CN103087969A - Bacterial surface demonstrating system for xylose dehydrogenase based on ice nucleating protein and application of system - Google Patents
Bacterial surface demonstrating system for xylose dehydrogenase based on ice nucleating protein and application of system Download PDFInfo
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- CN103087969A CN103087969A CN2012100073982A CN201210007398A CN103087969A CN 103087969 A CN103087969 A CN 103087969A CN 2012100073982 A CN2012100073982 A CN 2012100073982A CN 201210007398 A CN201210007398 A CN 201210007398A CN 103087969 A CN103087969 A CN 103087969A
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Abstract
The invention relates to the technical field of biotechnology and analysis, and in particular relates to a bacterial surface demonstrating system for xylose dehydrogenase based on ice nucleating protein and application of the system. The bacterial surface demonstrating system is a generic sequence xylB which codes xylose dehydrogenase of a target protein and a generic sequence inaPb-N in an N end structural domain of the ice nucleating protein which is responsible for positioning cross membrane and transferring. The bacterial surface demonstrating system is used for xylose detection. The method for detecting xylose is high in sensitivity, good in specificity and simple in method, is suitable for industrial fields of foods, fruits and vegetables, sugar and wine, beverages, meat, food additives, health-care products, medicines, cosmetics, toothpaste, cigarettes and the like, and is appropriate for monitoring xylose in biological process fields such as fermentation, biological conversion and biological energy resource.
Description
Technical field
The present invention relates to biotechnology and analysis technical field, specifically a kind of wood sugar desaturase bacterium surface exhibiting system and the application in the wood sugar rapid detection thereof based on ice nucleation protein.
Background technology
Wood sugar is a kind of important carbohydrate, has very important effect in people's productive life.At occurring in nature, natural D-wood sugar is that the form with polysaccharide is present in plant, their (for example crust of the cob of corn, stalk, cotton boll) content in the discarded part of agricultural-food is very high, and wood sugar is the important products in its natural degradation and artificial degraded.Utilize wood-sugar fermentation to produce ethanol and can improve the utilization ratio of raw material and the output of alcohol fuel, reduce the production cost of alcohol fuel, therefore be subjected at present increasing concern.What have in bacterium can utilize the D-wood sugar as carbon source for growth, and sheep almost can fully utilize wood sugar in animal.The D-wood sugar is connected with glycosidic link with the serine residue of polypeptide chain in the protein-polysaccharide of animal, becomes the bridging structure of polysaccharide side chain and protein.Xylitol is that wood sugar gets by Hydrogenation, and its purposes is very extensive.Be applied to feed for pet at developed country's wood sugar.Wood sugar can be used as non-caloric sweetener, nutrition agent and the therapeutical agent of diabetics's better tolerance.It can also as the heated food sweetener, be used for bakery product, ham, sausage, bacon, synthetic meat etc.Wood sugar also is widely used in the aspects such as meat-processing, Meat flavor aroma and preparation food antioxidant.In addition, wood sugar has been used for the multiple industries such as paint, toothpaste, cigarette, process hides, casting, gunpowder as tenderizer, ionic surface active agent, softening agent, moisture adjuster etc.Therefore, fast, highly sensitive, highly selective, real-time wood sugar detect heredity, human nutrition, food technology, medicine and comprise that the fields such as bioprocess of biofuel are of crucial importance.
At present, the detection method of wood sugar has reduction method, Enzymology method, chromatography, Raman spectroscopy and infrared spectroscopy etc.But there are various problems in these methods, and for example, when adopting reduction method to detect, its sensitivity is lower, and is vulnerable to the interference that other have color and reducing substances; In Enzymology method, the source of enzyme and purifying are vital problems; Chromatography comprises vapor-phase chromatography, liquid phase chromatography and ion chromatography, Main Problems is that the sample preparation process is complicated, during operational cost, error is large, need expensive device etc.Therefore, set up a kind of quick, sensitive, special wood sugar real-time detection method, imperative.
