CN101289687B - A kind of high vigor height product specificities sucrose isomerase produces the high-throughput screening method of bacterial strain - Google Patents
A kind of high vigor height product specificities sucrose isomerase produces the high-throughput screening method of bacterial strain Download PDFInfo
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Abstract
The invention discloses the high-throughput screening method that a kind of high vigor height product specificities sucrose isomerase produces bacterial strain, by sucrose isomerase enzyme-producing bacteria to be screened access containing shaking culture in the microwell plate of substratum, centrifugal segregation supernatant liquor, add sucrose solution and carry out enzymatic conversion reaction, after reaction terminates, the supernatant liquor in every hole is transferred to successively in another microwell plate, add colouring reagents, and detect light absorption value, filter out sucrose isomerase superior strain to carry out shaking flask again and sieve again, thin-layer chromatography is utilized to screen the good bacterial strain of product specificities, finally obtain the bacterial strain of high yield Palatinose or the bacterial strain of high yield trehalulose.This screening method utilizes color reaction intuitively, and the micro-cultivation of enzyme plate, continous sample adding apparatus and the volley of rifle fire effect, the treatment capacity of everyone 200 samples/more than day can be reached, for the screening of high product specificities sucrose isomerase producing strains provides easy, efficient, micro-screening method.
Description
Technical field
The invention belongs to fermentation engineering and technical field of enzyme engineering, be specifically related to a kind of selection producing the microorganism strains of sucrose isomerase (SIase).
Background technology
Sucrose isomerase is the important glucanotransferase of a class, and it is the key enzyme of preparation functional Sugar Alcohol-hydroxyl isomaltulose.Hydroxyl isomaltulose (Isomalt) is functional Sugar Alcohol emerging in the world in recent years, there is low-yield, low absorption, prevention of dental caries performance, can eat for diabetic population and fat-reducing personage, edible safety is high, it will be further appreciated that hydroxyl isomaltulose has extremely low water absorbability and pure taste, this is that other functional sugar alcohol difficulties such as Xylitol and maltose alcohol realize.Due to these features, hydroxyl isomaltulose becomes sucrose substitute most popular at present.
Hydroxyl isomaltulose is produced in two steps, the first step is by sucrose isomerase EC5.4.99.11 (Sucroseisomerase), or be Palatinose synthetic enzyme (Isomaltulosesyntheses), sucrose glucosyl group mutase (Sucroseglucosylmutase), alpha-glucosyl transferring enzyme (α-glucosyltransferase) catalysing sucrose generates Palatinose, and second step is Palatinose hydrogenation under catalyst action is hydroxyl isomaltulose.There will be a known some bacterial classifications and can produce sucrose isomerase.United States Patent (USP) 4,390,627 describe the method for producing Palatinose from the fixing sucrose mutase of Protaminobacterrubrum (protaminobacter ruber).United States Patent (USP) 4,670,387 describe use Erwiniarhapontici, Protaminobacterrubrum, the immobilized thallus of Serratiaplymuthica (serratia marcescens) produces the process of Palatinose, approximately can transform 70-95% sucrose.United States Patent (USP) 4,857,461 processes disclosing the immobilization obtained from Protaminobacterrubrum, Serratiaplymuthica and Erwiniacarotovora bacterium thick enzyme continuous seepage Palatinose.U.S. Patent No. 5,229,276 and No.5,336,617 describe the process of producing trehalulose and Palatinose with the immobilized thallus of Agrobacteriumradiobacter (radioactive soil bacillus), and Palatinose accounts for less than 10%.
Although above-mentioned several bacterium can be transformed into Palatinose sucrose, output is very unstable, and transformation efficiency is 8 ~ 85%.And except principal product, also there is the by product such as part trehalulose and a small amount of isomaltose, different melizitose, glucose and fructose in enzymatic conversion liquid, the ratio of general Palatinose and trehalulose is 3: 1 ~ 5: 1.Product specificities is not high, reduces the yield of object product on the one hand, and make downstream process become very difficult on the other hand, production cost increases greatly.Therefore current, the bottleneck of hydroxyl isomaltulose preparation technology is mainly that this process of Palatinose is prepared in sucrose isomerase enzyme catalysis.
It is worth noting by product trehalulose and Palatinose character similar, be also a kind of novel sweetener.Therefore screening obtains the sucrose isomerase producing strains of main product trehalulose, equally also has positive meaning to further development of new sweeting agent market.
