CN101845429B - High temperature resistant L-arabinose isomerase and application thereof - Google Patents
High temperature resistant L-arabinose isomerase and application thereof Download PDFInfo
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Abstract
The invention discloses a high temperature resistant L-arabinose isomerase which has an amino acid sequence shown in SEQ ID NO:2. The invention also discloses a gene sequence for encoding the high temperature resistant L-arabinose isomerase, a gene engineering bacterium and construction method thereof, an expression method for the L-arabinose isomerase, and application of the L-arabinose isomerase and the gene engineering bacterium in preparing D-tagatose. The reaction temperature of the high temperature resistant L-arabinose isomerase is 40 to 70 DEG C, and the reaction pH is 5.5 to 7.5. Therecombinase shows good heat stability and pH tolerance; the added low-concentration Mn2+, Co2+ ions can greatly improve enzyme activity and heat stability; and the high temperature resistant L-arabinose isomerase has wide application prospect and economic significance for industrial production of the novel functional sweetener D-tagatose.
Description
Technical field
The present invention relates to be derived from a kind of fire resistant L-arabinose isomerase of lactobacillus fermentum (Lactobacillus fermentum) NXTag-1, the structure of fire resistant L-arabinose isomerase engineering bacteria and the application of fire resistant L-arabinose isomerase, belong to technical field of bioengineering.
Background technology
Tagatose is the comparatively rare natural tagatose of a kind of occurring in nature, belongs to a kind of of rare sugar, and international rare sugared association (ISRS) is defined as " occurring in nature exists but a few class monose and the derivative thereof of content " to rare sugar.D-tagatose (D-tagatose) is " epimer " of fructose, extensively be present in occurring in nature, numerous food product is as all having a certain amount of tagatose in the yogurt of sterilization cow's milk, milk powder, hot cocoa, various cheese, some kind and the certain plants.The D-tagatose is the functional sweetener that has special nourishing function as a kind of, make through isomerization by the D-semi-lactosi in the industrial production, profile and sweet taste feature are similar to sucrose, there are not any undesired off taste or aftertaste, heat lower (1.5kcal/g), have different physiological roles such as the intestinal microflora of improvement, lowering blood glucose, anti-dental caries, and have advantages such as good Maillard reaction and caramelization.Calendar year 2001 is defined as generally recognized as safe food (GRAS) by FDA (FDA), and the D-tagatose is common to products such as healthy beverage and yogurt, fruit juice in the U.S. subsequently, as the surrogate of sucrose; European Union also goes on the market in Europe in approval D-tagatose in 2003.
L-arabinose isomerase (EC 5.3.1.4, L-arabinose isomerase, L-AI) not only catalysis L-arabinose isomery turns to the L-ribulose and can also generate the D-tagatose for catalysis D-semi-lactosi, be that the most effective enzyme of D-tagatose is produced in bio-transformation, therefore realize that by L-AI the industrialization of D-tagatose is just becoming the focus of people's research.1993, reported first such as Cheetham utilize L-AI catalysis D-semi-lactosi to produce the research of D-tagatose.Subsequently, multiple microbe-derived L-AI is found, and the L-AI encoding gene (araA) of multiple L-arabinose isomery enzyme-producing bacterias such as intestinal bacteria, plant lactobacillus, Bacillus subtilus, bacillus acidocldarius is cloned and heterogenous expression.Temperature of reaction is very important parameter in the biocatalysis aldehyde ketone sugar isomerization reaction, the balance temperature influence of the isomerization reaction between D-semi-lactosi and the D-tagatose is bigger, temperature of reaction is high more, and the isomerization reaction balance is partial to the D-tagatose more, and substrate conversion efficiency is high more; (more than 60 ℃) more help the generation of D-tagatose under the higher temperature conditions, help reducing the production cost of D-tagatose and improve product output.Although the araA gene of a lot of kinds is identified that the L-AI that is used to D-tagatose production research is also few, because the thermostability of L-AI enzyme is relatively poor mostly, optimal reactive temperature is lower, is unfavorable for industrial application.For example intestinal bacteria L-AI, M. smegmatics L-AI etc., its optimal reactive temperature only has 30～45 ℃.Therefore stable on heating L-arabinose isomerase will have the better application prospect.
Summary of the invention
Technical problem to be solved by this invention provides a kind of fire resistant L-arabinose isomerase.
The technical problem that the present invention also will solve provides the gene order of the above-mentioned L-arabinose isomerase of coding.
Another technical problem that the present invention also will solve provides genetic engineering bacterium and the construction process thereof that comprises above-mentioned L-arabinose isomerase.
Another technical problem that the present invention also will solve provides the expression method of above-mentioned L-arabinose isomerase.
Another technical problem that the present invention also will solve provides the application of above-mentioned L-arabinose isomerase in preparation D-tagatose.
The technical problem that the present invention will solve at last provides the application of genetic engineering bacterium in preparation D-tagatose of above-mentioned product L-arabinose isomerase.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of fire resistant L-arabinose isomerase, it has the aminoacid sequence shown in SEQ ID NO:2, comprises 474 amino acid.It derives from lactobacillus fermentum (Lactobacillus fermentum) NXTag-1CGMCCNO.2921 (number of patent application 200910025982.9).
The encoding gene of above-mentioned fire resistant L-arabinose isomerase, it has the nucleotide sequence shown in SEQ ID NO:1, and it contains the 1425bp base.
A kind of genetic engineering bacterium of fire resistant L-arabinose isomerase, it comprises the nucleotide sequence shown in SEQ ID NO:1.
