CN109628340A - A kind of Bacillus circulans bacterial strain and its selection producing high vigor beta galactosidase - Google Patents
A kind of Bacillus circulans bacterial strain and its selection producing high vigor beta galactosidase Download PDFInfo
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Abstract
The invention discloses a kind of Bacillus circulans bacterial strains and its selection for producing high vigor beta galactosidase, the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production is Bacillus circulans QHT-310-M481 (Bacillus circulans QHT-310-M481), on October 22nd, 2018 in China typical culture collection center preservation, deposit number is CCTCC NO:M 2018699, and preservation address is Wuhan University, Wuhan, China city.The Bacillus circulans bacterial strain of the high vigor beta galactosidase of production is to carry out mutagenic treatment through 1- methyl-3-nitro -1- nitrosoguanidine mutagenesis agent by Bacillus circulans starting strain to obtain, the beta galactosidase liquid that high fermentative activity can be produced, produces galactooligosaccharide for being industrially catalyzed galactosyl transfer reaction.
Description
Technical field
The invention belongs to Fermentation Engineerings and Microbial Breeding field, and in particular to a kind of high vigor beta galactosidase of production
Bacillus circulans bacterial strain and its selection.
Background technique
Galactooligosaccharide (Galactooligosaccharides, GOS) is that a kind of functionality with natural attribute is oligomeric
Sugar.Galactooligosaccharide is a kind of low energy sugar, is naturally present in animal cream and human milk.Galactooligosaccharide is double in human body intestinal canal
The fabulous nutrient source of the beneficial bacteriums such as discrimination bacillus, Lactobacillus acidophilus and effective proliferation factor, can improve disappearing for human body intestinal canal
Change absorption function.In addition, galactooligosaccharide can also improve lipid-metabolism, reduces serum cholesterol concentration, promotes mineral matter element
It absorbs, improves lactose intolerance, improves the functions such as immunity of organisms.
Galactooligosaccharide is widely used in food service industry, is applied to dairy products, bakery product, candy as prebiotic component
The fields such as processing and functional food.With the approval of regulation, galactooligosaccharide approval made from beta galactosidase becomes food
Product additive new varieties are used for dispensed food for baby and infant's cereal auxiliary food, are used alone or as a mixture.Along with complete
Two child's Policy Effect of face gives full play to, and birth level moderately improves.Baby is with milk powder, other dairy products and health care product as oligomeric
The field that galactolipin is mainly studied and applied has welcome new development peak.
In the industrial production, using lactose as raw material, galactosyl transfer reaction is catalyzed using microorganism beta galactosidase
To produce galactooligosaccharide.The molecular structure of galactooligosaccharide is on galactolipin or glucose molecule by β-(1 → 3) gala
Glycosidic bond, β-(1 → 4) galactolipin glycosidic bond or β-(1 → 6) galactoside are keyed 1-7 galactosyl.
Beta galactosidase is widely present in animals and plants and microorganism, its major function is that catalysing lactose hydrolysis generates
Glucose and galactolipin, the enzyme also have catalysis galactosyl transfer vigor, generate galactooligosaccharide.
In the industrial production, beta galactosidase substantially derives from microorganism, and has and be easy to largely prepare, stability
The features such as good.But the strain being directly separated from nature, it is however generally that its enzyme fermentation vigor be it is relatively low, cannot reach industry
The requirement of production, thus will according to the form of strain, physiologically the characteristics of, improve strain.The mutation of microorganism is in natural conditions
Under spontaneous can carry out, but the mutation rate of spontaneous mutation is very low, and the probability for obtaining satisfactory mutant strain is lower, and probability is only
It is 10-6~10-10.In order to make enzyme more suitable for practical application, the mutation of bacterial strain is improved using means such as physical chemical factors
Rate, make the mutant strain with beneficial traits screen a possibility that greatly increase.Chemical mutagen mainly by with nucleic acid base
Effect interferes DNA replication dna as base analogue or frameshift mutation agent, so that its inhereditary material is changed and is mutated.Mutagenesis
Agent 1- methyl-3-nitro -1- nitrosoguanidine is a kind of bifunctional alkylating agents that can be acted on nucleic acid base, can be with DNA molecular
Many positions are had an effect, and hydrogen atom active in DNA molecular is easily replaced, and are made the base on DNA molecular and are calculated part by alkane
Change, when DNA replication dna leads to base pairing mistakes and mutagenesis hinders in addition, it can also form covalent bond in DNA double interchain
DNA replication dna process double center chain is unlocked, and so as to cause mutation, can greatly improve the frequency of mutation.