Screening can the bacterial strain of High-efficient Production sucrose isomerase, while raising SIase enzyme is lived, research affects the internal factor of SIase product specificities and improves SIase product specificities, to the industrial production of Palatinose and sugar alcohol thereof and development of new sweeting agent significant.But the screening of microorganism strains is a heavy work, conventional screening method workload is very large, need to consume a large amount of manpower and materials, the screening cycle is very long, and often can not get object bacterial strain, the blindness of screening is very large, therefore sets up that one efficient, fast, trace, accurately screening method improve sucrose isomerase enzyme to live, improve the key of its product specificities.
Microwell plate is the experimental ware that a class is commonly used at bioengineering field, middle nineteen sixties, microtest plate is developed as the small and exquisite miniature surrogate of large volume test tube in clinical chemistry, successfully be used as the trusted platform of automatic search activeconstituents in recent years, be mainly used in pcr amplification, microplate reader detection, biolog identification systems etc., regular size has 48 orifice plates, 96 orifice plates, 384 orifice plates etc., has no the report of microplate application in bacterial strain screening.
Summary of the invention
Technical problem to be solved by this invention is to provide one conveniently high-throughput screening method, and to filter out high vigor, the specific sucrose isomerase of high product produces bacterial strain.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is as follows:
High vigor height product specificities sucrose isomerase produces a high-throughput screening method for bacterial strain, it is characterized in that the method comprises the following steps:
1, micro-cultivation
By sucrose isomerase enzyme-producing bacteria (as through the bacterial strain of chemomorphosis or the engineering bacteria of genetic modification) to be screened access containing in the microwell plate I of substratum, 25 ~ 35 DEG C, rotating speed 100 ~ 200rpm, shaking culture 8 ~ 20h.
2, enzymatic conversion
By microwell plate I centrifugal segregation supernatant liquor, every hole adds 0.1 ~ 2mL, and the sucrose solution of 50 ~ 500g/L carries out enzymatic conversion reaction.
3, primary dcreening operation
After conversion terminates, microwell plate I is centrifugal, is transferred to after the supernatant liquor in hole every in microwell plate I dilution 50 ~ 100 times successively in another microwell plate II.Colouring reagents is added in microwell plate II, as DNS reagent (3,5-dinitrosalicylic acid), microwell plate II is placed in boiling water bath reaction 5 minutes, DNS and reducing sugar generation redox reaction, 3-amino-5-nitro the salicylic acid generated is aobvious red-brown under boiling conditions, and within the scope of finite concentration shade and the proportional relation of reducing sugar content, around this principle discarding light-colored is the bacterial strain that total conversion rate is low.Then utilize spectrophotometer to detect the light absorption value of the darker bacterial strain conversion fluid of colour developing, the bacterial strain selecting transformation efficiency to improve more than 10% carries out multiple sieve.
4, multiple sieve
The bacterial strain obtained after primary dcreening operation is cultivated in shaking flask (or shaking pipe), add 50 ~ 500g/L sucrose solution, the add-on of sucrose solution is that 10 ~ 100mL/g wets bacterium, carry out enzymatic conversion reaction, conversion fluid point thin-layer chromatography (TLC), chromatographic condition: propyl carbinol: ethyl acetate: acetic acid: water=7: 10: 7: 6, developer: aniline-pentanoic-phosphoric acid.Palatinose and trehalulose develop the color difference with this understanding, the bacterial strain (spot is larger, color is darker, shows that the content of Palatinose or trehalulose is more) of high yield Palatinose or high yield trehalulose is screened according to the depth of color spot color and the size of spot.
High performance liquid chromatography detection by quantitative is carried out to the conversion fluid of the bacterial strain of the high yield Palatinose screened or high yield trehalulose, determines Palatinose or the trehalulose output of bacterial strain.Chromatographic condition: Agilent1200 type HPLC, differential refraction detector (ShodexR101), chromatographic column is ShodexSP0810, and moving phase is pure water, and flow velocity is 0.8mL/min, and column temperature is 80 DEG C.This method can detect the concentration of Palatinose or trehalulose simultaneously.
Wherein, step 2 or the centrifugal condition described in step 3 are: use enzyme plate whizzer, 5000 ~ 10000rpm, 10 ~ 30min.
Beneficial effect of the present invention: the screening method that invention describes a kind of high vigor, high product specificities sucrose isomerase production bacterial strain, the character (microwell plate rapid detection) of comprehensive micro-culture plate training method, zymetology reaction and the multiple sieve method of thin-layer chromatography, establish easy, efficient high-throughput screening method.This screening method utilizes color reaction intuitively, and the micro-cultivation of enzyme plate, continous sample adding apparatus and the volley of rifle fire effect, the treatment capacity of everyone 200 samples/more than day can be reached, for the screening of high product specificities sucrose isomerase producing strains provides easy, efficient, micro-screening method.
