CN107365713A - A kind of preparation of aspergillus oryzae protoplast for high yield lactase and renovation process - Google Patents
A kind of preparation of aspergillus oryzae protoplast for high yield lactase and renovation process Download PDFInfo
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- CN107365713A CN107365713A CN201710727884.4A CN201710727884A CN107365713A CN 107365713 A CN107365713 A CN 107365713A CN 201710727884 A CN201710727884 A CN 201710727884A CN 107365713 A CN107365713 A CN 107365713A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01108—Lactase (3.2.1.108)
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- C12R2001/66—Aspergillus
- C12R2001/69—Aspergillus oryzae
Abstract
The present invention relates to a kind of preparation of aspergillus oryzae protoplast for high yield lactase and renovation process, comprise the following steps：(1) aspergillus oryzae spore suspension is prepared；(2) physics and chemistry behavior, mutant strain is obtained；(3) enzymolysis prepares protoplast；(4) purifying of protoplast；(5) regeneration of protoplast.Protoplast quantity prepared by the present invention is more, and the vigor of the protoplast of acquisition is higher, and regeneration rate can reach 31.2%.Protolast's preparation rate height and protoplast activity keep preferably, being advantageous to the preparation of follow-up fusant, and the metabolic regulation theoretical research and raising lactose enzyme activity for aspergillus oryzae regulation and control lactase are all significant；The strain improvement also for lactase production provides new approach simultaneously.
Description
Technical field
The present invention relates to the field of biology of fungi, and in particular to a kind of system of the aspergillus oryzae protoplast of high yield lactase
Standby and renovation process.
Background technology
Lactase, the entitled β-D- galactosides galactohydrolase of system, or abbreviation beta galactosidase.Lactase is being eaten
Product industry, medical industry, environmental protection, analysis etc. are with a wide range of applications.Utilize the energy of lactose enzyme hydrolysis lactose
Power, production Low lactose milk product (such as lactose hydrolysis breast, low-lactose milk powder), can effectively solve " lactose intolerance " problem, this
It is current lactase in one of maximum purposes of food industry.
China's dairy products consumption figure has a long way to go compared with external, and the reason for causing this phenomenon is except the level of economic development
Outside, also it is exactly to have greatly to suffer from " lactose intolerance " in Chinese population.It is reported that it there are about 70% in the population of Asia
People with different degrees of " lactose intolerance ", the incidence of disease that China adult drinks lactose malabsorption after cow's milk is up to
86.7%, lactose intolerance index is 0.9, is mainly shown as indigestion, abdominal distension, borborygmus, vomiting, acute abdominalgia or diarrhoea
Deng.The long-term hazards of lactose intolerance are children easily show as bad calcium uptake, diarrhoea, osteomalacia, body weight is low and grows
It is slow, easily show as osteoporosis symptoms in the elderly's especially elderly woman.Due to the generally existing of " lactose intolerance ", make
Quite a few people without image of Buddha normal person equally receives milk, and this is natural, has food of good nutrition balance, and this turns into
Hinder one of major obstacle of China's dairy industry development.70%~80% lactose of general hydrolysis can solve " lactose intolerance "
The problem of, and the best method for hydrolyzing lactose is exactly enzymatic isolation method, it is low with the lactose in lactase hydrolysed milk or dairy products, production
Lactose products can effectively eliminate human body and not tolerate symptom to lactose, for developing China's dairy industry, improve people's
General level of the health tool is of great significance, and this is also current lactase in one of maximum purposes of food industry.
The research to lactase is started late both at home and abroad, just starts to grind lactase after the 1980s
Study carefully.Up to the present, it has been reported that some lactase producing bacterial strains, and to enzyme isolate and purify and zymologic property, mutagenesis are educated
Kind and producing enzyme optimization, enzyme immobilizatio and industrial applications etc. have carried out certain research.Because lactase is mainly used in
Relevant food and medicine industry of human body, health etc., so the bacterial strain used in production must be nontoxic safe bacterial strain,
Must have GRAS qualifications, therefore aspergillus niger, aspergillus oryzae, Kluyveromyces lactis and Kluyveromyces fragilis are generally used as work
The enzyme source of industry metaplasia production.Aspergillus oryzae lactase molecular weight of albumen is not required to mostly between 100kD~130kD, and be all ectoenzyme
The technology for broken wall of yeast lactase, purifying process is simple, and most suitable action pH is 4.5~5.0, and has higher heat endurance,
It is commonly used for working process milk, whey and production galactooligosaccharide.Although work of the aspergillus oryzae lactase in dairy products are handled
With very significantly, but the enzyme not commercially large-scale use at present, main reasons is that the specific yield of lactase is relatively low.
For problem above, in order to improve the enzyme activity of lactase, domestic and international researcher has carried out substantial amounts of spy from many aspects
Rope：(1) in terms of industrial fermentation, mainly medium optimization, the addition of inducible factor and fermentation condition and extraction process
Optimization；(2) superior strain is screened by mutation breeding；(3) immobilized lactase, its continuous use is made；(4) genetic engineering is passed through
Technology and the lactase gene that aspergillus and yeast sources are expressed using saccharomyces cerevisiae and filamentous fungi expression system.Although obtain
Certain progress, but expression quantity is still limited.Genome segment S9 technology provides new thinking for the solution of this problem.
