CN106520575A - Aspergillus niger strain capable of realizing high yield of catalase and application thereof - Google Patents

Aspergillus niger strain capable of realizing high yield of catalase and application thereof Download PDF

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CN106520575A
CN106520575A CN201611056550.0A CN201611056550A CN106520575A CN 106520575 A CN106520575 A CN 106520575A CN 201611056550 A CN201611056550 A CN 201611056550A CN 106520575 A CN106520575 A CN 106520575A
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aspergillus niger
fermentation
niger strain
catalase
strain
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CN106520575B (en
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王兴吉
贾仁洁
刘文龙
张�杰
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Shandong Longkete Enzyme Preparation Co Ltd
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    • C12R2001/685—Aspergillus niger
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01006—Catalase (1.11.1.6)

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Abstract

The invention especially relates to an Aspergillus niger strain capable of realizing high yield of catalase and a method for producing hydrogen peroxide by using the Aspergillus niger strain. The Aspergillus niger strain LDG1 has an accession number of CGMCC No. 13167. The invention also discloses the industrial production method for producing hydrogen peroxide by using the Aspergillus niger strain. The strain is subjected to seed culture and submerged fermentation, and the enzyme activity of fermentation broth reaches 152, 000 U/mL or above; and a catalase product prepared through extraction has low cost and resistance to alkali and high temperature, is extensively applicable to food, papermaking and textile industries and enables the production process of the above industries to be greener and environment-friendlier.

