CN101215554A - Fire resistant L-arabinose isomerase and gene sequence thereof - Google Patents
Fire resistant L-arabinose isomerase and gene sequence thereof Download PDFInfo
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- CN101215554A CN101215554A CNA2008100191094A CN200810019109A CN101215554A CN 101215554 A CN101215554 A CN 101215554A CN A2008100191094 A CNA2008100191094 A CN A2008100191094A CN 200810019109 A CN200810019109 A CN 200810019109A CN 101215554 A CN101215554 A CN 101215554A
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Abstract
High temperature resistant L-arabinose isomerase and the gene order belong to the technical field of bioengineering. The invention offers high temperature resistant L-arabinose isomerase which derives from bacillus stearothermophilus, the gene order is SEQ ID NO: 1, and the amino acid composition is SEQ ID NO: 2. The invention also discloses the construction of high temperature resistant L-arabinose isomerase gene engineering bacterial strain and the method for expressing with high efficiency and purify the high temperature resistant L-arabinose isomerase. The high temperature resistant L-arabinose isomerase which is provided by the invention has higher activity and stability under the higher temperature condition and has broad prospect and economic significance.
Description
Technical field
The present invention relates to be derived from a kind of fire resistant L-arabinose isomerase of bacstearothermophilus (Bacillus stearothermophilus) IAM11001 and the gene order of coding thereof, the structure of fire resistant L-arabinose isomerase engineering bacteria, and the method with purifying of efficiently expressing of fire resistant L-arabinose isomerase, belong to technical field of bioengineering.
Background technology
The D-tagatose is a kind of functional sweetener with special nourishing function of finding in recent years.The D-tagatose belongs to a kind of of natural rare sugar, all finds a small amount of the existence in numerous food product, as high-temperature sterilization milk, cheese, sour milk and other milk preparation etc.2000 FDA (FDA) determine that the D-tagatose is a general generally recognized as safe food (GRAS), and official approval is used for food beverage industry and pharmaceutical preparation as sweeting agent; The approval of the 57th meeting of the Food and Argriculture OrganizationFAO and the combination food additive council of the World Health Organization (JECFA) subsequently D-tagatose is as foodstuff additive; European Union also goes on the market in Europe in approval D-tagatose in 2003.
At present, domestic research for the production of D-tagatose also is in the starting stage, and comparatively deep to its research abroad, production method mainly contains two kinds: chemical method and biological process.But commercially available D-tagatose is all produced by chemical method at present.Because there are many unfavorable factors on the one hand in chemical method production, many as chemical pollutant, the isomerization reaction by product is many under the alkaline condition, separation difficulty etc.; On the other hand because the transformation of consumer spending idea is growing to the demand of whole food, therefore in recent years the research of tagatose production is concentrated on the biological process.Discovering that L-arabinose isomerase (EC 5.3.1.4 is abbreviated as L-AI) can be converted into the D-tagatose for catalysis D-semi-lactosi, is that present biological process is produced D-tagatose valid approach the most.The balance temperature influence of isomerization reaction is very big between D-semi-lactosi and the D-tagatose, and (more than 60 ℃) more help the generation of D-tagatose under the higher temperature conditions, and therefore stable on heating L-arabinose isomerase will have the better application prospect.
Summary of the invention
The gene order that the purpose of this invention is to provide a kind of fire resistant L-arabinose isomerase and coding thereof, the structure of fire resistant L-arabinose isomerase engineering bacteria, and the method with purifying of efficiently expressing of fire resistant L-arabinose isomerase.This fire resistant L-arabinose isomerase has preferably active and stable under the higher temperatures condition, is with a wide range of applications and economic implications for the suitability for industrialized production of new type functional sweetener D-tatai sugar.
Technical scheme of the present invention: a kind of fire resistant L-arabinose isomerase that derives from bacstearothermophilus (Bacillusstearothermophilus) IAM11001, its gene nucleotide series are SEQ ID NO:1.
[galactosidase gene is overexpression and IPTG inductive condition in intestinal bacteria at document the sixth of the twelve Earthly Branches for bacstearothermophilus (Bacillus stearothermophilus) IAM11001, Wuxi Light Industry Univ.'s journal the 21st volume in September, 2002 the 5th phase] in open, the inventor promises to undertake that this microorganism provided to the public in 20 years.
