CN109097349A - A kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase, its encoding gene and its expression and application - Google Patents
A kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase, its encoding gene and its expression and application Download PDFInfo
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Abstract
The present invention provides a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI, the base sequence of gene is as shown in SEQ ID No.1, 2- phenylacetyl-benzimidazole -7- the carboxylic acid synthetase obtained after 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI translation, it include that can synthesize 2- phenylacetyl-benzimidazole -7- carboxylic acid after 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombinant bacterium ferments, 2- phenylacetyl-benzimidazole -7- carboxylic acid has hydrophobic oleophobic property, it is a kind of uranidin, the dyestuff being used to prepare, color fastness is preferable, be not easy because and water, oil contact and fade.
Description
Technical field
The present invention relates to bioengineering fields, and in particular to a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase, its
Encoding gene and its expression and application.
Background technique
In recent years, with advances in technology with the raising of living standards of the people, dyestuff using more and more extensive, especially
Be it is increasing for the demand of asepsis environment-protecting dyestuff, more stringent requirements are proposed for this development to dye industry.Current dye
Material presses its source, can be divided mainly into two major classes: natural dye and synthetic dyestuffs.Natural dye application is more early, and mostly without poison ring
It protects, but the limitations such as that there is contents in nature is limited, extraction process is complicated, production cost is high.Synthetic dyestuffs and natural
Dyestuff compared to have the advantages that lovely luster, do not limited by raw material sources, production cost is low etc., but disadvantage is that synthetic dyestuffs are most
Structure is complicated, and bio-toxicity is high, is difficult to degrade in nature or degradation cost is very high, larger to ecological environment destruction.Naturally
The disadvantages mentioned above of dyestuff and synthetic dyestuffs seriously constrains the rapid development of dye industry and its further expansion of application field.
Summary of the invention
The object of the present invention is to provide a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase, its encoding gene and
It is expressed and application.
To achieve the above object, a kind of the technical solution used in the present invention: 2- phenylacetyl-benzimidazole -7- carboxylic acid conjunction
At enzyme StBI, amino acid sequence encodes 439 amino acid as shown in SEQ ID No.2.
The present invention also provides a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene, the gene coding amino acids
Sequence 2- phenylacetyl as shown in SEQ ID No.2-benzimidazole-7- carboxylic acid synthetase.
Preferably, the 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene base sequence such as SEQ ID No.1
It is shown, it is named as gene StBI, full length gene 1381bp.
The present invention also provides a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI preparation methods: with pure
The genomic DNA of Gibberella zeae (Fusarium graminearum PH-1) ATCC MYA-4620 of change makes as template
With primer A1:5 '-CGGGATCCCTCAGCGCCTGTACAAAGACAC-3 ' and draw A2:5 '-CGGAATTCCTAGTCATCCTGT
AAGCTGATGGTC-3 ' obtains 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene by polymerase chain reaction amplification
StBI。
Preferably, the method for the Gibberella zeae (Fusarium graminearum PH-1) of the purifying are as follows: use fungi
The Gibberella zeae Fusarium graminearum of genome DNA extracting reagent kit purifying strain number ATCC MYA-4620
The genomic DNA of PH-1.
Correspondingly, the present invention also provides include the 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI weight
The method of group carrier, recombinant bacterial strain and the expression 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase.
The present invention provides comprising the 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombinant vector,
Preparation method is the following steps are included: base sequence 2- phenylacetyl as shown in SEQ ID No.1-benzimidazole-7- carboxylic acid is closed
It is inserted between BamHI the and EcoRI restriction enzyme site on plasmid pYES2 at enzyme gene StBI, obtains recombinant plasmid
PYES2-StBI, then recombination recombinant plasmid pYES2-StBI is transferred to.
Include the 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombination the present invention also provides a kind of
Bacterium, preferred recombinant bacterium are engineering bacteria S (Saccharomyces cerevisiae), which was preserved on July 13rd, 2018
Guangdong Province's Culture Collection (GDMCC), address: Institute of Micro-biology of the province experiment of XianLie Middle Road, GuangZhou City, GuangDong Province 100
Lou Wulou, deposit number: GDMCCNO:60415.