The important sources that proof Bacillus circulans is beta galactosidase, and β-produced are reported in presently relevant research
Galactosidase has ideal changing effect to lactose.Have using Bacillus circulans production beta galactosidase significant
Application value.
Therefore, it is necessary to study the Bacillus circulans bacterial strain for being able to produce high vigor beta galactosidase and its breeding sides
Method.
Summary of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide a kind of ring-types for producing high vigor beta galactosidase
Bacillus strain, the bacterial strain can produce the beta galactosidase of high vigor, for being industrially catalyzed galactosyl transfer reaction
To produce galactooligosaccharide.The present invention also provides the breedings for the Bacillus circulans bacterial strain for producing high vigor beta galactosidase
Method, the selection are taken turns mutagenic treatment using 1- methyl-3-nitro -1- nitrosoguanidine as mutagens more, detect mutagenic strain
Beta galactosidase enzyme activity, to obtain the Bacillus circulans bacterial strain for producing high vigor beta galactosidase, selection
Simple and easy, mutagenesis screening success rate is high.
The technical solution adopted by the present invention is that: a kind of Bacillus circulans bacterial strain producing high vigor beta galactosidase,
The Bacillus circulans bacterial strain of the high vigor beta galactosidase of production is Bacillus circulans QHT-310-M481
(Bacillus circulans QHT-310-M481) is protected on October 22nd, 2018 in China typical culture collection center
Hiding, deposit number are CCTCC NO:M 2018699, and preservation address is Wuhan University, Wuhan, China city.
Specifically, the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production is gone out by Bacillus circulans
Bacterium germination strain carries out what mutagenic treatment obtained through mutagens, and the Bacillus circulans starting strain is Bacillus circulans
(Bacillus circulars) is purchased from American Type Culture Collecti (ATCC), number 31382.
Preferably, the mutagens are 1- methyl-3-nitro -1- nitroso guanidine solution, 1- methyl-3-nitro -1- nitrous
Base guanidine solution is the 1- methyl-3-nitro -1- nitrosoguanidine acetone soln that concentration is 0.2-1.0mg/mL.
Preferably, during mutagenic treatment, the 1- methyl-3-nitro -1- nitrosoguanidine acetone soln of 0.2-1.0mg/mL
Dosage be 0.5-1.5mL.
Preferably, the time of mutagenic treatment is 15-60min.
The selection of the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production, including following step
It is rapid:
1) Bacillus circulans starting strain is accessed in seed culture medium, after 37 DEG C of culture 4-6h, bacterium is collected by centrifugation
Body, is added the cleaning filtering of Sterile phosphate buffer solution, and adjustment bacteria suspension concentration is 105-107A/mL；
2) mutagens are added into the bacteria suspension of step 1), carries out mutagenic treatment, obtains mutagenesis bacterium solution, mutagenesis bacterium solution is applied
Solid medium tablets are distributed in, is cultivated 1-2 days at 37 DEG C, after growing bacterium colony, selects the bacterium colony that growing way is good, bacterium colony is big, are inoculated with
In 96 orifice plates containing fermentation medium, in 37 DEG C of culture 36-48h, M1 mutagenic strain is obtained, detects the β-of M1 mutagenic strain
Galactosidase enzyme activity calculates the Mutagenic Effect of M1 mutagenic strain；
3) the M1 mutagenic strain to the increase rate of beta galactosidase enzyme activity in step 2) greater than 30% carries out heredity
Stability analysis, continuous 7 secondary cultures, detects the beta galactosidase enzyme activity of mutagenic strain, it is best to select genetic stability
M1 mutagenic strain as mutagenesis female parent；
4) 1-5 wheel mutagenic treatment is carried out to the mutagenesis female parent that step 3) obtains, detects the beta galactosidase of mutagenic strain
Enzyme activity selects the highest mutagenic strain of beta galactosidase enzyme activity as the cyclic annular bud for producing high vigor beta galactosidase
Spore bacillus strain.
