CN104152361A - Screening method of yeast strains for highly producing beta-glucosaccharase - Google Patents
Screening method of yeast strains for highly producing beta-glucosaccharase Download PDFInfo
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Abstract
The invention relates to the technical field of a screening method of microorganisms, and particularly relates to a screening method of yeast strains for highly producing beta-glucosaccharase. Glucose and esculetin (6,7-dyhydroxy-coumarin) can be generated by esculin (6,7-dyhydroxy-coumarin-beta-D-glucoside) under the action of beta-glucosaccharase, and the esculin can react with Fe<3+> to display brownish black, so that a 96 pore plate developing technology of the strains for producing glycosidase is distinguished, a lot of strains can be efficiently and quickly screened simultaneously, meanwhile, the enzyme yield of the strains can be sensitively and accurately judged according to the color depth, and the dosage of a screened culture medium also can be reduced. The screening method of the yeast strains for highly producing the beta-glucosaccharase comprises the following operation steps: (1) preparing a culture medium; (2) purifying non-saccharomyces and obtaining a single colony; (3) activating the single colony; (4) adding the culture medium and the activated single colony to the 96 pore plate; and (5) screening the strains for highly producing glycosidase by virtue of colors.
Description
One, technical field:
The present invention relates to the screening method technical field of a kind of microorganism, be specifically related to the screening method of a kind of high-yield beta-glucosidase yeast strain.
Two, background technology:
Beta-glucosidase (EC3. 2. 1. 21), its English name is β-glucosidas, it is one of important composition composition in cellulolytic enzyme system, belong to hydrolase, its characteristic is β-D-glycosidic link that hydrolyzable is incorporated into end, irreducibility, discharges β-D-Glucose and corresponding aglucon simultaneously; In addition can also faintly be hydrolyzed p-nitrophenyl β-D-semi-lactosi and β-D-xyloside.The application of beta-glucosidase is very extensive, is used to flavouring, decomposes the aspects such as local flavor that cellulosic produces alcohol, improves fermented product.Beta-glucosidase widespread use in a lot of industries, has huge commercial value.Particularly more notable for flavouring effect vinous.For the Eurasian mature fruit of planting (Vitis vinifera) viticulture kind of the overwhelming majority of making wine, smell and there is no obvious characteristic perfume now, but the odour characteristics of each kind uniqueness of making wine.Reason is that most of grape variety of making wine fruits are containing the glucosides combination that has plenty of aroma component, human body olfactory organ cannot these nonvolatile aroma component glucosides of perception, wine brewing process promotes the release hydrolysis of these fragrance precursor substances, volatilizes free aroma component.Wine Aroma glucosides is the preparation storehouse of aroma component.Than many several times of the free state composition of their correspondences, even tens times.Grape fruit and yeast saccharomyces cerevisiae are the sources of enzymatic reaction Major Enzymes in brewing process, but typical Production of Wine condition, and the low pH value of high sugar degree high alcohol content and high density polyphenol substance etc. have limited the activity of Glycosylase in grape and yeast saccharomyces cerevisiae.So, from occurring in nature, directly screen and wine making environment, still have the yeast strain of the beta-glucosidase of efficient enzyme activity, become the most direct method of high-yield beta-glucosidase bacterial strain that obtains.
Due to microbes producing cellulase abundant species, enormous amount, so it is huge to screen the bacterial strain workload of high-yield beta-glucosidase by quantitative assay activity of beta-glucosidase.Research and develop now the developing technology of the qualitative analysis of several energy or evaluation beta-glucosidase, then carried out on this basis quantitative analysis, will greatly reduce workload, improved screening efficiency.The traditional prescreening method generally adopting mainly contains following several:
when polyacrylamide gel electrophoresis dyes, in p-nitrophenyl-β-D-Glucose glycosides solution, can be there is to the green stripes being caused by nitrophenol in the position of beta-glucosidase in gel insulation.But its accuracy is inadequate, and because nitrophenol has higher diffustivity, its band can fade away very soon.
rissler utilizes the insulation solution that the bromo-2-naphthyl β-D-Glucose of 6-glycosides is running gel and take geavy salt as effect reagent, and the degraded product Bromonaphthol of enzyme forms red precipitate under the effect of geavy salt.Due to the photosensitivity of geavy salt, this technology requires harsh to operating process.
use 4-methyl umbelliferone-D-Glucose glycosides as substrate, after beta-glucosidase effect, be decomposed into 4-methyl umbelliferone, the feature of utilizing 4-methyl umbelliferone to have intense fluorescence detects, thereby needs UV-light to observe, and this technological operation is complicated.
using p-nitrophenyl-beta-glucoside as substrate, after beta-glucosidase effect, be decomposed into p-NP, then add sodium carbonate to carry out color reaction, the bacterial strain that produces enzyme can present glassy yellow.The method is highly sensitive, and fast, but cost is expensive, and therefore the color similarity of transparent circle and substratum is not easy to differentiate.Aforesaid method can only be processed screening one strain bacterial strain simultaneously, so screening product Glycosylase bacterial strain workload is huge.
