CN101503681B - Mutant of cyclodextrin glucosyl transferase having highly alpha-cyclodextrin yielding property and mutation method - Google Patents
Mutant of cyclodextrin glucosyl transferase having highly alpha-cyclodextrin yielding property and mutation method Download PDFInfo
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Abstract
The invention relates to a mutant of a cyclodextrin glucosyltransferase with the capability of highly yielding alpha-cyclodextrin and a mutation method, which belong to the fields of gene engineering and enzyme engineering. The invention improves the specificity of products of the cyclodextrin glucosyltransferase (CGT enzyme for short), provides a mutant proposal for improving the capability of CGT enzyme from Peanibacillus macerans JFB05-01 (CCTCC NO: M 208063) for producing the alpha-cyclodextrin, and substitutes Asp on the 372 position of the CGT enzyme for Lys, and Tyr on the 89 position as Asp and Arg to respectively obtain single mutant enzyme D372K, Y89D and Y89R; the alpha-cyclodextrin production capacity of the obtained mutant enzyme is improved compared with wild type CGT enzymes; genetic fragments of the CGT enzyme with Lys 372 are substituted by corresponding genetic fragments of Y89R so as to obtain double mutant enzyme D372K/Y89R; and the yield of the alpha-cyclodextrin of the ouble mutant enzyme D372K/Y89R is improved by 1.5 times compared with the wild type CGT enzyme, while the yield of the beta-cyclodextrin is reduced by 57 percent. The mutants are more favorable for industrial production of the beta-cyclodextrin than the wild type CGT enzymes.
Description
Technical field
Mutant and mutation method with cyclomaltodextrin glucanotransferase of high yield alpha-cylodextrin ability the invention belongs to genetically engineered and enzyme engineering field.Specifically the present invention utilizes the rite-directed mutagenesis method of protein engineering to improve the specific technology of product of cyclomaltodextrin glucanotransferase (CGT enzyme).
Background technology
Cyclodextrin is the oligose irreducibility series of compounds with the hydrophobic conical structure of ring-type that is linked by six above glucose, wherein the most frequently used is α-, β-, γ-Huan Hujing, they are made of 6,7,8 glucose units respectively.At present, the suitability for industrialized production of cyclodextrin all adopts enzyme process synthetic, promptly under the CGT enzyme catalysis, by cyclization converted starch and the synthetic cyclodextrin of relevant matrix.Because cyclodextrin can form inclusion compound with many guest molecules, thereby change physics and chemical property such as solubleness, the stability etc. of guest molecule, therefore have in fields such as food, medicine, agriculturals widely and use.
The CGT enzyme is the enzyme of a multifunctional type, and four kinds of different reactions of its energy catalysis: three kinds are changeed glycosylation (disproportionation reaction, cyclization and coupled reaction) and hydrolysis reaction.The CGT enzyme can utilize the Starch Production cyclodextrin by cyclization, and this is the basis of its industrial application.With the CGT enzyme produce main unfavourable condition of cyclodextrin be all known wild-type CGT enzyme productions be α-, β-, the mixture of γ-Huan Hujing.This brings very big inconvenience not only for the separation and purification of product, and has improved production cost.At present, two kinds of technologies can be used for from the single cyclodextrin of cyclodextrin mixt purifying: selective crystallization of beta-cyclodextrin (beta-cyclodextrin solubleness is low relatively) and organic solvent selective complexation.Alpha-cylodextrin is generally used decyl alcohol, removes from the aqueous solution but this compound is very difficult, because its boiling point is up to 229 ℃; Generally γ-Huan Hujing is carried out complexing and selective precipitation with cyclododecanone, but this solvent for commerce make be used for saying too expensive, and, the unfavorable factor that organic solvent uses also comprises their toxicity and combustibility and needs solvent recovery process, therefore, at present α-, the utilization of γ-Huan Hujing is restricted to a great extent.Even the production technique of beta-cyclodextrin neither be very desirable, because should be used for saying for great majority, technology makes the production cost of beta-cyclodextrin still too high at present.Therefore, if a certain specific cyclodextrin ratios increases greatly in the cyclodextrin that resultant CGT enzyme is produced, so just can avoid problems such as the too high and organic solvent pollution of above-mentioned cost.