Description

One plant height produces catalatic Aspergillus niger strain and its application
Technical field：
The invention belongs to technical field of bioengineering, more particularly to a plant height produce catalatic Aspergillus niger strain and profit The method that hydrogen peroxide is produced with which.
Background technology：
Catalase is almost present in the internal of all aerobes, can catalyzing hydrogen peroxide be decomposed into water and oxygen Reaction, with remove free radical, protect cells from oxygen infringement etc. protection living organism function.Hydrogen peroxide is a kind of Strong oxidizer, is widely used in the bleaching and sterilization of weaving, food and medical field, but its residue is to production technology and body Body health has a significant impact.It is a kind of method of high-efficiency environment friendly using the removal hydrogen peroxide of hydrogen peroxide enzyme spcificity.
Catalatic production at present is mainly obtained by the method for fermentable, because in weaving, food and doctor The extensive application in treatment field, it is desirable to which catalase not only has acid-fast alkali-proof and heat-resisting etc. performance, enzyme activity of will also fermenting Level is high, low cost.At present, the catalase bacterium producing multi enzyme preparation of some documents and patent report, fermentation enzyme running water put down low, only note The application in highly basic hot environment in textile industry is focused on, the use environment in other industries, and the zymogenic bacteria of majority is not considered Strain is bacillus class.
It is used for the production of hydrogen peroxidase through fermenting, shaking flask liquid as Chinese patent CN103555620A discloses one plant of arthrobacterium The catalase running water of body submerged fermentation is flat to reach 49706U/mL；Chinese patent CN105483061A discloses one plant of solution Urea bacillus cereuss, the enzyme activity level of its shake flask fermentation can reach 41426U/mL.
Although these researchs have been also up to higher level, without one effective industrial process of proposition, Report is had no for the catalatic method of industrial fermentation production is carried out with aspergillus niger.
The content of the invention：
In order to solve above-mentioned technical problem, the present invention provides a plant height and produces catalatic Aspergillus niger strain and its industry Fermentation process.
One of technical scheme that the present invention is provided, is that a plant height produces catalatic Aspergillus niger strain, and the bacterial strain is The aspergillus niger LDG1 for obtaining is separated from the soil of the industrial park containing textile mills and food factory, identified its belongs to aspergillus niger (Aspergillus niger), the bacterial strain are preserved in Chinese microorganism strain preservation conservator on October 26th, 2016 Meeting common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postal 100101 are compiled, deposit number is CGMCC No.13167.
The two of technical scheme provided by the present invention, are that the Aspergillus niger strain CGMCC No.13167 ferment sends out hydrogen peroxide The method of enzyme, mainly comprises the steps：
A. seed tank culture：Strain is connected in seed culture medium, 33 DEG C, 180-300rpm, ferment 50h；
B. fermentor cultivation：Aseptically, according to 7% inoculum concentration, seed liquor is pressed into into fermentation tank, is fermented Tank culture, 33 DEG C, 300-800rpm ferments to 40h-110h, by control of additive raw material DE values between 55-60；When fermentation extremely During 110h-170h, DE values are controlled between 35-40 by feed supplement；, the enzyme activity serious to the self-dissolving of 170h-185h thalline when fermenting Nothing stops feed supplement when significantly improving；Tank is put when fermentation is reduced to 20 to 185h-195hDE values；
C. extract and refine：200ppm polyacrylamides and 2.5% perlite is added to be flocculated and sheet frame in fermentation liquid Filter pressing, then carries out being concentrated by ultrafiltration 6-8 times, and (addition is 0.1%- with liquid mass volume ratio is concentrated by ultrafiltration to be eventually adding preservative The potassium sorbate of 0.3% sodium nitrite, the sodium benzoate of 0.6%-0.8% and 0.3%-0.4%) and protective agent (addition with It is 7%-9% glucoses and 10%-15% glycerol that liquid mass volume ratio is concentrated by ultrafiltration) kieselguhr filtration sterilization is dissolved and uses, obtain To catalase finished product enzyme preparation；
Culture medium quality volume basis hundred constitute as follows：
Seed culture medium：Sucrose 4%-4.8%, glucose 1%-2%, magnesium sulfate 0.1%-0.2%, potassium chloride 0.05%-0.08%, sodium nitrate 0.3%-0.4%, potassium dihydrogen phosphate 0.2%-0.4%, remaining is water, pH 7.0；
Fermentation medium：Corn starch 9%-11%, Semen Maydis powder 5%-8%, soybean cake powder 2.5%-3%, calcium chloride 0.03- 0.04%, ammonium sulfate 1%-1.5%, potassium dihydrogen phosphate 0.5%-0.8%, sodium dihydrogen phosphate 0.4%-0.7%, sodium citrate 0.2%-0.4%, Semen Maydis pulp 1%-3%, remaining is water, pH 7.0；
Supplemented medium：Corn starch 25%, Semen Maydis pulp 2.3%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.2%- 0.5%, pH 7.0.
Fermented using above-mentioned Aspergillus niger strain CGMCC No.13167 and method, produced catalatic fermentation enzyme Running water is flat to can reach more than 152000U/mL；
Produced catalatic enzymatic property is as follows：
1st, optimal reactive temperature：55℃；
2nd, optimal reaction pH：9.0；
3rd, heat stability：1h is incubated under the conditions of 80 DEG C to remain to keep more than 90% activity；
4th, pH stability：Under conditions of pH7.0-12 after 24h, enzyme activity stills remain in more than 85%.
Beneficial effect：
