CN106434383A - Aspergillus oryza strain capable of highly producing fungal amylase - Google Patents
Aspergillus oryza strain capable of highly producing fungal amylase Download PDFInfo
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- CN106434383A CN106434383A CN201610935707.0A CN201610935707A CN106434383A CN 106434383 A CN106434383 A CN 106434383A CN 201610935707 A CN201610935707 A CN 201610935707A CN 106434383 A CN106434383 A CN 106434383A
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/69—Aspergillus oryzae
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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)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
- C12N9/242—Fungal source
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Abstract
The invention relates to the technical field of enzyme preparations, in particular to an aspergillus oryza strain capable of highly producing fungal amylase. A mutant strain-aspergillus oryzae TA20 obtained through ultraviolet-induced screening has a collection number of CCTCC NO:M 2016602; and after the strain is subjected to shaking flask fermentation for 5 days, the enzyme activity of the fungal amylase in fermentative supernatant liquid of the strain is 2,184 u/ml and is increased by 146% in comparison with that of a starting strain, and an unexpected technical effect is achieved. The mutant strain is in favor of reducing the production cost of the fungal amylase and facilitates the popularization and application of the fungal amylase in the food industry.
Description
Technical field
The invention belongs to gene engineering technology field, particular content is related to the aspergillus oryzae mutation that a plant height produces fungal amylase
Bacterial strain.
Technical background
Amylase is the general name of a group enzyme that energy catalytic starch hydrolysis become glucose, maltose and other oligosaccharide,
It is widely present in animals and plants and microorganism.Starch is the maximum amount of carbon hydrate that green plantss are obtained by photosynthesis
One of thing, rich reserves in nature.Starch is used as agriculture first big product, and its raw material deep processing is grain and food processing
In important component part.Starch is the primary raw material of fermentation industry, can produce numerous products by fermentation, such as ethanol,
Glycerol, organic acid, aminoacid, enzyme etc., it is also possible to produce biological preparation and the medicine of high added value.At the biochemistry to starch
Reason can improve the added value of starch product, and these are required for diastatic participation.Amylase is earliest for industrialized production
And be still purposes most one of enzyme preparation product of wide, yield maximum so far, before the number the year before last, the mankind just using high temperature with
Wine brewing, making maltose etc. are engaged in the effect of midrange thermal stable amylase.
Amylase divides predominantly three kinds according to the mechanism of action：1）α-amylase, from the internal hydrolyzing alpha-Isosorbide-5-Nitrae-glucosides of starch molecule
Key, and α -1 to starch molecule, 6- glycosidic bond can not be hydrolyzed, but it may span across α -1,6- glycosidic bond, to intramolecular α-Isosorbide-5-Nitrae -
Glycosidic bond works, and the precedence of hydrolyzing alpha-Isosorbide-5-Nitrae-glycosidic bond is irregular.2）Beta amylase, from the non-of starch molecule
Reducing end under neutral starts, and hydrolyzes α-Isosorbide-5-Nitrae glycosidic bond separately, cuts next maltose molecule successively.Beta amylase also can only
Hydrolyzing alpha-Isosorbide-5-Nitrae-glycosidic bond, and hydrolyzing alpha -1 is unable to, 6- glycosidic bond, and not across α -1,6- glycosidic bond, meet this key hydrolysis
Just stop.3）Glucoamylase, from the beginning of the non reducing end of starch molecule, gradually cuts glucose one by one.It is not
It is only capable of hydrolyzing alpha-Isosorbide-5-Nitrae-glycosidic bond, and energy hydrolyzing alpha -1,6- glycosidic bond and α -1,3- glycosidic bond, both speed after simply hydrolyzing
Degree is more slowly compared with the former, therefore, when glucoamylase acts on amylose and amylopectin, can hydrolyze all of which
For glucose.