Described fire resistant L-arabinose isomerase, its aminoacid sequence are SEQ ID NO:2.
The expression method of described fire resistant L-arabinose isomerase:
(1) structure contains fire resistant L-arabinose isomerase expression of gene plasmid:
Get the total dna solution 3 μ L of bacstearothermophilus IAM11001 as masterplate, as primer, obtain desired dna fragmentation through pcr amplification with the following nucleotide sequences of the restriction enzyme site that comprises BamHI and XhoI;
Primer 1:5 '-TGATGGATCCCATGCTGTCATTACGTCCT-3 '
Primer 2: 5 '-TATATACTCGAGTTACCGCCCCCGCCAGAATAC-3 '
The PCR reaction conditions is: 94 ℃ of 5min, a circulation; 94 ℃ of 60s, 56 ℃ of 60s, 72 ℃ of 90s, 35 circulations; Last 72 ℃ are extended 10min;
Reclaim the PCR product, carry out double digestion through restriction enzyme BamHI and XhoI, the plasmid pET-22b (+) with through same double digestion connects under the effect of T4 ligase enzyme, obtains recombinant plasmid pET-22b (+)-araA;
(2) with this recombinant plasmid transformed to host cell: recombinant plasmid pET-22b (+)-araA is converted in the competence e. coli bl21, coating contains on the antibiotic LB solid medium of 50 μ g/mL ammonia benzyls, cultivates 18-24h for 37 ℃ and obtains preliminary positive colony;
(3) obtain positive colony through the screening of resistance substratum: the preliminary positive colony of picking contains in the antibiotic LB liquid nutrient medium of 50 μ g/mL ammonia benzyls in 5mL respectively, 37 ℃, the 200rpm overnight incubation, extract plasmid, through restriction enzyme BamHI and XhoI digested plasmid, the plasmid of judging the dna fragmentation with sequence table SEQ ID NO:1 according to electrophoresis result is recombinant plasmid pET-22b (+)-araA, has the positive clone of bacterium colony of this plasmid, called after engineering bacteria BL21-AI;
Recombinant plasmid pET-22b (+)-araA is checked order, and the result shows that the insertion fragment is one and contains 1491bp, the protein of being made up of 496 amino acid of encoding.
Beneficial effect of the present invention: the gene order that the invention provides a kind of fire resistant L-arabinose isomerase and coding thereof, the structure of fire resistant L-arabinose isomerase engineering bacteria, and the method with purifying of efficiently expressing of fire resistant L-arabinose isomerase.This fire resistant L-arabinose isomerase has preferably active and stable under the higher temperatures condition, is with a wide range of applications and economic implications for the suitability for industrialized production of new type functional sweetener D-tatai sugar.
Description of drawings
The structure synoptic diagram of Fig. 1 recombinant plasmid pET-22b (+)-araA.
Embodiment
The extraction of embodiment 1 bacillus stearothermophilus (Bacillus stearothermophilus) the total DNA of IAM11001
Get fresh bacillus stearothermophilus (Bacillus stearothermophilus) IAM11001 wet thallus 20g, be suspended from the 10mL 50mM Tris-HCl damping fluid (pH8.0).
Add a small amount of N,O-Diacetylmuramidase and 8mL 0.25mM EDTA (pH8.0), 37 ℃ of static 20min behind the mixing.
Add 2mL 10% sodium laurylsulfonate (SDS), 55 ℃ of static 5min.
Use equal-volume phenol, each extracting of chloroform respectively successively once.
Get last supernatant, add 2 times of volume of ethanol, reclaim DNA.
With 70% ethanol, dehydrated alcohol washing precipitation successively respectively.
Precipitation is dissolved in 0.5mL TE damping fluid, and (10mM Tris, 1mM EDTA pH8.0), add 3 μ L10mg/mL RNase, 37 ℃ of insulation 1h.
Use equal-volume phenol, each extracting of chloroform respectively successively once, supernatant adds 2 times of volume of ethanol, reclaims DNA.
With 70% ethanol, dehydrated alcohol washing precipitation successively respectively.
Precipitation is dissolved in 0.5mL TE damping fluid, and (pH8.0) ,-20 ℃ of preservations are standby for 10mM Tris, 1mM EDTA.