The general principle of genome segment S9 is to simulate the mechanism that nature is evolved in laboratory conditions, by one
Fixed multifarious gene pool is recombinated to obtain different mosaic genes in vitro, and therefrom directed screening goes out to have expected property
The mutant of shape.This method is easily widely used in the modification of enzyme, the improvement of albumen, the optimization of vaccine etc..Genome
The method of reorganization is to select an original parental plant first, obtains what multiple phenotypes were improved by the method for mutation breeding of classics
Bacterial strain, structure mutation candidate strain library, the direct parental plant that the bacterial strain improved using these phenotypes merges as first run multi-parent strain, then
More parental plant fusions are carried out, its full-length genome is recombinated at random, obtain first generation model fusion；Therefrom selection phenotype is entered again
Direct parent merge as next round of bacterial strain that one step improves, the rest may be inferred carries out the more parental plants taken turns more and merges, finally from obtaining
The elevated purpose bacterial strain of character is filtered out in the mutant library obtained.This technology is in protein, enzyme and monoclonal antibody etc.
It is widely used in terms of orthogenesis, and is greatly promoted the development of living things catalysis engineering.Because enzyme is biological anti-
The special significance in approach is answered, therefore enzyme viability is improved by directed evolution method, even creates new enzyme as to generation
A kind of highly useful strategy that the approach of thanking is transformed.
Compared with traditional mutagenesis, using genome segment S9 technology can also very fast more efficient acquired character it is excellent
Recombinant, while negative mutation is rejected, this largely compensate for the deficiency of classic mutagenesis method.Can be with by genome segment S9
Improve microbial metabolism yield or improve quality, carry out the seed selection of superior strain.Genome segment S9 technology lures including protoplast
The contents such as change, preparation, regeneration, fusion, conversion, before protoplast fusion is carried out, can obtain original set out using mutagenesis
The mutagenesis body of bacterial strain, the starting strain that will be merged with the bacterial strain that positivity is mutated as the first round, is then passed through what is excessively taken turns
Pushing-type merges, so that the different genes recombinant with different forward mutation assays is into same cell.And the system of protoplast
Standby and regeneration is the precondition and key technology merged, therefore the preparation of aspergillus oryzae protoplast has with regeneration techniques
Important theory and practical value.The B of patent CN 103756917 provide a kind of preparation of the protoplast of catalpa bungei endophytic fungi
Method, for the transformation of follow-up thalline, improve its anticancer vigor and study its anticancer mechanism and provide foundation.Patent CN 102061266
B has invented preparation and the renovation process of a kind of Rhizopus oryzae protoplast for high yield L-lactic acid, prepare number height, regeneration rate compared with
Height, and the protoplast prepared fully meets the requirement of protoplast fusion, further to use protoplast fusion skill
The ferment strength that art improves L-lactic acid production by Rhizopus oryzae provides the foundation.Therefore, in order to further improve aspergillus oryzae galactopoiesis
The ability of carbohydrase, the Regulation Mechanism that lactase is also synthesized to inquire into aspergillus oryzae in next step provide basis, and the present invention changes from genome
Group set out structure high yield lactase aspergillus oryzae protoplast.
The content of the invention
In order that with the aspergillus oryzae strain of genome segment S9 technology breeding high-yield lactase, it is an object of the invention to provide one
The preparation method of the aspergillus oryzae protoplast of kind high yield lactase.
The present invention is achieved by the following technical solutions：
A kind of preparation method of the aspergillus oryzae protoplast of high yield lactase provided by the invention, comprises the following steps：
(1) activation culture of strain：
The aspergillus oryzae strain (BNCC338380) of lyophilized preservation is fully dissolved into bacteria suspension with sterile saline, by this
Bacterial suspension inoculation is activated in czapek agar medium inclined-plane, and 28 DEG C are cultivated 5~6 days, are connected again after yellow green spore is covered with
It is continuous to spread cultivation once.
The culture medium used when spreading cultivation is Czapek's medium.
(2) monospore suspension is prepared
After the yellow green spore sterile saline covered with Czapek's medium is eluted into spore, pour into equipped with bead
The sterile conical flasks of 250mL in, fully shake conical flask, in the presence of bead, the spore in bottle broken up, with sterile de-
Fat cotton filters spore suspension, the spore suspension for the mycelia that is removed, 10 is diluted to sterilized water6Individual/ml.
(3) ultraviolet and lithium chloride complex mutation obtains mutant strain
The bacteria suspension obtained by 5mL steps (2) is taken in the culture dish with magnetic rotor, it is molten to add 1.5~5%LiCl of 2mL
Liquid is uncapped after mixing carries out ultra violet lamp, 30cm, 30W 8~16min of ultra violet lamp, appropriate dilution Tu plate after dark processing,
4~6d of dark culturing in 30 DEG C of constant incubators.Meanwhile equally operated as control using the bacteria suspension without mutagenesis, calculate
Fatal rate.
