CN100582237C - Flat-plate sieving method of aesculin for high-yield beta-glucosidase producing strain - Google Patents
Flat-plate sieving method of aesculin for high-yield beta-glucosidase producing strain Download PDFInfo
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Abstract
The invention discloses a flat plate screening method for high yielding Beta-glucosidase production bacteria by using aesculin. The method comprises the steps of: 1. earth sample collection; 2. enrichment culture; 3. flat plate preparation of the aesculin; 4. flat plate primary screening aesculin; 5. separation and purification; 6. secondary screen by a shaking battle, etc. The invention can fast screen active strain possessing the Beta-glucosidase from samples which contain rich fibrin resources; compared with the method which is reported in published literature which screens Beta-glucosidase production bacteria by using a cellobiose flat plate method, the invention has the remarkable advantages at price; under the condition that obtains 100 strains by screening, the cost of the invention by using the aesculin is only one tenth of the cellobiose flat plate method; in addition, the invention has the advantages of simple operation, reliable result, etc.
Description
Technical field
The present invention relates to the plate screening that a kind of beta-glucosidase produces bacterium, relate in particular to a kind of method that Vitamin C2 is used for the plate screening of high yield β-grape Glycosylase generation bacterium.
Background technology
Natural cellulose is being hydrolyzed in the process of glucose, must relying on the synergy of endo cellulase, circumscribed cellulase and three kinds of enzymes of beta-glucosidase just can finish.Wherein beta-glucosidase is the rate-limiting enzyme of this process, and the height that its enzyme is lived directly influences the overall enzyme height alive of cellulase system.Most widely used cellulase production bacterial classification is the good mutant strain of Trichodermareesei (Trichoderma reesei) mostly at present.Though these bacterial strains can produce the inscribe and the circumscribed cellulase of high vigor, but because the vigor of beta-glucosidase is very low, in utilizing the cellulosic process of cellulase hydrolysis, cause the accumulation of cellobiose, cellobiose can form the intensive feedback inhibition to the katalysis of cellulase again.Therefore, improving the vigor of beta-glucosidase in the cellulase system, is one of key measure that improves cellulose hydrolysis yield and glucose yield.Produce the screening operation of bacterium for beta-glucosidase; traditional method mainly is to screen according to the size of transparent circle on the flat board that is sole carbon source with the cellobiose; because the cellobiose price is comparatively expensive; screen less feasible economically on a large scale; the beta-glucosidase enzyme bad judgement of height alive that produces by the transparent circle strain separated in addition also will be carried out shake flask fermentation usually one by one further to determine.
Summary of the invention
Problems such as comparatively expensive and method is more loaded down with trivial details at present beta-glucosidase screening method agents useful for same, and the present Research of in scientific effort, being badly in need of fast simple screening method, the problem to be solved in the present invention provides a kind of method that Vitamin C2 is used for the plate screening of beta-glucosidase generation bacterium.
The method that Vitamin C2 is used for the plate screening of high-yield beta-glucosidase generation bacterium of the present invention, step is the (as: farmland, place from the rich cellulose resource, forest, wood-working factory, the rural area straw is heaped ground, the compost place) gathers soil sample or select commercially available microbial strains, carry out enrichment culture, it is dull and stereotyped and with it pregnant solution microorganism is carried out primary dcreening operation that Vitamin C2 is added in the basic inorganic salt substratum preparation, becoming black with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies is that index is chosen the purpose bacterial strain and carried out shake flask fermentation again and sieve again, detect enzymic activity by DNS or Somogyi method, screening obtains the microorganism strains of high-yield beta-glucosidase; It is characterized in that: described enrichment culture be with Microcrystalline Cellulose or filter paper as sole carbon source, shaking table was cultivated 12～72 hours under 20～50 ℃, the condition of rotating speed 160～250r/min; The described method that the pregnant solution microorganism is carried out primary dcreening operation is that pregnant solution is carried out 10
-1～10
-7Gradient dilution, the diluent of drawing 0.1～0.3mL then respectively evenly is applied to and has added on the flat board of basic inorganic salt substratum that the quality volume percent is 0.01～3% Vitamin C2, under 20～50 ℃, cultivates 12～72 hours; The described bacterium colony that to choose method that purpose bacterial strain shake flask fermentation sieves again be picking becomes black with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies is connected to and produces in the enzyme substratum, shaking table was cultivated 1～7 day under the condition of 25～45 ℃ of temperature, rotating speed 150～250r/min, surveyed enzyme then and lived.