Preferably, a kind of selection for the Bacillus circulans bacterial strain producing high vigor beta galactosidase, including with
Lower step:
1) Bacillus circulans starting strain is accessed in seed culture medium, after 37 DEG C of culture 4-6h, bacterium is collected by centrifugation
Body, is added the cleaning filtering of Sterile phosphate buffer solution, and adjustment bacteria suspension concentration is 105-107A/mL；
2) it is sub- that the 1- methyl-3-nitro -1- that 1mL concentration is 0.2-1.0mg/mL is added into the bacteria suspension of 1mL step 1)
Nitroguanidine acetone soln carries out mutagenic treatment 30min, obtains mutagenesis bacterium solution I, take 100 μ L mutagenesis bacterium solution, I applying solid culture medium
Plate is cultivated 1-2 days at 37 DEG C, carries out bacterium colony counting, is calculated lethality I, is determined best mutagenesis concentration；
The 1- methyl-3-nitro-for the best mutagenesis concentration that 1mL step 2) determines is added into the bacteria suspension of 1mL step 1)
1- nitrosoguanidine acetone soln carries out mutagenic treatment 15-60min, obtains mutagenesis bacterium solution II, and 100 μ L mutagenesis bacterium solutions II is taken to be coated with
Solid medium tablets are cultivated 1-2 days at 37 DEG C, carry out bacterium colony counting, are calculated lethality II, are determined best mutation time；
The II applying solid culture medium flat plate of mutagenesis bacterium solution of obtained best mutation time is selected after growing bacterium colony
The bacterium colony that growing way is good, bacterium colony is big is inoculated in 96 orifice plates containing fermentation medium, in 37 DEG C of culture 36-48h, is obtained M1 and is lured
Become bacterial strain, detect the beta galactosidase enzyme activity of mutagenic strain, calculates the induced mutation rate of M1 mutagenic strain；
3) the M1 mutagenic strain to the increase rate of beta galactosidase enzyme activity in step 2) greater than 30% carries out heredity
Stability analysis, continuous 7 secondary cultures, detects the beta galactosidase enzyme activity of mutagenic strain, it is best to select genetic stability
M1 mutagenic strain as mutagenesis female parent；
4) 1-5 wheel mutagenic treatment is carried out to the mutagenesis female parent that step 3) obtains, detects the beta galactosidase of mutagenic strain
Enzyme activity selects the highest mutagenic strain of beta galactosidase enzyme activity as the cyclic annular bud for producing high vigor beta galactosidase
Spore bacillus strain.
Preferably, in step 1), seed culture medium is prepared from following methods: toward the prefabricated nutrient broth dry powder of 15-20g
Middle addition 1000mL distilled water dissolves by heating, and pH value does not need separately to adjust, cooling, obtains seed culture medium.
It is furthermore preferred that seed culture medium is prepared from following methods in step 1): toward the prefabricated nutrient broth dry powder of 18g
Middle addition 1000mL distilled water dissolves by heating, and pH value does not need separately to adjust, cooling, obtains seed culture medium.
Preferably, in step 1), Sterile phosphate buffer solution is the 0.01mol/L phosphate buffer solution that pH value is 6.
Preferably, in step 2), solid medium tablets are prepared from following methods: toward the prefabricated nutrient agar of 30-35g
1000mL distilled water is added in dry powder, dissolves by heating, pH value is not needed separately to adjust, be poured into culture dish, and it is cooling, obtain solid
Culture medium flat plate.