Include the 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombination the present invention also provides a kind of
Bacterial strain, preferred recombinant bacterium are the bacterial strain of engineering bacteria S (Saccharomyces cerevisiae).
Include the 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombination the present invention also provides a kind of
The preparation method of bacterium enters recombinant plasmid pYES2-StBI conversion in saccharomyces cerevisiae engineered yeast INVSc1, obtained recombinant bacterium,
It is named as engineering bacteria S (Saccharomyces cerevisiae).
The present invention also provides a kind of expression 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase StBI method, the methods
Are as follows:, in culture medium top fermentation, 2- benzene will be made comprising 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombinant bacterium
Acetyl-benzimidazole -7- carboxylic acid synthetase expression, recycles after fermentation and purifies expressed 2- phenylacetyl-benzimidazole -
7- carboxylic acid synthetase.
Preferably, described comprising being work comprising 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombinant bacterium
Journey bacterium S (Saccharomyces cerevisiae).
Preferably, the cultivation and fermentation method comprises the steps of:
(1) recombinant bacterium is drawn, i.e. the seed culture fluid of engineering bacteria S (Saccharomyces cerevisiae) is added to training
It supports among base, in 25-30 DEG C, shaker fermentation culture；
(2) in fermentation process, the absorbance of fermentation liquid under 600nm wavelength is detected, to absorbance under fermentation liquid 600nm wavelength
When reaching 0.4-1.0, adds final concentration of 1% yeast extract and 2% peptone is placed in into fermentation liquid, and by fermentation liquid
25-30 DEG C, fermented and cultured in shaking table.
Preferably, the culture medium is SC culture medium, and the SC nutrient media components include: the selection for accounting for SC culture medium quality
Acidic amino acid mixture (- Ura) 0.062%, without amino acid yeast nitrogen basis 6.4%, glucose 2%, the SC culture medium
PH5.5.
Preferably, after final concentration of 1% yeast extract of addition and 2% peptone being added, shaker fermentation culture 12-
120 hours.
Preferably, when absorbance reaches 0.8 under fermentation liquid 600nm wavelength, addition final concentration of 1% yeast extract and
2% peptone is into fermentation liquid.
Preferably, the temperature of fermented and cultured is 28 DEG C.
Preferably, after final concentration of 1% yeast extract of addition and 2% peptone being added, the time of fermented and cultured is
72 hours.
Preferably, the frequency of shaking table is 200rpm in fermentation process.
Preferably, the method for the seed culture fluid of preparation engineering bacterium S (Saccharomyces cerevisiae) are as follows: from guarantor
Have on the solid medium of engineering bacteria S that picking single bacterium colony is among the SC culture medium of 5mL, in 28 DEG C, 200rpm shaking table
The culture 24 hours seed culture fluids to get engineering bacteria S.
The present invention also provides a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase StBI in preparation 2- phenylacetyl-benzo
Application in imidazoles -7- carboxylic acid.
Exist the present invention also provides a kind of comprising 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombinant bacterium
Prepare the application in 2- phenylacetyl-benzimidazole -7- carboxylic acid.
The present invention also provides a kind of engineering bacteria S (Saccharomyces cerevisiae) in preparation 2- phenylacetyl-benzo
Application in imidazoles -7- carboxylic acid.
It is preferably, described to prepare 2- phenylacetyl-benzimidazole -7- carboxylic acid application method, comprising the following steps:
(1) comprising 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombinant bacterium in culture medium top fermentation,
Make 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase expression；
(2) it recycles and purifies 2- phenylacetyl-benzimidazole -7- carboxylic acid.
Preferably, it recycles and purifies 2- phenylacetyl-benzimidazole -7- carboxylic acid method the following steps are included: (1) uses chloroform
The fermentation liquid that recombinant bacterium cell is removed after extractive fermentation, obtains comprising 2- phenylacetyl-benzimidazole -7- carboxylic acid crude extract；(2)
It will be dry to constant weight comprising 2- phenylacetyl-benzimidazole -7- carboxylic acid crude extract；(3) crude extract after drying crosses silicagel column, uses
Chloroform elutes and collects fraction；(4) chloroform fraction is dry, by the solid being dried to obtain successively use methanol, ethyl acetate, just oneself
Alkane removes organic impurities using washing, centrifugation, the method for abandoning supernatant and obtains pure 2- phenylacetyl-benzimidazole -7- carboxylic acid.