The above-mentioned method that Vitamin C2 is used for the plate screening of high-yield beta-glucosidase generation bacterium: the collection principle of described soil sample is to contain bacterium, fungi or the actinomycetes that produce beta-glucosidase; Described commercially available microbial strains is meant bacterium, fungi or the actinomycetes that can produce beta-glucosidase.
The above-mentioned method that Vitamin C2 is used for the plate screening of high-yield beta-glucosidase generation bacterium: the culture medium prescription of described enrichment culture (in the quality concentration of volume percent) is as follows: (NH
4)
2SO
40.4%, MgSO
47H
2O0.05%, NaCl0.2%, CaCO
30.4%, KH
2PO
40.1%, Vitamin C2 0.5%, Streptomycin sulphate is an amount of.
The above-mentioned method that Vitamin C2 is used for the plate screening of high-yield beta-glucosidase generation bacterium: described basic inorganic salt substratum (Basal Salts Medium, prescription BSM) is:
In per 1000 ml distilled waters,, add ironic citrate 0.005～0.5%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 0.5～4.0% in the quality concentration of volume percent.
Wherein: the formula optimization of described basic inorganic salt substratum is:
In per 1000 ml distilled waters,, add ironic citrate 0.01～0.2%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 1.5～3.0% in the quality concentration of volume percent.
Above-mentioned Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium: the described amount that adds Vitamin C2 in the basic inorganic salt culture medium flat plate is quality volume percent 0.1%～1.0% preferably.
The above-mentioned method that Vitamin C2 is used for the plate screening of high-yield beta-glucosidase generation bacterium: preferably 25～45 ℃ of the temperature of described enrichment culture, incubation time preferably 24～60 hours.
Above-mentioned Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium: the described culture temperature that microorganism in the pregnant solution is carried out the dull and stereotyped primary dcreening operation of Vitamin C2 is preferably 25～40 ℃, preferred 18～48 hours of incubation time.Further preferred culture temperature is 25～37 ℃, and the microorganism of the product beta-glucosidase of screening can grow as sole carbon source with Vitamin C2 on the basic inorganic salt substratum.
The above-mentioned method that Vitamin C2 is used for the plate screening of high-yield beta-glucosidase generation bacterium: the prescription of described product enzyme substratum (in the quality concentration of volume percent) is: grass meal 2.0%, wheat bran 2.0%, peptone 0.25%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.
Above-mentioned Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium: described preferably 25～40 ℃ of temperature that purpose bacterial strain shake flask fermentation sieves again, rotating speed preferably 180～220r/min, the shaking table incubation time preferably 2～5 days chosen.
Above-mentioned Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium: described the pregnant solution microorganism is carried out in the primary dcreening operation, the bacterium colony that the substratum color with dull and stereotyped dorsal view periphery of bacterial colonies of picking becomes black can move to receive in the basic inorganic salt slant medium that contains Vitamin C2 preserves standby or repurity.
Before above-mentioned substratum uses, all under 115 ℃ of conditions, sterilized 30 minutes.
The present invention has adopted a kind of quick, new easily screening method-Vitamin C2 flat band method, it promptly is the method for microorganism of sole carbon source screening high-yield beta-glucosidase with the Vitamin C2, can be from the sample of rich cellulose resource rapid screening go out to have the bacterial strain of beta-glucosidase vigor, with reported in the document of having delivered carry out method for screening with the cellobiose flat board and compare, has tangible price advantage, obtain under the situation of 100 bacterial strains in same screening, the cost of Vitamin C2 method is just with 1/50th of cellobiose flat band method, and this method also tool is simple to operate, many advantages such as credible result.