Amylase is important enzyme preparation, is widely used in the production of maltose, syrup, dextrin etc., in textile industry,
Amylase can be catalyzed the starch size on fabric, due to diastatic high efficiency and specificity, the desizing rate height of enzyme desizing, desizing
Hurry up, pollution is few, and product is more soft than acid system, alkaline process, and does not damage fiber.Mycete used in Alcohol Production and brewing industry is formed sediment
Powder enzyme can effectively improve conversion coefficient, the growth of Rate of producing alcohol and enzyme mother.Amylase applies also for out in starch production process
Waste water, mitigate pollution to environment.Therefore now focus is become to diastatic research.At present, diastatic production is main
Using fermentable.Amylase producing strains mainly have antibacterial（Such as bacillus subtilis, Bacillus licheniformis etc.）And funguses（As black fermented preparation
Mould, aspergillus oryzae and rhizopus etc.）, filter out high yield, hereditary stability is good, vitality is vigorous amylase production bacterial strain, be to open up
The industrial important channel of wide amylase.
Content of the invention
The present invention for solve prior art problem, there is provided a plant height produce fungal amylase aspergillus oryzae mutant strain and its
Application.Applicant obtains a plant mutant bacterial strain by the method screening of ultraviolet mutagenesis, and the expression of fungal amylase is obtained significantly
Improve.
One aspect of the present invention is related to a kind of mutant strain aspergillus oryzae TA20（Aspergillus oryzaeTA20）, in
On October 31st, 2016 is preserved in the China typical culture collection center of Wuhan, China Wuhan University, and deposit number is CCTCC
NO:M2016602.
The invention further relates to application of the above-mentioned aspergillus oryzae TA20 in fungal amylase production.
A kind of fermentation process for producing fungal amylase, be with above-mentioned aspergillus oryzae TA20 as fermentation strain.
The each component of fermentation medium used in described fermentation process and its mass percent are respectively：Maltose 12%；
Semen Maydis pulp 1%；Ammonium sulfate 0.5%；Potassium dihydrogen phosphate 0.35%；Dipotassium hydrogen phosphate 0.75%；Magnesium sulfate heptahydrate 0.03%.
In described fermentation process, fermentation temperature selects 30 DEG C.
The mutant strain aspergillus oryzae TA20 that the present invention is obtained by Uv-induced screening, after its shake flask fermentation 5d, bacterial strain is sent out
In ferment supernatant, fungal amylase enzyme activity 2184u/ml, improves 146% than starting strain, achieves unexpected technology effect
Really.The mutant strain advantageously reduces the production cost of fungal amylase, promotes fungal amylase pushing away in food service industry
Extensively with application.
Specific embodiment
The present invention has used routine techniquess and the method that genetic engineering and biology field are used, for example
MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT
Described method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These generality are with reference to text
Offer there is provided definition well known by persons skilled in the art and method.But, those skilled in the art can be remembered in the present invention
On the basis of the technical scheme of load, other conventional methods, experimental program and reagent using this area, and it is not limited to present invention tool
The restriction of body embodiment.
Describe the present invention with reference to specific embodiment.
1 fungal amylase bacterial strain shake flask fermentation of embodiment and Enzyme activity assay
By fungal amylase bacterium producing multi enzyme preparation aspergillus oryzae TA01（Aspergillus oryzaeTA01）（The bacterial strain is by inventor Xu
Xiao Dong is screened from the soil of Qingdao city Laoshan District forest farm in September, 2014）It is inoculated into fresh PDA plate, 30 DEG C of cultures
5-7d.
The truffle of 2cm × 2cm size is extracted, is seeded to 50ml liquid submerged culture base（Maltose 12%；Semen Maydis pulp 1%；Sulfur
Sour ammonium 0.5%；Potassium dihydrogen phosphate 0.35%；Dipotassium hydrogen phosphate 0.75%；Magnesium sulfate heptahydrate 0.03%）Middle fermentation, 30 DEG C of culture 5d；From
Heart thalline, obtains supernatant and is crude enzyme liquid, fermented supernatant fluid is carried out fungal amylase enzyme activity determination, its fungal amylase
Enzyme activity is 885 u/ml.
1. fungal amylase enzyme activity detection
（1）The definition of fungal amylase enzyme-activity unit
At 40 DEG C, under the conditions of pH is 5.0,30min is reacted, is produced in reactant liquor needed for the reducing sugar equivalent to 10mg glucose
Enzyme amount, is defined as a unit of activity（IU）.