The Peanibacillus of deriving from macerans JFB 05-01 used in the present invention (CCTCC NO:M208063) though the CGT enzyme mainly produce alpha-cylodextrin at the enzymatic conversion reaction initial stage, but the content of later stage alpha-cylodextrin is on the low side, under with an organic solvent situation not, the content of beta-cyclodextrin even be higher than alpha-cylodextrin in the reaction mixture, therefore, the product alpha-cylodextrin ability that further improves this enzyme is favourable relatively to the production of industrial alpha-cylodextrin.
Summary of the invention
An object of the present invention is to provide and improve the sudden change scheme that P.macerans JFB 05-01 CGT enzyme produces the alpha-cylodextrin ability.
Another object of the present invention is the mutant enzyme D372K that proposition has higher alpha-cylodextrin throughput, Y89D, Y89R and D372K/Y89R, and construction process.
Technical scheme of the present invention:
Derive from the cyclomaltodextrin glucanotransferase of softening series bacillus (Peanibacillus macerans) JFB 05-01, be called for short the CGT enzyme, three kinds of single-mutant enzyme D372K, Y89D and Y89R: the 372nd aspartic acid Asp in the CGT enzyme gene has been mutated into Methionin Lys, called after D372K; The 89th tyrosine Tyr in the CGT enzyme gene aspartic acid Asp or arginine Arg have been mutated into respectively, respectively called after Y89D and Y89R; They have higher product alpha-cylodextrin ability than wild-type CGT enzyme.
Three kinds of single-mutant enzyme D372K, the preparation method of Y89D and Y89R, according to P.macerans JFB05-01CGT enzyme gene order, design and synthesize respectively and introduce D372K, the primer of Y89D or Y89R sudden change carries out rite-directed mutagenesis to CGT enzyme gene, measure dna sequence dna, identify the 372nd Asp codon respectively and become the Lys codon, the 89th Tyr codon becomes the mutant of Asp or Arg codon respectively, and carries out escherichia coli expression.
A kind of double-mutant enzyme D372K/Y89R of P.macerans JFB 05-01 CGT enzyme: the 89th tyrosine Tyr in the single-mutant enzyme D372K gene has been mutated into arginine Arg, called after D372K/Y89R, it has higher product alpha-cylodextrin ability than wild-type CGT enzyme.
The preparation method of a kind of double-mutant enzyme D372K/Y89R, with single-mutant enzyme D372K gene is template, design and synthesize and introduce the Arg codon in 89 rite-directed mutagenesis primer, the D372K gene is carried out rite-directed mutagenesis, measure sequence, the Tyr that identifies 89 is mutated into the mutant of Arg, and carries out escherichia coli expression.
Described mutant enzyme D372K, Y89D, the preparation of Y89R and D372K/Y89R:
(1) rite-directed mutagenesis
Single-mutant enzyme D372K, the rite-directed mutagenesis of Y89D and Y89R: utilizing the fast PCR technology, is template with expression vector cgt/pET-20b (+),
The rite-directed mutagenesis primer of introducing the D372K codon is:
Forward primer: 5 '-GACCGGCGATGGC
AAACCCAACAACC-3 ' (underscore is a mutating alkali yl)
Reverse primer: 5 '-GGTTGTTGGG
TTTGCCATCGCCGGTC-3 ' (underscore is a mutating alkali yl)
The rite-directed mutagenesis primer of introducing the Y89D codon is:
Forward primer: 5 '-CTCCGTCATCAAG
GATTCCGGCGTTA-3 ' (underscore is a mutating alkali yl)
Reverse primer: 5 '-TAACGCCGGA
ATCCTTGATGACGGAG-3 ' (underscore is a mutating alkali yl)
The rite-directed mutagenesis primer of introducing the Y89R codon is:
Forward primer: 5 '-CTCCGTCATCAAG
CGTTCCGGCGTTA-3 ' (underscore is a mutating alkali yl)
Reverse primer: 5 '-TAACGCCGGA
ACGCTTGATGACGGAG-3 ' (underscore is a mutating alkali yl)
The rite-directed mutagenesis of double-mutant enzyme D372K/Y89: utilizing the fast PCR technology, is template with single-mutant enzyme D372K gene,
The rite-directed mutagenesis primer of introducing the Y89R codon is:
Forward primer: 5 '-CTCCGTCATCAAG
CGTTCCGGCGTTA-3 ' (underscore is a mutating alkali yl)
Reverse primer: 5 '-TAACGCCGGA
ACGCTTGATGACGGAG-3 ' (underscore is a mutating alkali yl)
The PCR reaction system is: 10 * Ex Taq buffer, 5 μ L, and 2.5mM dNTPs 5 μ L, forward primer (10 μ M) 1 μ L, reverse primer (10 μ M) 1 μ L, template DNA 1 μ L, Ex Taq HS (5U/ μ L) 0.25 μ L adds distilled water to 50 μ L.