Three, summary of the invention:
The present invention is in order to solve the weak point in above-mentioned background technology, the screening method of a kind of high-yield beta-glucosidase yeast strain is provided, it is according to polychrom (6,7-dihydroxyl-tonka bean camphor-β-D-Glucose glycosides) under the effect of beta-glucosidase, can generate glucose and aesculetin (6,7-dihydroxyl-tonka bean camphor), Vitamin C2 proper energy and Fe
3+effect presents brownish black and distinguishes the 96 orifice plate developing technology of producing Glycosylase bacterial strain, can to a large amount of bacterial strains, efficiently screen fast simultaneously, simultaneously according to the sensitive accurate judgement strain enzyme-producing height of shade energy, can also reduce the consumption of screening culture medium.
For achieving the above object, the technical solution used in the present invention is: the screening method of a kind of high-yield beta-glucosidase yeast strain, is characterized in that: described screening method comprises following operation steps: the preparation of (1) substratum; (2) purifying of non-Saccharomyces, obtains single bacterium colony; (3) activation of single bacterium colony; (4) in 96 orifice plates, add the single bacterium colony after substratum and activation; (5) by dithering high yield Glycosylase bacterial strain.
The preparation of described step (1) substratum: the Vitamin C2 that is 0.3% by weight percent, the ironic citrate that weight percent is 0.05%, the NaCl that weight percent is 0.2%, the MgSO that weight percent is 0.05%
4.7H
2o, the KH that weight percent is 0.1%
2pO
4after adding water and mix with the weight percent agar that is 2%, at 121 ℃ of sterilizing 20min, obtain substratum;
The purifying of described step (2) non-Saccharomyces: non-Saccharomyces is first cultivated 5 days on WL nutrient agar, obtained single bacterium colony;
The activation of the single bacterium colony of described step (3): according to the difference of single colony colour, bacterium colony size, projection, edge and form thereof, the different single bacterium colony of difference picking, in the test tube that adds YPD liquid nutrient medium, activate, every test tube adds YPD liquid nutrient medium 5mL, activates two days;
Described step (4) is added the single bacterium colony after substratum and activation in 96 orifice plates: in 96 orifice plates, 250 μ l substratum are added in every hole; Add the single bacterium colony 15 μ l after activated, the single bacterium colony after each activation adds two holes again, and in contrast, coating simultaneously evenly, good each the single bacterium colony numbering corresponding with 96 orifice plates of record;
Described step (5) is passed through dithering high yield Glycosylase bacterial strain: cultivate after 3 days, select to present saturate brownish black list bacterium colony, obtain rhodotorula mucilaginosa (depositary institution's title: Chinese Typical Representative culture collection center; Depositary institution address: China, Wuhan, Wuhan University; Preservation date: on December 13rd, 2013; Deposit number: CCTCC NO:M2013660; Classification And Nomenclature: 29(Rhodotorula mucilaginosa north, rhodotorula mucilaginosa north 29).) and film uncut jade pichia spp (depositary institution's title: Chinese Typical Representative culture collection center, depositary institution address: China, Wuhan, Wuhan University, preservation date: on December 13rd, 2013, deposit number: CCTCC NO:M2013659; Classification And Nomenclature: 13(Pichia membranifaciens south, film uncut jade pichia spp south 13).), rhodotorula mucilaginosa (depositary institution's title: Chinese Typical Representative culture collection center; Depositary institution address: China, Wuhan, Wuhan University; Preservation date: on December 13rd, 2013; Deposit number: CCTCC NO:M2013660; Classification And Nomenclature: 29(Rhodotorula mucilaginosa north, rhodotorula mucilaginosa north 29).) and film uncut jade pichia spp (depositary institution's title: Chinese Typical Representative culture collection center, depositary institution address: China, Wuhan, Wuhan University, preservation date: on December 13rd, 2013, deposit number: CCTCC NO:M2013659; Classification And Nomenclature: 13(Pichia membranifaciens south, film uncut jade pichia spp south 13).) be high-yield beta-glucosidase yeast strain.