It is furthermore preferred that solid medium tablets are prepared from following methods in step 2): toward the prefabricated nutrient agar of 33g
1000mL distilled water is added in dry powder, dissolves by heating, pH value is not needed separately to adjust, be poured into culture dish, and it is cooling, obtain solid
Culture medium flat plate.
Preferably, in step 2), fermentation medium includes following components in percentage by weight: 0.6-1.6% soybean protein
Peptone, 0.35-0.65% yeast extract, 0.2-0.5% disodium hydrogen phosphate, 0.12-0.25% sodium carbonate, 0.12-0.55% sulphur
Sour magnesium, 1.0-1.5% lactose and 95.5-97.5% water, fermentation medium pH value are 6.0-6.5.
In industrial application, our purpose is the unit of activity of the total enzyme activity of fermenting and producing in unit volume
It maximizes.The Enzyme activities of the beta galactosidase of Bacillus circulans, be by DNA, RNA in bacterial body and protein etc. no
The non-linear regulated and control network of same level is controlled.Strain variation caused by every wheel mutagenesis, the part of the only regulated and control network of influence
Region, for example, beta-galactosidase gene expression regulation element is enhanced, the transcription product of beta-galactosidase gene it is steady
It is qualitative significantly improve, positive change occurs for the corresponding gene order of the key amino acid of the enzymatic activity pocket of beta galactosidase.
A kind of Breeding Process for the Bacillus circulans bacterial strain producing high vigor beta galactosidase, is the total enzyme activity power with fermentation liquid
It significantly improves to be oriented to, according to the actual conditions that the total enzyme activity of the beta galactosidase of the bacterial strain of every wheel mutagenesis improves, flexibly
The screening criteria for setting every wheel bacterial strain, finally obtains a kind of bacterial strain currently optimized.
Compared with prior art, the present invention having the following beneficial effects:
1. a kind of Bacillus circulans bacterial strain for producing high vigor beta galactosidase of the invention can produce high fermentation and live
The beta galactosidase liquid of power, produces galactooligosaccharide for being industrially catalyzed galactosyl transfer reaction.
2. the selection of the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production of the invention, with 1- first
Base -3- nitro -1- nitrosoguanidine is mutagens, is taken turns mutagenic treatment more, obtains the ring-type for producing high vigor beta galactosidase
Bacillus strain, 1- methyl-3-nitro -1- nitrosoguanidine mutagenesis agent are a kind of bifunctional alkylating agents that can be acted on nucleic acid base
Agent can have an effect with many positions of DNA molecular, easily replace hydrogen atom active in DNA molecular, make the alkali on DNA molecular
Base and nucleic acid moiety are by alkanisation, and when DNA replication dna leads to base pairing mistakes and mutagenesis, in addition, it can also be in DNA double interchain
Covalent bond is formed, unlocking for DNA replication dna process double center chain is hindered, so as to cause mutation, the frequency of mutation can be greatly improved.
3. the selection is significantly improving as guiding with the total enzyme activity power of fermentation liquid, according to the bacterial strain of every wheel mutagenesis
The actual conditions that the total enzyme activity of beta galactosidase improves flexibly set the screening criteria of every wheel bacterial strain, finally obtain one kind
The bacterial strain currently optimized, method is flexibly pragmatic, simple and easy, and mutagenesis screening success rate is high, is more advantageous to the positive mutagenesis of acquisition
As a result, obtaining the Bacillus circulans bacterial strain for producing high vigor beta galactosidase.
Detailed description of the invention
Fig. 1 is the M4 mutagenic strain beta galactosidase enzyme activity testing result figure of embodiment 1.
Fig. 2 is the thallus microscopy figure of the M4-81 mutagenic strain of embodiment 1.
Fig. 3 is the colonial morphology figure of the M4-81 mutagenic strain of embodiment 1.
Specific embodiment
Below with reference to embodiment, the present invention will be further described.