The pcr amplification condition is: 94 ℃ of pre-sex change 4min; Carry out 35 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 6min) subsequently; Last 72 ℃ of insulation 10min.
The PCR product is through Dpn I (available from Canadian Fermentas company) digestion, transformed into escherichia coli JM 109 competent cells, competent cell is after LB solid medium (containing 100 μ g/mL penbritins) overnight incubation, choosing mono-clonal cultivates in LB liquid nutrient medium (containing 100 μ g/mL penbritins), plasmid is extracted in the back, mutant plasmid is transformed expression host e. coli BL21 (DE3) competent cell, and all mutant plasmids all check order correctly.
(2) expression and purification of mutant:
Picking changes the mono-clonal of expressive host e. coli bl21 (DE3) in LB liquid nutrient medium (containing 100 μ g/mL penbritins) growth 8～10h, by 4% inoculum size seed fermentation liquid is received TB liquid nutrient medium (containing 100 μ g/mL penbritins); Intestinal bacteria are cultured to OD at 30 ℃ of shaking tables
600=0.6, the IPTG that adds the 0.01mM final concentration induces born of the same parents to express outward, and continues cultivation and fermentation after 90 hours at 25 ℃ of shaking tables, fermented liquid is removed thalline in 4 ℃, the centrifugal 20min of 10000rpm, collect supernatant liquor and purifying, obtain mutant enzyme D372K respectively, Y89D, Y89R and D372K/Y89R goods.
Softening series bacillus (Peanibacillus macerans) JFB 05-01 (CCTCC NO:M208063), open at Chinese patent CN101294149A, open day on October 29th, 2008.
Beneficial effect of the present invention: the present invention has made up 4 significant mutant, has all realized the specific raising of alpha-cylodextrin product, is more conducive to the suitability for industrialized production of alpha-cylodextrin than wild-type CGT enzyme.
Description of drawings
Three kinds of cyclodextrin output of Fig. 1 wild-type CGT enzyme and mutant enzyme.A, wild-type CGT enzyme; B, mutant enzyme D372K; C, mutant enzyme Y89D; D, mutant enzyme Y89R; E, mutant enzyme D372K/Y89R.Wherein ◆ the expression alpha-cylodextrin; ■ represents beta-cyclodextrin; ▲ expression γ-Huan Hujing.
Embodiment
Embodiment 1: this example explanation mutant enzyme D372K, Y89D, Y89R, the preparation of D372K/Y89R.