（2）Enzyme activity determination method
（2.1）Reagent and solution：
30% liquor kalii iodide：Potassium iodide 150g is dissolved in 350ml water, is stored in brown reagent bottle；
25% sulfuric acid solution：Sulphuric acid 125g is dissolved in 375ml water；
0.05mol/L hypo solution：By the 0.1mol/L thiosulfuric acid mother liquid of sodium 500ml of quantitative analyses with boiling
Cooling water dilution, the cumulative volume of resulting solution be.After preparing, should be demarcated, be obtained concentration correction coefficientf
Value；
1mol/L acetic acid-sodium acetate buffer solution（pH 5.0）：1mol/L sodium acetate solution is added to the acetic acid solution of 1mol/L
In, adjust pH to 5.0；
Soluble starch solution（pH 5.0）：Weigh after soluble starch is dry 4h at 105 DEG C and calculate water content.Then basis
The water content of soluble starch weighs 0.50g（Give money as a gift）Soluble starch, be slowly added in boiling water 50ml, boil 5min,
After originally water cooling, 1mol/L acetic acid-sodium acetate buffer solution is added（pH 5.0）5ml, is settled to 100ml with water；
Fehling's reagent：
Copper solution：Copper sulfate 34.66g is dissolved in water, and is settled to 500ml；
Sodium potassium tartrate tetrahydrate aqueous slkali：Sodium potassium tartrate tetrahydrate 173g and sodium hydroxide 50g are dissolved in water, and are settled to 500ml；
Using front, accurately take isopyknic copper solution and sodium potassium tartrate tetrahydrate aqueous slkali is sufficiently mixed.
（2.2）The preparation of sample solution：
Dilute with water enzyme sample, so as to get enzyme liquid（T0-T30）×f× 1.62 values are 3mg ~ 6mg glucose amount.
Example：Fungal alpha-amylase：[n=50000] dilutes 1ml 250ml, then dilutes 1ml 200ml.
（2.3）Enzyme activity determination：
Soluble starch solution 10ml is added in 100ml Erlenmyer triangular flask, is placed in 40 DEG C of waters bath with thermostatic control, preheating 10
~ 15min, adds enzyme liquid 1ml for having diluted.After accurate response 30min, Fehling's reagent 4ml is added to inactivate enzyme.Triangular flask is straight
It is connected on electric furnace after heating 2min, is immediately placed in tap water and cools down.Subsequently, 30% liquor kalii iodide 2ml and 25% sulphuric acid are added
Solution 2ml, dissociated the iodine for the titration of 0.05mol/L hypo solution, is disappeared as titration end-point T using blue30（ml）.
Blank is tested：Enzyme liquid is replaced with water, is determined with above-mentioned same operating procedure in another triangular flask empty
White control value T0（ml）, when terminal is closed on, add 1% soluble starch solution 1 that ~ 2 drops are dripped, disappeared as titration end-point using blue.
Enzyme activity computing formula：
Enzyme activity（U/ml or U/g）=(T0-T30)×f×1.62×0.1×n
In formula：T0 --- blank solution titration consumes the volume of sodium thiosulfate standard solution, ml；
T30 --- enzyme reaction solution titration consumes the volume of sodium thiosulfate standard solution, ml；
f--- the correction coefficient of 0.05mol/L hypo solution concentration；
1.62 conversion coefficient；
The constant of 0.1 analysis method（Reducing sugar equivalent to 10mg glucose）；
N --- the extension rate of enzyme sample；
The mutagenesis screening of 2 fungal amylase bacterial strain of embodiment
Determine fatality rate：Above-mentioned aspergillus oryzae TA01 is inoculated in PDA plate, 30 DEG C of culture 5-7d.Treat that bacterium colony surface produces a large amount of
During spore, the aseptic water elution of 5ml is drawn, spore liquid is obtained, resuspended with sterilized water after centrifugation, counted with blood counting chamber.Take one
Individual 90mm culture dish, the spore suspension for adding 5ml to dilute（Concentration about 1 × 107Individual/mL）, add rotor and stir in magnetic force
Mixing stirring on device makes spore liquid in uniform state.In aseptic superclean bench, with the uviol lamp that power is 9w in vertical away from
Irradiate from the top of 20cm, irradiate 30s, 45s, 60s, 75s, 90s, 105s, 120s respectively, take the spore liquid dilution after irradiating
10th, 100,1000 times, 100ul coating PDA plate is taken, is counted after 30 DEG C of culture 2-3d, with non-irradiated spore liquid as control, meter
Calculate fatality rate.When wherein irradiating 120s, fatality rate is that 95%, choosing the irradiation time carries out follow-up Mutagenesis experiments.