1st, present invention firstly provides a plant height produces catalatic Aspergillus niger strain, industrialization is carried out using the bacterial strain The fermentation enzyme running water of fermenting and producing is flat to can reach more than 152000U/mL, greatly reduces production cost, is conducive to hydrogen peroxide The large-scale application of enzyme, energy-saving and emission-reduction.
2nd, the catalase optimal reactive temperature produced by this bacterial strain is 55 DEG C, is incubated 1h and remains to keep under the conditions of 80 DEG C More than 90% activity；Optimal reaction pH is 9.0, and under conditions of pH7.0-12 after 24h, enzyme activity is stilled remain in More than 85%；The alkaline-resisting resistant to elevated temperatures characteristic of the above, makes the catalase for preparing using the bacterial strain and process matched therewith can be with The industry such as food and weaving is widely suitable for, therefore the bacterial strain and technique of present invention offer are with a wide range of applications and show The economic benefit of work.
Description of the drawings：
Catalase enzyme activity force curve under Fig. 1 different temperatures；
The heat-resisting linearity curve of Fig. 2 catalases；
Catalase enzyme activity force curve under Fig. 3 difference pH；
Fig. 4 catalase resistance to acids and bases curves.
Specific embodiment：
More detailed description is done to the present invention below by way of specific embodiment, case is embodied as by way of example only, no As the restriction to the scope of the present invention.
1 catalase enzyme activity determination method of embodiment
One standard enzyme-activity unit (1U) is defined as：Under the conditions of 37 DEG C, 1min decomposes 1umolH2O2(extinction coefficient are Enzyme amount needed for 39.4L/mol/cm).
H2O2Decomposition rate with ultra-violet and visible spectrophotometer S53 in 240nm, be measured under the conditions of 30 DEG C.Reaction Cumulative volume is 3mL, and enzyme liquid containing 0.1mL and 2.9mL contain 120mmol/L H2O2Potassium dihydrogen phosphate, phosphoric acid hydrogen with 50mmol/L Dipotassium buffer (pH7.0).
The screening of 2 bacterium producing multi enzyme preparation of embodiment
Screening Jing flat board cultures, Deca H from the industrial park soil containing textile mills and food factory2O2Aerogenesis steeps feelings Condition is screened again, and is compared hydrogen peroxide activated size and obtained.
Soil is cultivated through the increment of Cha Shi fluid mediums twice, is coated with Czapek's medium flat board, Deca 0.2mL after culture 8% hydrogen peroxide on uniform bacterium colony selects aerogenesis bubble rapid and the persistent period is long bacterium colony carries out test tube fermentation training Support, by the capacity of water for comparing decomposition of hydrogen peroxide, final screening obtains bacterial strain CGMCC No.13167.The identified bacterial strain Belong to aspergillus niger (Aspergillus niger).
3 liquid fermentation of embodiment produces catalase and its extraction
(1) seed tank culture：Strain is connected in seed culture medium, 33 DEG C, 180rpm, ferment 50h；
(2) fermentor cultivation：Aseptically, according to 7% inoculum concentration, seed liquor is pressed into into fermentation tank, carry out send out Fermentation tank culture, 33 DEG C, 300rpm ferments to 40h-110h, by control of additive raw material DE values between 55-60；When fermentation to 110h- During 170h, DE values are controlled between 35-40 by feed supplement；When fermentation is serious to the self-dissolving of 170h thalline, enzyme activity is without significantly improving When stop feed supplement；Tank is put when fermentation is reduced to 20 to 185h DE values；
(3) extract and refine：200ppm polyacrylamides and 2.5% perlite is added to be flocculated and sheet frame in fermentation liquid Filter pressing, then carries out being concentrated by ultrafiltration 6 times, and (addition is 0.1% nitrous with liquid mass volume ratio is concentrated by ultrafiltration to be eventually adding preservative Sour sodium, 0.6% sodium benzoate and 0.3% potassium sorbate) and protective agent (addition is 7% Portugal with liquid mass volume ratio is concentrated by ultrafiltration Grape sugar and 10% glycerol) kieselguhr filtration sterilization is dissolved and uses, obtain catalase finished product enzyme preparation；
(4) culture medium quality volume basis hundred constitute as follows：
Seed culture medium：Sucrose 4%, glucose 1%, magnesium sulfate 0.1%, potassium chloride 0.05%, sodium nitrate 0.3%, phosphorus Acid dihydride potassium 0.2%, pH 7.0；
Fermentation medium：Corn starch 9%, Semen Maydis powder 5%, soybean cake powder 2.5%, calcium chloride 0.03%, ammonium sulfate 1%, Potassium dihydrogen phosphate 0.5%, sodium dihydrogen phosphate 0.4%, sodium citrate 0.2%, Semen Maydis pulp 1%, pH 7.0；
Supplemented medium：Corn starch 25%, Semen Maydis pulp 2.3%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.2%, pH 7.0；
Using said method, when fermentation period is 185 hours, catalase fermentation broth enzyme vigor is：152884U/mL.
4 liquid fermentation of embodiment produces catalase and its extraction
(1) seed tank culture：Strain is connected in seed culture medium, 33 DEG C, 300rpm, ferment 50h；
(2) fermentor cultivation：Aseptically, according to 7% inoculum concentration, by seed zymotic fluid be pressed into fermentation tank, carry out Fermentor cultivation, 33 DEG C, 800rpm ferments to 40h-110h, by control of additive raw material DE values between 55-60；When fermentation extremely During 110h-170h, DE values are controlled between 35-40 by feed supplement；When fermentation is serious to the self-dissolving of 185h thalline, enzyme activity is without obvious Stop feed supplement during raising；Tank is put when fermentation is reduced to 20 to 195h DE values；