The clone of embodiment 2 fire resistant L-arabinose isomerase genes
1. get the total dna solution 3 μ L of bacstearothermophilus as masterplate, as primer (restriction enzyme site that comprises BamHI and XhoI), obtain desired dna fragmentation through pcr amplification with following nucleotide sequences.
Primer sequence is as follows:
Primer 1:5 '-TGATGGATCCCATGCTGTCATTACGTCCT-3 '
Primer 2: 5 '-TATATACTCGAGTTACCGCCCCCGCCAGAATAC-3 '
The PCR reaction conditions is: 94 ℃ of 5min, a circulation; 94 ℃ of 60s, 56 ℃ of 60s, 72 ℃ of 90s, 35 circulations; Last 72 ℃ are extended 10min.
2. reclaim the PCR product, carry out double digestion through restriction enzyme BamHI and XhoI, the plasmid pET-22b (+) with through same double digestion connects under the effect of T4 ligase enzyme, obtains recombinant plasmid pET-22b (+)-araA.
3. recombinant plasmid pET-22b (+)-araA is converted in the competence e. coli bl21, coating contains on the antibiotic LB solid medium of 50 μ g/mL ammonia benzyls, cultivates 18-24h for 37 ℃ and obtains preliminary positive colony.
4. the preliminary positive colony of picking contains in the antibiotic LB liquid nutrient medium of 50 μ g/mL ammonia benzyls in 5mL respectively, 37 ℃, the 200rpm overnight incubation, extract plasmid, through restriction enzyme BamHI and XhoI double digestion plasmid, judge that according to electrophoresis result the plasmid with this dna fragmentation (SEQ ID NO:1 in the sequence table) is recombinant plasmid pET-22b (+)-araA, have the positive clone of bacterium colony of this plasmid, called after engineering bacteria BL21-AI.
5. recombinant plasmid pET-22b (+)-araA is checked order, the result shows that the insertion fragment is one and contains 1491 bp, the protein of being made up of 496 amino acid of encoding.
Embodiment 3 fire resistant L-arabinose isomerase genes efficiently expressing in engineering bacteria
Engineering bacteria BL21-AI is in containing the antibiotic LB liquid nutrient medium of 50 μ g/mL ammonia benzyls, 37 ℃, the 200rpm overnight incubation, be forwarded in the antibiotic LB liquid nutrient medium of fresh ammonia benzyl, 37 ℃, when 200rpm was cultured to the OD value for 0.6-0.8, the adding final concentration was that 30 ℃ of abduction delivering 4 h of isopropyl-(IPTG) of 0.5mM reach the highest amount of inducing.
The purifying and the characteristic thereof of embodiment 4 recombination high temperature-resistant L-arabinose isomerases
The gained thalline is in 50mM pH7.5 Tris-HCl damping fluid behind the abduction delivering, ultrasonic cell-break, and the supernatant liquor after centrifugal is reorganization L-arabinose isomerase crude enzyme liquid.Crude enzyme liquid is 10 through the molecular weight that dams, after the ultra-filtration membrane of 000Da concentrates, concentrated solution carries out purifying through DEAE-Sepharose Fast Flow and SepharoseCL-6B chromatography column to recombinant protein again, purifying is single band after SDS-PAGE detects, and show that the proteic molecular weight of reorganization L-arabinose isomerase is 59KDa, the temperature of recombinase reactivity is 60-80 ℃, and pH is 7.0-8.0.