The calculation method of beta galactosidase enzyme activity:
1. preparation of reagents
(1) Z- buffer
16.1g disodium hydrogen phosphate, 5.5g sodium dihydrogen phosphate, 0.75g potassium chloride, 0.246g sulfuric acid are dissolved in 800mL water
The sodium hydroxide solution of 2mol/L is added in magnesium and 2.7mL 2 mercapto ethanol, adjusts pH to 6.0 ± 0.05, is detected with pH instrument.It will be molten
Liquid is transferred to 1000mL volumetric flask, with water constant volume, mixes.
(2) ortho-nitrophenyl-β-D- galactopyranoside (ONPG) solution
With the ONPG of 75mL Z- buffer solution 250.0mg, solution is transferred to 100mL volumetric flask, it is fixed with Z- buffer
Hold, as substrate.
(3) stop bath
It takes water as a solvent, after dissolving 10g sodium carbonate, is transferred to 100mL volumetric flask constant volume.
(4) prepared by test specimens
Setup test enzyme sample, so that the beta galactosidase containing 0.05~0.25 unit in the final solution of every mL.
2. detecting step
The teat glass of specification 20*150mm a series of is placed in 50 ± 0.1 DEG C of water-bath, then is distinguished with liquid-transfering gun
The ONPG solution of 0.25mL is inhaled into teat glass, constant temperature water bath.
The sample to be tested (water of 0.25mL is added in contrast test tube) of 0.25mL is added with pipettor gun rapidly, then opens
Dynamic oscillation, presses stopwatch and starts timing；The reaction solution of 0.5mL is sucked out after oscillation 10min from each test tube, is added to and is equipped with
In the test tube of 0.5mL stop bath, then stir and evenly mix.
Sample is moved into ELISA Plate, Detection wavelength 405nm, using the solution in contrast test tube as control, detection is each
The absorbance of sample.
3. the drafting of standard curve
139.0mg o-nitrophenol (ONP) is rotated into 1000mL volumetric flask, is dissolved before turning with 95% alcohol, it is fixed with water
Hold, mixes.The solution of 2.5,5,12.5 and 25mL is inhaled respectively with liquid-transfering gun to the volumetric flask of 100mL, with the sodium carbonate of 10wt%
Solution constant volume mixes.Every mL contains 0.025,0.05,0.125,0.25 micromolar ONP respectively in these solution.
Standard solution is loaded into the quartz ampoule of a 1cm, surveys absorbance under the conditions of wavelength 405nm, is control with water,
The concentration of ONP is abscissa, and the absorbance of each concentration standard object is ordinate, an available straight line by origin.
4. calculating
It is computed analysis and obtains standard curve: Y=4.0688X+0.0152, R2=0.9991
(Y is absorbance value at 405nm, and X is the concentration of ONP)
Beta galactosidase vigour-testing method an are as follows: fcc lactase units (LacU) is defined as under the conditions of this method
Enzyme activity amount needed for 1 micromole ONP of release per minute.
Enzyme activity (U/mL)=4*DF*X/10
5. 0.25mL, which is diluted the enzyme activity in enzyme solution, converts the enzymatic activity for 1mL；
DF: extension rate；
X:ONP concentration (μm ol)；
10: reaction time, 10min.
Used seed culture medium is prepared from following methods in embodiment: toward the prefabricated nutrient broth dry powder of 15-20g
Middle addition 1000mL distilled water dissolves by heating, and pH value does not need separately to adjust, cooling, obtains seed culture medium.
Sterile phosphate buffer solution is the 0.01mol/L phosphate buffer solution that pH value is 6.
Solid medium tablets are prepared from following methods: 1000mL is added into the prefabricated nutrient agar dry powder of 30-35g
Distilled water dissolves by heating, and pH value is not needed separately to adjust, be poured into culture dish, cooling, obtains solid medium tablets.
Fermentation medium includes following components in percentage by weight: 0.6-1.6% soy peptone, 0.35-0.65% ferment
Female extract, 0.2-0.5% disodium hydrogen phosphate, 0.12-0.25% sodium carbonate, 0.12-0.55% magnesium sulfate, 1.0-1.5% cream
Sugar and 95.5-97.5% water, fermentation medium pH value are 6.0-6.5.