There is the characteristic of hydrophobic oleophobic using the 2- phenylacetyl being prepared-benzimidazole -7- carboxylic acid, be used to prepare
Dyestuff, color fastness is preferable, be not easy because with water, oil contact and fade.
It is furthermore preferred that washing, being centrifuged in the step (4), the method for the centrifugation of abandoning supernatant is 21000 × g centrifugation 10
Minute.
Preferably, the method that the fermentation liquid of recombinant bacterium cell is removed after fermentation is: 850 × g is centrifuged 5 minutes.
Preferably, temperature range dry during preferred recovery purifying is 37~45 DEG C.
The beneficial effects of the present invention are: the present invention utilizes Gibberella zeae (the Fusarium graminearum purified
PH-1) genomic DNA of ATCC MYA-4620 extends to obtain 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI,
And it is transferred to engineering bacteria, what is obtained ferments comprising 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI recombinant bacterium
2- phenylacetyl-benzimidazole -7- carboxylic can be synthesized, it is one that 2- phenylacetyl-benzimidazole -7- carboxylic acid, which has hydrophobic oleophobic property,
Kind of uranidin, the dyestuff being used to prepare, color fastness is preferable, be not easy because with water, oil contact and fade.
Detailed description of the invention
Fig. 1 is compound 2- phenylacetyl-benzimidazole -7- carboxylic acid structure chart.
Fig. 2 is uranidin natural dye 2- phenylacetyl-benzimidazole -7- carboxylic acid of utilizing works bacterium S of the present invention synthesis
DMSO solution；Left side is DMSO solvent blank comparative diagram.
Fig. 3 is 2- phenylacetyl-benzimidazole -7- carboxylic acid ultraviolet absorption spectrum.
Fig. 4 is 2- phenylacetyl-benzimidazole -7- carboxylic acid Mass Spectrometer Method figure [M-H] -.
Fig. 5 is 2 result figure of embodiment.
Fig. 6 is 3 result figure of embodiment.
Fig. 7 is 4 result figure of embodiment.
Fig. 8 is 5 result figure of embodiment.
Fig. 9 is 6 result figure of embodiment.
Figure 10 is 7 result figure of embodiment.
Figure 11 is 8 result figure of embodiment.
Specific embodiment
The present invention is specifically described below with reference to embodiment, but not limited to this.
Embodiment 1
2- phenylacetyl-benzimidazole -7- carboxylic acid preparation method.
(1) experimental material
Gibberella zeae (Fusarium graminearum PH-1) is purchased from Unite States Standard biology product collecting center
(ATCC), number is ATCC MYA-4620；Fungal genomic DNA extracts kit and pYES2 plasmid vector are silent winged purchased from match
Scientific and technological (China) Co., Ltd of generation that；Restriction endonuclease BamHI and EcoRI are purchased from precious bioengineering (Dalian) limited public affairs
Department；Primer A1 and A2 are ordered from Sangon Biotech (Shanghai) Co., Ltd.) limited liability company.
(2) preparation of engineering bacteria S (Saccharomyces cerevisiae).
(1) purify the Gibberella zeae of strain number ATCC MYA-4620 with fungal genomic DNA extracts kit
The genomic DNA of Fusarium graminearum PH-1；
(2) using the Gibberella zeae genomic DNA of purifying as template, primer A1:5 '-is used
CGGGATCCCTCAGCGCCTGTACAAAGACAC-3 ' and primer A2:5 '-CGGAATTCCTAGTCATCCTGTAAGCTGATGG
TC-3 ' obtains 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI by polymerase chain reaction amplification, expands
The StBI gene order arrived are as follows: SEQ ID No.1；
(3) amplification obtains the 2- benzene second obtained after 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI translation
Acyl-benzimidazole -7- carboxylic acid synthetase, is named as StBI, and sequence is SEQ ID No.2；
(4) amplification obtains 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene StBI and passes through restriction endonuclease
BamHI and EcoRI is connected in pYES2 plasmid vector to arrive recombinant plasmid pYES2-StBI；
(5) method introduced referring to " molecular biology experiment guidance " (second edition), the recombinant plasmid pYES2- that will be obtained
StBI conversion enters in saccharomyces cerevisiae engineered yeast INVSc1, and obtaining engineering bacteria is S (Saccharomyces cerevisiae).