Described non-Saccharomyces is that the strange yeast of U.S. utmost point plum, Sucus Vitis viniferae have spore debaryomyces hansenii, Issatchenkia orientalis or candiyeast.
Compared with prior art, the present invention has advantage and effect are as follows:
1, the present invention utilizes 96 orifice plates to replace classic flat-plate to screen bacterium producing multi enzyme preparation, can to a large amount of bacterial strains, screen simultaneously, has reduced the workload of bacterial strain screening, qualitatively judges strain enzyme-producing height by contrast shade simultaneously.
2, the present invention carries out novelty application to 96 orifice plates, and 96 orifice plates are the flat board for culturing cell originally, in the present invention for bacterium, can screen a large amount of bacterial strains by screening culture medium seldom.
3, the preparation of screening culture medium of the present invention, can make color reaction (brownish black) obvious with screening culture medium (transparent color) difference, so it is comparatively sensitive and accurate that screening is produced to Glycosylase bacterial strain.
Four, accompanying drawing explanation:
Fig. 1 is developing technology schema of the present invention.
Five, embodiment:
Referring to Fig. 1: the present invention joins screening culture medium in 96 orifice plates, then add the bacterial strain of activation, finally by colour developing, filter out high yield Glycosylase bacterial strain.Concrete implementation step is as follows:
The preparation of described step (1) substratum: the Vitamin C2 that is 0.3% by weight percent, the ironic citrate that weight percent is 0.05%, the NaCl that weight percent is 0.2%, the MgSO that weight percent is 0.05%
4.7H
2o, the KH that weight percent is 0.1%
2pO
4after adding water and mix with the weight percent agar that is 2%, at 121 ℃ of sterilizing 20min, obtain substratum;
The purifying of described step (2) non-Saccharomyces: bring interference for fear of impure bacterial strain, obtain purer single bacterium colony bacterial strain, non-Saccharomyces is first cultivated 5 days on WL nutrient agar, obtain single bacterium colony;
Described non-Saccharomyces is that the strange yeast of U.S. utmost point plum, Sucus Vitis viniferae have spore debaryomyces hansenii, Issatchenkia orientalis or candiyeast.
The activation of the single bacterium colony of described step (3): according to the difference of single colony colour, bacterium colony size, projection, edge and form thereof, the different single bacterium colony of difference picking, in the test tube that adds YPD liquid nutrient medium, activate, every test tube adds YPD liquid nutrient medium 5mL, activates two days;
Described step (4) is added the single bacterium colony after substratum and activation in 96 orifice plates: in 96 orifice plates, 250 μ l substratum are added in every hole; Add the single bacterium colony 15 μ l after activated, the single bacterium colony after each activation adds two holes again, and in contrast, coating simultaneously evenly, good each the single bacterium colony numbering corresponding with 96 orifice plates of record;
Described step (5) is passed through dithering high yield Glycosylase bacterial strain: cultivate after 3 days, select to present saturate brownish black list bacterium colony, obtain rhodotorula mucilaginosa (depositary institution's title: Chinese Typical Representative culture collection center; Depositary institution address: China, Wuhan, Wuhan University; Preservation date: on December 13rd, 2013; Deposit number: CCTCC NO:M2013660; Classification And Nomenclature: 29(Rhodotorula mucilaginosa north, rhodotorula mucilaginosa north 29).) and film uncut jade pichia spp (depositary institution's title: Chinese Typical Representative culture collection center, depositary institution address: China, Wuhan, Wuhan University, preservation date: on December 13rd, 2013, deposit number: CCTCC NO:M2013659; Classification And Nomenclature: 13(Pichia membranifaciens south, film uncut jade pichia spp south 13).), rhodotorula mucilaginosa (depositary institution's title: Chinese Typical Representative culture collection center; Depositary institution address: China, Wuhan, Wuhan University; Preservation date: on December 13rd, 2013; Deposit number: CCTCC NO:M2013660; Classification And Nomenclature: 29(Rhodotorula mucilaginosa north, rhodotorula mucilaginosa north 29).) and film uncut jade pichia spp be high-yield beta-glucosidase yeast strain.
Rhodotorula mucilaginosa (depositary institution's title: Chinese Typical Representative culture collection center; Depositary institution address: China, Wuhan, Wuhan University; Preservation date: on December 13rd, 2013; Deposit number: CCTCC NO:M2013660; Classification And Nomenclature: 29(Rhodotorula mucilaginosa north, rhodotorula mucilaginosa north 29).) and film uncut jade pichia spp (depositary institution's title: Chinese Typical Representative culture collection center, depositary institution address: China, Wuhan, Wuhan University, preservation date: on December 13rd, 2013, deposit number: CCTCC NO:M2013659; Classification And Nomenclature: 13(Pichia membranifaciens south, film uncut jade pichia spp south 13).) be high yield Glycosylase bacterial strain, can be used for mixed fermentation or produce Glycosylase increasing Wine Aroma.Through quantivative approach analysis: rhodotorula mucilaginosa inulinase-producing activity is 42.1U/ml * 10
-3; Film uncut jade pichia spp 42.51U/ml * 10
-3.