(4) primary dcreening operation of mutant strain
By the colony inoculation of the different shape feature grown after mutagenesis into primary dcreening operation culture medium, 30 DEG C of culture 3d~6d.To
Spray concentration is 10%Na in primary dcreening operation tubule2CO3Solution, hang on, the bacterial strain of the more yellow tubule of color is therefrom found out, according to secondary
Sequence is transferred on slant medium, and culture is ripe to spore in the incubator that temperature is 30 DEG C, in 4 DEG C of preservations of refrigerator.
Described primary dcreening operation culture medium is：Lactose 1g, (NH4)2SO41g, KH2PO41.5g, agar 2g, 2g wheat brans are weighed, are added
Enter 100mL distilled water, boil 10min, with four layers of filtered through gauze, filtrate moisturizing to 100mL, pH is adjusted to sterilize at 4.8,121 DEG C
60 DEG C are cooled to after 20min, adds degerming 0.1% ONPG (ortho-nitrophenyl β-D- synthesis) solution of micro-filtration.Shake
In the centrifuge tube for the 1.0mL that sterilizing is added on super-clean bench with the dropper of sterilizing after even, it is standby to be placed in refrigerator cold-storage.
(5) secondary screening of mutant strain
After the bacterial strain activation that primary dcreening operation is obtained, solid-state fermentation culture medium is seeded to, 30 DEG C of culture 6d, adds 5 at room temperature
Times volume pH4.5 acetate buffer solution 200r/min vibration 1h, then with three layers of filtered through gauze, collect filtrate, 4 DEG C,
15min is centrifuged in 10000r/min high speed freezing centrifuge, takes supernatant to determine lactose enzyme activity, filters out superior strain.
Described solid-state fermentation culture medium is：Lactose 10g, (NH4)2SO410g, KH2PO415g, distilled water 1000mL match somebody with somebody
Solution is set to, adjusts pH4.8, then wheatfeed and above-mentioned solution are mixed in 1: 1.1 (w/v) ratio, sterilized at 121 DEG C
20min。
(6) the mycelial culture of mutant strain and pretreatment
The ripe spore of aspergillus oryzae mutant strain is eluted from inclined-plane with sterile saline, transferred into PDA liquid
In culture medium, 10~36h of quiescent culture in 28 DEG C of incubators, then 4000r/min centrifugations 10min collection mycelium, are used in combination
0.6mol/L sodium chloride homeo-osmosis agent washing.
The ripe spore of described aspergillus oryzae mutant strain is the aspergillus oryzae mutant strain picking that will be obtained by primary dcreening operation, secondary screening
The ring of mycelia one, is inoculated in solid slope made of potato dextrose agar, obtained by 28 DEG C of incubated 6d of temperature.
(7) preparation of protoplast
Take 0.3mg mycelium, add the mixed enzyme solution that 1mL concentration is 1%, slight oscillatory digests at 20~40 DEG C of temperature
1.5~6h；0.6~0.8mol/L sodium chloride homeo-osmosis agent enzymolysis reactions are added, obtain enzymolysis liquid, observe protoplast
Preparation situation, protoplast number is calculated with blood cell counting plate, calculates preparation rate.
Described complex enzyme hydrolysis liquid is the cellulase of+1% glusulase of 1% lysozyme+1%.
(8) purifying of protoplast
After enzymolysis, enzymolysis liquid is filtered with 3 layers of sterile lens paper, filtrate 3000r/min low-temperature centrifugation 10min, is removed
Supernatant, it is suspended in appropriate homeo-osmosis agent, is purified after being washed with 0.6mol/L sodium chloride homeo-osmosis agents
Protoplast suspension.
(9) regeneration of protoplast
Using homeo-osmosis agent by the concentration dilution of protoplast to 105Individual/mL, then draw 0.2mL protoplasts
It is coated on regenerated solids culture medium, 28 DEG C of incubated 2~3d, you can see regeneration bacterium colony.
Described regenerated solids culture medium is to prepare potato dextrose agar, i.e. horse with 0.6mol/L sodium chloride
Bell potato 200g/L, glucose 20g/L, agar 20g/L, sodium chloride 35.1g/L.
Protoplast obtained by the preparation method of the aspergillus oryzae protoplast of high yield lactase of the present invention prepares number height,
Regeneration rate is high, is the reliable method that aspergillus oryzae protoplast is prepared and regenerated, and is the aspergillus oryzae protoplast of high yield lactase
Prepare and provide Technical Reference with regeneration.The protoplast prepared using the present invention fully meets the condition of protoplast fusion, is
The expression quantity that aspergillus oryzae fermenting and producing lactase is further improved using genome segment S9 technology is provided the foundation, also to be further
The regulatory mechanism of research aspergillus oryzae production lactase provides basis early stage.
Embodiment
Below by way of specific implementation, the present invention is further illustrated.
Embodiment 1：A kind of preparation of aspergillus oryzae protoplast for high yield lactase and renovation process, including following step
Suddenly：
(1) activation culture of strain：
The aspergillus oryzae strain (BNCC338380) of lyophilized preservation is fully dissolved into bacteria suspension with sterile saline, by this
Bacterial suspension inoculation is activated in czapek agar medium inclined-plane, and 28 DEG C are cultivated 6 days, standby after yellow green spore is covered with, even
It is continuous to spread cultivation once.