Accompanying drawing explanation
Fig. 1 is the schema that high product specificities sucrose isomerase of the present invention produces the high-throughput screening method of bacterial strain.
Embodiment
Embodiment 1: the bacterial strain of screening high yield Palatinose.
Sucrose isomerase producing strains is cultured to logarithmic phase, and collected by centrifugation thalline makes bacteria suspension (about 10
8individual), add chemical mutagen ethyl sulfate stoste 0.2ml, 30 DEG C, termination reaction after 150rpm shaking culture 30min, washes twice with potassium phosphate buffer (0.1mol/L, pH7.0), get different extent of dilution spread plates after centrifugal, put in 30 DEG C of incubators and cultivate.Obtain 410 strain list bacterium colonies altogether.
By single bacterium colony access containing in 96 orifice plates of liquid nutrient medium, 30 DEG C, rotating speed 120rpm, shaking culture 12h.
By centrifugal for 96 orifice plates, remove supernatant liquor, the sucrose solution that every hole adds 1mL, 100g/L transforms.
Conversion terminates rear centrifuging and taking supernatant liquor, after diluting 50 times successively, respectively getting 100uL goes in another 96 orifice plate, add isopyknic sterilized water and DNS reagent (3,5-dinitrosalicylic acid), fully react 5 minutes in boiling water bath, discard the light-colored bacterial strain that namely total conversion rate is low, primary dcreening operation is operated in one day and completes, and obtains the strain of total conversion rate good bacterial strain 112.Then utilize spectrophotometer to detect the light absorption value of this 112 strain conversion fluid, the bacterial strain choosing 55 strain total conversion rates raisings more than 10% carries out shaking flask and sieves again.
The bacterial strain obtained after primary dcreening operation is cultivated in shaking flask, add 100g/L sucrose solution 5mL and carry out enzymatic conversion reaction, conversion fluid point thin-layer chromatography (TLC), chromatographic condition: propyl carbinol: ethyl acetate: acetic acid: water=7: 10: 7: 6, developer: aniline-pentanoic-phosphoric acid.With this understanding, Palatinose shows green and trehalulose is aobvious red, according to the depth of color spot color and the size of spot, and bacterial strain 19 strain of screening acquisition high yield Palatinose or trehalulose, wherein bacterial strain 11 strain of object bacterial strain and high yield Palatinose.
Finally carry out liquid chromatography detection by quantitative to 11 strain bacterial strains, Agilent1200 type HPLC, differential refraction detector (ShodexR101), chromatographic column is ShodexSP0810, and moving phase is pure water, and flow velocity is 0.8mL/min, and column temperature is 80 DEG C.Final acquisition produces Palatinose: bacterial strain 6 strain that trehalulose is greater than 8: 1.Whole screening operation amounts to 5 days.
Embodiment 2: the bacterial strain of screening high yield trehalulose.
Design pair of primers, forward primer: 5 '-TTAAGCTTCCATGGATTCTCAAGGATT-3 '
Reverse primer: 5 '-TTGGATCCCTCGAGCGGATTAAGTTTATAAATG-3 '
By pcr amplification reaction (pcr amplification condition: 95 DEG C, 3min, 94 DEG C, 30s, 50 DEG C, 30s, 72 DEG C, 5min, totally 30 circulations) obtain in ErwiniarhaponticiNCPPB1579 sucrose isomerase gene, after NcoI and XhoI double digestion, be connected with carrier PET-22b, obtain recombinant plasmid, transformation of E. coli DH5 α, build the genetic engineering bacterium of correction sucrose isomerase, by comparing with the sucrose isomerase gene of other Pseudomonas, and with reference to pertinent literature, the multiple conserved sequences of Fast Fixed-point mutagenesis kit (TAKALA) to this sucrose isomerase gene are utilized to carry out random mutation, then express in bacillus coli DH 5 alpha, obtain bacterial strain 317 strain that enzyme is lived.
By single bacterium colony access containing in 96 orifice plates of liquid nutrient medium, 35 DEG C, rotating speed 200rpm, shaking culture 20h.
By centrifugal for 96 orifice plates, remove supernatant liquor, the sucrose solution that every hole adds 0.2mL, 500g/L transforms.
