CN102634456A

CN102634456A - Method for rapidly screening heat-resistant beta-glucanase production strains

Info

Publication number: CN102634456A
Application number: CN2011100378308A
Authority: CN
Inventors: 邓开健
Original assignee: KAISHENG BIOLOGICAL SCIENCE AND TECHNOLOGY Co Ltd GUANGZHOU
Current assignee: KAISHENG BIOLOGICAL SCIENCE AND TECHNOLOGY Co Ltd GUANGZHOU
Priority date: 2011-02-14
Filing date: 2011-02-14
Publication date: 2012-08-15

Abstract

The invention discloses a method for rapidly screening heat-resistant beta-glucanase production strains. According to the method, beta-glucanase can generate a transparent ring after hydrolyzing carboxymethyl cellulose in a solid phase on a flat plate containing proper amount of congo red. As microorganism strains can not survive under high temperature, nylon membranes used by bacterial colony is subjected to in-situ transferring, the original flat plate continues to culture so as to select the garget strain later. The nylon membranes with different bacterial colonies can be incubated under higher temperature, and the transparent ring and bacterial colonies with larger specific value are selected by being taken as heat-resistant strains. According to the method, the high throughput screening can be conducted, and the result is accurate and easy to distinguish.

Description

A kind of method of rapid screening thermotolerance beta-glucan enzyme-producing bacteria

Technical field

The invention belongs to technical field of bioengineering, more specifically, the present invention relates to a kind of method of screening thermotolerance beta-glucan enzyme-producing bacteria.

Background technology

In brewing industry, abroad some countries have adopted beta-glucanase as one of main zymin.Excessive beta-glucan is to cause the unsettled major reason of beer, causes vaporific muddiness of beer and gel precipitation.From the source analysis that exists of beta-glucanase, it is the Primary Actor of degraded starch albuminous cell wall, and is relevant with the Fructus Hordei Germinatus leaching yield.Add beta-glucanase during malt amylase and can decompose beta-glucan gel wherein, diastatic malt juice reduces viscosity; Reduce gelatinous precipitate in the beer; Improve the filtering velocity and the yield of wheat juice in the beer manufacturing, thereby help improving beer flavor, improve beer quality.

Be in the animal cultivation of feedstuff raw material with the wheat class, beta-glucan can form thick substances in animal intestinal influence animal to absorption of nutrient ingredients, is called as ANFs.Beta-glucanase reduces its wetting ability and viscosity through the degraded beta-glucan, promptly reduces the viscosity of intestinal contents, effectively improves gastrointestinal tract environment and the animal assimilated efficiency to nutritive substance, improves efficiency of feed utilization, removes the effect of ANFs.Beta-glucanase can with the receptors bind on the bacteria cell wall; Thereby stop bacterium to combine with glycosyl on the animal intestine mucous membrane; The integrity of the 26S Proteasome Structure and Function of protection intestines mucosa; Have the harmful microorganism of inhibition and safeguard the effect of animal intestinal microecological balance, and reduce traditional antibiotic usage quantity.It is thus clear that beta-glucanase all has important economic worth in beer fermentation industry and fodder enzyme preparation industry, has been acknowledged as effective fodder additives, enzyme food preparation.

The high temperature of brewage Mashing process is 70-80 ℃, and the temperature of feed granulation is generally also about 80 ℃.Yet most beta-glucanases are this high temperature of ability not.Therefore, the active the bacterial classification high and beta-glucanase that thermotolerance is strong of screening is significant from produce beta-glucanase bacterial classification library or selection by mutation.

For the thermostability mutant choice, at present commonly used is 96 orifice plates, plate screening method and superthin layer agar plate diffusion process, but these methods use not only and waste time and energy, and costliness.Because LSD can produce transparent circle behind the CMC 99.5 in the hydrolysis solid phase on containing Congo red in right amount flat board, therefore can use the plate screening strategy.Its principle is: Congo redly become red compound with the beta-glucan energy coupling; Cultivate after 2-3 days for 30 ℃; The beta-glucanase decomposition back color that produces via bacterium colony shoals; Present transparent circle on the flat board, the Congo red transparent circle diameter that fades is directly proportional with the logarithmic value of enzymic activity, thereby can judge the strain enzyme-producing situation according to the size and the transparency of transparent circle.Yet microbial strains can not at high temperature survive, and therefore will filter out resistant to elevated temperatures LSD and come just must reasonably improve existing method, and screening process and condition are optimized.