The culture medium used when spreading cultivation is Czapek's medium.
(2) monospore suspension is prepared
After the yellow green spore sterile saline covered with Czapek's medium is eluted into spore, pour into equipped with bead
The sterile conical flasks of 250ml in, fully shake conical flask, in the presence of bead, the spore in bottle broken up, with sterile de-
Fat cotton filters spore suspension, the spore suspension for the mycelia that is removed, 3.5 × 10 is diluted into sterilized water6Individual/
ml。
(3) ultraviolet and lithium chloride complex mutation obtains mutant strain
The bacteria suspension obtained by 5mL steps (2) is taken in the culture dish with magnetic rotor, adds 2mL 1.5%LiCl solution
Uncapped after mixing and carry out ultra violet lamp, 30cm, 30W ultra violet lamp 10min, appropriate dilution Tu plate after dark processing, 30
4~6d of dark culturing in DEG C constant incubator.Meanwhile equally operated as control using the bacteria suspension without mutagenesis, calculate lethal
Rate.
(4) primary dcreening operation of mutant strain
By the colony inoculation of the different shape feature grown after mutagenesis into primary dcreening operation culture medium, 30 DEG C of 3~6d of culture.To first
It is 10%Na to sieve spray concentration in tubule2CO3Solution, hang on, the bacterial strain of the more yellow tubule of color is therefrom found out, according to order
It is transferred on slant medium, culture is ripe to spore in the incubator that temperature is 30 DEG C, in 4 DEG C of preservations of refrigerator.
Described primary dcreening operation culture medium is：Lactose 1g, (NH4)2SO41g, KH2PO41.5g, agar 2g, 2g wheat brans are weighed, are added
Enter 100mL distilled water, boil 10min, with four layers of filtered through gauze, filtrate moisturizing to 100mL, pH is adjusted to sterilize at 4.8,121 DEG C
60 DEG C are cooled to after 20min, adds degerming 0.1% ONPG (ortho-nitrophenyl β-D- synthesis) solution of micro-filtration.Shake
In the centrifuge tube for the 1.0mL that sterilizing is added on super-clean bench with the dropper of sterilizing after even, it is standby to be placed in refrigerator cold-storage.
(5) secondary screening of mutant strain
After the bacterial strain activation that primary dcreening operation is obtained, solid-state fermentation culture medium is seeded to, 30 DEG C of culture 6d, adds 5 at room temperature
Times volume pH4.5 acetate buffer solution 200r/min vibration 1h, then with three layers of filtered through gauze, collect filtrate, 4 DEG C,
15min is centrifuged in 10000r/min high speed freezing centrifuge, takes supernatant to determine lactose enzyme activity, filters out superior strain.
Described solid-state fermentation culture medium is：Lactose 10g, (NH4)2SO410g, KH2PO415g, distilled water 1000mL match somebody with somebody
Solution is set to, adjusts pH4.8, then wheatfeed and above-mentioned solution are mixed in 1: 1.1 (w/v) ratio, sterilized at 121 DEG C
20min。
(6) the mycelial culture of mutant strain and pretreatment
The ripe spore of aspergillus oryzae mutant strain is eluted from inclined-plane with sterile saline, transferred into PDA liquid
In culture medium, the quiescent culture 10h in 28 DEG C of incubators, then 4000r/min centrifuges 10min and collects mycelium, and uses 0.6mol/
L sodium chloride homeo-osmosis agent washing.
The ripe spore of described aspergillus oryzae mutant strain is the aspergillus oryzae mutant strain picking that will be obtained by primary dcreening operation, secondary screening
The ring of mycelia one, is inoculated in solid slope made of potato dextrose agar, obtained by 28 DEG C of incubated 6d of temperature.
(7) preparation of protoplast
Take 0.3mg mycelium, add the mixed enzyme solution that 1mL concentration is 1%, slight oscillatory digests at 30 DEG C of temperature
2.5h；0.6mol/L sodium chloride homeo-osmosis agent enzymolysis reactions are added, obtain enzymolysis liquid, observation protoplast prepares feelings
Condition, protoplast number is calculated with blood cell counting plate, calculates preparation rate.
Described complex enzyme hydrolysis liquid is the cellulase of+1% glusulase of 1% lysozyme+1%.
(8) purifying of protoplast
After enzymolysis, enzymolysis liquid is filtered with 3 layers of sterile lens paper, filtrate 3000r/min low-temperature centrifugation 10min, is removed
Supernatant, it is suspended in appropriate homeo-osmosis agent, is purified after being washed with 0.6moL/L sodium chloride homeo-osmosis agents
Protoplast suspension.
(9) regeneration of protoplast
Using homeo-osmosis agent by the concentration dilution of protoplast to 1.5 × 105Individual/mL, it is primary then to draw 0.2mL
Plastid is coated on regenerated solids culture medium, 28 DEG C of incubated 2d, you can see regeneration bacterium colony.
Described regenerated solids culture medium is to prepare potato dextrose agar, i.e. horse with 0.6moL/L sodium chloride
Bell potato 200g/L, glucose 20g/L, agar 20g/L, sodium chloride 35.1g/L.