The advantage of Enzymology method be highly sensitive, specificity good, but the purifying of enzyme and stability are insoluble problems.Wood sugar desaturase (XDH) (EC 1.1.1.175) is the key enzyme of xylose metabolism approach in some microorganisms.Wood sugar is under the catalysis of wood sugar desaturase, by means of coenzyme NAD
+, be oxidized to the xylosic acid lactone, coenzyme NAD
+Be reduced to NADH.
It is the important branch that the microorganism surface presents system that bacterium surface presents system, and its elementary tactics is also in bacterium surface with foreign protein or polypeptide and bacterium surface protein fusion or chimeric and activity expression.Since finding that escherichia coli outer membrane protein LamB, OmpA and PhoE can be expression vector as the surface of foreign protein, this technology has obtained fast development, now become the focus of research, be widely applied in the basic and applied research in a plurality of fields such as microbiology, molecular biology, vaccinology.Ice nucleation protein (ice-nucleation protein, INP)) be the outer membrane protein of the bacterial strain such as pseudomonas syringae, the ice crystal that can accelerate pure water forms.INP is anchored on bacterium surface by glycosyl-phosphatidyl inositol, and the surface that is beneficial to high molecular weight protein presents.The ice nucleation protein bacterium comprises Rhodopseudomonas, erwinia and Xanthomonas campestris etc.At present, utilize total length ice nucleation protein, remove in the middle of tumor-necrosis factor glycoproteins series winding N end, C end structure territory albumen or only all successfully showed foreign protein on the intestinal bacteria surfaces with the albumen in N end structure territory, such as green fluorescent protein, chitinase and phosphate binding protein etc.In recent years, find a kind of new ice nucleation protein gene inaPb in Pseudomonas borealis bacterial strain, it only has with the ice nucleation protein of having known～66% homology.
Summary of the invention
The purpose of this invention is to provide a kind of bacterium surface exhibiting system based on ice nucleation protein and the application in wood sugar detects thereof.
For achieving the above object, the technical solution used in the present invention is:
A kind of wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein: bacterium surface exhibiting system is the gene order inaPb-N in the ice nucleation protein N end structure territory of the gene order xylB of coding target protein wood sugar desaturase and responsible cross-film location and transhipment.
The gene order inaPb-N in the ice nucleation protein N end structure territory of the gene order xylB of coding target protein wood sugar desaturase and responsible cross-film location and transhipment merges, be expressed in host cell surface, obtain the wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein.
Application based on the wood sugar desaturase bacterium surface exhibiting system of ice nucleation protein: described bacterium surface exhibiting system is used for high specific, the high-sensitivity detection of wood sugar.Described bacterium surface exhibiting system is used for high specific, the high-sensitivity detection of D-wood sugar.
Detect principle and be wood sugar under the catalysis of the wood sugar desaturase on thalline surface, by means of coenzyme Reduced nicotinamide-adenine dinucleotide (NAD
+) be oxidized to xylosic acid lactone, coenzyme NAD
+Be reduced to the Reduced nicotinamide-adenine dinucleotide (NADH) of reduction-state.NADH has charateristic avsorption band at the 340nm place, therefore can try to achieve according to the light absorption value at 340nm place the content of wood sugar.
Effect of the present invention is:
1. the present invention utilizes the ice nucleation protein surface display system wood sugar desaturase stably to be illustrated in the surface of bacterium, thereby has solved the difficult problem that the intracellular enzyme leaching process is complicated and stability is low.
2. the wood sugar desaturase enzyme of bacterium surface displaying of the present invention is alive high, compares good stability with the wood sugar desaturase in born of the same parents in original strain.
3. the wood sugar desaturase specificity of bacterium surface displaying of the present invention is good, other carbohydrates, as all noiseless to the mensuration of D-wood sugar in D-Glucose, cellobiose, D-semi-lactosi, D-MANNOSE, D-ribose, D-Fructose, D-sucrose, D-Xylitol, D-Maltose and L-arabinose.