The construction process of said gene engineering bacteria, this method comprises the steps:
(1) structure contains fire resistant L-arabinose isomerase expression of gene plasmid:
Get lactobacillus fermentum (Lactobacillus fermentum) NXTag-1CGMCC NO.2921 genomic dna as masterplate, as primer, carry out pcr amplification with the following nucleotide sequences of the restriction enzyme site that comprises EcoRI and XhoI:
Primer 1:5 '-AGAGAATTCATGCGTAAGATGCAAGATTAC-3 '
Primer 2: 5 '-AAGCTCGAGCTACTTGATGTTGATAAAGT-3 '
The pcr amplification system is: genomic dna 2 μ L, each 1 μ L of primer 1 and primer 2, dNTP2 μ L, 10 * Tag damping fluid, 2.5 μ L, ExTag polysaccharase 0.5 μ L, ddH
2O 14 μ L;
The PCR response procedures is: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 2min; 60 ℃ of annealing 30s then, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ are extended 1min;
Reclaim pcr amplification product, through restriction enzyme EcoRI and XhoI double digestion, the plasmid pET-28a (+) with through same double digestion connects under the effect of T4 ligase enzyme, obtains recombinant plasmid pET-araA
(2) this recombinant plasmid pET-araA is converted in the host cell:
Recombinant plasmid pET-araA is converted in the competence e. coli bl21 (DE3), and coating contains on the LB solid medium of 25 μ g/mL kantlex, 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 LB liquid nutrient medium of 25 μ g/mL kantlex in 5mL respectively, 37 ℃, the 200rpm overnight incubation, extract plasmid, through restriction enzyme EcoRI 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-araA, has the positive clone of bacterium colony of this plasmid, is genetic engineering bacterium.PET-araA checks order to recombinant plasmid, and the result shows that the insertion fragment is one and contains 1425bp, the protein that 474 amino acid of encoding are formed.
The expression method of above-mentioned fire resistant L-arabinose isomerase is inoculated in the genetic engineering bacterium that comprises the nucleotide sequence shown in SEQ ID NO:1 in the LB liquid nutrient medium that has added 25 μ g/mL kantlex 37 ℃ of shaking table overnight incubation; Inoculum size with 5% (v/v) is transferred in the LB substratum that contains 25 μ g/mL kantlex again, and 37 ℃ of fermentation culture 2～3 hours are to OD
600Be to add the isopropyl-(IPTG) of 0.2～1mmol/L or the lactose of 0.5～2g/L at 0.6 o'clock, continue abduction delivering after 6～12 hours, centrifugal collection thalline.
The purification process of above-mentioned fire resistant L-arabinose isomerase, the genetic engineering bacterium that will comprise the nucleotide sequence shown in SEQ ID NO:1 is suspended from the damping fluid (being pH 7.0 potassium phosphate buffers), utilize the ultrasonic disruption cell, centrifuged supernatant is a crude enzyme liquid, crude enzyme liquid is behind 0.2 μ m membrane filtration, use the affine resin of Ni-NTA to carry out purifying, purifying is single band after SDS-PAGE detects, and molecular weight is about 55kDa (Fig. 3).
The application of above-mentioned fire resistant L-arabinose isomerase in preparation D-tagatose.Be specially: the D-semi-lactosi with 1～100g/L is a substrate, fire resistant L-arabinose isomerase behind the adding purifying carries out enzymatic conversion reaction, the consumption of fire resistant L-arabinose isomerase is 10～500mg/L, 40～70 ℃ of temperature of reaction, transformation time 8～15h measures D-tagatose growing amount in the conversion fluid by halfcystine-carbazole method.Preferred mode is: the D-semi-lactosi with 1～100g/L is a substrate, adds 0.5～2mmol/L Mn
2+Ion and 1～3mmol/L Co
2+Ion, the fire resistant L-arabinose isomerase behind the adding purifying carries out enzymatic conversion reaction, and the consumption of fire resistant L-arabinose isomerase is 10～500mg/L, 40～70 ℃ of temperature of reaction, transformation time 8～15h.
The genetic engineering bacterium of above-mentioned product fire resistant L-arabinose isomerase can not need directly genetic engineering bacterium to be applied to the preparation of D-tagatose through extracting the process of pure enzyme.Be specially: the D-semi-lactosi with 1～100g/L is a substrate, the genetic engineering bacterium of the product fire resistant L-arabinose isomerase of adding after above-mentioned expression method is induced carries out conversion reaction, the consumption that produces the genetic engineering bacterium of fire resistant L-arabinose isomerase is 10～100g/L (wet thallus), 40～70 ℃ of temperature of reaction, transformation time 12～48h.Measure D-tagatose growing amount in the conversion fluid by halfcystine-carbazole method.Preferred mode is: the D-semi-lactosi with 1～100g/L is a substrate, adds 0.5～2mmol/L Mn
2+Ion and 1～3mmol/L Co
2+Ion, the genetic engineering bacterium that adds the product fire resistant L-arabinose isomerase after inducing carries out conversion reaction, the consumption that produces the genetic engineering bacterium of fire resistant L-arabinose isomerase is 10～100g/L (wet thallus), 40～70 ℃ of temperature of reaction, transformation time 12～48h.
Beneficial effect: the temperature of reaction of fire resistant L-arabinose isomerase of the present invention is 40～70 ℃, and reaction pH is 5.5～7.5.This recombinase demonstrates good thermostability and pH tolerance, retains 80～90% enzyme activities behind 75 ℃ of thermal treatment 1～3h, and pH 6.5 little sour conditions are handled the free of losses of 0～48h enzymic activity.