(3) 2- phenylacetyl-benzimidazole -7- carboxylic acid preparation.
A. from picking single bacterium colony on the solid medium for preserving engineering bacteria S among the SC culture medium of 5mL, in 28
DEG C, 24 hours seed culture fluids to get engineering bacteria S are cultivated in 200rpm shaking table；Dosage of each component accounts in the SC culture medium
The percentage of SC culture medium quality dosage is respectively as follows: selective ispol (- Ura) 0.062%；Without amino acid yeast nitrogen
Source basis 6.4%；Glucose 2%；pH5.5；
B. the seed culture fluid for drawing engineering bacteria S is added among SC culture medium, in 28 DEG C, shaker fermentation culture, with point
The absorbance that the fermentation liquid is detected under light photometer and 600nm wavelength, reaches 0.8 to absorbance under fermentation liquid 600nm wavelength
When, final concentration of 1% yeast extract and 2% peptone are added into fermentation liquid, fermentation liquid are placed in 28 DEG C, 200rpm shakes
Fermented and cultured 72 hours in bed；
C. the above-mentioned fermentation liquid of 200mL 850 × g in centrifuge is taken to be centrifuged the cell of 5 minutes removal engineering bacteria S；It will removal
Fermentation liquid after cell is extracted with the chloroform of 200mL, this process is repeated 2 times for extracting the crude extract in fermentation liquid completely；It will
Crude extract is chromatographed by silica gel (200-300 mesh) column, collects the component of 100% chloroform elution, and be somebody's turn to do by the way that rotary evaporation is dry
Component；Dry component is vibrated with 10mL methanol and is suspended, then 21000 × g is centrifuged 10 minutes, abandons supernatant, this process repeats 2
It is secondary to be used to remove organic impurities；2 components are respectively washed using same method ethyl acetate and n-hexane later, from
And obtain neat compounds.
(4) identification of compound
1, UV pop Scanning Detction is carried out.UV absorb response as shown in figure 3, feature UV absorption wavelength be 242nm,
280nm, 376nm, 387nm.
2, liquid chromatogram, Mass Spectrometer Method are carried out.
With methanol dissolved compound；It is detected under 380nm wavelength with high performance liquid chromatography, high performance liquid chromatography elution process
For gradient elution 35min, 30-90% (acetonitrile: water), target peak is detected in 20.13min, as shown in Fig. 5 (II).
Mass spectrogram [M-H]-As shown in Figure 4, it is known that its molecular weight is 280.
3, nuclear magnetic resonance (NMR) identification is carried out.Carbon spectrum and hydrogen spectrum are as shown in table 1, table 2.
The 13C NMR data (100MHz) of compound prepared by table 1 in DMSO-d6
Compound prepared by table 2 is in DMSO-d61H NMR data (400MHz)
The structure for determining this neat compounds is composed according to nuclear magnetic resonance (NMR) carbon spectrum and hydrogen are as follows:
For 2- phenylacetyl-benzimidazole -7- carboxylic acid.
(4) test of compound solubility
At 25 DEG C, weigh 100mL solution respectively: ethyl alcohol, methanol, acetone, ethyl acetate, chloroform, n-hexane, chloroform,
Glycerol, grease and DMSO are put into beaker.Weigh the uranidin solid powder of 20g respectively, and be separately added into fill solution ethanol,
Methanol, acetone, ethyl acetate, chloroform, n-hexane, chloroform, glycerol, grease and DMSO beaker in.It is solid to be gradually added uranidin
The solute of body powder, is stirred continuously, and sufficiently dissolves.Guaranteeing solution supersaturation, the undissolved solid yellow of filtering taking-up is plain, and
55 DEG C dry 24 hours, guarantee that solvent volatilizees completely.Undissolved solid gross mass is weighed respectively, and by formula, (20g- is not molten
Solution pigment quality=dissolved chromatoplasm amount), the solid masses dissolved in 100mL solvent is found out, as at this temperature should
The solubility of solid.It is as shown in Figure 2 in the solution of DMSO and blank control.
