the first batch after 5 batches are transformed. It can be seen that the introduction of disulfide bonds improves the thermostability of L-isoleucine hydroxylase.
Therefore, the thermal stability of the L-isoleucine hydroxylase mutant T181C obtained by the method is improved, the operation stability and the recycling batch of the L-isoleucine hydroxylase mutant in a catalytic process are improved, and the industrial application value of the L-isoleucine hydroxylase mutant is improved; meanwhile, the rational design of the disulfide bond can also be used for the improvement of the thermal stability of other hydroxylases, and provides theoretical guidance for producing other high value-added hydroxylated amino acids.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> L-isoleucine hydroxylase mutant with improved heat stability and enzyme activity
<160> 4
<170> PatentIn version 3.3
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Val His Glu Phe Glu Ser Lys Gly Phe Leu Glu Ile Ser Asn Glu Ile
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Phe Leu Gln Glu Glu Glu Asn His Arg Leu Leu Thr Gln Ala Gln Leu
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Asp Asn Ser Asn Asp Tyr Phe Gln Ser Lys Glu Tyr Asn Tyr Asp Asp
85 90 95
Gly Gly Lys Val Arg Gln Phe Asn Ser Ile Asn Asp Ser Phe Leu Cys
100 105 110
Asn Pro Leu Ile Gln Asn Ile Val Arg Phe Asp Thr Glu Phe Ala Phe
115 120 125
Lys Thr Asn Ile Ile Asp Thr Ser Lys Asp Leu Ile Ile Gly Leu His
130 135 140
Gln Val Arg Tyr Lys Ala Thr Lys Glu Arg Pro Ser Phe Ser Ser Pro
145 150 155 160
Ile Trp Leu His Lys Asp Asp Glu Pro Val Val Phe Leu His Leu Met
165 170 175
Asn Leu Ser Asn Thr Ala Ile Gly Gly Asp Asn Leu Ile Ala Asn Ser
180 185 190
Pro Arg Glu Ile Asn Gln Phe Ile Ser Leu Lys Glu Pro Leu Glu Thr
195 200 205
Leu Val Phe Gly Gln Lys Val Phe His Ala Val Thr Pro Leu Gly Thr
210 215 220
Glu Cys Ser Thr Glu Ala Phe Arg Asp Ile Leu Leu Val Thr Phe Ser
225 230 235 240
Tyr Lys Glu Thr Lys
245
<210> 3
<211> 738
<212> DNA
<213> Artificial Synthesis
<400> 3
atgacctttg tgctgagcaa aatgaatggt tttagcattg aagaaaaagt gcatgaattt 60
gaaagtaagg gctttttaga gatcagcaac gagatctttt tacaagaaga agaaaaccat 120
cgtctgctga cccaagctca gctggactac tacaatctgg aagacgatgc ctatggcgaa 180
tgccgcgcac gcagctacag ccgctacatc aaatacgtgg acagcccgga ctacatttta 240
gataatagca atgattattt tcagagcaaa gaatacaatt atgatgatgg cggcaaggtg 300
cgccagttca acagcattaa tgatagcttt ctgtgcaacc cgctgatcca gaatatcgtg 360
cgcttcgaca ccgagtttgc ctttaaaaca aatattattg ataccagcaa agatttaatt 420
atcggtttac atcaagttcg ctataaagcc accaaagaac gcccgagctt cagcagccct 480
atctggctgc acaaagatga tgaaccggtg gtgtttttac atttaatgaa tttaagcaac 540
tgtgccattg gcggcgataa tttaatcgcc aacagtccgc gcgagatcaa tcagttcatc 600
tctttaaagg agccgctgga aactttagtt tttggccaga aagtgttcca cgccgtgacc 660
ccgctgggta ccgaatgcag caccgaagca tttcgtgaca ttttactggt gaccttcagc 720
tacaaagaaa ccaaataa 738
<210> 4
<211> 245
<212> PRT
<213> Artificial Synthesis
<400> 4
Met Thr Phe Val Leu Ser Lys Met Asn Gly Phe Ser Ile Glu Glu Lys
1 5 10 15
Val His Glu Phe Glu Ser Lys Gly Phe Leu Glu Ile Ser Asn Glu Ile
20 25 30
Phe Leu Gln Glu Glu Glu Asn His Arg Leu Leu Thr Gln Ala Gln Leu
35 40 45