Description of drawings
The growing state of Fig. 1 aspergillus Asp-1 on the Vitamin C2 flat board.
The growing state of Fig. 2 aspergillus Asp-2 on the Vitamin C2 flat board.
The growing state of Fig. 3 aspergillus Asp-3 on the Vitamin C2 flat board.
The growing state of Fig. 4 mould Peni-1 on the Vitamin C2 flat board.
The growing state of Fig. 5 mould Peni-8 on the Vitamin C2 flat board.
Embodiment
Embodiment 1: screening obtains aspergillus Asp-1
The dead twigs and withered leaves that reaches the woods from the rural area mow is down gathered 10 of soil samples down, accurately taking by weighing each 0.5g of soil sample then is dissolved in the 25ml distilled water, fully respectively draw 1ml in enrichment medium behind the concussion mixing, place 30 ℃, carry out enrichment culture on the 180r/min shaking table two days, and then it was diluted 10 respectively
1, 10
2, 10
3, 10
4, 10
5, 10
6Doubly, the diluent of drawing 0.1mL more respectively evenly is applied to and has added on the flat board of basic inorganic salt substratum that the quality volume percent is 0.1% Vitamin C2, put in 30 ℃ of thermostat containers and cultivated 36 hours, carry out primary dcreening operation, get extension rate suitable, colony growth is dull and stereotyped uniformly, with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies to become black the be the index screening picking saturate bacterium colony in the dull and stereotyped back side of one strain, called after aspergillus Asp-1 (dull and stereotyped cultivation results is seen Fig. 1).Bacterium colony Asp-1 is connected in the product enzyme substratum, and 30 ℃ of temperature, shaking table was cultivated 2 days under the condition of rotating speed 180r/min, surveyed enzyme then and lived.
The enzyme biopsy is surveyed and is used the DNS method: get 0.5ml and set dilution sample liquid, the saligenin (being dissolved in the 0.1MpH=4.8 acetate buffer solution) of adding 1%, behind 50 ℃ of insulation 30min, add 2ml DNS reagent, in boiling water, boil 10min behind the thorough mixing, the cooling back to 12.5ml, is surveyed the OD value with distilled water diluting under 540nm photoabsorption condition on the microplate spectrograph; According to same treatment process with heat-killed beta-glucosidase as blank.It is enzyme unit (IU) alive that the definition per minute produces the required enzyme amount of 1 μ mol reducing sugar (with glucose meter) by substrate.
The result is surveyed in the enzyme biopsy: aspergillus Asp-1 can produce higher beta-glucosidase, and enzyme is lived and is 5.3u/mL.
Confirm to use the aspergillus Asp-1 that the inventive method screening has obtained to produce higher beta-glucosidase.
Above-mentionedly be used to screen beta-glucosidase to produce the enrichment culture based formulas (in the quality concentration of volume percent) of bacterium as follows: (NH
4)
2SO
40.4%, MgSO
47H
2O0.05%, NaCl0.2%, CaCO
30.4%, KH
2PO
40.1%, Vitamin C2 0.5%, Streptomycin sulphate is an amount of.
The prescription that above-mentioned dull and stereotyped primary dcreening operation is cultivated used basic inorganic salt substratum is:
In per 1000 ml distilled waters,, add ironic citrate 0.1%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 2.5% in the quality concentration of volume percent.
It is that 0.1% Vitamin C2 is as sole carbon source that above-mentioned basic inorganic salt substratum has added mass percent.
The prescription of above-mentioned product enzyme substratum (in the quality concentration of volume percent) is: grass meal 2.0%, wheat bran 2.0%, peptone 0.25%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.