4. the wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein of the present invention's structure is highly sensitive, has wider wood sugar sensing range (5-900 μ M) and lower detectability (2 μ M).
5. the wood sugar desaturase that builds of the present invention shows that bacterial strain can be fixed on electrode, builds electrochemica biological sensor, realizes the detection of wood sugar, and this thalline can repeatedly use, good stability.
6. the wood sugar desaturase of the present invention's structure shows that bacterial strain can be developed into a kind of biological catalyst, is used for the fields such as biofuel cell.
7. set up the method for wood sugar rapid detection by bacterium surface exhibiting system provided by the invention.This method is highly sensitive, specificity good, simple to operate, cost is low, can be used for food, fruits and vegetables, sugar wine beverage, meat, foodstuff additive, healthcare products, medicine, with industrial circles such as makeup, toothpaste, cigarette, and the monitoring of wood sugar in the bioprocess such as fermentation, bio-transformation, bioenergy field.
Description of drawings
The PCR product glue recovery figure of the wood sugar dehydrogenase gene xdh that Fig. 1 provides for the embodiment of the present invention.A is the DL2000 molecular weight standard; B is that the PCR product glue of gene xdh reclaims band.
The PCR product glue recovery figure of the wood sugar dehydrogenase gene inaPb-N that Fig. 2 provides for the embodiment of the present invention.A is the DL2000 molecular weight standard; B is that the PCR product glue of gene inaPb-N reclaims band.
The vector construction schema of the wood sugar desaturase bacterium surface exhibiting system that Fig. 3 provides for the embodiment of the present invention.
The canonical plotting of the UV spectrophotometer measuring D-wood sugar that Fig. 4 provides for the embodiment of the present invention.
The object of the invention, function and advantage are described further with reference to accompanying drawing in connection with embodiment.
Embodiment
The present invention builds based on wood sugar desaturase bacterium surface exhibiting system, utilizes the UV spectrophotometer measuring signal, thereby obtains the content of wood sugar, and then be used for the rapid detection of wood sugar, is specially:
1. build the wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein;
2. make the typical curve that wood sugar detects;
3. the wood sugar in the mensuration actual sample calculates the content of wood sugar according to typical curve.
Embodiment 1
The acquisition of cell surface display wood sugar desaturase thalline:
1. cultivate the crescent handle bacillus strain (Caulobacter crescentus NA1000) (commercial product) of wood sugar desaturase: above-mentioned bacterial strains is inoculated in mid-30 ℃ of substratum, 200rpm, shaking table was cultivated 24 hours.
Medium component: phytone 5.0g, yeast extract 2.5g, K
2HPO
43.7g, KH
2PO
41.3g, MgSO
47H
2O 0.5g, NaCl 1.0g, distilled water 1.0L, pH 7.2-7.5, high-temperature sterilization, stand-by;
2. cultivate ice nucleation protein bacterium Pseudomonas borealis (commercial product): with above-mentioned bacterial strains in substratum mid-22 ℃, 200rpm, shaking table was cultivated 16 hours.
Medium component is: Tryptones 1.5% (g/100ml), soy peptone 0.5% (g/100ml), sodium-chlor 0.5% (g/100ml), formulated with distilled water, regulating pH is 7.2 ± 0.2, after 121 ℃ of pressuresteam sterilizations, stand-by.
3. the structure of recombinant expression vector.
1) design and synthesize primer take crescent handle bacillus (C.crescentus NA1000) genomic dna as template, carry out pcr amplification, the gene xylB of the wood sugar desaturase that obtains encoding.
Primer is P1 (5 ' → 3 '): CG
GGATCCATGTCCTCAGCCATCTATCCCAGCC and P2 (5 ' → 3 '): CCC
AAGCTTACGCCAGCCGGCGTCGATCCAGT.