Asp Tyr Tyr Asn Leu Glu Asp Asp Ala Tyr Gly Glu Cys Arg Ala Arg
50 55 60
Ser Tyr Ser Arg Tyr Ile Lys Tyr Val Asp Ser Pro Asp Tyr Ile Leu
65 70 75 80
Asp Asn Ser Asn Asp Tyr Phe Gln Ser Lys Glu Tyr Asn Tyr Asp Asp
85 90 95
Gly Gly Lys Val Arg Gln Phe Asn Ser Ile Asn Asp Ser Phe Leu Cys
100 105 110
Asn Pro Leu Ile Gln Asn Ile Val Arg Phe Asp Thr Glu Phe Ala Phe
115 120 125
Lys Thr Asn Ile Ile Asp Thr Ser Lys Asp Leu Ile Ile Gly Leu His
130 135 140
Gln Val Arg Tyr Lys Ala Thr Lys Glu Arg Pro Ser Phe Ser Ser Pro
145 150 155 160
Ile Trp Leu His Lys Asp Asp Glu Pro Val Val Phe Leu His Leu Met
165 170 175
Asn Leu Ser Asn Cys Ala Ile Gly Gly Asp Asn Leu Ile Ala Asn Ser
180 185 190
Pro Arg Glu Ile Asn Gln Phe Ile Ser Leu Lys Glu Pro Leu Glu Thr
195 200 205
Leu Val Phe Gly Gln Lys Val Phe His Ala Val Thr Pro Leu Gly Thr
210 215 220
Glu Cys Ser Thr Glu Ala Phe Arg Asp Ile Leu Leu Val Thr Phe Ser
225 230 235 240
Tyr Lys Glu Thr Lys
245
Claims (10)
1. An L-isoleucine hydroxylase mutant with improved heat stability and enzyme activity is characterized in that the amino acid sequence of the mutant is shown as SEQ ID No. 4.
2. A gene encoding the L-isoleucine hydroxylase mutant according to claim 1.
3. A vector comprising the gene of claim 2.
4. A cell expressing the L-isoleucine hydroxylase mutant of claim 1.
5. A genetically engineered bacterium is characterized in that a Bacillus subtilis (Bacillus subtilis) is used as a host to express an L-isoleucine hydroxylase mutant shown as SEQ ID No. 4.
6. The genetically engineered bacterium of claim 5, wherein Bacillus subtilis168 is used as a host.
7. The genetically engineered bacterium of claim 5 or 6, wherein pMA5 is used as an expression vector.
8. A method for improving the stability of L-isoleucine hydroxylase is characterized in that the 181 th serine of the L-isoleucine hydroxylase shown in SEQ ID No.2 is mutated into cysteine.
9. A method for producing L-isoleucine hydroxylase, comprising the steps of:
(1) inoculating the single colony of the genetically engineered bacteria of any one of claims 5 to 7 into an LB liquid culture medium, and culturing at the temperature of 35-39 ℃ and under the condition of 120-180rpm for 8-12h to obtain a seed solution;
(2) inoculating the seed solution obtained in the step (1) into a new LB liquid culture medium in an inoculation amount of 1-5%, and culturing for 12-24h at the temperature of 25-37 ℃ and under the condition of 120-180rpm to obtain a culture solution of the genetic engineering bacteria;
(3) and (3) crushing the culture solution obtained in the step (2) and then centrifuging to obtain crude enzyme liquid of the genetically engineered bacteria.
10. Use of the L-isoleucine hydroxylase mutant according to claim 1 and the genetically engineered bacterium according to any one of claims 5 to 7 for producing a product containing 4-hydroxyisoleucine.
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| RU2007147436A (en) * | 2007-12-21 | 2009-06-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) | BACTERIA-PRODUCER (2S, 3R, 4S) -4-HYDROXY-L-ISOLEUCIN AND METHOD FOR PRODUCTION (2S, 3R, 4S) -4-HYDROXY-L-ISOLEUCIN |
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