Embodiment 2：A kind of preparation of aspergillus oryzae protoplast for high yield lactase and renovation process, including following step
Suddenly：
(1) activation culture of strain：
The aspergillus oryzae strain (BNCC338380) of lyophilized preservation is fully dissolved into bacteria suspension with sterile saline, by this
Bacterial suspension inoculation is activated in czapek agar medium inclined-plane, and 28 DEG C are cultivated 5 days, standby after yellow green spore is covered with, even
It is continuous to spread cultivation once.
The culture medium used when spreading cultivation is Czapek's medium.
(2) monospore suspension is prepared
After the yellow green spore sterile saline covered with Czapek's medium is eluted into spore, pour into equipped with bead
The sterile conical flasks of 250ml in, fully shake conical flask, in the presence of bead, the spore in bottle broken up, with sterile de-
Fat cotton filters spore suspension, the spore suspension for the mycelia that is removed, 2.3 × 10 is diluted into sterilized water6Individual/
ml。
(3) ultraviolet and lithium chloride complex mutation obtains mutant strain
The bacteria suspension obtained by 5mL steps (2) is taken in the culture dish with magnetic rotor, 2mL 2%LiCl solution is added and mixes
Uncapped after even and carry out ultra violet lamp, 30cm, 30W ultra violet lamp 12min, appropriate dilution Tu plate after dark processing, at 30 DEG C
Dark culturing 4-6d in constant incubator.Meanwhile equally operated as control using the bacteria suspension without mutagenesis, calculate fatal rate.
(4) primary dcreening operation of mutant strain
By the colony inoculation of the different shape feature grown after mutagenesis into primary dcreening operation culture medium, 30 DEG C of 3~6d of culture.To first
It is 10%Na to sieve spray concentration in tubule2CO3Solution, hang on, the bacterial strain of the more yellow tubule of color is therefrom found out, according to order
It is transferred on slant medium, culture is ripe to spore in the incubator that temperature is 30 DEG C, in 4 DEG C of preservations of refrigerator.
Described primary dcreening operation culture medium is：Lactose 1g, (NH4)2SO41g, KH2PO41.5g, agar 2g, 2g wheat brans are weighed, are added
Enter 100mL distilled water, boil 10min, with four layers of filtered through gauze, filtrate moisturizing to 100mL, pH is adjusted to sterilize at 4.8,121 DEG C
60 DEG C are cooled to after 20min, adds degerming 0.1% ONPG (ortho-nitrophenyl β-D- synthesis) solution of micro-filtration.Shake
In the centrifuge tube for the 1.0mL that sterilizing is added on super-clean bench with the dropper of sterilizing after even, it is standby to be placed in refrigerator cold-storage.
(5) secondary screening of mutant strain
After the bacterial strain activation that primary dcreening operation is obtained, solid-state fermentation culture medium is seeded to, 30 DEG C of culture 6d, adds 5 at room temperature
Times volume pH4.5 acetate buffer solution 200r/min vibration 1h, then with three layers of filtered through gauze, collect filtrate, 4 DEG C,
15min is centrifuged in 10000r/min high speed freezing centrifuge, takes supernatant to determine lactose enzyme activity, filters out superior strain.
Described solid-state fermentation culture medium is：Lactose 10g, (NH4)2SO410g, KH2PO415g, distilled water 1000mL match somebody with somebody
Solution is set to, adjusts pH4.8, then wheatfeed and above-mentioned solution are mixed in 1: 1.1 (w/v) ratio, sterilized at 121 DEG C
20min。
(6) the mycelial culture of mutant strain and pretreatment
The ripe spore of aspergillus oryzae mutant strain is eluted from inclined-plane with sterile saline, transferred into PDA liquid
In culture medium, the quiescent culture 12h in 28 DEG C of incubators, then 4000r/min centrifuges 10min and collects mycelium, and uses 0.6moL/
L sodium chloride homeo-osmosis agent washing.
The ripe spore of described aspergillus oryzae mutant strain is the aspergillus oryzae mutant strain picking that will be obtained by primary dcreening operation, secondary screening
The ring of mycelia one, is inoculated in solid slope made of potato dextrose agar, obtained by 28 DEG C of incubated 6d of temperature.
(7) preparation of protoplast
Take 0.3mg mycelium, add the mixed enzyme solution that 1mL concentration is 1%, the slight oscillatory enzymolysis 2h at 35 DEG C of temperature；
0.7moL/L sodium chloride homeo-osmosis agent enzymolysis reactions are added, obtain enzymolysis liquid, observation protoplast prepares situation, uses blood
Cell counting count board calculates protoplast number, calculates preparation rate.
Described complex enzyme hydrolysis liquid is the cellulase of+1% glusulase of 1% lysozyme+1%.
(8) purifying of protoplast
After enzymolysis, enzymolysis liquid is filtered with 3 layers of sterile lens paper, filtrate 3000r/min low-temperature centrifugation 10min, is removed
Supernatant, it is suspended in appropriate homeo-osmosis agent, is purified after being washed with 0.6moL/L sodium chloride homeo-osmosis agents
Protoplast suspension.