The PCR reaction system is: ddH
2O 34.7 μ L, template DNA 1 μ L, 10 * PCR Buffer (Mg
2+Free) 5 μ L, MgCl
2(25mmol/L) 3 μ L, primer P1 (20pmol/ μ L) 1 μ L, primer P2 (20pmol/ μ L) 1 μ L, dNTPMixture (each 2.5mmol/L) 4 μ L, TaKaRa Taq
TM(5U/ μ L) 0.3 μ L.Reaction parameter is: 95 ℃ of denaturation 5min, and 94 ℃ of 30s, 59 ℃ of 30s, 72 ℃ of 60s, 30 circulations, last 72 ℃ are extended 10min.
PCR product purifying after agarose gel electrophoresis is identified is reclaimed (referring to Fig. 1), be connected in pMD-19T simple carrier, construction recombination plasmid, called after pMDXdh.
2) take ice nucleation protein bacterium Pseudomonas borealis strain gene group DNA as template, design and synthesize primer, carry out pcr amplification, the gene inaPb-N in the ice nucleation protein N end structure territory that obtains encoding.
Primer is P3 (5 ' → 3 '): CATG
CCATGGGCATGAACGATGACAAAGTTTTGGTC and P4 (5 ' → 3 '): CG
GGATCCCACCGCTGTCTCCAGCGTTTGTG.
The PCR reaction system is: ddH
2O 34.7 μ L, template DNA 1 μ L, 10 * PCR Buffer (Mg
2 +Free) 5 μ L, MgCl
2(25mmol/L) 3 μ L, primer P3 (20pmol/ μ L) 1 μ L, primer P4 (20pmol/ μ L) 1 μ L, dNTPMixture (each 2.5mmol/L) 4 μ L, TaKaRa Taq
TM(5U/ μ L) 0.3 μ L.Reaction parameter is: 95 ℃ of denaturation 5min, and 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 60s, 30 circulations, last 72 ℃ are extended 10min.
PCR product purifying after agarose gel electrophoresis is identified is reclaimed (referring to Fig. 2), be connected in pMD-19T simple carrier.Then the recombinant vectors that obtains is connected on the carrier pET28a (+) of same double digestion with restriction enzyme Nco I and BamH I double digestion the recombinant vectors called after pTInaPb-N that obtains.
3) with above-mentioned carrier pMDXdh restriction enzyme BamH I and Hind III double digestion, then be connected with the carrier pTInaPb-N of same double digestion, obtain the fusion gene of xylB and inaPb-N;
4. the expression of recombinant vectors.Recombinant vectors is transformed intestinal bacteria Escherichia coli BL21 (DE3), namely obtain the thalline (referring to Fig. 3) of cell surface display wood sugar desaturase.Enzyme is inoculated in the fresh LB liquid nutrient medium that contains kantlex (30 μ g/ml) after cutting and identifying correctly, and shaking culture is to absorbancy (OD
600) be～0.4 o'clock, adding IPTG (sec.-propyl-β-D-sulfo-galactopyranoside) to make its final concentration is 1mmol/L, 25 ℃ of inducing culture 24h are stand-by.
Intestinal bacteria E.coli BL21 (DE3) cultivates in the LB substratum, and the LB medium component is: 5g/L yeast extract paste, 10g/L peptone, 10g/L NaCl.
Embodiment 2
Utilize the wood sugar desaturase bacterium surface exhibiting system that builds, carry out the rapid detection of wood sugar by ultraviolet spectrophotometer:
1. the drafting of typical curve: above-described embodiment gained cell surface display wood sugar desaturase thalline is carried out abduction delivering, gather in the crops thalline after 24h, with 50mM PBS damping fluid (pH 8.0) washing three times, cell density is adjusted to OD
600=1.0.The bacterium liquid, the NAD that add above-mentioned 100 μ L in the 1ml reaction solution (50mM PBS damping fluid, pH 8.0)
+The D-xylose solution of (final concentration is 1mM) and different concns (final concentration is 0-1000 μ M), carry out parallel running, mixing is placed in 30 ℃ of water-baths, takes out after 10 minutes, 12000rpm/min, centrifugal 1 minute, get supernatant liquor, move in micro-cuvette, wavelength 340nm carries out spectrometry in the place, returns to zero with 50mMPBS damping fluid (pH 8.0).At last take xylose concentration as X-coordinate, the light absorption value at wavelength 340nm place is ordinate zou, and the drawing standard curve can get straight line, obtains its slope, is the working standard rate of curve (referring to Fig. 4) of D-wood sugar.