1) rite-directed mutagenesis
Utilize the fast PCR technology, single-mutant enzyme D372K, the rite-directed mutagenesis of Y89D and Y89R: with expression vector cgt/pET-20b (+) is template,
The rite-directed mutagenesis primer of introducing the D372K codon is:
Forward primer: 5 '-GACCGGCGATGGC
AAACCCAACAACC-3 ' (underscore is a mutating alkali yl)
Reverse primer: 5 '-GGTTGTTGGG
TTTGCCATCGCCGGTC-3 ' (underscore is a mutating alkali yl)
The rite-directed mutagenesis primer of introducing the Y89D codon is:
Forward primer: 5 '-CTCCGTCATCAAG
GATTCCGGCGTTA-3 ' (underscore is a mutating alkali yl)
Reverse primer: 5 '-TAACGCCGGA
ATCCTTGATGACGGAG-3 ' (underscore is a mutating alkali yl)
The rite-directed mutagenesis primer of introducing the Y89R codon is:
Forward primer: 5 '-CTCCGTCATCAAG
CGTTCCGGCGTTA-3 ' (underscore is a mutating alkali yl)
Reverse primer: 5 '-TAACGCCGGA
ACGCTTGATGACGGAG-3 ' (underscore is a mutating alkali yl)
The rite-directed mutagenesis of double-mutant enzyme D372K/Y89: utilizing the fast PCR technology, is template with single-mutant enzyme D372K gene,
The rite-directed mutagenesis primer of introducing the Y89R codon is:
Forward primer: 5 '-CTCCGTCATCAAG
CGTTCCGGCGTTA-3 ' (underscore is a mutating alkali yl)
Reverse primer: 5 '-TAACGCCGGA
ACGCTTGATGACGGAG-3 ' (underscore is a mutating alkali yl)
The PCR reaction system is: 10 * Ex Taq buffer, 5 μ L, and dNTPs (2.5mM) 5 μ L, forward primer (10 μ M) 1 μ L, reverse primer (10 μ M) 1 μ L, template DNA 1 μ L, Ex Taq HS (5U/ μ L) 0.25 μ L adds distilled water to 50 μ L.
The pcr amplification condition is: 94 ℃ of pre-sex change 4min; Carry out 35 circulations (94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 6min) subsequently; Last 72 ℃ of insulation 10min.
The PCR product is through Dpn I (available from Canadian Fermentas company) digestion, transformed into escherichia coli JM109 competent cell, competent cell is after LB solid medium (containing 100 μ g/mL penbritins) overnight incubation, choosing mono-clonal cultivates in LB liquid nutrient medium (containing 100 μ g/mL penbritins), extract plasmid, mutant plasmid transforms expresses host e. coli BL21 (DE3) competent cell, and mutant plasmid all checks order correctly.
2) expression and purification of mutant enzyme:
Picking changes the mono-clonal of expressive host e. coli bl21 (DE3) in LB liquid nutrient medium (containing 100 μ g/mL penbritins) growth 8～10h, by 4% inoculum size seed fermentation liquid is received TB liquid nutrient medium (containing 100 μ g/mL penbritins); Intestinal bacteria are cultured to OD at 30 ℃ of shaking tables
600=0.6, the IPTG that adds the 0.01mM final concentration induces born of the same parents to express outward, and continues cultivation and fermentation after 90 hours at 25 ℃ of shaking tables, and fermented liquid is removed thalline in 4 ℃, the centrifugal 20min of 10000rpm, collects supernatant liquor.
Adding 70% solid ammonium sulfate is saltoutd and is spent the night in the supernatant liquor, 4 ℃, the centrifugal 20min of 10000rpm, taking precipitate dissolves with the buffer A that contains 20mM sodium phosphate, 0.5M sodium-chlor, 20mM imidazoles, pH 7.4 in right amount, and in buffer A after the dialysed overnight, by making all product behind the 0.22 μ m membrane filtration; After the Ni affinity column is used the buffer A balance, to go up all product and suck the Ni post, after making it to adsorb fully, respectively with buffer A, contain the 20-480mM imidazoles buffer A, contain the buffer A wash-out of 480mM imidazoles, flow velocity 1mL/min, the detection wavelength is 280nm, and fraction collection contains the elutriant that CGT enzyme enzyme is lived; The vigor component after the dialysed overnight, obtains purified mutant body enzyme D372K, Y89D, Y89R and D372K/Y89R respectively in 50mM sodium phosphate buffer (pH=6).
Embodiment 2: present embodiment explanation enzyme is lived and is analyzed.