Embodiment 2: screening obtains aspergillus Asp-2
Evergreen holly is gathered 8 of soil samples down from the campus, accurately take by weighing each 0.5g of soil sample then and be dissolved in the 25ml distilled water, respectively draw 1ml in enrichment medium after fully shaking mixing, place 20 ℃, carry out enrichment culture on the 200r/min shaking table two days, and then it was diluted 10 respectively
1, 10
2, 10
3, 10
4, 10
5, 10
6Doubly, the diluent of drawing 0.3mL more respectively evenly is applied to and has added on the flat board of basic inorganic salt substratum that the quality volume percent is 0.3% Vitamin C2, put in 20 ℃ of thermostat containers and cultivated 72 hours, carry out primary dcreening operation, get extension rate suitable, colony growth is dull and stereotyped uniformly, with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies to become black the be the index screening picking saturate bacterium colony in the dull and stereotyped back side of one strain, called after aspergillus Asp-2 (dull and stereotyped cultivation results is seen Fig. 2).Bacterium colony Asp-2 is connected in the product enzyme substratum, and 25 ℃ of temperature, shaking table was cultivated 7 days under the condition of rotating speed 200r/min, surveyed enzyme then and lived.
The enzyme biopsy is surveyed and is used the DNS method: get 0.5ml and set dilution sample liquid, the saligenin (being dissolved in the 0.1MpH=4.8 acetate buffer solution) of adding 1%, behind 50 ℃ of insulation 30min, add 2ml DNS reagent, in boiling water, boil 10min behind the thorough mixing, the cooling back to 12.5ml, is surveyed the OD value with distilled water diluting under 540nm photoabsorption condition on the microplate spectrograph; According to same treatment process with heat-killed beta-glucosidase as blank.It is enzyme unit (IU) alive that the definition per minute produces the required enzyme amount of 1 μ mol reducing sugar (with glucose meter) by substrate.
The result is surveyed in the enzyme biopsy: aspergillus Asp-2 can produce higher beta-glucosidase, and enzyme is lived and is 2.6u/mL.
Confirm to use the aspergillus Asp-2 that the inventive method screening has obtained to produce higher beta-glucosidase.
Above-mentionedly be used to screen beta-glucosidase to produce the enrichment culture based formulas (in the quality concentration of volume percent) of bacterium as follows: (NH
4)
2SO
40.4%, MgSO
47H
2O0.05%, NaCl0.2%, CaCO
30.4%, KH
2PO
40.1%, Vitamin C2 0.5%, Streptomycin sulphate is an amount of.
The prescription that above-mentioned dull and stereotyped primary dcreening operation is cultivated used basic inorganic salt substratum is:
In per 1000 ml distilled waters,, add ironic citrate 0.2%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 1.5% in the quality concentration of volume percent.
It is that 0.3% Vitamin C2 is as sole carbon source that above-mentioned basic inorganic salt substratum has added mass percent.
The prescription of above-mentioned product enzyme substratum (in the quality concentration of volume percent) is: grass meal 2.0%, wheat bran 2.0%, peptone 0.25%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.
Embodiment 3: screening obtains aspergillus Asp-3
Gather 12 of soil samples down from Mount Taishan back forest, accurately take by weighing each 0.5g of soil sample then and be dissolved in the 25ml distilled water, respectively draw 1ml in enrichment medium after fully shaking mixing, place 50 ℃, carry out enrichment culture on the 220r/min shaking table 20 hours, and then it was diluted 10 respectively
1, 10
2, 10
3, 10
4, 10
5, 10
6Doubly, the diluent of drawing 0.2mL more respectively evenly is applied to and has added on the flat board of basic inorganic salt substratum that the quality volume percent is 3% Vitamin C2, put in 50 ℃ of thermostat containers and cultivated 12 hours, carry out primary dcreening operation, get extension rate suitable, colony growth is dull and stereotyped uniformly, with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies to become black the be the index screening picking saturate bacterium colony in the dull and stereotyped back side of one strain, called after aspergillus Asp-3 (dull and stereotyped cultivation results is seen Fig. 3).Bacterium colony Asp-3 is connected in the product enzyme substratum, and 45 ℃ of temperature, shaking table was cultivated 1.5 days under the condition of rotating speed 250r/min, surveyed enzyme then and lived.