Embodiment 1
1. producing the preliminary screening of the Bacillus circulans of high vigor beta galactosidase, comprising the following steps:
1) starting strain activates: by Bacillus circulans, (Bacillus circulars is purchased from American Type Culture Collecti
(ATCC, number 31382) is used as starting strain, carries out scribing line culture, and starting strain passes through inclined-plane nutrient agar culture three times
After base activation culture, be inoculated in 100mL fermentation medium (composition of fermentation medium are as follows: the soy peptone of 1.2wt%,
The yeast extract of 0.65wt%, the disodium hydrogen phosphate of 0.35wt%, the sodium carbonate of 0.25wt%, 0.55wt% magnesium sulfate,
The lactose of 1.5wt% and the water of 95.50wt%, pH6.0-6.5), it is centrifuged after shaking flask culture 48h at 37 DEG C, takes supernatant, carried out
Beta galactosidase enzyme enzyme activity determination, measurement result are as shown in table 1:
1 Bacillus circulans starting strain beta galactosidase enzyme activity of table
2) one ring slant strains of picking are in seed culture medium (prefabricated nutrient broth medium), after 37 DEG C of culture 6h,
10000rpm is centrifuged 10min, collects thallus, is resuspended in pH6.0 phosphate buffer solution, is placed on dress through absorbent cotton filtering
Have in the small triangular flask of bead, shaken well, being prepared into unicellular bacteria suspension concentration is 105~107A/mL.
3) take 1mL concentration be 0.25,0.5,0.75, the 1- methyl-3-nitro -1- nitrosoguanidine acetone of 1.0mg/mL it is molten
Liquid is added separately in 1mL bacteria suspension, is carried out mutagenic treatment 30min, is obtained mutagenesis bacterium solution I, and 100 μ L mutagenesis bacterium solutions I is taken to be coated with
Solid medium tablets carry out bacterium colony counting in 37 DEG C of culture 48h, calculate lethality I, determine that best mutagenesis concentration is
0.5mg/mL.The results are shown in Table 2.
2 1- methyl-3-nitro -1- nitrosoguanidine mutagenesis concentration of table
| Mutagenesis concentration (mg/mL) | 0.25 | 0.5 | 0.75 | 1 |
| Lethality I (%) | 82.6 | 99.0 | 99.9 | 99.9 |
4) taking 1mL concentration is the 1- methyl-3-nitro -1- nitrosoguanidine acetone soln of 0.5mg/mL, and it is outstanding to be added to 1mL bacterium
In liquid, under the conditions of 37 DEG C, 15,30,45,60min are vibrated respectively, obtain mutagenesis bacterium solution II, take the coating of 100 μ L mutagenesis bacterium solutions II solid
Body culture medium flat plate calculates lethality II in 37 DEG C of culture 48h, determines that best mutation time is 30min.As a result such as 3 institute of table
Show.
The 3 1- methyl-3-nitro -1- nitrosoguanidine mutagenesis time of table
| Mutation time (min) | 15 | 30 | 45 | 60 |
| Lethality II (%) | 80.3 | 99.0 | 99.9 | 99.9 |
5) the mutagenesis bacterium solution II of mutagenic treatment 30min in 100 μ L steps 4), applying solid culture medium flat plate, at 37 DEG C are taken
Cultivate 48h.
6) after growing bacterium colony, select the bacterium colony that growing way is good, bacterium colony is big, with sterile toothpick one by one dibbling label bacterium colony in
(2mL deep-well plates, every hole liquid amount are 96 orifice plates containing fermentation medium (composition of fermentation medium is identical as step 1))
800uL), 96 orifice plates are placed in shaking table and are cultivated, 37 DEG C, humidity 80%, 300r/min culture 48h obtain M1 mutagenic strain, uses
Microplate reader detects the beta galactosidase enzyme activity of M1 mutagenic strain, calculates the Mutagenic Effect of M1 mutagenic strain.