(3) extract and refine：200ppm polyacrylamides and 2.5% perlite is added to be flocculated and sheet frame in fermentation liquid Filter pressing, then carries out being concentrated by ultrafiltration 8 times, and (addition is 0.3% nitrous with liquid mass volume ratio is concentrated by ultrafiltration to be eventually adding preservative Sour sodium, 0.8% sodium benzoate and 0.4% potassium sorbate) and protective agent (addition is 9% Portugal with liquid mass volume ratio is concentrated by ultrafiltration Grape sugar and 15% glycerol) kieselguhr filtration sterilization is dissolved and uses, obtain catalase finished product enzyme preparation；
(4) culture medium quality volume basis hundred constitute as follows：
Seed culture medium：Sucrose 8%, glucose 2%, magnesium sulfate 0.2%, potassium chloride 0.08%, sodium nitrate 0.4%, phosphorus Acid dihydride potassium 0.4%, pH 7.0；
Fermentation medium：Corn starch 11%, Semen Maydis powder 8%, soybean cake powder 3%, calcium chloride 0.04%, ammonium sulfate 1.5%, Potassium dihydrogen phosphate 0.8%, sodium dihydrogen phosphate 0.7%, sodium citrate 0.4%, Semen Maydis pulp 3%, pH 7.0；
Supplemented medium：Corn starch 25%, Semen Maydis pulp 2.3%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.5%, pH 7.0。
Using said method, when fermentation period is 195 hours, catalase fermentation broth enzyme vigor is：152965U/mL.
5 liquid fermentation of embodiment produces catalase and its extraction
(1) seed tank culture：Strain is connected in seed culture medium, 33 DEG C, 240rpm, ferment 50h；
(2) fermentor cultivation：Aseptically, according to 7% inoculum concentration, seed liquor is pressed into into fermentation tank, carry out send out Fermentation tank culture, 33 DEG C, 500rpm ferments to 40h-110h, by control of additive raw material DE values between 55-60；When fermentation to 110h- During 170h, DE values are controlled between 35-40 by feed supplement；When fermentation is serious to the self-dissolving of 180h thalline, enzyme activity is without significantly improving When stop feed supplement；Tank is put when fermentation is reduced to 20 to 190h DE values；
(3) extract and refine：200ppm polyacrylamides and 2.5% perlite is added to be flocculated and sheet frame in fermentation liquid Filter pressing, then carries out being concentrated by ultrafiltration 7 times, and (addition is 0.2% nitrous with liquid mass volume ratio is concentrated by ultrafiltration to be eventually adding preservative Sour sodium, 0.7% sodium benzoate and 0.35% potassium sorbate) and protective agent (addition is 8% Portugal with liquid mass volume ratio is concentrated by ultrafiltration Grape sugar and 13% glycerol) kieselguhr filtration sterilization is dissolved and uses, obtain catalase finished product enzyme preparation；
(4) culture medium quality volume basis hundred constitute as follows：
Seed culture medium：Sucrose 4.5%, glucose 1.5%, magnesium sulfate 0.15%, potassium chloride 0.06%, sodium nitrate 0.35%, potassium dihydrogen phosphate 0.3%, pH 7.0；
Fermentation medium：Corn starch 10%, Semen Maydis powder 6%, soybean cake powder 3%, calcium chloride 0.04%, ammonium sulfate 1.5%, Potassium dihydrogen phosphate 0.6%, sodium dihydrogen phosphate 0.5%, sodium citrate 0.3%, Semen Maydis pulp 2%, pH 7.0；
Supplemented medium：Corn starch 25%, Semen Maydis pulp 2.3%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.4%, pH 7.0；
Using said method, when fermentation period is 190 hours, catalase fermentation broth enzyme vigor is：153780U/mL.
6 optimal reactive temperature of embodiment
The catalase finished product enzyme preparation that treating excess syndrome example 5 is obtained, with normal condition pH7.0, the enzyme activity of 55 DEG C of measure of temperature Power is that enzyme activity is determined under 100%, pH7.0, different temperatures, calculates enzyme activity.As a result as shown in figure 1, optimal reaction temperature Spend for 55 DEG C.
7 thermostability of embodiment
The catalase finished product enzyme preparation that treating excess syndrome example 5 is obtained, with 55 DEG C, determines under the conditions of being incubated 1h under the conditions of pH7.0 Enzyme activity be 100%, calculate enzyme activity.By the test tube for filling 9mL pH7.0 buffer be respectively placed in 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, preheat 10min in 90 DEG C of water-bath, point Not Jia Ru 1mL catalases, shake up rapidly and accurate timing 1h, take out rapidly and cool down in ice-water bath, after cooling, use pH7.0 Buffer continues to be diluted to corresponding multiple, determines enzyme activity.Measurement result is remained to as shown in Fig. 2 1h is incubated under the conditions of 80 DEG C Keep more than 90% activity.
8 optimal reaction pH of embodiment
The catalase finished product enzyme preparation that treating excess syndrome example 5 is obtained, at 55 DEG C, different pH value 3,3.5,4,4.5,5,5.5,6, 6.5th, under the conditions of 7,7.5,8,8.5,9,9.5,10,10.5,11, determine the enzyme activity of same liquid enzyme preparation sample.It is chosen at 55 DEG C, the enzyme activity determined under the conditions of pH9.0 is 100%, calculates relative enzyme activity.As a result as shown in figure 3, optimum pH is 9.0.
9 resistance to acids and bases of embodiment
The catalase finished product enzyme preparation that treating excess syndrome example 5 is obtained, with pH9.0, the enzyme activity determined under the conditions of 55 DEG C is 100%, calculate enzyme activity.The buffer for taking 9ml difference pH respectively (prepares the disodium hydrogen phosphate and phosphorus of 0.2mol/L respectively Acid dihydride sodium, takes corresponding volume and adjusts to required pH) in 21 test tubes, add 1ml catalases to shake up, room temperature decentralization After putting 24h, shake up continuation pH7.0 buffer and be diluted to corresponding multiple, determine enzyme activity.Measurement result as shown in figure 4, Under conditions of pH7.0-12 after 24h, enzyme activity stills remain in more than 85%.