(9) regeneration of protoplast
Using homeo-osmosis agent by the concentration dilution of protoplast to 2.5 × 105Individual/mL, it is primary then to draw 0.2mL
Plastid is coated on regenerated solids culture medium, 28 DEG C of incubated 3d, you can see regeneration bacterium colony.
Described regenerated solids culture medium is to prepare potato dextrose agar, i.e. horse with 0.6moL/L sodium chloride
Bell potato 200g/L, glucose 20g/L, agar 20g/L, sodium chloride 35.1g/L.
Embodiment 3：A kind of preparation method of aspergillus oryzae protoplast for high yield lactase, comprises the following steps：
(1) activation culture of strain：
The aspergillus oryzae strain (BNCC338380) of lyophilized preservation is fully dissolved into bacteria suspension with sterile saline, by this
Bacterial suspension inoculation is activated in czapek agar medium inclined-plane, and 28 DEG C are cultivated 5 days, standby after yellow green spore is covered with, even
It is continuous to spread cultivation once.
The culture medium used when spreading cultivation is Czapek's medium.
(2) monospore suspension is prepared
After the yellow green spore sterile saline covered with Czapek's medium is eluted into spore, pour into equipped with bead
The sterile conical flasks of 250ml in, fully shake conical flask, in the presence of bead, the spore in bottle broken up, with sterile de-
Fat cotton filters spore suspension, the spore suspension for the mycelia that is removed, 2.3 × 10 is diluted into sterilized water6Individual/
ml。
(3) ultraviolet and lithium chloride complex mutation obtains mutant strain
The bacteria suspension obtained by 5mL steps (2) is taken in the culture dish with magnetic rotor, adds 2mL 2.5%LiCl solution
Uncapped after mixing and carry out ultra violet lamp, 30cm, 30W ultra violet lamp 14min, appropriate dilution Tu plate after dark processing, 30
4~6d of dark culturing in DEG C constant incubator.Meanwhile equally operated as control using the bacteria suspension without mutagenesis, calculate lethal
Rate.
(4) primary dcreening operation of mutant strain
By the colony inoculation of the different shape feature grown after mutagenesis into primary dcreening operation culture medium, 30 DEG C of 3~6d of culture.To first
It is 10%Na to sieve spray concentration in tubule2CO3Solution, hang on, the bacterial strain of the more yellow tubule of color is therefrom found out, according to order
It is transferred on slant medium, culture is ripe to spore in the incubator that temperature is 30 DEG C, in 4 DEG C of preservations of refrigerator.
Described primary dcreening operation culture medium is：Lactose 1g, (NH4)2SO41g, KH2PO41.5g, agar 2g, 2g wheat brans are weighed, are added
Enter 100mL distilled water, boil 10min, with four layers of filtered through gauze, filtrate moisturizing to 100mL, pH is adjusted to sterilize at 4.8,121 DEG C
60 DEG C are cooled to after 20min, adds degerming 0.1% ONPG (ortho-nitrophenyl β-D- synthesis) solution of micro-filtration.Shake
In the centrifuge tube for the 1.0mL that sterilizing is added on super-clean bench with the dropper of sterilizing after even, it is standby to be placed in refrigerator cold-storage.
(5) secondary screening of mutant strain
After the bacterial strain activation that primary dcreening operation is obtained, solid-state fermentation culture medium is seeded to, 30 DEG C of culture 6d, adds 5 at room temperature
Times volume pH4.5 acetate buffer solution 200r/min vibration 1h, then with three layers of filtered through gauze, collect filtrate, 4 DEG C,
15min is centrifuged in 10000r/min high speed freezing centrifuge, takes supernatant to determine lactose enzyme activity, filters out superior strain.
Described solid-state fermentation culture medium is：Lactose 10g, (NH4)2SO410g, KH2PO415g, distilled water 1000mL match somebody with somebody
Solution is set to, adjusts pH4.8, then wheatfeed and above-mentioned solution are mixed in 1: 1.1 (w/v) ratio, sterilized at 121 DEG C
20min。
(6) the mycelial culture of mutant strain and pretreatment
The ripe spore of aspergillus oryzae mutant strain is eluted from inclined-plane with sterile saline, transferred into PDA liquid
In culture medium, the quiescent culture 14h in 28 DEG C of incubators, then 4000r/min centrifuges 10min and collects mycelium, and uses 0.6moL/
L sodium chloride homeo-osmosis agent washing.
The ripe spore of described aspergillus oryzae mutant strain is the aspergillus oryzae mutant strain picking that will be obtained by primary dcreening operation, secondary screening
The ring of mycelia one, is inoculated in solid slope made of potato dextrose agar, obtained by 28 DEG C of incubated 6d of temperature.
(7) preparation of protoplast
Take 0.3mg mycelium, add the mixed enzyme solution that 1mL concentration is 1%, slight oscillatory digests at 25 DEG C of temperature
2.5h；0.8mol/L sodium chloride homeo-osmosis agent enzymolysis reactions are added, obtain enzymolysis liquid, observation protoplast prepares feelings
Condition, protoplast number is calculated with blood cell counting plate, calculates preparation rate.
Described complex enzyme hydrolysis liquid is the cellulase of+1% glusulase of 1% lysozyme+1%.