7) increase rate for the beta galactosidase enzyme activity for coming out step 6) picking is greater than 30% M1 mutagenic strain
It is rechecked, culture transferring cultivates 48h in solid medium tablets, then transposing, in 96 orifice plate fermented and cultureds, every plant is done 5 and tested in parallel
Card, in 37 DEG C of culture 48h, is measured beta galactosidase enzyme activity, and wherein the enzyme activity of M1-38, M1-63 improve the most
Significantly, 30.35% and 30.22% is promoted than starting strain respectively；
2. producing the genetic stability analysis of the Bacillus circulans of high vigor beta galactosidase
M1-38, M1-63 mutagenic strain are accessed in inclined-plane solid medium, in 37 DEG C of culture 48h, the sterile phosphorus of 9mL is added
Acid buffering solution, elution, dilution spread solid medium tablets select the single bacterium that growing way is good, bacterium colony is big after growing bacterium colony
It falls, carries out continuous 7 secondary cultures, and detect the beta galactosidase enzyme activity of bacterial strain, determine that bacterial strain is producing high activity beta-
Genetic stability in terms of galactosidase.Wherein M1-38 has good genetic stability, chooses M1-38 mutagenic strain and makees
For mutagenesis female parent, specific data are shown in Table 4.
4 M1 of table is verified for genetic stability
| Enzyme activity (U/mL) | It is primary | 1st generation | 2nd generation | 3rd is low | 4th generation | 5th generation | 6th generation | 7th generation |
| M1-38 | 19.94 | 19.92 | 19.98 | 19.89 | 19.95 | 19.94 | 19.96 | 19.91 |
| M1-63 | 18.82 | 19.45 | 17.88 | 15.71 | 15.24 | 14.95 | 15.36 | 14.81 |
3. producing the more round mutagenesis of Bacillus circulans of high vigor beta galactosidase
The M1-38 mutagenic strain for choosing genetic stability is that mutagenesis is maternal, and one ring slant strains of picking are in fresh seed
In culture medium, after 37 DEG C of culture 6h, 10000rpm is centrifuged 10min, collects thallus, is resuspended in pH6.0 phosphate buffer solution
In, it is placed in the small triangular flask equipped with bead through absorbent cotton filtering, shaken well, being prepared into unicellular bacteria suspension concentration is
105~107A/mL, taking 1mL concentration is the 1- methyl-3-nitro -1- nitrosoguanidine acetone soln of 0.5mg/mL, is added to 1mL
In bacteria suspension, mutagenesis bacterium solution applying solid culture medium flat plate culture is selected the good single colonie of growing way, utilizes 96 by processing 30min
Orifice plate cultivating system, fermented and cultured simultaneously detect bacterial strain biomass and beta galactosidase enzyme activity.It repeats mutagenesis steps 3 times,
It obtains offspring's mutagenic strain and is respectively labeled as M2, M3, M4.At 4 round 1- methyl-3-nitro -1- nitrosoguanidine mutagenesis agent
Reason, it is the Bacillus circulans bacterial strain for producing high vigor beta galactosidase, M4 generation production that finishing screen, which selects M4-81 mutagenic strain,
The vigor of enzyme see that Fig. 1, horizontal axis are strain numbers, the longitudinal axis is enzyme activity.
As shown in Figure 1, forth generation mutant strain M4-81, enzyme activity 39.86U/mL are the vigor of starting strain institute producing enzyme
2.60 times of (Bacillus circulans ATCC, No.31382), are the bacterial strains currently optimized.It is cyclic annular gemma by the Strain Designation
Bacillus QHT-310-M481 (Bacillus circulans QHT-310-M481), on October 22nd, 2018 in Chinese Typical Representative
Culture collection preservation, deposit number are CCTCC NO:M 2018699, and preservation address is Wuhan University, Wuhan, China city.
A kind of form such as Fig. 2 and Fig. 3 institute of the Bacillus circulans bacterial strain for producing high vigor beta galactosidase
Show, bacterium colony is glassy yellow, regular shape, surface wettability, neat in edge；Thallus is rod-shaped, single to arrange, and gemma end is raw, and packing is not
It expands, Gram-positive.