Sequence table
<210>SEQ?ID?NO：1
<211>1491
<212>DNA
＜213〉bacillus stearothermophilus (Bacillus stearothermophilus) IAM11001
<400>1
atgctgtcat?tacgtcctta?tgaattttgg?tttgtgacag?gaagtcagca?cttgtacgga?60
gaagaagcgt?taaaacaagt?cgaagaacat?tcaagaatca?tggtcaatga?atggaatcgc?120
gattcggtgt?ttccgttccc?attcgttttc?aaatcagtcg?tgacgacgcc?agaggaaatc?180
cggcgcgttt?gccttgaggc?gaatgcgagc?gaacaatgtg?ccggcgttgt?cacatggatg?240
catacgttct?cgccagcgaa?aatgtggatt?ggcggccttt?tggagttgag?aaaaccgtta?300
ttgcatcttc?acacccagtt?taaccgtggt?attccgtggg?acagcatcga?tatggacttt?360
atgaacttaa?accaatcagc?tcacggtgac?cgagaatacg?gatttatcgg?tgcgagaatg?420
ggagttgccc?ggaaagtggt?cgttggtcat?tgggaagacc?cagaagtccg?cgagcggctg?480
gcgaaatgga?tgcggacggc?cgtcgccttt?gcggaaagcc?gacatcttaa?agttgcccgt?540
ttcggcgata?acatgcgtga?agtggcggta?acggaagggg?acaaagtggg?agcgcaaatt?600
caattcggct?ggtcgatcaa?cggttatggc?attggggatt?tggtgcaatc?tattcgcgat?660
gtttctgaac?aaagcgttaa?cgaactgctt?gatgaatatg?ccgaactgta?tgacattgta?720
cctgctggcc?gtcaagatgg?accggttcgc?gagtcgatcc?gtgagcaggc?gcggattgag?780
cttgggttaa?aagccttttt?gcaagacggg?aacttcactg?cctttacgac?gacgtttgaa?840
gatttgcacg?gcatgaagca?acttccagga?ctagcggttc?aacggcttat?ggcagaggga?900
tatggatttg?gcggcgaagg?cgactggaaa?acggctgctc?tcgttcggtt?gatgaaagtc?960
atggcggatg?gcaaaggaac?atcgttcatg?gaagactaca?cgtaccactt?tgagccgggc?1020
aacgaactga?ttcttggcgc?gcatatgctc?gaagtatgcc?cgacgattgc?cgcaacccgg?1080
ccgcgcatcg?aagtccatcc?gctttcgatc?ggtggaaaag?aagatccagc?ccgtctcgtg?1140
tttgacggcg?gcgagggtgc?tgcggtcaat?gcttcgttga?ttgacttagg?acaccgtttc?1200
cgtctcattg?tcaatgaagt?cgatgcagtg?aaaccagaac?atgagatgcc?gaaactgcct?1260
gttgcgcgca?ttctatggaa?gccgcgccca?tcgctccgcg?attcggccga?agcatggatt?1320
ttagccggcg?gagcgcatca?cacatgcttc?tcgtttgcgg?tcacggctga?acagctgcaa?1380
gactttgcgg?aaatggcggg?cattgaatgc?gtcgtgatca?atgaacatac?gtccgtctcg?1440
tccttcaaaa?atgaactgaa?atggaacgaa?gtattctggc?gggggcggta?a 1491
<210>SEQ?ID?NO：2
<211>496
<212>PRT
＜213〉bacillus stearothermophilus (Bacillus stearothermophilus) IAM11001
<400>2
Met?Leu?Ser?Leu?Arg?Pro?Tyr?Glu?Phe?Trp?Phe?Val?Thr?Gly?Ser
5 10 15
Gln?His?Leu?Tyr?Gly?Glu?Glu?Ala?Leu?Lys?Gln?Val?Glu?Glu?His
20 25 30
Ser?Arg?Ile?Met?Val?Ash?Glu?Trp?Asn?Arg?Asp?Ser?Val?Phe?Pro
35 40 45
Phe?Pro?Phe?Val?Phe?Lys?Ser?Val?Val?Thr?Thr?Pro?Glu?Glu?Ile
50 55 60
Arg?Arg?Val?Cys?Leu?Glu?Ala?Asn?Ala?Ser?Glu?Gln?Cys?Ala?Gly