Therefore, along with beta-glucanase is applied in more and more in the every field and to the raising of its request for utilization, is needed a kind of method of screening the beta-glucanase bacterial classification of high temperatures, for the screening of beta-glucanase provide new by way of.

Summary of the invention

Picking list bacterium colony is after the cultivation of seed stage; Be inoculated in by 1% inoculum size and 100mL to be housed to produce on the enzyme substratum and (to produce enzyme substratum (w/v): 4% oatmeal, 1% peptone, sodium-chlor 0.5%; PH nature) in the 250mL triangular flask, cultivated 24-48 hour in 28-37 ℃, 150-200r/min.Bacterium liquid is coated after suitably diluting on the product enzyme substratum; Make the colony count that forms on the product enzyme substratum when 50 left and right sides; Independent between the transparent circle of each bacterium colony, big or small suitable, regular shape on the flat board; But when colony count is above above 50, then there is the transparent circle of part bacterium colony to link together easily, like this size of judging transparent circle is prone to produce and disturbs.28-37 ℃ is continued to cultivate appropriate time, places 1-2h for 4 ℃, and bacterium colony carries out the original position transfer printing with nylon membrane, and former flat board continues at cultivation, so that later picking purpose bacterium.To aseptic nylon membrane (under the indicia face) be laid on the flat board during original position transfer printing; And contact with bacterium colony; Soak (should prevent to produce bubble between nylon membrane and agar plate) fully until nylon membrane, take nylon membrane off (should carry out azimuth mark on the nylon membrane) from the agar face with one-time action stably immediately, with the bacterium colony of nylon membrane towards last; Place on the new product enzyme substratum, cultivate appropriate time down for 28-37 ℃.Use cell walls lysate (25mmol/L Tris-HCl (p H8.0) then; Include 1mg/mL N,O-Diacetylmuramidase and 1mmol/L EDTA (pH8.0)), cytolemma lysate (10mmol/L Tris-HCl (p H8.0); Include 0.1% (w/v) Triton-X-100) and level pad (50mmol/LTris-HCl (p H8.0)) soak 3 filter paper respectively; Be placed on after the taking-up in the clean petridish; Rush unnecessary liquid preventing that bacterium colony from being broken up with glass rod, and the order that nylon membrane is faced up by cell walls lysate, cytolemma lysate, level pad places on 3 filter paper successively, and room temperature is handled 30min respectively.Then the bacterium colony of nylon membrane is faced down and is laid on that screening is dull and stereotyped and goes up (composition of screening culture medium is following: with 15-25g agar powder, 5g Xylo-Mucine and 1L0.1mol/L imidazoles-o-2 potassium acid hydrogen potassium damping fluid mixing and autoclaving, add fall behind the 2.5mL penbritin dull and stereotyped).Incubation temperature is that 28-75 ℃, time are 1-3h during screening.Throw off nylon membrane, on flat board, add an amount of 0.1% Congo red colour developing 15min, discard Congo redly, in flat board, add an amount of 1mol/LNaCl decolouring 15min again, the situation of observation transparent circle.Selecting the bigger bacterium colony of transparent circle and bacterium colony ratio selects as heat-resisting bacterial strain.

From final effect, the method that this paper set up is quick, accurate, workable, goes for the screening that heat-resisting polysaccharide hydrolase produces bacterium in the general born of the same parents.

Embodiment

Embodiment 1: the single bacterium colony of picking lichens bud robe bacillus is after the cultivation of seed stage; Be inoculated in by 1% inoculum size and 100mL to be housed to produce on the enzyme substratum and (to produce enzyme substratum (w/v): 4% oatmeal, 1% peptone, sodium-chlor 0.5%; PH nature) in the 250mL triangular flask, cultivated 24 hours in 37 ℃, 200r/min.With bacterium liquid dilution 10 ^-7After coat and produce on the enzyme substratum, be 56 producing the colony count that forms on the enzyme substratum.Continue to cultivate 15 hours for 37 ℃, place 1-2h for 4 ℃, bacterium colony carries out the original position transfer printing with nylon membrane, and the bacterium colony of nylon membrane towards last, is placed on the new product enzyme substratum, cultivates appropriate time down for 37 ℃.Use the cell walls lysate then; Cytolemma lysate and level pad soak 3 filter paper respectively; Be placed on after the taking-up in the clean petridish; Rush unnecessary liquid preventing that bacterium colony from being broken up with glass rod, and the order that nylon membrane is faced up by cell walls lysate, cytolemma lysate, level pad places on 3 filter paper successively, and room temperature is handled 30min respectively.Then the bacterium colony of nylon membrane is faced down and be laid on the screening flat board.Incubation temperature is that 75 ℃, time are 3h during screening.Throw off nylon membrane, on flat board, add an amount of 0.1% Congo red colour developing 15min, discard Congo redly, in flat board, add an amount of 1mol/LNaCl decolouring 15min again, the situation of observation transparent circle.