Solubility table (25 DEG C, unit: g/100mL solvent)
Embodiment 2
The verifying of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthesis StBI gene enzyme.
1, the empty plasmid pYES2 for being not inserted into enzyme gene StBI is transferred to saccharomyces cerevisiae engineered yeast INVSc1, such as implemented
1 identical method culture of example 72 hours, takes fermentation liquid spare.
2, the fermentation liquid and above-mentioned fermentation liquid of the engineering bacteria S (Saccharomyces cerevisiae) of Example 1, into
The detection of row liquid phase, 380nm wavelength, elution process are gradient elution 35min, 30-90% (acetonitrile: water).
As a result as shown in figure 4, the empty plasmid pYES2 for being not inserted into enzyme gene StBI is transferred to saccharomyces cerevisiae engineered yeast
The fermentation liquid of INVSc1 such as shown in (I), shows that 2- phenylacetyl-benzimidazole -7- carboxylic acid is not detected；With recombinant plasmid
The hair of the saccharomyces cerevisiae engineered yeast INVSc1 of pYES2-StBI plasmid, i.e. engineering bacteria S (Saccharomyces cerevisiae)
Zymotic fluid such as shown in (II), shows to detect 2- phenylacetyl-benzimidazole -7- carboxylic acid；Prove that StBI gene enzyme is synthesis 2- benzene second
Acyl-benzimidazole -7- carboxylic acid key enzyme.
Embodiment 3
As embodiment 1 method prepare engineering bacteria S (Saccharomyces cerevisiae), such as the 2- of embodiment 1
Phenylacetyl-benzimidazole -7- carboxylic acid preparation method, engineering bacteria S (Saccharomyces cerevisiae) is fermented respectively
12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours are cultivated,
As a result 2- phenylacetyl-benzimidazole -7- carboxylic of isolated 0.31g~1.98g is distinguished from the 1L culture of recombination engineering S
Acid, as shown in Figure 6.
Embodiment 4
As embodiment 1 method prepare engineering bacteria S (Saccharomyces cerevisiae), such as the 2- of embodiment 1
Phenylacetyl-benzimidazole -7- carboxylic acid preparation method, by engineering bacteria S (Saccharomyces cerevisiae) fermented and cultured.
When difference is that absorbance reaches 0.4 under fermentation liquid 600nm wavelength, final concentration of 1% yeast extract and 2% albumen are added
Peptone.
Fermented and cultured 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, it is 108 small
When, 120 hours, as a result from the 1L culture of recombination engineering S distinguish isolated 0.11g~1.29g 2- phenylacetyl-benzene
And imidazoles -7- carboxylic acid, as shown in Figure 7.
Embodiment 5
As embodiment 1 method prepare engineering bacteria S (Saccharomyces cerevisiae), such as the 2- of embodiment 1
Phenylacetyl-benzimidazole -7- carboxylic acid preparation method, by engineering bacteria S (Saccharomyces cerevisiae) fermented and cultured.
When difference is that absorbance reaches 0.6 under fermentation liquid 600nm wavelength, final concentration of 1% yeast extract and 2% albumen are added
Peptone.
Fermented and cultured 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, it is 108 small
When, 120 hours, as a result from the 1L culture of recombination engineering S distinguish isolated 0.26g~1.52g 2- phenylacetyl-benzene
And imidazoles -7- carboxylic acid, as shown in Figure 8.
Embodiment 6
As embodiment 1 method prepare engineering bacteria S (Saccharomyces cerevisiae), such as the 2- of embodiment 1
Phenylacetyl-benzimidazole -7- carboxylic acid preparation method, by engineering bacteria S (Saccharomyces cerevisiae) fermented and cultured.
When difference is that absorbance reaches 1.0 under fermentation liquid 600nm wavelength, final concentration of 1% yeast extract and 2% albumen are added
Peptone.
Fermented and cultured 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, it is 108 small
When, 120 hours, as a result from the 1L culture of recombination engineering S distinguish isolated 0.32g~1.79g 2- phenylacetyl-benzene
And imidazoles -7- carboxylic acid, as shown in Figure 9.