Lower concentration Mn
2+And Co
2+Ionic adds can significantly improve enzyme activity and thermostability, adds 0.5～2mmol/L MnCl
2, 1～3mmol/L CoCl
2The 65 ℃ of following enzyme activities in back improve 2 times than the control group that does not add metal ion, (the reorganization L-arabinose isomerase enzyme of unit U alive is defined as under 65 ℃ to reach 10U/mg, pH is the enzyme amount that per minute generates the D-tagatose of 1 μ mol under 6.5 the condition), 65 ℃ of thermal treatment 6h enzyme activity losses are lower than 10%.
This fire resistant L-arabinose isomerase has preferably active and stable under higher temperature conditions, 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
Fig. 1 is the structure synoptic diagram of recombinant plasmid pET-araA.
Fig. 2 is that single double digestion of recombinant plasmid pET-araA is identified (8g/L agarose gel electrophoresis).Wherein swimming lane 1 is an EcoRI single endonuclease digestion product, and swimming lane 2 is the standard DNA molecular weight, and the top-down band is respectively (kb): 15.0,10.0,7.5,5.0,2.5,1.0, and swimming lane 3 is EcoRI and XhoI double digestion product.
Fig. 3 is reorganization fire resistant L-arabinose isomerase SDS-PAGE electrophorogram.Swimming lane 1 is the standard protein molecular weight, and the top-down band is respectively (kDa): 116.0,66.2,45.0,35.0,25.0.
Fig. 4 is MnCl
2And CoCl
2Interpolation concentration to the influence of reorganization fire resistant L-arabinose isomerase enzymic activity.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that embodiment is described only to be used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the extraction of lactobacillus fermentum (Lactobacillus fermentum) NXTag-1 genomic dna.
The working instructions that provide according to the manufacturer, with Genomic DNA Purification Kit (Takara, Dalian) extracting is in the genomic dna of lactobacillus fermentum (Lactobacillus fermentum) the NXTag-1CGMCC NO.2921 of logarithmic phase, and with the 8g/L agarose gel electrophoresis bacterial genomes that obtains is detected.
The clone of embodiment 2:L-Arabinose isomerase encoding gene (araA) and reorganization bacterium make up.
2.1araA the pcr amplification of gene
According to the sequence of having reported lactobacillus fermentum L-AI gene on the Gene Bank, utilization Vector NTI software design primer Primer1 and Primer2, primer sequence is:
Primer1：5’-AGAGAATTCATGCGTAAGATGCAAGATTAC-3’
Primer2：5’-AAGCTCGAGCTACTTGATGTTGATAAAGT-3’
The genomic dna of the lactobacillus fermentum that obtains with embodiment 1 is a template, amplification lactobacillus fermentum gene fragment.
The pcr amplification system is: genomic dna 2 μ L, each 1 μ L of primer Primer1 and Primer2, dNTP 2 μ L, 10 * Tag damping fluid, 2.5 μ L, ExTag polysaccharase 0.5 μ L, ddH
2O 14 μ L.
The PCR response procedures is: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 2min; 60 ℃ of annealing 30s then, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ are extended 1min.
Amplified band cut reclaim test kit with the pillar rubber tapping of Axygen company behind the glue and reclaim, be connected on the pMD18-T of the Takara company carrier and transformed into escherichia coli JM109.By on penbritin LB flat board, verifying, identify positive colony, and carry out sequencing in Nanjing Jin Sirui biotechnology company in conjunction with plasmid list double digestion.To record full length sequence and in the GenBank database, analyze, and by the definite entire reading frame wherein of Vector NTI software.
2.2araA expression of gene
Utilize pET-28a (+) plasmid (Novagen), construction of expression vector is expressed goal gene, further confirms the exactness of gene clone.
2.2.1 the restriction enzyme digestion reaction, purifying and ligation
PCR product after purified, the pairing enzyme of restriction enzyme site carries out endonuclease reaction in primer sequence with designing in advance.In this experiment, used enzyme is EcoRI and XhoI.The enzyme system of cutting is: PCR product or plasmid solution 100 μ L, EcoRI 3 μ L, XhoI 3 μ L, 10 * damping fluid, 10 μ L, ddH
2O 34 μ L, cumulative volume 100 μ L.
Because selected two restriction enzyme sites close proximity (about 30bp) on pET-28a (+) empty plasmid, therefore, PCR product after enzyme is cut and plasmid vector only need can reach the purpose of purifying through PCR cleaning agents box.
PCR product and plasmid vector after enzyme is cut purifying can be used for ligation.The ligation system is: enzyme is cut the PCR product 4 μ L of purifying, and enzyme is cut the plasmid 4 μ L of purifying, T4 ligase enzyme 1 μ L, 10 * ligase enzyme damping fluid, 1 μ L.Connect the back and obtain recombinant plasmid pET-araA, its primary structure as shown in Figure 1.
2.2.2 plasmid preparation and conversion
Plasmid extraction adopts plasmid extraction kit, operates with reference to manufacturer's specification sheets.
The plasmid transformant uses Calcium Chloride Method.
2.2.3 the conversion of recombinant plasmid pET-araA
(1) gets 0.1-1 μ g recombinant plasmid pET-araADNA in 200 μ L competent cells, ice bath 30 minutes.
(2) 42 ℃ of water-bath heat shocks 90 seconds placed 1-3 minute on ice fast.
(3) add fresh LB liquid nutrient medium 800 μ L, in 37 ℃ of shaking culture 45 minutes.
(4) get 200 μ L thalline and coat selectivity LB solid culture primary surface.Cultivate 12-16 hour to single bacterium colony appearance for 37 ℃.