1) enzyme activity determination method:
The tropeolin-D method is measured the method for α-cyclisation vigor: the enzyme liquid 0.1mL that gets suitable dilution, add and to be equipped with in 3% (w/v) Zulkovsky starch solution that 0.9mL uses 50mM phosphoric acid buffer (pH6.5) preparation in advance, behind reaction 10min under 40 ℃, the hydrochloric acid stopped reaction that adds 1.0mL 1.0N, add the 0.1mM tropeolin-D of 1.0mL again with the preparation of 50mM phosphoric acid buffer, be incubated 20min down at 16 ℃, under 505nm, measure absorbancy.An enzyme unit definition alive generates the required enzyme amount of 1 μ mol alpha-cylodextrin for per minute under this condition.
The phenolphthalein method is measured the method for β-cyclisation vigor: the enzyme liquid 0.1mL that gets suitable dilution, add and to be equipped with in the test tube of 3% (w/v) Zulkovsky starch solution that 0.9mL uses 50mM phosphoric acid buffer (pH6.5) preparation in advance, behind reaction 10min under 40 ℃, add 3.5mL 30mM NaOH and 0.5mL by 5mM Na
2CO
30.02% (w/v) phenolphthalein stopped reaction of solution preparation at room temperature is incubated 20min, measures absorbancy under 550nm.Enzyme unit definition of living generates the required enzyme amount of 1 μ mol beta-cyclodextrin for per minute under these conditions.
The method that bromine cresols chlorine method is measured γ-cyclisation vigor is as follows: the enzyme liquid 0.1mL that gets suitable dilution, add and to be equipped with in the test tube of 3% (w/v) Zulkovsky starch solution that 0.9mL uses 50mM phosphoric acid buffer (pH6.5) preparation in advance, behind reaction 10min under 40 ℃, the hydrochloric acid stopped reaction that adds 50 μ L 1.0N, add 2mL 0.2M citrate buffer solution (pH 4.2) and 100 μ L 5mM bromine cresols chlorine solution again, at room temperature be incubated 20min, under 630nm, measure absorbancy.An enzyme unit definition alive is that per minute generates the required enzyme amount of 1 μ mol γ-Huan Hujing under these conditions.
2) enzyme is lived relatively: experimental result is listed in table 1, and the pure enzyme preparation of mutant is compared with the pure enzyme preparation of wild-type, can find: though the α of single-mutant enzyme D372K-cyclisation vigor rises to some extent, β-cyclisation vigor has descended 51%; The α of Y89D-cyclisation vigor has risen 10%, and β-cyclisation vigor also has slight rising; The α of Y89R-cyclisation vigor has descended 18%, and β-cyclisation vigor has reduced by 56%; The α of double-mutant enzyme D372K/Y89R-cyclisation vigor has risen 24% than wild-type enzyme, and β-cyclisation vigor has descended 67%.Y89D, total cyclisation vigor of Y89R and D372K/Y89R has risen about 10%.And, mutant enzyme D372K, Y89D, the ratio that the α of Y89R and D372K/Y89RY89D-cyclisation vigor accounts for total cyclisation vigor is higher than wild-type enzyme.
Table 1
Embodiment 3: present embodiment explanation HPLC methods analyst cyclodextrin growing amount.
(w/v) Zulkovsky starch solution is as substrate for wet basis, water content 8%, and the 5g starch dissolution is settled to 100mL in 90mL sodium phosphate buffer (pH6.0), boil 30min in boiling water in preparation 5%.Add a certain amount of wild CGT enzyme, mutant enzyme D372K respectively, Y89D, Y89R or D372K/Y89R make the enzyme 0.2U/mL of being alive in the reaction system, place 40 ℃ of reaction 40h down, the interval 600 μ L that take a sample, the centrifugal 10min of 12000rpm gets supernatant 500 μ L, adds 5 μ L saccharifying enzyme (70U/mL), at 30 ℃ of saccharification 1h, 10min boils deactivation, and the centrifugal 30min of 12000rpm gets and gets that HPLC analyzes on the 20 μ L behind the supernatant 0.45 μ m ultrafiltration membrance filter.