The enzyme biopsy is surveyed and is used the DNS method: get 0.5ml and set dilution sample liquid, the saligenin (being dissolved in the 0.1MpH=4.8 acetate buffer solution) of adding 1%, behind 50 ℃ of insulation 30min, add 2ml DNS reagent, in boiling water, boil 10min behind the thorough mixing, the cooling back to 12.5ml, is surveyed the OD value with distilled water diluting under 540nm photoabsorption condition on the microplate spectrograph; According to same treatment process with heat-killed beta-glucosidase as blank.It is enzyme unit (IU) alive that the definition per minute produces the required enzyme amount of 1 μ mol reducing sugar (with glucose meter) by substrate.
The result is surveyed in the enzyme biopsy: aspergillus Asp-3 can produce higher beta-glucosidase, and enzyme is lived and is 4.6u/mL.
Confirm to use the aspergillus Asp-3 that the inventive method screening has obtained to produce higher beta-glucosidase.
Above-mentionedly be used to screen beta-glucosidase to produce the enrichment culture based formulas (in the quality concentration of volume percent) of bacterium as follows: (NH
4)
2SO
40.4%, MgSO
47H
2O0.05%, NaCl0.2%, CaCO
30.4%, KH
2PO
40.1%, Vitamin C2 0.5%, Streptomycin sulphate is an amount of.
The prescription that above-mentioned dull and stereotyped primary dcreening operation is cultivated used basic inorganic salt substratum is:
In per 1000 ml distilled waters,, add ironic citrate 0.5%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 4% in the quality concentration of volume percent.
It is that 3% Vitamin C2 is as sole carbon source that above-mentioned basic inorganic salt substratum has added mass percent.
The prescription of above-mentioned product enzyme substratum (in the quality concentration of volume percent) is: grass meal 2.0%, wheat bran 2.0%, peptone 0.25%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.
Embodiment 4: screening obtains mould Peni-1
Evergreen holly is gathered 8 of soil samples down from the campus, accurately take by weighing each 0.5g of soil sample then and be dissolved in the 25ml distilled water, respectively draw 1ml in enrichment medium after fully shaking mixing, place 30 ℃, carry out enrichment culture on the 200r/min shaking table 48 hours, and then it was diluted 10 respectively
1, 10
2, 10
3, 10
4, 10
5, 10
6Doubly, the diluent of drawing 0.2mL more respectively evenly is applied to and has added on the flat board of basic inorganic salt substratum that the quality volume percent is 0.3% Vitamin C2, put in 35 ℃ of thermostat containers and cultivated 48 hours, carry out primary dcreening operation, get extension rate suitable, colony growth is dull and stereotyped uniformly, with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies to become black the be the index screening picking saturate bacterium colony in the dull and stereotyped back side of one strain, called after mould Peni-1 (dull and stereotyped cultivation results is seen Fig. 4).Bacterium colony Peni-1 is connected in the product enzyme substratum, and 35 ℃ of temperature, shaking table was cultivated 5 days under the condition of rotating speed 200r/min, surveyed enzyme then and lived.
The enzyme biopsy is surveyed and is used the DNS method: get 0.5ml and set dilution sample liquid, the saligenin (being dissolved in the 0.1MpH=4.8 acetate buffer solution) of adding 1%, behind 50 ℃ of insulation 30min, add 2ml DNS reagent, in boiling water, boil 10min behind the thorough mixing, the cooling back to 12.5ml, is surveyed the OD value with distilled water diluting under 540nm photoabsorption condition on the microplate spectrograph; According to same treatment process with heat-killed beta-glucosidase as blank.It is enzyme unit (IU) alive that the definition per minute produces the required enzyme amount of 1 μ mol reducing sugar (with glucose meter) by substrate.
The result is surveyed in the enzyme biopsy: mould Peni-1 can produce higher beta-glucosidase, and enzyme is lived and is 8.7u/mL.
Confirm to use the mould Peni-1 that the inventive method screening has obtained to produce higher beta-glucosidase.