(8) purifying of protoplast
After enzymolysis, enzymolysis liquid is filtered with 3 layers of sterile lens paper, filtrate 3000r/min low-temperature centrifugation 10min, is removed
Supernatant, it is suspended in appropriate homeo-osmosis agent, is purified after being washed with 0.6moL/L sodium chloride homeo-osmosis agents
Protoplast suspension.
(9) regeneration of protoplast
Using homeo-osmosis agent by the concentration dilution of protoplast to 2.5 × 105Individual/mL, it is primary then to draw 0.2mL
Plastid is coated on regenerated solids culture medium, 28 DEG C of incubated 3d, you can see regeneration bacterium colony.
Described regenerated solids culture medium is to prepare potato dextrose agar, i.e. horse with 0.6moL/L sodium chloride
Bell potato 200g/L, glucose 20g/L, agar 20g/L, sodium chloride 35.1g/L.
Claims (7)
1. a kind of preparation of aspergillus oryzae protoplast for high yield lactase and renovation process, it is characterised in that：
(1) activation culture of strain：
The aspergillus oryzae strain (BNCC338380) of lyophilized preservation is fully dissolved into bacteria suspension with sterile saline, this bacterium is hanged
Liquid is inoculated in czapek agar medium inclined-plane and activated, and 28 DEG C are cultivated 5~6 days, continuous again after yellow green spore is covered with to expand
Training is once.
(2) monospore suspension is prepared
After the yellow green spore sterile saline covered with Czapek's medium is eluted into spore, pour into equipped with bead
In the sterile conical flasks of 250mL, conical flask is fully shaken, in the presence of bead, the spore in bottle is broken up, uses sterile absorbent
Cotton filters spore suspension, the spore suspension for the mycelia that is removed, 10 is diluted to sterilized water6Individual/mL.
(3) ultraviolet and lithium chloride complex mutation obtains mutant strain
The bacteria suspension obtained by 5mL steps (2) is taken in the culture dish with magnetic rotor, 2mL 1.5~5%LiCl solution is added and mixes
Uncapped after even and carry out ultra violet lamp, 30cm, 30W 8~16min of ultra violet lamp, appropriate dilution Tu plate after dark processing, 30
4~6d of dark culturing in DEG C constant incubator.Meanwhile equally operated as control using the bacteria suspension without mutagenesis, calculate lethal
Rate.
(4) primary dcreening operation of mutant strain
By the colony inoculation of the different shape feature grown after mutagenesis into primary dcreening operation culture medium, 30 DEG C of 3~6d of culture.It is small to primary dcreening operation
Spray concentration is 10%Na in pipe2CO3Solution, hang on, therefrom find out the bacterial strain of the more yellow tubule of color, transferred according to order
Onto slant medium, culture is ripe to spore in the incubator that temperature is 30 DEG C, in 4 DEG C of preservations of refrigerator.
(5) secondary screening of mutant strain
After the bacterial strain activation that primary dcreening operation is obtained, solid-state fermentation culture medium is seeded to, 30 DEG C of culture 6d, adds 5 times of bodies at room temperature
Product pH4.5 acetate buffer solution 200r/min vibration 1h, then with three layers of filtered through gauze, collect filtrate, in 4 DEG C, 10000r/
15min is centrifuged in min high speed freezing centrifuge, takes supernatant to determine lactose enzyme activity, filters out superior strain.
(6) the mycelial culture of mutant strain and pretreatment
The ripe spore of aspergillus oryzae mutant strain is eluted from inclined-plane with sterile saline, transferred into PDA Liquid Cultures
In base, 10~36h of quiescent culture in 28 DEG C of incubators, then 4000r/min centrifuges 10min and collects mycelium, and uses 0.6moL/
L sodium chloride homeo-osmosis agent washing.
(7) preparation of protoplast
Take 0.3mg mycelium, add the mixed enzyme solution that 1mL concentration is 1%, the slight oscillatory enzymolysis 1.5 at 20~40 DEG C of temperature
~6h；0.6~0.8moL/L sodium chloride homeo-osmosis agent enzymolysis reactions are added, obtain enzymolysis liquid, observe plasm system
Standby situation, protoplast number is calculated with blood cell counting plate, calculates preparation rate.
(8) purifying of protoplast
After enzymolysis, enzymolysis liquid is filtered with 3 layers of sterile lens paper, filtrate 3000r/min low-temperature centrifugation 10min, removes supernatant
Liquid, it is suspended in after being washed with 0.6moL/L sodium chloride homeo-osmosis agents in appropriate homeo-osmosis agent, what is purified is primary
Plastid suspension.
(9) regeneration of protoplast
Using homeo-osmosis agent by the concentration dilution of protoplast to 105Individual/mL, then draw 0.2mL protoplasts and be coated on
On regenerated solids culture medium, 28 DEG C of incubated 2~3d, you can see regeneration bacterium colony.
2. preparation and the renovation process of a kind of aspergillus oryzae protoplast for high yield lactase according to claim 1,
Characterized in that, the culture medium used when spreading cultivation described in step (1) is Czapek's medium.