α in the reaction solution-, β-and the concentration of γ-Huan Hujing adopt HPLC to measure, the chromatographic condition that adopts HPLC to carry out product analysis is: Waters 600HPLC chromatographic instrument, Waters automatic sampler, chromatographic column Lichrosorb NH
2(4.6mm * 150mm), Waters2410 differential detector; Moving phase (V/V) is 65% acetonitrile solution, flow velocity 1mL min
-130 ℃ of column temperatures.
Experimental result is listed in Fig. 1, and the pure enzyme preparation of mutant that the said mutation body surface is reached acquisition is compared with the pure enzyme preparation of wild-type, can find, and mutant D372K, Y89D, Y89R, D372K/Y89R have realized the raising of alpha-cylodextrin throughput.Table 3 as can be seen, after cultivating by 40h, the alpha-cylodextrin output of single-mutant enzyme D372K has increased by 29%, beta-cyclodextrin output has reduced 23%; The alpha-cylodextrin output of Y89D has increased by 12%, and beta-cyclodextrin output has reduced 10%, and the alpha-cylodextrin output of Y89R has risen 35%, and beta-cyclodextrin output has descended 30%; Double-mutant enzyme D372K/Y89R is better than wild-type enzyme and single-mutant enzyme, alpha-cylodextrin output has increased by 50%, beta-cyclodextrin rate ratio wild-type has reduced 43%, the final cyclodextrin ratios of wild-type enzyme is 41.8: 54.4: 3.8, and the final cyclodextrin ratios of double-mutant enzyme is 65: 31.9: 3.1.
Table 3
Sequence table
<210>SEQ?ID?NO：1
Forward primer: 5 '-GACCGGCGATGGCAAACCCAACAACC-3 '
Reverse primer: 5 '-GGTTGTTGGGTTTGCCATCGCCGGTC-3 '
<210>SEQ?ID?NO：2
Forward primer: 5 '-CTCCGTCATCAAGGATTCCGGCGTTA-3 '
Reverse primer: 5 '-TAACGCCGGAATCCTTGATGACGGAG-3 '
<210>SEQ?ID?NO：3
Forward primer: 5 '-CTCCGTCATCAAGCGTTCCGGCGTTA-3 '
Reverse primer: 5 '-TAACGCCGGAACGCTTGATGACGGAG-3 '
Claims (4)
1. derive from the preparation method of single-mutant enzyme D372K of the cyclomaltodextrin glucanotransferase of softening series bacillus (Peanibacillus macerans) JFB 05-01, it is characterized in that:
(1) rite-directed mutagenesis
Utilizing the fast PCR technology, is template with expression vector cgt/pET-20b (+),
The rite-directed mutagenesis primer of introducing the D372K codon is:
Forward primer: 5 '-GACCGGCGATGGC
AAACCCAACAACC-3 ', underscore are mutating alkali yl,
Reverse primer: 5 '-GGTTGTTGGG
TTTGCCATCGCCGGTC-3 ', underscore are mutating alkali yl,
The PCR reaction system is: 10 * Ex Taq buffer, 5 μ L, and 2.5mM dNTPs 5 μ L, 10 μ M forward primers, 1 μ L, 10 μ M reverse primers, 1 μ L, template DNA 1 μ L, 5U/ μ L Ex Taq HS 0.25 μ L adds distilled water to 50 μ L;
The pcr amplification condition is: 94 ℃ of pre-sex change 4min; Carry out 94 ℃ of 30s subsequently, 55 ℃ of 30s, 35 circulations of 72 ℃ of 6min; Last 72 ℃ of insulation 10min;
The PCR product digests through Dpn I, transformed into escherichia coli JM109 competent cell, competent cell is after the LB solid medium overnight incubation that contains 100 μ g/mL penbritins, choosing mono-clonal cultivates in the LB liquid nutrient medium that contains 100 μ g/mL penbritins, plasmid is extracted in the back, mutant plasmid is transformed expression host e. coli BL21 (DE3) competent cell, and the mutant plasmid order-checking is correct;
(2) expression and purification of mutant:
Picking changes the mono-clonal of expressive host e. coli bl21 (DE3) in the LB liquid culture basal growth 8～10h that contains 100 μ g/mL penbritins, seed fermentation liquid is received the TB liquid nutrient medium that contains 100 μ g/mL penbritins by 4% inoculum size; Intestinal bacteria are cultured to OD at 30 ℃ of shaking tables
600=0.6, the IPTG that adds the 0.01mM final concentration induces born of the same parents to express outward, and continues cultivation and fermentation after 90 hours at 25 ℃ of shaking tables, and fermented liquid is removed thalline in 4 ℃, the centrifugal 20min of 10000rpm, collects supernatant liquor and purifying, obtains mutant enzyme D372K goods.