Claims (7)

1. a plant height produces catalatic Aspergillus niger strain, it is characterised in that the bacterial strain is specially aspergillus niger (Aspergillus niger) LDG1, deposit number are CGMCC No.13167.
2. the catalatic method of Aspergillus niger strain fermenting and producing described in claim 1, it is characterised in that step is as follows：
A. seed liquor culture：Strain is connected in seed culture medium, 33 DEG C, 180-300rpm, ferment 50h；
B. fermentor cultivation：Aseptically, according to 7% inoculum concentration, seed liquor is pressed into into fermentation tank, fermentation tank training is carried out Support, 33 DEG C, 300-800rpm ferments to 40h-110h, by control of additive raw material DE values between 55-60；When fermentation to 110h- During 170h, DE values are controlled between 35-40 by feed supplement；When fermentation is serious to the self-dissolving of 170h-185h thalline, enzyme activity is without obvious Stop feed supplement during raising；Tank is put when fermentation is reduced to 20 to 185h-195h DE values；
C. extract and refine：200ppm polyacrylamides and 2.5% perlite is added to be flocculated and filter press in fermentation liquid, Then carry out being concentrated by ultrafiltration 6-8 times, be eventually adding preservative and protective agent dissolves and use kieselguhr filtration sterilization, obtain peroxidating Hydrogen enzyme finished product enzyme preparation.
3. the catalatic method of Aspergillus niger strain fermenting and producing described in claim 2, it is characterised in that culture medium mass body Product very constitutes as follows：
Seed culture medium：Sucrose 4%-4.8%, glucose 1%-2%, magnesium sulfate 0.1%-0.2%, potassium chloride 0.05%- 0.08%, sodium nitrate 0.3%-0.4%, potassium dihydrogen phosphate 0.2%-0.4%, pH 7.0；
Fermentation medium：Corn starch 9%-11%, Semen Maydis powder 5%-8%, soybean cake powder 2.5%-3%, calcium chloride 0.03- 0.04%, ammonium sulfate 1%-1.5%, potassium dihydrogen phosphate 0.5%-0.8%, sodium dihydrogen phosphate 0.4%-0.7%, sodium citrate 0.2%-0.4%, Semen Maydis pulp 1%-3%, pH 7.0；
Supplemented medium：Corn starch 25%, Semen Maydis pulp 2.3%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.2%-0.5%, pH 7.0。
4. the catalatic method of Aspergillus niger strain fermenting and producing described in claim 2, it is characterised in that preservative addition For sodium nitrite 0.1%-0.3%, sodium benzoate 0.6%-0.8%, potassium sorbate 0.3%-0.4%.
5. the catalatic method of Aspergillus niger strain fermenting and producing described in claim 2, it is characterised in that protective agent addition For 7%-9% glucoses and 10%-15% glycerol.
6. the application described in the catalatic claim 1 of high yield described in claim 1.
7. the catalase produced by Aspergillus niger strain described in claim 1.
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Publication number Priority date Publication date Assignee Title
CN107384886A (en) * 2017-08-28 2017-11-24 王艺璇 A kind of new catalase and its application
CN107384814A (en) * 2017-08-28 2017-11-24 王艺璇 A kind of bacterial strain for recombinantly expressing catalase and its application
CN110527628A (en) * 2019-07-30 2019-12-03 南京农业大学 A kind of protective agent and its preparation method and application of Acetamiprid degradation bacteria

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107384886A (en) * 2017-08-28 2017-11-24 王艺璇 A kind of new catalase and its application
CN107384814A (en) * 2017-08-28 2017-11-24 王艺璇 A kind of bacterial strain for recombinantly expressing catalase and its application
CN107384814B (en) * 2017-08-28 2020-03-31 王艺璇 Bacterial strain for recombinant expression of catalase and application thereof
CN110527628A (en) * 2019-07-30 2019-12-03 南京农业大学 A kind of protective agent and its preparation method and application of Acetamiprid degradation bacteria

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