Above-mentionedly be used to screen beta-glucosidase to produce the enrichment culture based formulas (in the quality concentration of volume percent) of bacterium as follows: (NH
4)
2SO
40.4%, MgSO
47H
2O0.05%, NaCl0.2%, CaCO
30.4%, KH
2PO
40.1%, Vitamin C2 0.5%, Streptomycin sulphate is an amount of.
The prescription that above-mentioned dull and stereotyped primary dcreening operation is cultivated used basic inorganic salt substratum is:
In per 1000 ml distilled waters,, add ironic citrate 0.2%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 1.5% in the quality concentration of volume percent.
It is that 0.3% Vitamin C2 is as sole carbon source that above-mentioned basic inorganic salt substratum has added mass percent.
The prescription of above-mentioned product enzyme substratum (in the quality concentration of volume percent) is: grass meal 2.0%, wheat bran 2.0%, peptone 0.25%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.
Embodiment 5: screening obtains mould peni-8
The dead twigs and withered leaves that reaches the woods from the rural area mow is down gathered 15 of soil samples down, accurately taking by weighing each 0.5g of soil sample then is dissolved in the 25ml distilled water, fully respectively draw 1ml in enrichment medium behind the concussion mixing, place 40 ℃, carry out enrichment culture on the 180r/min shaking table two days, and then it was diluted 10 respectively
1, 10
2, 10
3, 10
4, 10
5, 10
6Doubly, the diluent of drawing 0.1mL more respectively evenly is applied to and has added on the flat board of basic inorganic salt substratum that the quality volume percent is 0.5% Vitamin C2, put in 40 ℃ of thermostat containers and cultivated 48 hours, carry out primary dcreening operation, get extension rate suitable, colony growth is dull and stereotyped uniformly, with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies to become black the be the index screening picking saturate bacterium colony in the dull and stereotyped back side of one strain, called after mould peni-8 (dull and stereotyped cultivation results is seen Fig. 5).Bacterium colony peni-8 is connected in the product enzyme substratum, and 40 ℃ of temperature, shaking table was cultivated 3 days under the condition of rotating speed 180r/min, surveyed enzyme then and lived.
The enzyme biopsy is surveyed and is used the DNS method: get 0.5ml and set dilution sample liquid, the saligenin (being dissolved in the 0.1MpH=4.8 acetate buffer solution) of adding 1%, behind 50 ℃ of insulation 30min, add 2ml DNS reagent, in boiling water, boil 10min behind the thorough mixing, the cooling back to 12.5ml, is surveyed the OD value with distilled water diluting under 540nm photoabsorption condition on the microplate spectrograph; According to same treatment process with heat-killed beta-glucosidase as blank.It is enzyme unit (IU) alive that the definition per minute produces the required enzyme amount of 1 μ mol reducing sugar (with glucose meter) by substrate.
The result is surveyed in the enzyme biopsy: mould peni-8 can produce higher beta-glucosidase, and enzyme is lived and is 3.8u/mL.
Confirm to use the mould peni-8 that the inventive method screening has obtained to produce higher beta-glucosidase.
Above-mentionedly be used to screen beta-glucosidase to produce the enrichment culture based formulas (in the quality concentration of volume percent) of bacterium as follows: (NH
4)
2SO
40.4%, MgSO
47H
2O0.05%, NaCl0.2%, CaCO
30.4%, KH
2PO
10.1%, Vitamin C2 0.5%, Streptomycin sulphate is an amount of.
The prescription that above-mentioned dull and stereotyped primary dcreening operation is cultivated used basic inorganic salt substratum is:
In per 1000 ml distilled waters,, add ironic citrate 0.3%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 3.0% in the quality concentration of volume percent.
It is that 0.5% Vitamin C2 is as sole carbon source that above-mentioned basic inorganic salt substratum has added mass percent.
The prescription of above-mentioned product enzyme substratum (in the quality concentration of volume percent) is: grass meal 2.0%, wheat bran 2.0%, peptone 0.25%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.