2. derive from the preparation method of single-mutant enzyme Y89D of the cyclomaltodextrin glucanotransferase of softening series bacillus (Peanibacillus macerans) JFB 05-01, it is characterized in that:
(1) rite-directed mutagenesis
Utilizing the fast PCR technology, is template with expression vector cgt/pET-20b (+),
The rite-directed mutagenesis primer of introducing the Y89D codon is:
Forward primer: 5 '-CTCCGTCATCAAG
GATTCCGGCGTTA-3 ', underscore are mutating alkali yl,
Reverse primer: 5 '-TAACGCCGGA
ATCCTTGATGACGGAG-3 ', underscore are mutating alkali yl,
The PCR reaction system is: 10 * Ex Taq buffer, 5 μ L, and 2.5mM dNTPs 5 μ L, 10 μ μ M forward primers, 1 μ L, 10 μ M reverse primers, 1 μ L, template DNA 1 μ L, 5U/ μ L Ex Taq HS 0.25 μ L adds distilled water to 50 μ L;
The pcr amplification condition is: 94 ℃ of pre-sex change 4min; Carry out 94 ℃ of 30s subsequently, 55 ℃ of 30s, 35 circulations of 72 ℃ of 6min; Last 72 ℃ of insulation 10min;
The PCR product digests through Dpn I, transformed into escherichia coli JM109 competent cell, competent cell is after the LB solid medium overnight incubation that contains 100 μ g/mL penbritins, choosing mono-clonal cultivates in the LB liquid nutrient medium that contains 100 μ g/mL penbritins, plasmid is extracted in the back, mutant plasmid is transformed expression host e. coli BL21 (DE3) competent cell, and the mutant plasmid order-checking is correct;
(2) expression and purification of mutant:
Picking changes the mono-clonal of expressive host e. coli bl21 (DE3) in the LB liquid culture basal growth 8～10h that contains 100 μ g/mL penbritins, seed fermentation liquid is received the TB liquid nutrient medium that contains 100 μ g/mL penbritins by 4% inoculum size; Intestinal bacteria are cultured to OD at 30 ℃ of shaking tables
600=0.6, the IPTG that adds the 0.01mM final concentration induces born of the same parents to express outward, and continues cultivation and fermentation after 90 hours at 25 ℃ of shaking tables, and fermented liquid is removed thalline in 4 ℃, the centrifugal 20min of 10000rpm, collects supernatant liquor and purifying, obtains mutant enzyme Y89D goods.