Embodiment 2: picking black mold list bacterium colony is after the cultivation of seed stage; Be inoculated in by 1% inoculum size and 100mL to be housed to produce on the enzyme substratum and (to produce enzyme substratum (w/v): 4% oatmeal, 1% peptone, sodium-chlor 0.5%; PH nature) in the 250mL triangular flask, cultivated 48 hours in 30 ℃, 200r/min.With bacterium liquid dilution 10 ^-6After coat and produce on the enzyme substratum, be 53 producing the colony count that forms on the enzyme substratum.Continue to cultivate 36 hours for 30 ℃, place 1-2h for 4 ℃, bacterium colony carries out the original position transfer printing with nylon membrane, and the bacterium colony of nylon membrane towards last, is placed on the new product enzyme substratum, cultivates appropriate time down for 30 ℃.Use the cell walls lysate then; Cytolemma lysate and level pad soak 3 filter paper respectively; Be placed on after the taking-up in the clean petridish; Rush unnecessary liquid to prevent that bacterium colony from being broken up with glass rod, nylon membrane is faced up put successively on 3 filter paper by the order of cell walls lysate, cytolemma lysate, level pad, room temperature is handled 30min respectively.Then the bacterium colony of nylon membrane is faced down and be laid on the screening flat board.Incubation temperature is that 70 ℃, time are 2h during screening.Throw off nylon membrane, on flat board, add an amount of 0.1% Congo red colour developing 15min, discard Congo redly, in flat board, add an amount of 1mol/LNaCl decolouring 15min again, the situation of observation transparent circle.

Claims

1. the method for the production bacterium of the stable on heating beta-glucanase of screening is characterized in that, said method comprising the steps of:

(1) picking list bacterium colony bacterial classification is after the cultivation of seed stage, is inoculated in by 1% inoculum size and 100mL is housed produces on the enzyme substratum;

(2) bacterium colony carries out the original position transfer printing with nylon membrane;

(3) handle the nylon membrane that has bacterium respectively with cell walls lysate, cytolemma lysate and level pad, the bacterium colony of nylon membrane is faced down is laid on the screening flat board then;

(4) throw off nylon membrane, on flat board, add an amount of 0.1% Congo red colour developing 15min, discard Congo redly, in flat board, add an amount of 1mol/L NaCl decolouring 15min again, the situation of observation transparent circle.

2. the method for claim 1 is characterized in that, said bacterial classification is bacterium, yeast and mould.

3. the method for claim 1 is characterized in that, producing the enzyme substratum is (w/v): 4% oatmeal, 1% peptone, sodium-chlor 0.5%, pH nature.

4. the method for claim 1 is characterized in that, the incubation temperature of heat-resisting bacterial classification is that 28～75 ℃, time are 1～3h during screening.

5. the method for claim 1 is characterized in that, the cell walls lysate is: 25mmol/L Tris-HCl (p H8.0), include 1mg/mL N,O-Diacetylmuramidase and 1mmol/L EDTA (p H8.0); The cytolemma lysate is: 10mmol/L Tris-HCl (p H8.0) includes 0.1% (w/v) Triton-X-100; Level pad is: 50mmol/L Tris-HCl (pH 8.0).

6. the method for claim 1; It is characterized in that; The composition of screening culture medium is following: with 15～25g agar powder, 5g Xylo-Mucine and 1L 0.1mol/L imidazoles-o-2 potassium acid hydrogen potassium damping fluid mixing and autoclaving, fall dull and stereotyped behind the adding 2.5mL penbritin.