Mutagenesis screening：A 90mm culture dish is taken, the spore suspension for adding 5ml to dilute（Concentration is 1 × 107）, add
Rotor and on magnetic stirring apparatuss stirring make spore liquid in uniform state.In aseptic superclean bench, it is 9w's with power
Uviol lamp is irradiated in the top of vertical dimension 20cm, dilutes 1000 times after irradiating 120s, takes 100ul coating PDA plate, 30 DEG C of trainings
Foster 2-3d.
200 piece PDA plate are coated with altogether, and after 30 DEG C of culture 2-3d, each flat board grows 30-50 bacterium colony, first passes through bacterium colony
Form, screens the mutant of short branch, the mutant 126 that picking colony form is less, mycelia is fine and close, periphery of bacterial colonies fine hair is shorter
Individual be inoculated into PDA plate, 30 DEG C culture 5-7d.Each transformant extracts the truffle of 2cm × 2cm size, is inoculated in 50ml respectively
Liquid submerged culture base（Maltose 12%；Semen Maydis pulp 1%；Ammonium sulfate 0.5%；Potassium dihydrogen phosphate 0.35%；Dipotassium hydrogen phosphate 0.75%；
Magnesium sulfate heptahydrate 0.03%）Middle fermentation, after 30 DEG C of culture 5d, centrifugation thalline obtains supernatant and is crude enzyme liquid.By to acquisition
Crude enzyme liquid carries out fungal amylase enzyme activity detection, and applicant finally filters out a fungal strain amylase production highest mutant bacteria
Strain, is named as aspergillus oryzae TA20（Aspergillus oryzaeTA20）, the strain fermentation supernatant fungal amylase enzyme activity
2184u/ml, improves 146% than starting strain, achieves unexpected technique effect.
Applicant is on October 31st, 2016 by aspergillus oryzae TA20（Aspergillus oryzaeTA20）In being preserved in
The China typical culture collection center of Wuhan Wuhan University of state, deposit number is CCTCC NO:M2016602.
Claims (5)
1. aspergillus oryzae, it is characterised in that its deposit number be:M2016602.
2. application of the aspergillus oryzae according to claim 1 in fermenting and producing fungal amylase.
3. a kind of fermentation process for producing fungal amylase, it is characterised in that with aspergillus oryzae as claimed in claim 1 as fermentation
Bacterial strain.
4. fermentation process according to claim 3, it is characterised in that the fermentation medium used in the fermentation process is each
Component and its mass percent are respectively：Maltose 12%；Semen Maydis pulp 1%；Ammonium sulfate 0.5%；Potassium dihydrogen phosphate 0.35%；Phosphoric acid hydrogen
Dipotassium 0.75%；Magnesium sulfate heptahydrate 0.03%.
5. the fermentation process according to claim 3 or 4, it is characterised in that described fermentation temperature be.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828666A (en) * | 2017-11-21 | 2018-03-23 | 佛山市海天调味食品股份有限公司 | One Aspergillus oryzae ZA160 and its application |
| CN108795904A (en) * | 2018-05-18 | 2018-11-13 | 南京林业大学 | A method of the high enzyme activity amylase of expression |
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| CN105524839A (en) * | 2014-09-29 | 2016-04-27 | 湖南新鸿鹰生物工程有限公司 | Trichoderma reesei of high-yield and high-temperature-resistant fungal alpha-amylase |
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2016
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828666A (en) * | 2017-11-21 | 2018-03-23 | 佛山市海天调味食品股份有限公司 | One Aspergillus oryzae ZA160 and its application |
| CN107828666B (en) * | 2017-11-21 | 2018-10-09 | 佛山市海天调味食品股份有限公司 | One Aspergillus oryzae ZA160 and its application |
| CN108795904A (en) * | 2018-05-18 | 2018-11-13 | 南京林业大学 | A method of the high enzyme activity amylase of expression |
| CN108795904B (en) * | 2018-05-18 | 2020-12-15 | 南京林业大学 | Method for expressing amylase with high enzyme activity |
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Application publication date: 20170222 |