Embodiment 6 beta-glucosidases produce the screening of bacterium
Enrichment medium after the inoculation soil sample is placed 28 ℃, cultivated two days on the 180r/min shaking table, dilute 10 respectively then
1, 10
2, 10
3, 10
4, 10
5, 10
6Doubly, separate application is on 0.2% the Vitamin C2 primary dcreening operation flat board in the Vitamin C2 mass percentage content, in 28 ℃ of thermostat containers, cultivate after 20 hours, it is dull and stereotyped uniformly to get the suitable colony growth of extension rate, the saturate bacterium colony in the dull and stereotyped back side of picking, through 28 ℃, the 180r/min shake flask fermentation detects enzyme and lives after 3 days, sieves to such an extent that can produce the microorganism of higher beta-glucosidase.
Above-mentioned dull and stereotyped primary dcreening operation substratum uses the basic minimal medium of liquid, adds mass percent and be 0.2% Vitamin C2 as sole carbon source.
The basic minimal medium of solid is that aforesaid liquid minimal medium composition interpolation quality volume percent is 2.5% agar powder.
Embodiment 7 beta-glucosidases produce the screening of bacterium
Enrichment medium after the inoculation soil sample is placed 30 ℃, cultivated two days on the 200r/min shaking table, dilute 10 respectively then
1, 10
2, 10
3, 10
4, 10
5, 10
6Doubly, separate application is on 0.5% the Vitamin C2 primary dcreening operation flat board in the Vitamin C2 mass percentage content, in 32 ℃ of thermostat containers, cultivate after 3 days, it is dull and stereotyped uniformly to get the suitable colony growth of extension rate, the saturate bacterium colony in the dull and stereotyped back side of picking, through 30 ℃, the 200r/min shake flask fermentation detects enzyme and lives after 4 days, sieves to such an extent that can produce the microorganism of higher beta-glucosidase.
Above-mentioned dull and stereotyped primary dcreening operation substratum uses the basic minimal medium of liquid, adds mass percent and be 0.5% Vitamin C2 as sole carbon source.
The basic minimal medium of solid is that aforesaid liquid minimal medium composition interpolation quality volume percent is 2.5% agar powder.
Embodiment 8 high-yield beta-glucosidases produce the screening of bacterium
The enrichment medium that inoculation can be produced behind the microbial strains (commercially available) of beta-glucosidase places 32 ℃, cultivates two days on the 220r/min shaking table, dilutes 10 respectively then
1, 10
2, 10
3, 10
4, 10
5, 10
6Doubly, separate application is on 0.7% the Vitamin C2 primary dcreening operation flat board in the Vitamin C2 mass percentage content, in 35 ℃ of thermostat containers, cultivate after 3 days, it is dull and stereotyped uniformly to get the suitable colony growth of extension rate, the saturate bacterium colony in the dull and stereotyped back side of picking, through 32 ℃, the 220r/min shake flask fermentation detects enzyme and lives after 5 days, sieve can high-yield beta-glucosidase microorganism.
Above-mentioned dull and stereotyped primary dcreening operation substratum uses the basic minimal medium of liquid, adds mass percent and be 0.7% Vitamin C2 as sole carbon source.
The basic minimal medium of solid is that aforesaid liquid minimal medium composition interpolation quality volume percent is 2.5% agar powder.
Claims (10)
1. one kind is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium with Vitamin C2, step is to gather soil sample or select commercially available microbial strains from the place of rich cellulose resource, carry out enrichment culture, it is dull and stereotyped and with it pregnant solution microorganism is carried out primary dcreening operation that Vitamin C2 is added in the basic inorganic salt substratum preparation, becoming black with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies is that index is chosen the purpose bacterial strain and carried out shake flask fermentation again and sieve again, detect enzymic activity by DNS or Somogyi method, screening obtains the microorganism strains of high-yield beta-glucosidase; It is characterized in that: described enrichment culture be with Microcrystalline Cellulose or filter paper as sole carbon source, shaking table was cultivated 12～72 hours under 20～50 ℃, the condition of rotating speed 160～250r/min; The described method that the pregnant solution microorganism is carried out primary dcreening operation is that pregnant solution is carried out 10
-1～10
-7Gradient dilution, the diluent of drawing 0.1～0.3mL then respectively evenly is applied to and has added on the flat board of basic inorganic salt substratum that the quality volume percent is 0.01～3% Vitamin C2, under 20～50 ℃, cultivates 12～72 hours; The described bacterium colony that to choose method that purpose bacterial strain shake flask fermentation sieves again be picking becomes black with the substratum color of dull and stereotyped dorsal view periphery of bacterial colonies is connected to and produces in the enzyme substratum, shaking table was cultivated 1～7 day under the condition of 25～45 ℃ of temperature, rotating speed 150～250r/min, surveyed enzyme then and lived.