The working standard curve of D-wood sugar as shown in Figure 4, as seen from the figure, the R of this working standard curve
2Value reaches 0.999, and Linear Quasi is right fine, and its slope is 0.0029.
2.D-the testing process of wood sugar.The bacterium liquid, the NA that add above-mentioned 100 μ L in the reaction solution
+(ultimate density is 1mM) and the testing sample that contains the D-wood sugar through diluting, carry out parallel running, mixing, place in 30 ℃ of water-baths, take out after 10 minutes, centrifugal, get supernatant liquor in its absorbancy of spectrophotometer wavelength 340nm place's measurement, return to zero with 50mM PBS damping fluid (pH 8.0).According to the absorbancy that records, utilize D-wood sugar working standard opisometer to calculate the content of D-wood sugar.
Embodiment 3
The mensuration of D-Xylose Content in degraded product after stalk is processed through physical chemistry:
1. sample preparation: the degradation solution of stalk after physical chemistry is processed carried out filtration treatment with the disposable filter membrane of 0.22 μ m, dilute (Dilution ratio is respectively 10 times, 100 times, 1000 times) with 50mM PBS damping fluid (pH 8.0), standby.
2. the thalline that the wood sugar desaturase is arranged is showed in preparation: above-described embodiment gained cell surface display wood sugar desaturase thalline is inoculated in the fresh LB liquid nutrient medium that contains kantlex (30 μ g/ml), and shaking culture is to absorbancy (OD
600) be～0.4 o'clock, adding IPTG to make its final concentration is 1mmol/L, 25 ℃ of inducing culture 24h, the results thalline, washing is adjusted to OD with 50mM PBS damping fluid (pH 8.0) with cell density
600=1.0.
3.D-wood sugar testing process: concrete steps are seen embodiment 2.
4.D-the calculating of xylose concentration: the light absorption value of getting the Dilution ratio of light absorption value in standard curve range calculates, the calculation formula that the substitution typical curve draws, multiply by at last corresponding extension rate, thereby the concentration that obtains D-wood sugar in former straw degradative liquid is 84.87 ± 1.45mM.D-xylose concentration calculation formula is:
Embodiment 4
The mensuration of D-Xylose Content in Tsingtao beer:
1. sample preparation: with Tsingtao beer, carry out filtration treatment with the disposable filter membrane of 0.22 μ m, dilute (Dilution ratio is respectively 10 times, 100 times, 1000 times) with 50mM PBS damping fluid (pH 8.0), standby.
2. the thalline that the wood sugar desaturase is arranged is showed in preparation: above-described embodiment gained cell surface display wood sugar desaturase thalline is inoculated in the fresh LB liquid nutrient medium that contains kantlex (30 μ g/ml), and shaking culture is to absorbancy (OD
600) be～0.4 o'clock, adding IPTG to make its final concentration is 1mmol/L, 25 ℃ of inducing culture 24h, the results thalline, washing is adjusted to OD with 50mM PBS damping fluid (pH 8.0) with cell density
600=1.0.
3.D-wood sugar testing process: the bacterium liquid, the NAD that add above-mentioned 100 μ L in the 1ml reaction solution (50mM PBS damping fluid, pH 8.0)
+(final concentration is 1mM) and the beer that diluted, mixing, place in 30 ℃ of water-baths, take out 12000rpm/min, centrifugal 1 minute after 10 minutes, get supernatant liquor, move in micro-cuvette, wavelength 340nm carries out spectrometry in the place, take 50mM PBS damping fluid (pH 8.0) as blank.