Embodiment 7
As embodiment 1 method prepare engineering bacteria S (Saccharomyces cerevisiae), such as the 2- of embodiment 1
Phenylacetyl-benzimidazole -7- carboxylic acid preparation method, by engineering bacteria S (Saccharomyces cerevisiae) fermented and cultured.
Difference is that cultivation temperature is 25 DEG C.
Fermented and cultured 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, it is 108 small
When, 120 hours, as a result from the 1L culture of recombination engineering S distinguish isolated 0.27g~1.61g 2- phenylacetyl-benzene
And imidazoles -7- carboxylic acid, as shown in Figure 10.
Embodiment 8
As embodiment 1 method prepare engineering bacteria S (Saccharomyces cerevisiae), such as the 2- of embodiment 1
Phenylacetyl-benzimidazole -7- carboxylic acid preparation method, by engineering bacteria S (Saccharomyces cerevisiae) fermented and cultured.
Difference is that cultivation temperature is 30 DEG C.
Fermented and cultured 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, it is 108 small
When, 120 hours, as a result from the 1L culture of recombination engineering S distinguish isolated 0.35g~1.87g 2- phenylacetyl-benzene
And imidazoles -7- carboxylic acid, as shown in figure 11.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>Traditional Chinese Medicine University Of Guangzhou
<120>a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase, its encoding gene and its expression and application
<130> 20180720
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1381
<212> DNA
<213>artificial synthesized
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agctaagaac ttggacacgc aattgcaatc tcaaggtttt ccacagccat catttgaagc 180
tgatggtcca acatacgttg ttcccaagga tgcccccaaa gcagcccacg aagcccgtgt 240
ggcaaccgct gaggcagccc tgaaactgtt caatcttgta tctggaccca gcgagcttct 300
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Met Gly Ser Ile Ser Ser Pro Ser Leu Ile Ile Asp Leu Ala Asn Ala
1 5 10 15
Val Ser Ser Ala Ala Lys Asn Leu Asp Thr Gln Leu Gln Ser Gln Gly
20 25 30
Phe Pro Gln Pro Ser Phe Glu Ala Asp Gly Pro Thr Tyr Val Val Pro
35 40 45
Lys Asp Ala Pro Lys Ala Ala His Glu Ala Arg Val Ala Thr Ala Glu
50 55 60
Ala Ala Leu Lys Leu Phe Asn Leu Val Ser Gly Pro Ser Glu Leu Leu
65 70 75 80
Pro Asn Met Thr Ala Ser Tyr His Thr Ile Phe Ala Leu Gln Trp Leu
85 90 95
His His Phe Asp Val Phe Ser His Ile Pro Leu Asp Gly Ser Leu Ser
100 105 110
Tyr Glu Lys Leu Ala Thr Lys Ala Asn Val Pro Glu Ser Leu Leu Lys
115 120 125
Ser Val Ala Arg Met Ala Met Thr Ser Asn Ile Leu Ala Glu Pro Thr
130 135 140
Thr Gly Gln Val Ala His Ser Ala Asn Ser Ala Met Phe Val Lys Phe
145 150 155 160
Pro Asn Met Arg Asp Trp Ala Ser Tyr Met Phe Thr Ala Ser Ile Pro
165 170 175
Thr Ala Ala Ala Met Val Gln Ala Thr Glu Lys Trp Pro Gly Ser Val
180 185 190
Lys Lys Thr Glu Thr Ala Tyr Asn Ile Ala Phe Asn His Asp Leu Pro
195 200 205
Phe Phe Asp His Leu Ser Gln Ser Pro Val Met Thr Lys Gln Phe Ser
210 215 220
Gly Tyr Met Arg Ser Val Thr Asp Gly Gln Gly Met Asp Leu Ser His
225 230 235 240
Leu Val Asn Gly Phe Asp Trp Ala Ser Leu Pro Asp Lys Ser Leu Ile
245 250 255
Val Asp Ile Gly Gly Ser Ala Gly His Ala Ser Tyr Ala Leu Ala Ala
260 265 270
Ala Tyr Pro His Leu Arg Phe Glu Val Gln Asp Leu Asp Thr Val Val
275 280 285
Asn Gly Glu Lys Ala Ala Lys Glu His Glu Glu Ala Val Ser Lys His
290 295 300
Val Ile Gly Thr Asp Asn Arg Val Thr Phe Lys Ala His Asn Phe Phe
305 310 315 320
Glu Ala Gln Pro Thr Lys Asp Ala Thr Val Tyr Met Leu Arg Met Ile