Conversion terminates rear centrifuging and taking supernatant liquor, after diluting 100 times successively, respectively getting 50uL goes in another 96 orifice plate, add isopyknic sterilized water and DNS reagent (3,5-dinitrosalicylic acid), fully react 5 minutes in boiling water bath, discard the light-colored bacterial strain that namely total conversion rate is low, primary dcreening operation is operated in one day and completes, and obtains the strain of total conversion rate good bacterial strain 98.Then utilize spectrophotometer to detect the light absorption value of this 98 strain conversion fluid, the bacterial strain choosing 48 strain total conversion rates raisings more than 10% carries out shaking flask and sieves again.
The bacterial strain obtained after primary dcreening operation is cultivated shaking in pipe, add 500g/L sucrose solution 2mL and carry out enzymatic conversion reaction, conversion fluid point thin-layer chromatography (TLC), chromatographic condition: propyl carbinol: ethyl acetate: acetic acid: water=7: 10: 7: 6, developer: aniline-pentanoic-phosphoric acid.With this understanding, Palatinose shows green and trehalulose is aobvious red, according to the depth of color spot color and the size of spot, and bacterial strain 15 strain of screening acquisition high yield Palatinose or trehalulose, wherein bacterial strain 7 strain of object bacterial strain and high yield trehalulose.
Finally liquid chromatography detection by quantitative is carried out, chromatographic condition to 7 strain bacterial strains: Agilent1200 type HPLC, differential refraction detector (ShodexR101), chromatographic column is ShodexSP0810, moving phase is pure water, and flow velocity is 0.8mL/min, and column temperature is 80 DEG C.Final acquisition produces trehalulose: bacterial strain 3 strain that Palatinose is greater than 8: 1.Whole screening operation amounts to 5 days.
Claims (9)
1. high vigor height product specificities sucrose isomerase produces a high-throughput screening method for bacterial strain, it is characterized in that the method comprises the following steps:
(1) sucrose isomerase enzyme-producing bacteria to be screened access is contained in the microwell plate I of substratum, shaking culture;
(2) by microwell plate I centrifugal segregation supernatant liquor, add sucrose solution and carry out enzymatic conversion reaction;
(3) after conversion terminates, microwell plate I is centrifugal, is transferred in microwell plate II successively, adds colouring reagents, and detect light absorption value, filter out sucrose isomerase superior strain in microwell plate II after being diluted by the supernatant liquor in hole every in microwell plate I;
(4) by sucrose isomerase superior strain amplification culture, add sucrose solution and carry out enzymatic conversion reaction, detect the proportion of products in sucrose inversion liquid with thin-layer chromatography, obtain the bacterial strain of high yield Palatinose or the bacterial strain of high yield trehalulose; The colouring reagents used during described thin-layer chromatography detects is aniline-pentanoic-phosphoric acid.
2. high vigor height product specificities sucrose isomerase according to claim 1 produces the high-throughput screening method of bacterial strain, the shaking culture condition described in step (1) that it is characterized in that is: 25 ~ 35 DEG C, rotating speed 100 ~ 200rpm, shaking culture 8 ~ 20h.
3. high vigor height product specificities sucrose isomerase according to claim 1 produces the high-throughput screening method of bacterial strain, it is characterized in that step (2) or the centrifugal condition described in step (3) are: use enzyme plate whizzer, 5000 ~ 10000rpm, 10 ~ 30min.
4. high vigor height product specificities sucrose isomerase according to claim 1 produces the high-throughput screening method of bacterial strain, it is characterized in that the colouring reagents described in step (3) is DNS reagent.
5. high vigor height product specificities sucrose isomerase according to claim 1 produces the high-throughput screening method of bacterial strain, it is characterized in that the sucrose isomerase superior strain filtered out in step (3) is the bacterial strain that transformation efficiency improves more than 10%.
6. high vigor height product specificities sucrose isomerase according to claim 1 produces the high-throughput screening method of bacterial strain, it is characterized in that step (2) or the sucrose solution concentration described in step (4) are 50 ~ 500g/L.
7. high vigor height product specificities sucrose isomerase according to claim 6 produces the high-throughput screening method of bacterial strain, it is characterized in that in step (2), sucrose solution every hole add-on is 0.1 ~ 2mL.
8. high vigor height product specificities sucrose isomerase according to claim 6 produces the high-throughput screening method of bacterial strain, it is characterized in that the add-on of sucrose solution in step (4) is that 10 ~ 100mL/g wets bacterium.
9. high vigor height product specificities sucrose isomerase according to claim 1 produces the high-throughput screening method of bacterial strain, it is characterized in that the bacterial strain amplification culture described in step (4) is shake-flask culture or shakes pipe cultivation.
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