3. derive from the preparation method of single-mutant enzyme Y89R of the cyclomaltodextrin glucanotransferase of softening series bacillus (Peanibacillus macerans) JFB 05-01, it is characterized in that:
(1) rite-directed mutagenesis
Utilizing the fast PCR technology, is template with expression vector cgt/pET-20b (+),
The rite-directed mutagenesis primer of introducing the Y89R codon is:
Forward primer: 5 '-CTCCGTCATCAAG
CGTTCCGGCGTTA-3 ', underscore are mutating alkali yl,
Reverse primer: 5 '-TAACGCCGGA
ACGCTTGATGACGGAG-3 ', underscore are mutating alkali yl,
The PCR reaction system is: 10 * Ex Taq buffer, 5 μ L, and 2.5mM dNTPs 5 μ L, 10 μ M forward primers, 1 μ L, 10 μ M reverse primers, 1 μ L, template DNA 1 μ L, 5U/ μ L Ex Taq HS 0.25 μ L adds distilled water to 50 μ L;
The pcr amplification condition is: 94 ℃ of pre-sex change 4min; Carry out 94 ℃ of 30s subsequently, 55 ℃ of 30s, 35 circulations of 72 ℃ of 6min; Last 72 ℃ of insulation 10min;
The PCR product digests through Dpn I, transformed into escherichia coli JM109 competent cell, competent cell is after the LB solid medium overnight incubation that contains 100 μ g/mL penbritins, choosing mono-clonal cultivates in the LB liquid nutrient medium that contains 100 μ g/mL penbritins, plasmid is extracted in the back, mutant plasmid is transformed expression host e. coli BL21 (DE3) competent cell, and the mutant plasmid order-checking is correct;
(2) expression and purification of mutant:
Picking changes the mono-clonal of expressive host e. coli bl21 (DE3) in the LB liquid culture basal growth 8～10h that contains 100 μ g/mL penbritins, seed fermentation liquid is received the TB liquid nutrient medium that contains 100 μ g/mL penbritins by 4% inoculum size; Intestinal bacteria are cultured to OD at 30 ℃ of shaking tables
600=0.6, the IPTG that adds the 0.01mM final concentration induces born of the same parents to express outward, and continues cultivation and fermentation after 90 hours at 25 ℃ of shaking tables, and fermented liquid is removed thalline in 4 ℃, the centrifugal 20min of 10000rpm, collects supernatant liquor and purifying, obtains mutant enzyme Y89R goods.
4. derive from the preparation of double-mutant enzyme D372K/Y89R of the cyclomaltodextrin glucanotransferase of softening series bacillus (Peanibacillus macerans) JFB 05-01, it is characterized in that:
(1) rite-directed mutagenesis
Utilizing the fast PCR technology, is template with the described single-mutant enzyme D372K of claim 1 gene, and the rite-directed mutagenesis primer of introducing the Y89R codon is:
Forward primer: 5 '-CTCCGTCATCAAG
CGTTCCGGCGTTA-3 ', underscore are mutating alkali yl,
Reverse primer: 5 '-TAACGCCGGA
ACGCTTGATGACGGAG-3 ', underscore are mutating alkali yl,
The PCR reaction system is: 10 * Ex Taq buffer, 5 μ L, and 2.5mM dNTPs 5 μ L, 10 μ M forward primers, 1 μ L, 10 μ M reverse primers, 1 μ L, template DNA 1 μ L, 5U/ μ L Ex Taq HS 0.25 μ L adds distilled water to 50 μ L;
The pcr amplification condition is: 94 ℃ of pre-sex change 4min; Carry out 94 ℃ of 30s subsequently, 55 ℃ of 30s, 35 circulations of 72 ℃ of 6min; Last 72 ℃ of insulation 10min;
The PCR product digests through Dpn I, transformed into escherichia coli JM109 competent cell, competent cell is after the LB solid medium overnight incubation that contains 100 μ g/mL penbritins, choosing mono-clonal cultivates in the LB liquid nutrient medium that contains 100 μ g/mL penbritins, plasmid is extracted in the back, mutant plasmid is transformed expression host e. coli BL21 (DE3) competent cell, and the mutant plasmid order-checking is correct;
(2) expression and purification of mutant:
Picking changes the mono-clonal of expressive host e. coli bl21 (DE3) in the LB liquid culture basal growth 8～10h that contains 100 μ g/mL penbritins, seed fermentation liquid is received the TB liquid nutrient medium that contains 100 μ g/mL penbritins by 4% inoculum size; Intestinal bacteria are cultured to OD at 30 ℃ of shaking tables
600=0.6, the IPTG that adds the 0.01mM final concentration induces born of the same parents to express outward, and continues cultivation and fermentation after 90 hours at 25 ℃ of shaking tables, and fermented liquid is removed thalline in 4 ℃, the centrifugal 20min of 10000rpm, collects supernatant liquor and purifying, obtains mutant enzyme D372K/Y89R goods.
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| Bart A. van der Veen1等.Rational Design of Cyclodextrin Glycosyltransferase from Bacillus circulans Strain 251 to Increase a-Cyclodextrin Production.《J. Mol. Biol》.2000,第296卷1027－1038. * |
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