3. preparation and the renovation process of a kind of aspergillus oryzae protoplast for high yield lactase according to claim 1,
Characterized in that, the primary dcreening operation culture medium described in step (4) is：Lactose 1g, (NH4)2SO41g, KH2PO41.5g, agar 2g, claim
2g wheat brans are taken, 100mL distilled water is added, boils 10min, 4.8 are adjusted to four layers of filtered through gauze, filtrate moisturizing to 100mL, pH,
60 DEG C are cooled to after sterilizing 20min at 121 DEG C, adds the degerming 0.1% ONPG (ortho-nitrophenyl β-D- gala pyranoses of micro-filtration
Glycosides) solution.In the centrifuge tube for the 1.0mL that sterilizing is added on super-clean bench with the dropper of sterilizing after shaking up, it is cold to be placed in refrigerator
Hide standby.
4. preparation and the renovation process of a kind of aspergillus oryzae protoplast for high yield lactase according to claim 1,
Characterized in that, the solid-state fermentation culture medium described in step (5) is：Lactose 10g, (NH4)2SO410g, KH2PO415g, distillation
Water 1000mL is configured to solution, adjusts pH4.8, then mixes wheatfeed and above-mentioned solution in 1: 1.1 (w/v) ratio, 121
Sterilize 20min at DEG C.
5. preparation and the renovation process of a kind of aspergillus oryzae protoplast for high yield lactase according to claim 1,
Characterized in that, the ripe spore of the aspergillus oryzae mutant strain described in step (6) is dashed forward by the aspergillus oryzae for obtaining primary dcreening operation, secondary screening
Become the ring of bacterial strain picking mycelia one, solid slope made of potato dextrose agar is inoculated in, in 28 DEG C of constant temperature of temperature
Cultivate obtained by 6d.
6. preparation and the renovation process of a kind of aspergillus oryzae protoplast for high yield lactase according to claim 1,
Characterized in that, the complex enzyme hydrolysis liquid described in step (7) is the cellulase of+1% glusulase of 1% lysozyme+1%.
7. preparation and the renovation process of a kind of aspergillus oryzae protoplast for high yield lactase according to claim 1,
Characterized in that, the regenerated solids culture medium described in step (9) is to prepare potato dextrose agar with 0.6mol/L sodium chloride
Culture medium, i.e. potato 200g/L, glucose 20g/L, agar 20g/L, sodium chloride 35.1g/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531405A (en) * | 2018-04-09 | 2018-09-14 | 保定康而沃生物科技有限公司 | Promote the resistant to pollution complex enzyme of edible fungus culturing energy-saving and production-increase and preparation method |
| CN110272825A (en) * | 2019-07-05 | 2019-09-24 | 四川省丹丹郫县豆瓣集团股份有限公司 | The fermenting microbe method of inspection for bean paste |
| CN110810733A (en) * | 2019-11-27 | 2020-02-21 | 周伟光 | Process for preparing Yangjiang fermented soya beans by pure aspergillus oryzae fermentation |
| CN114540329A (en) * | 2020-11-25 | 2022-05-27 | 潍坊康地恩生物科技有限公司 | Lactase mutant |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996000786A1 (en) * | 1994-06-29 | 1996-01-11 | Genencor International, Inc. | INCREASED PRODUCTION OF β-GALACTOSIDASE IN ASPERGILLUS ORYZAE |
| CN102061266A (en) * | 2010-11-23 | 2011-05-18 | 合肥工业大学 | Method for preparing and regenerating rhizopus oryzae protoplast for producing L-lactic acid at high yield |
-
2017
- 2017-08-23 CN CN201710727884.4A patent/CN107365713A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996000786A1 (en) * | 1994-06-29 | 1996-01-11 | Genencor International, Inc. | INCREASED PRODUCTION OF β-GALACTOSIDASE IN ASPERGILLUS ORYZAE |
| CN102061266A (en) * | 2010-11-23 | 2011-05-18 | 合肥工业大学 | Method for preparing and regenerating rhizopus oryzae protoplast for producing L-lactic acid at high yield |
Non-Patent Citations (3)
| Title |
|---|
| 刘源慧等: "米曲霉F16原生质体的制备和再生研究", 《海峡科学》 * |
| 刘芳宁等: "紫外线与氯化锂诱变选育高产乳糖酶米曲霉", 《食品工业科技》 * |
| 宋鹏等: "米曲霉菌种选育中原生质体制备方法的研究", 《食品工业》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531405A (en) * | 2018-04-09 | 2018-09-14 | 保定康而沃生物科技有限公司 | Promote the resistant to pollution complex enzyme of edible fungus culturing energy-saving and production-increase and preparation method |
| CN110272825A (en) * | 2019-07-05 | 2019-09-24 | 四川省丹丹郫县豆瓣集团股份有限公司 | The fermenting microbe method of inspection for bean paste |
| CN110810733A (en) * | 2019-11-27 | 2020-02-21 | 周伟光 | Process for preparing Yangjiang fermented soya beans by pure aspergillus oryzae fermentation |
| CN114540329A (en) * | 2020-11-25 | 2022-05-27 | 潍坊康地恩生物科技有限公司 | Lactase mutant |
| CN114540329B (en) * | 2020-11-25 | 2024-04-02 | 潍坊康地恩生物科技有限公司 | Lactase mutant |
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