325 330 335
Ile His Asp Trp Pro Asp Ala Glu Ala Lys Thr Ile Leu Gly Asn Leu
340 345 350
Val Pro Ala Leu Glu Ser Ala Lys Ala Thr Leu Leu Ile Met Asp Thr
355 360 365
Val Leu Pro Ser Pro Gly Ser Ile Pro Ser Val Arg Glu Arg Val Ile
370 375 380
Arg Thr Arg Asp Leu Thr Met Arg Gln Val Phe Asn Ala Lys Glu Arg
385 390 395 400
Gly Val Asp Asp Trp Glu Ala Ile Leu Arg Glu Thr Asp Ser Arg Leu
405 410 415
Thr Leu Lys Asn Leu Arg Gln Pro Glu Gly Ser Asn Met Cys Leu Leu
420 425 430
Thr Ile Ser Leu Gln Asp Asp
435
<210> 3
<211> 30
<212> DNA
<213>unknown
<400> 3
cgggatccct cagcgcctgt acaaagacac 30
<210> 4
<211> 33
<212> DNA
<213>unknown
<400> 4
cggaattcct agtcatcctg taagctgatg gtc 33
Claims (9)
1. a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase, which is characterized in that its amino acid sequence such as SEQ ID No.2
It is shown.
2. a kind of encode 2- phenylacetyl as described in claim 1-benzimidazole-7- carboxylic acid synthetase gene, which is characterized in that
The base sequence of the gene is as shown in SEQ ID No.1.
3. a kind of 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase gene preparation method: with the base of the Gibberella zeae of purifying
Because group DNA is as template, primer A1:5 '-CGGGATCCCTCAGCGCCTGTACAAAGACAC-3 ' and primer A2:5 '-C are used
GGAATTCCTAGTCATCCTGTAAGCTGATGGTC-3 ' obtains 2- phenylacetyl-benzo miaow by polymerase chain reaction amplification
Azoles -7- carboxylic acid synthetase gene.
4. a kind of include 2- phenylacetyl as claimed in claim 2-benzimidazole-7- carboxylic acid synthetase gene recombinant vector.
5. recombinant vector according to claim 4, which is characterized in that the preparation method of the recombinant vector includes following step
It is rapid: base sequence 2- phenylacetyl as shown in SEQ ID No.1-benzimidazole-7- carboxylic acid synthetase gene is inserted into plasmid
Between BamHI and EcoRI restriction enzyme site on pYES2.
6. a kind of engineering bacteria S, deposit number are as follows: GDMCCNO:60415.
7. a kind of include 2- phenylacetyl as claimed in claim 2-benzimidazole-7- carboxylic acid synthetase gene recombinant bacterial strain.
8. a kind of expression 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase method, the method are as follows: will include 2- phenylacetyl -
The recombinant bacterium of benzimidazole -7- carboxylic acid synthetase gene closes 2- phenylacetyl-benzimidazole -7- carboxylic acid in culture medium top fermentation
At expression of enzymes, recycles after fermentation and purify expressed 2- phenylacetyl-benzimidazole -7- carboxylic acid synthetase.
9. a kind of engineering bacteria S as claimed in claim 6 is preparing the application in 2- phenylacetyl-benzimidazole -7- carboxylic acid.
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| CN102952813A (en) * | 2012-10-26 | 2013-03-06 | 东北电力大学 | Synthetic method and extraction process of blue natural dye |
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| CN102952813A (en) * | 2012-10-26 | 2013-03-06 | 东北电力大学 | Synthetic method and extraction process of blue natural dye |
Non-Patent Citations (2)
| Title |
|---|
| CUOMO,C.A. ET AL.: "Accession number：XM_011319933，Fusarium graminearum PH-1 hypothetical protein partial mRNA", 《GENBANK》 * |
| CUOMO,C.A. ET AL.: "Accession number：XP_011318235，hypothetical protein FGSG_02326 [Fusarium graminearum PH-1]", 《GENBANK》 * |
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