Described step (6) quantitative verification high yield Glycosylase bacterial strain is active:
A extracts crude enzyme liquid: by rhodotorula mucilaginosa (depositary institution's title: Chinese Typical Representative culture collection center of screening; Depositary institution address: China, Wuhan, Wuhan University; Preservation date: on December 13rd, 2013; Deposit number: CCTCC NO:M2013660; Classification And Nomenclature: 29(Rhodotorula mucilaginosa north, rhodotorula mucilaginosa north 29).) and film uncut jade pichia spp (depositary institution's title: Chinese Typical Representative culture collection center, depositary institution address: China, Wuhan, Wuhan University, preservation date: on December 13rd, 2013, deposit number: CCTCC NO:M2013659; Classification And Nomenclature: 13(Pichia membranifaciens south, film uncut jade pichia spp south 13).) inoculum size by 10% is linked into fermention medium (the bottled 20ml substratum of 300mL triangle, 121 ℃ of deactivation 20min), temperature is 30 ℃, under shaking table 150rpm/min condition, cultivate 72h, take out fermented liquid 1.0ml in 1.5ml centrifuge tube, the centrifugal 10min degerming of 8000rpm/min, collects supernatant liquor, is beta-glucosidase crude enzyme liquid.
B wavelength is selected: 0.1, the p-nitrophenyl phenol solution of 0.3,0.5 mmol/L, get respectively 5ml, and by spectrophotometer scanning maximum absorption band wavelength, the condition of scanning: range of absorbency is 0.00-3.00, wavelength region is 390-415nm.Select maximum absorption wavelength.
The mensuration that beta-glucosidase enzyme is lived: the preparation of (1) p-NP standardized solution takes 0.0209g p-NP, dissolves with distilled water, transfers in the volumetric flask of 100ml, and constant volume, shakes up, and is mixed with the p-nitrophenyl phenol solution of 1.5mmol/L.Be mixed with respectively 0.05mmol/L, 0.1 mmol/L, 0.2 mmol/L, 0.3 mmol/L, 0.4 mmol/L, 0.5mmol/L, 0.6 mmol/L, 0.7 mmol/L, the p-nitrophenyl phenol solution of 0.8 mmol/L.
P-NP typical curve is got 10 25ml graduated tubes, add respectively 0.05mmol/L, 0.1 mmol/L, 0.2 mmol/L, 0.3mmol/L, 0.4 mmol/L, 0.5mmol/L, 0.6 mmol/L, 0.7 mmol/L, 0.8 mmol/L p-nitrophenyl phenol solution 0.4ml, then add the sodium carbonate solution of 2.0ml 1 mmol/L and the distilled water of 10ml, room temperature shakes up after placing 5min.Using distilled water as blank, in above-mentioned maximum absorption wave strong point, measure light absorption value A.Take light absorption value as ordinate zou, and the concentration of p-NP is X-coordinate, formulates typical curve.
(2) the corresponding pH value of mensuration that beta-glucosidase enzyme is lived adds 750 μ L citric acid-Sodium phosphate dibasic damping fluids, the thick zyme extract that adds 200ul, add 40 ℃ of p-nitrophenyl-beta-glucosides (pNPG) of 250 μ L1 mmol/L to process 60min, then add the 1mol/L Na of 1.0 ml
2cO
3termination reaction.In above-mentioned maximum absorption wave strong point, survey absorbancy, distilled water is blank.Each sample repeats twice.Beta-glucosidase enzyme activity unit (IU) is defined as: at 40 ℃, in 1min, catalysis generates the desired enzyme amount of 1 μ mol p-NP.
Claims (3)
1. a screening method for high-yield beta-glucosidase yeast strain, is characterized in that: described screening method comprises following operation steps: the preparation of (1) substratum; (2) purifying of non-Saccharomyces, obtains single bacterium colony; (3) activation of single bacterium colony; (4) in 96 orifice plates, add the single bacterium colony after substratum and activation; (5) by dithering high yield Glycosylase bacterial strain.