2.2.4 the evaluation of recon
Positive bacterium colony is inoculated in the LB liquid nutrient medium that contains kantlex (25 μ g/mL) cultivates and extract plasmid, cut system and condition is carried out list-double digestion evaluation with EcoRI, EcoRI and XhoI to recombinant plasmid respectively according to the enzyme in " restriction enzyme digestion reaction; purifying and ligation ", enzyme is cut product and is carried out the agarose gel electrophoresis evaluation, the results are shown in Figure 2, the recombinant plasmid that the experimental result explanation obtains is correct.
Confirm that through electrophoresis result this positive colony bacterium colony contains dna fragmentation and inserts plasmid pET-araA, contains the recombination bacillus coli of this recombinant plasmid pET-araA, is the recombination bacillus coli BL21-AI of conversion.Sequencing result shows that the insertion fragment contains the open reading frame of a long 1425bp.
The abduction delivering of embodiment 3:L-Arabinose isomerase.
Recombination bacillus coli BL21-AI is inoculated in 5mL has added in the LB liquid nutrient medium of 25 μ g/mL kantlex 37 ℃ of shaking table overnight incubation; Be transferred to the 500mL that 100mL LB substratum (containing 25 μ g/mL kantlex) is housed with 5% inoculum size again and shake in the bottle, 37 ℃ of shaking tables were cultivated 2～3 hours, to OD
600Be about 0.6 o'clock adding IPTG and induce (IPTG final concentration 1mmol/L), perhaps add the 1g/L lactose and induce, continue abduction delivering then after 6 hours, centrifugal collection thalline.
Embodiment 4: the purifying of reorganization L-arabinose isomerase.
The thalline of the reorganization bacterium E.coli BL21-AI that obtains is suspended from the potassium phosphate buffer (pH 7.0), clean twice with physiological saline, and use ultrasonic disruption instrument smudge cells (400W, 30min), the centrifugal 10min of 12000rpm, gained supernatant liquor are solubility target protein (crude enzyme liquid).Crude enzyme liquid adds to sample in the affine resin of Ni-NTA behind 0.2 μ m membrane filtration, and the control flow velocity is about 15mL/h, with 10 times of column volume Wash-Buffer (300mM NaCl, 50mM NaH
2PO
4, the 10mM imidazoles) and flushing, use 10 times of column volume Elution-Buffer (300mMNaCl, 50mM NaH at last
2PO
4, the 250mM imidazoles) wash-out and collect target protein, with under 4 ℃ of the target protein solution in potassium phosphate buffer (pH 7.0) dialyzed overnight.The purified target protein enzyme activity reaches 10U/mg and (adds 1mmol/L MnCl
2With 2mmol/L CoCl
2), detecting through SDS-PAGE is single band, and shows that the proteic molecular weight of reorganization L-arabinose isomerase is 55kDa.
Embodiment 5: the experiment of reorganization L-arabinose isomerase thermostability.
The reorganization L-arabinose isomerase 100 μ L (10mg/mL) that get behind the purifying handle 3h in 40 ℃, 50 ℃, 60 ℃, 65 ℃, 70 ℃ and 75 ℃ of differing temps water-baths, joining 900 μ L then contains in the reaction system of 100mM potassium phosphate buffer (pH 6.5), add the D-semi-lactosi to final concentration 100mM, 65 ℃ of water-bath 30min measure D-tagatose growing amount by halfcystine-carbazole method.Record and the results are shown in Table 1 (the reorganization L-arabinose isomerase enzyme of the unit U that lives is defined as under 65 ℃, and pH is the enzyme amount that per minute generates the D-tagatose of 1 μ mol under 6.5 the condition).
Table 1
Embodiment 6: reorganization L-arabinose isomerase pH stability experiment.
The reorganization L-arabinose isomerase 10 μ L (10mg/mL) that get behind the purifying are respectively room temperature treatment 0～48h in 5.5,6.0,6.5 and 7.5 the 100mM potassium phosphate buffer in 90 μ L pH, joining 900 μ L then contains in the reaction system of 100mM potassium phosphate buffer (pH 6.5), add the D-semi-lactosi to final concentration 100mM, 65 ℃ of water-bath 30min measure D-tagatose growing amount by halfcystine-carbazole method.Record and the results are shown in Table 2.
Table 2
Embodiment 7:Mn
2+And Co
2+Ion adds concentration to the active influence of reorganization L-arabinose isomerase enzyme.
(pH 7.0 in 10mL 100mM potassium phosphate buffer to get reorganization L-arabinose isomerase 2mg behind the purifying, contain 10mM EDTA), 4 ℃ of dialyzed overnights place 100mM potassium phosphate buffer dialysis 12h that EDTA is removed then, add 0～5mM MnCl to enzyme liquid respectively
2And CoCl
2, and adding the D-semi-lactosi to final concentration 100mM, 65 ℃ of water-bath 30min measure D-tagatose growing amount by halfcystine-carbazole method.Record result such as Fig. 4 (with 1mM Mn
2+Record enzyme activity under the ion condition and be set at 100%.
Embodiment 8: the application I of reorganization L-arabinose isomerase.
The cumulative volume of enzymatic conversion reaction is 10mL, with 10g/L D-semi-lactosi as conversion of substrate, be dissolved in 100mmol/L potassium phosphate buffer (pH 6.5), get reorganization L-arabinose isomerase behind 2mg embodiment 4 purifying in system, 65 ℃ of reaction 12h measure D-tagatose growing amount by halfcystine-carbazole method.After measured, D-tagatose concentration is 5.5g/L in the conversion fluid, and reorganization L-arabinose isomerase reaches 55% to the transformation efficiency of substrate D-semi-lactosi.