65 70 75
Val?Val?Thr?Trp?Met?His?Thr?Phe?Ser?Pro?Ala?Lys?Met?Trp?Ile
80 85 90
Gly?Gly?Leu?Leu?Glu?Leu?Arg?Lys?Pro?Leu?Leu?His?Leu?His?Thr
95 100 105
Gln?Phe?Ash?Arg?Gly?Ile?Pro?Trp?Asp?Ser?Ile?Asp?Met?Asp?Phe
110 115 120
Met?Ash?Leu?Ash?Gln?Ser?Ala?His?Gly?Asp?Arg?Glu?Tyr?Gly?Phe
125 130 135
Ile?Gly?Ala?Arg?Met?Gly?Val?Ala?Arg?Lys?Val?Val?Val?Gly?His
140 145 150
Trp?Glu?Asp?Pro?Glu?Val?Arg?Glu?Arg?Leu?Ala?Lys?Trp?Met?Arg
155 160 165
Thr?Ala?Val?Ala?Phe?Ala?Glu?Ser?Arg?His?Leu?Lys?Val?Ala?Arg
170 175 180
Phe?Gly?Asp?Asn?Met?Arg?Glu?Val?Ala?Val?Thr?Glu?Gly?Asp?Lys
185 190 195
Val?Gly?Ala?Gln?Ile?Gln?Phe?Gly?Trp?Ser?Ile?Asn?Gly?Tyr?Gly
200 205 210
Ile?Gly?Asp?Leu?Val?Gln?Ser?Ile?Arg?Asp?Val?Ser?Glu?Gln?Ser
215 220 225
Val?Asn?Glu?Leu?Leu?Asp?Glu?Tyr?Ala?Glu?Leu?Tyr?Asp?Ile?Val
230 235 240
Pro?Ala?Gly?Arg?Gln?Asp?Gly?Pro?Val?Arg?Glu?Ser?Ile?Arg?Glu
245 250 255
Gln?Ala?Arg?Ile?Glu?Leu?Gly?Leu?Lys?Ala?Phe?Leu?Gln?Asp?Gly
260 265 270
Ash?Phe?Thr?Ala?Phe?Thr?Thr?Thr?Phe?Glu?Asp?Leu?His?Gly?Met
275 280 285
Lys?Gln?Leu?Pro?Gly?Leu?Ala?Val?Gln?Arg?Leu?Met?Ala?Glu?Gly
290 295 300
Tyr?Gly?Phe?Gly?Gly?Glu?Gly?Asp?Trp?Lys?Thr?Ala?Ala?Leu?Val
305 310 315
Arg?Leu?Met?Lys?Val?Met?Ala?Asp?Gly?Lys?Gly?Thr?Ser?Phe?Met
320 325 330
Glu?Asp?Tyr?Thr?Tyr?His?Phe?Glu?Pro?Gly?Asn?Glu?Leu?Ile?Leu
335 340 345
Gly?Ala?His?Met?Leu?Glu?Val?Cys?Pro?Thr?Ile?Ala?Ala?Thr?Arg
350 355 360
Pro?Arg?Ile?Glu?Val?His?Pro?Leu?Ser?Ile?Gly?Gly?Lys?Glu?Asp
365 370 375
Pro?Ala?Arg?Leu?Val?Phe?Asp?Gly?Gly?Glu?Gly?Ala?Ala?Val?Asn
380 385 390
Ala?Ser?Leu?Ile?Asp?Leu?Gly?His?Arg?Phe?Arg?Leu?Ile?Val?Asn
395 400 405
Glu?Val?Asp?Ala?Val?Lys?Pro?Glu?His?Glu?Met?Pro?Lys?Leu?Pro
410 415 420
Val?Ala?Arg?Ile?Leu?Trp?Lys?Pro?Arg?Pro?Ser?Leu?Arg?Asp?Ser
425 430 435
Ala?Glu?Ala?Trp?Ile?Leu?Ala?Gly?Gly?Ala?His?His?Thr?Cys?Phe
440 445 450
Ser?Phe?Ala?Val?Thr?Ala?Glu?Gln?Leu?Gln?Asp?Phe?Ala?Glu?Met
455 460 465
Ala?Gly?Ile?Glu?Cys?Val?Val?Ile?Asn?Glu?His?Thr?Ser?Val?Ser
470 475 480
Ser?Phe?Lys?Asn?Glu?Leu?Lys?Trp?Asn?Glu?Val?Phe?Trp?Arg?Gly
485 490 495
Arg
496
Claims (3)
1. fire resistant L-arabinose isomerase that derives from bacstearothermophilus (Bacillus stearothermophilus) IAM11001, its gene nucleotide series is SEQ ID NO:1.
2. fire resistant L-arabinose isomerase as claimed in claim 1, its aminoacid sequence are SEQ IDNO:2.