4.D-the calculating of Xylose Content: the light absorption value of getting the Dilution ratio of light absorption value in standard curve range calculates, the calculation formula that the substitution typical curve draws, multiply by at last corresponding extension rate, thereby the content that obtains D-wood sugar in raw sample is 1.81 ± 0.13mM.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (4)
1. wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein is characterized in that: bacterium surface exhibiting system is the gene order inaPb-N in the ice nucleation protein N end structure territory of the gene order xylB of coding target protein wood sugar desaturase and responsible cross-film location and transhipment.
2. by the wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein claimed in claim 1, it is characterized in that: the gene order inaPb-N in the ice nucleation protein N end structure territory of the gene order xylB of coding target protein wood sugar desaturase and responsible cross-film location and transhipment merges, be expressed in host cell surface, obtain the wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein.
3. the application of the wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein claimed in claim 1 is characterized in that: described bacterium surface exhibiting system is used for high specific, the high-sensitivity detection of wood sugar.
4. the application of the wood sugar desaturase bacterium surface exhibiting system based on ice nucleation protein claimed in claim 3 is characterized in that: described bacterium surface exhibiting system is used for high specific, the high-sensitivity detection of D-wood sugar.
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| CN104804068A (en) * | 2014-01-26 | 2015-07-29 | 中国科学院青岛生物能源与过程研究所 | Polypeptide, application thereof, and method for covalently connecting polypeptide to solid phase interface |
| CN105624077A (en) * | 2014-11-05 | 2016-06-01 | 中国科学院青岛生物能源与过程研究所 | Sequential enzyme surface co-display system and use thereof |
| CN105624265A (en) * | 2014-10-30 | 2016-06-01 | 中国科学院青岛生物能源与过程研究所 | Method for determining L-glutamic acid content through whole-cell of which surface displays glutamate dehydrogenase |
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| CN114958891A (en) * | 2022-05-24 | 2022-08-30 | 郑州大学 | Escherichia coli recombinant expression vector and application thereof |
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| CN104804068A (en) * | 2014-01-26 | 2015-07-29 | 中国科学院青岛生物能源与过程研究所 | Polypeptide, application thereof, and method for covalently connecting polypeptide to solid phase interface |
| CN104804068B (en) * | 2014-01-26 | 2017-12-19 | 中国科学院青岛生物能源与过程研究所 | A kind of polypeptide and its application and the method that polypeptid covalence is connected to solid phase interface |
| CN105624265A (en) * | 2014-10-30 | 2016-06-01 | 中国科学院青岛生物能源与过程研究所 | Method for determining L-glutamic acid content through whole-cell of which surface displays glutamate dehydrogenase |
| CN105624077A (en) * | 2014-11-05 | 2016-06-01 | 中国科学院青岛生物能源与过程研究所 | Sequential enzyme surface co-display system and use thereof |
| CN105624077B (en) * | 2014-11-05 | 2020-09-18 | 中国科学院青岛生物能源与过程研究所 | Sequential enzyme surface co-display system and application thereof |
| CN108303539A (en) * | 2018-01-31 | 2018-07-20 | 刘双萍 | A kind of breast cancer cell detection biological reagent and application |
| CN108303539B (en) * | 2018-01-31 | 2020-08-11 | 刘双萍 | Biological reagent for detecting breast cancer cells and application thereof |
| CN114958891A (en) * | 2022-05-24 | 2022-08-30 | 郑州大学 | Escherichia coli recombinant expression vector and application thereof |
| CN114990038A (en) * | 2022-05-24 | 2022-09-02 | 郑州大学 | Bacterial outer membrane vesicle and application thereof in preparation of preeclampsia treatment medicine |
| CN114958891B (en) * | 2022-05-24 | 2023-04-14 | 郑州大学 | Escherichia coli recombinant expression vector and application thereof |
| CN114990038B (en) * | 2022-05-24 | 2023-06-27 | 郑州大学 | Bacterial outer membrane vesicle and application thereof in preparation of preeclampsia treatment drug |
| CN116240193A (en) * | 2023-05-06 | 2023-06-09 | 北京量维生物科技研究院有限公司 | Choline kinase mutant and application thereof in production of citicoline |
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