2. the screening method of a kind of high-yield beta-glucosidase yeast strain according to claim 1, is characterized in that:
The preparation of described step (1) substratum: the Vitamin C2 that is 0.3% by weight percent, the ironic citrate that weight percent is 0.05%, the NaCl that weight percent is 0.2%, the MgSO that weight percent is 0.05%
4.7H
2o, the KH that weight percent is 0.1%
2pO
4after adding water and mix with the weight percent agar that is 2%, at 121 ℃ of sterilizing 20min, obtain substratum;
The purifying of described step (2) non-Saccharomyces: non-Saccharomyces is first cultivated 5 days on WL nutrient agar, obtained single bacterium colony;
The activation of the single bacterium colony of described step (3): according to the difference of single colony colour, bacterium colony size, projection, edge and form thereof, the different single bacterium colony of difference picking, in the test tube that adds YPD liquid nutrient medium, activate, every test tube adds YPD liquid nutrient medium 5mL, activates two days;
Described step (4) is added the single bacterium colony after substratum and activation in 96 orifice plates: in 96 orifice plates, 250 μ l substratum are added in every hole; Add the single bacterium colony 15 μ l after activated, the single bacterium colony after each activation adds two holes again, and in contrast, coating simultaneously evenly, good each the single bacterium colony numbering corresponding with 96 orifice plates of record;
Described step (5) is passed through dithering high yield Glycosylase bacterial strain: cultivate after 3 days, selection presents saturate brownish black list bacterium colony, obtain rhodotorula mucilaginosa and film uncut jade pichia spp, rhodotorula mucilaginosa and film uncut jade pichia spp are high-yield beta-glucosidase yeast strain.
3. the screening method of a kind of high-yield beta-glucosidase yeast strain according to claim 1, is characterized in that: described non-Saccharomyces is that the strange yeast of U.S. utmost point plum, Sucus Vitis viniferae have spore debaryomyces hansenii, Issatchenkia orientalis or candiyeast.
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Cited By (6)
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| CN108179119A (en) * | 2018-03-07 | 2018-06-19 | 中国农业大学 | A kind of zymotechnique for improving ice-wine flavouring essence quality using Non-Saccharomyces |
| CN109295040A (en) * | 2018-11-29 | 2019-02-01 | 石河子开发区天易特色果蔬加工生产力促进中心（有限责任公司） | A kind of preparation method of Pichia pastoris beta-glucosidase enzyme preparation |
| CN109749956A (en) * | 2019-01-08 | 2019-05-14 | 甘肃农业大学 | A kind of Oenococcus Oeni bacterial strain high-throughput screening method based on 48 hole deep-well plates |
| CN112625928A (en) * | 2021-01-15 | 2021-04-09 | 江南大学 | Hansenula polymorpha strain capable of increasing wine brewing aroma |
| CN112746029A (en) * | 2021-01-22 | 2021-05-04 | 西北农林科技大学 | Hansenula polymorpha strain QTX22 for producing aroma substances at high yield and application thereof |
| CN112826076A (en) * | 2020-12-31 | 2021-05-25 | 国投中鲁果汁股份有限公司 | Body-building type sugar-cored apple flavor enzyme |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108179119A (en) * | 2018-03-07 | 2018-06-19 | 中国农业大学 | A kind of zymotechnique for improving ice-wine flavouring essence quality using Non-Saccharomyces |
| CN108179119B (en) * | 2018-03-07 | 2021-05-11 | 中国农业大学 | Fermentation process for improving aroma quality of ice wine by using non-saccharomyces cerevisiae |
| CN109295040A (en) * | 2018-11-29 | 2019-02-01 | 石河子开发区天易特色果蔬加工生产力促进中心（有限责任公司） | A kind of preparation method of Pichia pastoris beta-glucosidase enzyme preparation |
| CN109749956A (en) * | 2019-01-08 | 2019-05-14 | 甘肃农业大学 | A kind of Oenococcus Oeni bacterial strain high-throughput screening method based on 48 hole deep-well plates |
| CN112826076A (en) * | 2020-12-31 | 2021-05-25 | 国投中鲁果汁股份有限公司 | Body-building type sugar-cored apple flavor enzyme |
| CN112625928A (en) * | 2021-01-15 | 2021-04-09 | 江南大学 | Hansenula polymorpha strain capable of increasing wine brewing aroma |
| CN112746029A (en) * | 2021-01-22 | 2021-05-04 | 西北农林科技大学 | Hansenula polymorpha strain QTX22 for producing aroma substances at high yield and application thereof |
| CN112746029B (en) * | 2021-01-22 | 2022-07-29 | 西北农林科技大学 | Hansenula polymorpha strain QTX22 for producing aroma substances in grape juice and application of strain |
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