Embodiment 9: the application II of reorganization L-arabinose isomerase.
The cumulative volume of enzymatic conversion reaction is 10mL, as conversion of substrate, is dissolved in 100mmol/L potassium phosphate buffer (pH 6.5) with 10g/L D-semi-lactosi, gets reorganization L-arabinose isomerase behind 2mg embodiment 4 purifying in system, and adds MnCl
2(final concentration 1mmol/L) and CoCl
2(final concentration 2mmol/L), 65 ℃ of reaction 12h measure D-tagatose growing amount by halfcystine-carbazole method.After measured, D-tagatose concentration is 6.5g/L in the conversion fluid, and reorganization L-arabinose isomerase reaches 65% to the transformation efficiency of substrate D-semi-lactosi.
Embodiment 10: the application I that produces the genetic engineering bacterium of reorganization L-arabinose isomerase.
The cumulative volume of conversion reaction is 100mL, with 100g/L D-semi-lactosi as conversion of substrate, be suspended in 100mmol/L potassium phosphate buffer (pH 6.5), the genetic engineering bacterium of getting the product reorganization L-arabinose isomerase that 5g (wet thallus) embodiment 3 obtains is in reaction system, 65 ℃ of reaction 36h measure D-tagatose growing amount by halfcystine-carbazole method.After measured, D-tagatose concentration is 60g/L in the conversion fluid, and reorganization L-arabinose isomerase reaches 60% to the transformation efficiency of substrate D-semi-lactosi.
Embodiment 11: the application II that produces the genetic engineering bacterium of reorganization L-arabinose isomerase.
The cumulative volume of conversion reaction is 100mL, with 100g/L D-semi-lactosi as conversion of substrate, be suspended in 100mmol/L potassium phosphate buffer (pH 6.5), the genetic engineering bacterium of getting the product reorganization L-arabinose isomerase that 5g (wet thallus) embodiment 3 obtains and adds MnCl in reaction system
2(final concentration 1mmol/L) and CoCl
2(final concentration 2mmol/L), 65 ℃ of reaction 36h measure D-tagatose growing amount by halfcystine-carbazole method.After measured, D-tagatose concentration is 70g/L in the conversion fluid, and reorganization L-arabinose isomerase reaches 70% to the transformation efficiency of substrate D-semi-lactosi.
Nucleotide or aminoacid sequence table
SEQUENCE?LISTING
＜110〉Nanjing University of Technology
＜120〉a kind of fire resistant L-arabinose isomerase and application thereof
<130>njut100413
<160>4
<170>PatentIn?version?3.3
<210>1
<211>1425
<212>DNA
＜213〉lactobacillus fermentum (Lactobacillus fermentum) NXTag-1
<220>
<221>CDS
<222>(1)..(1425)
<400>1
atg?cgt?aag?atg?caa?gat?tac?aag?ttc?tgg?ttt?gtt?gtt?ggt?agc?caa 48
Met?Arg?Lys?Met?Gln?Asp?Tyr?Lys?Phe?Trp?Phe?Val?Val?Gly?Ser?Gln
1 5 10 15
ccg?ctt?tac?ggc?ccg?gaa?gcg?ctg?gca?gag?gtt?gaa?aag?gac?gct?cgc 96
Pro?Leu?Tyr?Gly?Pro?Glu?Ala?Leu?Ala?Glu?Val?Glu?Lys?Asp?Ala?Arg
20 25 30
aag?ctc?gtt?gat?ggc?tta?aac?aaa?ggc?ggt?aag?ctt?gac?tac?ccg?gtt 144
Lys?Leu?Val?Asp?Gly?Leu?Asn?Lys?Gly?Gly?Lys?Leu?Asp?Tyr?Pro?Val
35 40 45
gaa?ttt?aag?ctg?gtt?gct?acg?acg?gcc?gac?agc?atc?acg?aag?ttc?atg 192
Glu?Phe?Lys?Leu?Val?Ala?Thr?Thr?Ala?Asp?Ser?Ile?Thr?Lys?Phe?Met
50 55 60