Claims (10)
1. a kind of Bacillus circulans bacterial strain for producing high vigor beta galactosidase, which is characterized in that the high vigor of production
The Bacillus circulans bacterial strain of beta galactosidase is Bacillus circulans QHT-310-M481 (Bacillus circulans
QHT-310-M481), on October 22nd, 2018 in China typical culture collection center preservation, deposit number CCTCC
NO:M 2018699, preservation address are Wuhan University, Wuhan, China city.
2. the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production according to claim 1, which is characterized in that
The Bacillus circulans bacterial strain of the high vigor beta galactosidase of production is by Bacillus circulans starting strain through mutagens
Carry out what mutagenic treatment obtained.
3. the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production according to claim 2, which is characterized in that
The mutagens are 1- methyl-3-nitro -1- nitroso guanidine solution, and 1- methyl-3-nitro -1- nitroso guanidine solution is that concentration is
1- methyl-3-nitro -1- nitrosoguanidine the acetone soln of 0.2-1.0mg/mL.
4. the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production according to claim 2, which is characterized in that
During mutagenic treatment, the dosage of mutagens is 0.5-1.5mL.
5. the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production according to claim 2, which is characterized in that
The time of mutagenic treatment is 15-60min.
6. the Bacillus circulans bacterial strain of the high vigor beta galactosidase of production according to any one of claims 1-5
Selection, which comprises the following steps:
1) Bacillus circulans starting strain is accessed in seed culture medium and is cultivated, thalline were collected by centrifugation, and sterile phosphorus is added
The cleaning filtering of acid buffering solution, adjustment bacteria suspension to debita spissitudo；
2) mutagens are added into the bacteria suspension of step 1), carries out mutagenic treatment, obtains mutagenesis bacterium solution, mutagenesis bacterium solution is coated on
Solid medium tablets are cultivated, and after growing bacterium colony, are selected the bacterium colony that growing way is good, bacterium colony is big, are inoculated in fermentation medium
In cultivated, obtain M1 mutagenic strain, detect the beta galactosidase enzyme activity of M1 mutagenic strain, calculate M1 mutagenic strain
Mutagenic Effect；
3) the M1 mutagenic strain to the increase rate of beta galactosidase enzyme activity in step 2) greater than 30% carries out inheritance stability
Analysis, continuous 7 secondary cultures detect the beta galactosidase enzyme activity of mutagenic strain, select the best M1 of genetic stability
Mutagenic strain is as mutagenesis female parent；
4) 1-5 wheel mutagenic treatment is carried out to the mutagenesis female parent that step 3) obtains, detects the beta galactosidase enzyme activity of mutagenic strain
Power selects the highest mutagenic strain of beta galactosidase enzyme activity as the cyclic annular gemma bar for producing high vigor beta galactosidase
Bacteria strain.
7. selection according to claim 6, which is characterized in that in step 1), seed culture medium is by following methods system
It is standby to obtain: 1000mL distilled water is added into the prefabricated nutrient broth dry powder of 15-20g, dissolves by heating, it is cooling, obtain seed culture
Base.
8. selection according to claim 6, which is characterized in that in step 1), Sterile phosphate buffer solution is pH value
For 6 0.01mol/L phosphate buffer solution.
9. selection according to claim 6, which is characterized in that in step 2), solid medium tablets are by with lower section
Method is prepared: 1000mL distilled water is added into the prefabricated nutrient agar dry powder of 30-35g, dissolves by heating, pours into culture dish,
It is cooling, obtain solid medium tablets.
10. selection according to claim 6, which is characterized in that in step 2), fermentation medium includes following weight
The component of percentage: 0.6-1.6% soy peptone, 0.35-0.65% yeast extract, 0.2-0.5% disodium hydrogen phosphate,
0.12-0.25% sodium carbonate, 0.12-0.55% magnesium sulfate, 1.0-1.5% lactose and 95.5-97.5% water, fermentation medium pH
Value is 6.0-6.5.
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