2. according to claim 1 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: the collection principle of described soil sample is to contain bacterium, fungi or the actinomycetes that produce beta-glucosidase; Described commercially available microbial strains is meant bacterium, fungi or the actinomycetes that can produce beta-glucosidase.
3. according to claim 1 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: the prescription of described basic inorganic salt substratum is:
In per 1000 ml distilled waters,, add ironic citrate 0.005～0.5%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 0.5～4.0% in the quality concentration of volume percent.
4. as described in claim 3 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: the prescription of described basic inorganic salt substratum is:
In per 1000 ml distilled waters,, add ironic citrate 0.01～0.2%, peptone 0.5%, potassium primary phosphate 0.1%, sal epsom 0.05%, agar 1.5～3.0% in the quality concentration of volume percent.
5. according to claim 1 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: the described amount that adds Vitamin C2 in the basic inorganic salt culture medium flat plate is a quality volume percent 0.1%～1.0%.
6. according to claim 1 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: the temperature of described enrichment culture is 25～45 ℃, and incubation time is 24～60 hours.
7. according to claim 1 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: the described culture temperature that microorganism in the pregnant solution is carried out the dull and stereotyped primary dcreening operation of Vitamin C2 is 25～40 ℃, and incubation time is 18～48 hours.
8. according to claim 1 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: the prescription of described product enzyme substratum is counted with the quality concentration of volume percent: grass meal 2.0%, wheat bran 2.0%, peptone 0.25%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.
9. according to claim 1 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: described to choose temperature that purpose bacterial strain shake flask fermentation sieves again be that 25～40 ℃, rotating speed are that 180～220r/min, shaking table incubation time are 2～5 days.
10. according to claim 1 Vitamin C2 is used for the method that high-yield beta-glucosidase produces the plate screening of bacterium, it is characterized in that: described the pregnant solution microorganism is carried out in the primary dcreening operation, the bacterium colony that the substratum color with dull and stereotyped dorsal view periphery of bacterial colonies of picking becomes black can also move to receive in the basic inorganic salt slant medium that contains Vitamin C2 preserves standby or repurity.
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| CN102399695B (en) * | 2011-12-28 | 2014-01-15 | 昆明理工大学 | Method for separating microorganisms capable of purifying bean curd wastewater |
| CN102732597B (en) * | 2012-04-26 | 2014-03-05 | 杭州师范大学 | Method for screening beta-glucosidase from fosmid library by using esculin and 4-MUG |
| CN104152361A (en) * | 2014-03-04 | 2014-11-19 | 西北农林科技大学 | Screening method of yeast strains for highly producing beta-glucosaccharase |
| CN112143682B (en) * | 2020-09-28 | 2022-06-17 | 广东海洋大学深圳研究院 | Color development solid culture medium of nocardia seriolae and preparation method and application thereof |
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Non-Patent Citations (4)
| Title |
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| β-葡萄糖苷酶产生菌及其发酵培养基的优化. 郭海风.扬州职业大学学报，第3卷第4期. 1999 |
| β-葡萄糖苷酶产生菌及其发酵培养基的优化. 郭海风.扬州职业大学学报，第3卷第4期. 1999 * |
| β-葡萄糖苷酶产生菌的分离筛选. 杨胜远等.工业微生物，第32卷第4期. 2002 |
| β-葡萄糖苷酶产生菌的分离筛选. 杨胜远等.工业微生物，第32卷第4期. 2002 * |
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