aag?gaa?gcc?aac?tac?aat?gat?gat?gta?gct?ggg?gta?att?act?tgg?atg 240
Lys?Glu?Ala?Asn?Tyr?Asn?Asp?Asp?Val?Ala?Gly?Val?Ile?Thr?Trp?Met
65 70 75 80
cac?acc?ttc?tca?ccg?gcc?aag?aac?tgg?atc?cgg?ggg?acg?gaa?ctc?ctg 288
His?Thr?Phe?Ser?Pro?Ala?Lys?Asn?Trp?Ile?Arg?Gly?Thr?Glu?Leu?Leu
85 90 95
caa?aag?ccg?ctc?ctg?cac?ttg?gca?acc?caa?ttc?tta?aac?aac?att?ccg 336
Gln?Lys?Pro?Leu?Leu?His?Leu?Ala?Thr?Gln?Phe?Leu?Asn?Asn?Ile?Pro
100 105 110
ttt?gac?tcc?atc?gat?atg?gac?tat?atg?aac?ttg?cac?caa?agt?gcc?cac 384
Phe?Asp?Ser?Ile?Asp?Met?Asp?Tyr?Met?Asn?Leu?His?Gln?Ser?Ala?His
115 120 125
ggg?gac?cgc?gag?tac?gcc?tac?att?aac?tcc?cgg?ctc?aat?gtt?ccg?gca 432
Gly?Asp?Arg?Glu?Tyr?Ala?Tyr?Ile?Asn?Ser?Arg?Leu?Asn?Val?Pro?Ala
130 135 140
gcc?agc?gtt?tac?ggc?tgg?tgg?ggc?gat?gca?gac?gtt?caa?gaa?caa?att 480
Ala?Ser?Val?Tyr?Gly?Trp?Trp?Gly?Asp?Ala?Asp?Val?Gln?Glu?Gln?Ile
145 150 155 160
gcg?gac?tgg?caa?cac?gtt?gcg?gtt?gct?tac?aac?gaa?tcc?ttc?cac?att 528
Ala?Asp?Trp?Gln?His?Val?Ala?Val?Ala?Tyr?Asn?Glu?Ser?Phe?His?Ile
165 170 175
aag?atc?gcc?cgt?ttt?gga?gac?acg?atg?cgt?gac?gtg?gcc?gtt?acg?gaa 576
Lys?Ile?Ala?Arg?Phe?Gly?Asp?Thr?Met?Arg?Asp?Val?Ala?Val?Thr?Glu
180 185 190
ggt?gac?aag?gtg?gcc?gct?caa?atc?aag?ctt?ggt?tgg?aca?gtt?gat?tac 624
Gly?Asp?Lys?Val?Ala?Ala?Gln?Ile?Lys?Leu?Gly?Trp?Thr?Val?Asp?Tyr
195 200 205
tac?ccg?acc?aac?gaa?ttg?gta?gcg?gtg?gta?aac?gga?atc?gcc?gaa?gac 672
Tyr?Pro?Thr?Asn?Glu?Leu?Val?Ala?Val?Val?Asn?Gly?Ile?Ala?Glu?Asp
210 215 220
gaa?atc?gac?gcc?gct?tac?aag?gac?ttg?gaa?gct?aac?tat?gac?ttg?gtt 720
Glu?Ile?Asp?Ala?Ala?Tyr?Lys?Asp?Leu?Glu?Ala?Asn?Tyr?Asp?Leu?Val
225 230 235 240
gaa?ggt?gac?aac?gac?cac?gaa?aag?tac?gtt?cac?aac?gtt?cgc?tac?caa 768
Glu?Gly?Asp?Asn?Asp?His?Glu?Lys?Tyr?Val?His?Asn?Val?Arg?Tyr?Gln
245 250 255
ctc?cgt?gaa?tac?ctg?ggg?atc?aag?aag?ttc?ttg?gat?gac?aat?ggc?tac 816
Leu?Arg?Glu?Tyr?Leu?Gly?Ile?Lys?Lys?Phe?Leu?Asp?Asp?Asn?Gly?Tyr
260 265 270
gat?gcc?ttc?acc?gac?aac?ttc?caa?gac?ctg?gaa?ggc?tta?gaa?caa?ctg 864
Asp?Ala?Phe?Thr?Asp?Asn?Phe?Gln?Asp?Leu?Glu?Gly?Leu?Glu?Gln?Leu
275 280 285
cca?ggg?ctg?gcc?gtt?caa?ctg?ttg?atg?att?gat?ggt?tac?ggc?ttt?ggt 912
Pro?Gly?Leu?Ala?Val?Gln?Leu?Leu?Met?Ile?Asp?Gly?Tyr?Gly?Phe?Gly
290 295 300
cct?gaa?ggg?gac?ttc?aag?atg?gcc?ggc?tta?acc?cgc?ttg?ctt?aag?att 960
Pro?Glu?Gly?Asp?Phe?Lys?Met?Ala?Gly?Leu?Thr?Arg?Leu?Leu?Lys?Ile
305 310 315 320
gcc?gcc?gac?aac?aag?caa?acc?gcc?ctg?atg?gaa?gac?tac?acg?ctg?gac 1008
Ala?Ala?Asp?Asn?Lys?Gln?Thr?Ala?Leu?Met?Glu?Asp?Tyr?Thr?Leu?Asp
325 330 335
ctt?cgt?cac?ggt?cac?gaa?gcc?atc?atg?ggt?tcg?cac?atg?cta?gaa?gtt 1056
Leu?Arg?His?Gly?His?Glu?Ala?Ile?Met?Gly?Ser?His?Met?Leu?Glu?Val
340 345 350
gac?cca?acc?ctg?gcc?tcc?gac?aag?ccg?cgc?gtg?gaa?gtt?cac?cca?ctg 1104
Asp?Pro?Thr?Leu?Ala?Ser?Asp?Lys?Pro?Arg?Val?Glu?Val?His?Pro?Leu
355 360 365
ggg?att?ggt?ggc?aag?gac?gac?ccg?gcg?cgc?ctg?gtc?ttc?act?ggg?gcc 1152
Gly?Ile?Gly?Gly?Lys?Asp?Asp?Pro?Ala?Arg?Leu?Val?Phe?Thr?Gly?Ala
370 375 380
gaa?ggg?aag?ggt?tac?gac?att?acc?ctg?tct?tac?ttc?gat?gat?ggc?tac 1200
Glu?Gly?Lys?Gly?Tyr?Asp?Ile?Thr?Leu?Ser?Tyr?Phe?Asp?Asp?Gly?Tyr
385 390 395 400
aag?ttc?att?ggc?tac?ccg?gtt?gac?tgc?aag?acg?cca?gaa?gcc?gaa?atg 1248
Lys?Phe?Ile?Gly?Tyr?Pro?Val?Asp?Cys?Lys?Thr?Pro?Glu?Ala?Glu?Met
405 410 415