3. the expression method of fire resistant L-arabinose isomerase as claimed in claim 1 is characterized in that:
(1) structure contains fire resistant L-arabinose isomerase expression of gene plasmid:
Get the total dna solution 3 μ L of bacstearothermophilus IAM11001 as masterplate, as primer, obtain desired dna fragmentation through pcr amplification with the following nucleotide sequences of the restriction enzyme site that comprises BamHI and XhoI;
Primer 1:5 '-TGATGGATCCCATGCTGTCATTACGTCCT-3 '
Primer 2: 5 '-TATATACTCGAGTTACCGCCCCCGCCAGAATAC-3 '
The PCR reaction conditions is: 94 ℃ of 5min, a circulation; 94 ℃ of 60s, 56 ℃ of 60s, 72 ℃ of 90s, 35 circulations; Last 72 ℃ are extended 10min;
Reclaim the PCR product, carry out double digestion through restriction enzyme BamHI and XhoI, the plasmid pET-22b (+) with through same double digestion connects under the effect of T4 ligase enzyme, obtains recombinant plasmid pET-22b (+)-araA;
(2) with this recombinant plasmid transformed to host cell: recombinant plasmid pET-22b (+)-araA is converted in the competence e. coli bl21, coating contains on the antibiotic LB solid medium of 50 μ g/mL ammonia benzyls, cultivates 18-24h for 37 ℃ and obtains preliminary positive colony;
(3) obtain positive colony through the screening of resistance substratum: the preliminary positive colony of picking contains in the antibiotic LB liquid nutrient medium of 50 μ g/mL ammonia benzyls in 5mL respectively, 37 ℃, the 200rpm overnight incubation, extract plasmid, through restriction enzyme BamHI and XhoI digested plasmid, the plasmid of judging the dna fragmentation with sequence table SEQ ID NO:1 according to electrophoresis result is recombinant plasmid pET-22b (+)-araA, has the positive clone of bacterium colony of this plasmid, called after engineering bacteria BL21-AI;
Recombinant plasmid pET-22b (+)-araA is checked order, and the result shows that the insertion fragment is one and contains 1491bp, the protein of being made up of 496 amino acid of encoding.
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| CNA2008100191094A CN101215554A (en) | 2008-01-08 | 2008-01-08 | Fire resistant L-arabinose isomerase and gene sequence thereof |
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|---|---|---|---|
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845429A (en) * | 2010-04-21 | 2010-09-29 | 南京工业大学 | High temperature resistant L-arabinose isomerase and application thereof |
| CN101768581B (en) * | 2010-02-20 | 2012-05-23 | 江南大学 | Mutant enzyme L20A of L-arabinose isomerase with D-tagatose high-yield capability and mutation method thereof |
| CN110951717A (en) * | 2019-12-28 | 2020-04-03 | 浙江工业大学 | L-arabinose isomerase isomer and application thereof |
| CN111235137A (en) * | 2020-03-02 | 2020-06-05 | 江苏大学 | L-arabinose isomerase and application thereof |
| CN112813088A (en) * | 2021-01-08 | 2021-05-18 | 上海咏科生物科技有限公司 | Preparation method of recombinant DpnI restriction endonuclease |
-
2008
- 2008-01-08 CN CNA2008100191094A patent/CN101215554A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101768581B (en) * | 2010-02-20 | 2012-05-23 | 江南大学 | Mutant enzyme L20A of L-arabinose isomerase with D-tagatose high-yield capability and mutation method thereof |
| CN101845429A (en) * | 2010-04-21 | 2010-09-29 | 南京工业大学 | High temperature resistant L-arabinose isomerase and application thereof |
| CN101845429B (en) * | 2010-04-21 | 2011-08-31 | 南京工业大学 | High temperature resistant L-arabinose isomerase and application thereof |
| CN110951717A (en) * | 2019-12-28 | 2020-04-03 | 浙江工业大学 | L-arabinose isomerase isomer and application thereof |
| CN110951717B (en) * | 2019-12-28 | 2023-08-18 | 浙江工业大学 | L-arabinose isomerase isomer and application thereof |
| CN111235137A (en) * | 2020-03-02 | 2020-06-05 | 江苏大学 | L-arabinose isomerase and application thereof |
| CN112813088A (en) * | 2021-01-08 | 2021-05-18 | 上海咏科生物科技有限公司 | Preparation method of recombinant DpnI restriction endonuclease |
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