ccg?aag?ttg?ccg?gtc?gct?aag?caa?atg?tgg?acg?cca?gaa?att?ggg?ttg 1296
Pro?Lys?Leu?Pro?Val?Ala?Lys?Gln?Met?Trp?Thr?Pro?Glu?Ile?Gly?Leu
420 425 430
gct?gag?ggt?gct?aag?caa?tgg?atg?aag?tac?ggt?ggt?ggt?cac?cac?acc 1344
Ala?Glu?Gly?Ala?Lys?Gln?Trp?Met?Lys?Tyr?Gly?Gly?Gly?His?His?Thr
435 440 445
gtc?ttg?acc?cta?gcc?cta?agc?gaa?gaa?caa?tta?gaa?caa?ttg?gca?cgt 1392
Val?Leu?Thr?Leu?Ala?Leu?Ser?Glu?Glu?Gln?Leu?Glu?Gln?Leu?Ala?Arg
450 455 460
ttg?ttc?aag?gtc?gac?ttt?atc?aac?atc?aag?tag 1425
Leu?Phe?Lys?Val?Asp?Phe?Ile?Asn?Ile?Lys
465 470
<210>2
<211>474
<212>PRT
＜213〉lactobacillus fermentum (Lactobacillus fermentum) NXTag-1
<400>2
Met?Arg?Lys?Met?Gln?Asp?Tyr?Lys?Phe?Trp?Phe?Val?Val?Gly?Ser?Gln
1 5 10 15
Pro?Leu?Tyr?Gly?Pro?Glu?Ala?Leu?Ala?Glu?Val?Glu?Lys?Asp?Ala?Arg
20 25 30
Lys?Leu?Val?Asp?Gly?Leu?Asn?Lys?Gly?Gly?Lys?Leu?Asp?Tyr?Pro?Val
35 40 45
Glu?Phe?Lys?Leu?Val?Ala?Thr?Thr?Ala?Asp?Ser?Ile?Thr?Lys?Phe?Met
50 55 60
Lys?Glu?Ala?Asn?Tyr?Asn?Asp?Asp?Val?Ala?Gly?Val?Ile?Thr?Trp?Met
65 70 75 80
His?Thr?Phe?Ser?Pro?Ala?Lys?Asn?Trp?Ile?Arg?Gly?Thr?Glu?Leu?Leu
85 90 95
Gln?Lys?Pro?Leu?Leu?His?Leu?Ala?Thr?Gln?Phe?Leu?Asn?Asn?Ile?Pro
100 105 110
Phe?Asp?Ser?Ile?Asp?Met?Asp?Tyr?Met?Asn?Leu?His?Gln?Ser?Ala?His
115 120 125
Gly?Asp?Arg?Glu?Tyr?Ala?Tyr?Ile?Asn?Ser?Arg?Leu?Asn?Val?Pro?Ala
130 135 140
Ala?Ser?Val?Tyr?Gly?Trp?Trp?Gly?Asp?Ala?Asp?Val?Gln?Glu?Gln?Ile
145 150 155 160
Ala?Asp?Trp?Gln?His?Val?Ala?Val?Ala?Tyr?Asn?Glu?Ser?Phe?His?Ile
l65 170 175
Lys?Ile?Ala?Arg?Phe?Gly?Asp?Thr?Met?Arg?Asp?Val?Ala?Val?Thr?Glu
180 185 190
Gly?Asp?Lys?Val?Ala?Ala?Gln?Ile?Lys?Leu?Gly?Trp?Thr?Val?Asp?Tyr
195 200 205
Tyr?Pro?Thr?Asn?Glu?Leu?Val?Ala?Val?Val?Asn?Gly?Ile?Ala?Glu?Asp
210 215 220
Glu?Ile?Asp?Ala?Ala?Tyr?Lys?Asp?Leu?Glu?Ala?Asn?Tyr?Asp?Leu?Val
225 230 235 240
Glu?Gly?Asp?Asn?Asp?His?Glu?Lys?Tyr?Val?His?Asn?Val?Arg?Tyr?Gln
245 250 255
Leu?Arg?Glu?Tyr?Leu?Gly?Ile?Lys?Lys?Phe?Leu?Asp?Asp?Asn?Gly?Tyr
260 265 270
Asp?Ala?Phe?Thr?Asp?Asn?Phe?Gln?Asp?Leu?Glu?Gly?Leu?Glu?Gln?Leu
275 280 285
Pro?Gly?Leu?Ala?Val?Gln?Leu?Leu?Met?Ile?Asp?Gly?Tyr?Gly?Phe?Gly
290 295 300
Pro?Glu?Gly?Asp?Phe?Lys?Met?Ala?Gly?Leu?Thr?Arg?Leu?Leu?Lys?Ile
305 310 315 320
Ala?Ala?Asp?Asn?Lys?Gln?Thr?Ala?Leu?Met?Glu?Asp?Tyr?Thr?Leu?Asp
325 330 335
Leu?Arg?His?Gly?His?Glu?Ala?Ile?Met?Gly?Ser?His?Met?Leu?Glu?Val
340 345 350
Asp?Pro?Thr?Leu?Ala?Ser?Asp?Lys?Pro?Arg?Val?Glu?Val?His?Pro?Leu
355 360 365
Gly?Ile?Gly?Gly?Lys?Asp?Asp?Pro?Ala?Arg?Leu?Val?Phe?Thr?Gly?Ala
370 375 380
Glu?Gly?Lys?Gly?Tyr?Asp?Ile?Thr?Leu?Ser?Tyr?Phe?Asp?Asp?Gly?Tyr
385 390 395 400
Lys?Phe?Ile?Gly?Tyr?Pro?Val?Asp?Cys?Lys?Thr?Pro?Glu?Ala?Glu?Met
405 410 415
Pro?Lys?Leu?Pro?Val?Ala?Lys?Gln?Met?Trp?Thr?Pro?Glu?Ile?Gly?Leu
420 425 430
Ala?Glu?Gly?Ala?Lys?Gln?Trp?Met?Lys?Tyr?Gly?Gly?Gly?His?His?Thr
435 440 445
Val?Leu?Thr?Leu?Ala?Leu?Ser?Glu?Glu?Gln?Leu?Glu?Gln?Leu?Ala?Arg
450 455 460
Leu?Phe?Lys?Val?Asp?Phe?Ile?Asn?Ile?Lys
465 470
<210>3
<211>30
<212>DNA
<213>Artificial
<220>
<223>Primer1
<400>3
agagaattca?tgcgtaagat?gcaagattac 30
<210>4
<211>29
<212>DNA
<213>Artificial
<220>
<223>Primer2
<400>4
aagctcgagc?tacttgatgt?tgataaagt 29
Claims (10)
1. fire resistant L-arabinose isomerase, the aminoacid sequence that it is characterized in that it is shown in SEQ ID NO:2.
2. the encoding gene of fire resistant L-arabinose isomerase shown in the claim 1, the nucleotide sequence that it is characterized in that it is shown in SEQ IDNO:1.
3. genetic engineering bacterium that produces fire resistant L-arabinose isomerase is characterized in that it comprises nucleotide sequence shown in SEQ IDNO:1.
4. the construction process of the described genetic engineering bacterium of claim 3 is characterized in that this method comprises the steps:
(1) structure contains fire resistant L-arabinose isomerase expression of gene plasmid:
Get lactobacillus fermentum (Lactobacillus fermentum) NXTag-1CGMCC NO.2921 genomic dna as masterplate, as primer, carry out pcr amplification with the following nucleotide sequences of the restriction enzyme site that comprises EcoRI and XhoI:
Primer 1:5 '-AGAGAATTCATGCGTAAGATGCAAGATTAC-3 '
Primer 2: 5 '-AAGCTCGAGCTACTTGATGTTGATAAAGT-3 '
The pcr amplification system is: genomic dna 2 μ L, each 1 μ L of primer 1 and primer 2, dNTP2 μ L, 10 * Tag damping fluid, 2.5 μ L, ExTag polysaccharase 0.5 μ L, ddH
2O 14 μ L;
The PCR response procedures is: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 2min; 60 ℃ of annealing 30s then, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ are extended 1min;
Reclaim pcr amplification product, through restriction enzyme EcoRI and XhoI double digestion, the plasmid pET-28a (+) with through same double digestion connects under the effect of T4 ligase enzyme, obtains recombinant plasmid pET-araA;
(2) this recombinant plasmid pET-araA is converted in the host cell:
Recombinant plasmid pET-araA is converted in the competence e. coli bl21 (DE3), and coating contains on the LB solid medium of 25 μ g/mL kantlex, 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 LB liquid nutrient medium of 25 μ g/mL kantlex in 5mL respectively, 37 ℃, the 200rpm overnight incubation, extract plasmid, through restriction enzyme EcoRI 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-araA, has the positive clone of bacterium colony of this plasmid, is genetic engineering bacterium.
5. the expression method of a fire resistant L-arabinose isomerase, it is characterized in that the genetic engineering bacterium that will comprise the nucleotide sequence shown in SEQ ID NO:1 is inoculated in the LB liquid nutrient medium that has added 25 μ g/mL kantlex 37 ℃ of shaking table overnight incubation; Inoculum size with 5% (v/v) is transferred in the LB substratum that contains 25 μ g/mL kantlex again, and 37 ℃ of fermentation culture 2～3 hours are to OD
600Be to add the isopropyl-of 0.2～1mmol/L or the lactose of 0.5～2g/L at 0.6 o'clock, continue abduction delivering after 6～12 hours, centrifugal collection thalline.
6. the application of the described fire resistant L-arabinose isomerase of claim 1 in preparation D-tagatose.
7. application according to claim 6, it is characterized in that the D-semi-lactosi with 1～100g/L is a substrate, add fire resistant L-arabinose isomerase and carry out enzymatic conversion reaction, the consumption of fire resistant L-arabinose isomerase is 10～500mg/L, 40～70 ℃ of temperature of reaction, transformation time 8～15h.
8. the application of the genetic engineering bacterium of the described product fire resistant L-arabinose isomerase of claim 3 in preparation D-tagatose.
9. application according to claim 8, it is characterized in that the D-semi-lactosi with 1～100g/L is a substrate, the genetic engineering bacterium of the product fire resistant L-arabinose isomerase of adding behind abduction delivering carries out conversion reaction, produce the consumption of the genetic engineering bacterium of fire resistant L-arabinose isomerase and count 10～100g/L with wet thallus, 40～70 ℃ of temperature of reaction, transformation time 12～48h.
10. application according to claim 9 is characterized in that the D-semi-lactosi with 1～100g/L is a substrate, adds 0.5～2mmol/L Mn
2+Ion and 1～3mmol/L Co
2+Ion, the genetic engineering bacterium of the product fire resistant L-arabinose isomerase of adding behind abduction delivering carries out conversion reaction, produce the consumption of the genetic engineering bacterium of fire resistant L-arabinose isomerase and count 10～100g/L with wet thallus, 40～70 ℃ of temperature of reaction, transformation time 12～48h.
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| CN105483108B (en) * | 2016-01-29 | 2019-07-09 | 南京工业大学 | A kind of L-arabinose isomerase and its application in the production of L- ribulose |
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| CN110951717B (en) * | 2019-12-28 | 2023-08-18 | 浙江工业大学 | L-arabinose isomerase isomer and application thereof |
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