CN106801045A - A kind of method of recycled fiber element enzyme in fermentation of Aspergillus niger liquid from ocean - Google Patents
A kind of method of recycled fiber element enzyme in fermentation of Aspergillus niger liquid from ocean Download PDFInfo
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- CN106801045A CN106801045A CN201710080722.6A CN201710080722A CN106801045A CN 106801045 A CN106801045 A CN 106801045A CN 201710080722 A CN201710080722 A CN 201710080722A CN 106801045 A CN106801045 A CN 106801045A
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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
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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Abstract
The method of recycled fiber element enzyme in a kind of fermentation of Aspergillus niger liquid from ocean of present invention offer, the cellulase produced through cellulase-producing microbial fermentation using improved tannin precipitation method separation and Extraction, and it is prepared into powdery cellulase product with vacuum freeze-drying method, the rate of recovery of cellulase is set to reach 90 95%, it is short with the used time, energy consumption is small, simple to operate, the advantage of cellulase high income.Instant invention overcomes the separation and Extraction cellulase stage in whole lignocellulosic ethanol the problem such as high cost, complex operation, it is adaptable to from the aspergillus niger of ocean recycled fiber element enzyme industrialization prepare on a large scale.The ocean aspergillus niger that the present invention is selected has the characteristics of salt-resistance, enzyme system are abundant, expression quantity is high, secretion capacity is strong.The inventive method is reasonable in design, and operation is simple and feasible, used device low cost, high income, it is adaptable to the industrialized production of extensive separation and Extraction cellulase.
Description
Technical field
Method the invention belongs to separate cellulase in industry, is related to one kind to be reclaimed from the fermentation of Aspergillus niger liquid of ocean fine
The method of the plain enzyme of dimension.
Background technology
Cellulase is that most wide, the most most abundant polysaccharose substance of cheap, content is distributed on the earth, in case of human, it
It is simultaneously again the maximum regenerative resource of nature quantity, its degraded is the key link of nature carbon cycle.Cellulase
Cellulosic material can thoroughly be degraded by hydrolysis, its hydrolysate can further fermentative production of ethanol, hydrogen and
Produce raw oil material of biodiesel etc..Therefore, cellulose is converted using cellulase to be polluted for solving current world environments
And tool is of great significance the problems such as energy crisis.At present, cellulase has been widely used for detergent industry, numb cotton
The industry of blend fabric Final finishing, food industry, feed industry, aquaculture industry, and achieve good effect.
The microorganism for producing cellulase is varied, there is bacterium, fungi, actinomyces etc., and terrestrial is substantially at present
Microorganism, with the cellulase of terrestrial micro-organisms, its stability and environmental resistance are not typically especially desirable, and fine
The degraded for tieing up extract plant typically is required to be carried out under conditions of rough, and its cellulose degradation efficiency is subjected to limitation.Ocean is micro-
Biology caused by its environment, with the unique technical ability different from terrestrial microorganism.Ocean oligotrophy, the unique environments of high salt are pregnant
The microorganism for being markedly different from terrestrial microbiologic properties is brought out there is provided a special environment.Live in micro- life of oligotrophy environment
Thing often has enzyme system of the degraded macromolecular substances as itself available nutriment, and being degraded to for cellulase is available
Carbohydrate be the limited acquisition nutrition that microorganism in oligotrophy environment uses major way.
The preparation method of cellulase has two kinds：There is liquid fermentation and solid fermentation, solid fermentation liquid fermentation not have
Advantage, mycelia can effectively be attached to solid fermentation matrix surface, and it is high to improve fermentation efficiency, specific enzyme activity, the life of FU volume
Production capacity power is high, therefore solid fermentation turns into the industrialized important directions of fermenting and producing.
From zymotic fluid in terms of the technical grade preparation of separation and Extraction cellulase, column chromatography for separation mode due to high cost with
The reasons such as the complexity of operation are difficult effectively to be amplified to commercial scale.At present, the separation method of industrial cellulase is main
There are ammonium sulfate precipitation and ethanol precipitation etc..But the more difficult removing of substantial amounts of salt can be produced after ammonium sulfate precipitation and is reclaimed, and ethanol
The cost of precipitation is again of a relatively high, so as to constrain the heavy industrialization application of lignocellulosic material and cellulase.
The content of the invention
The drawbacks of it is an object of the invention to be directed to general extraction cellulase method, provide a kind of black from microorganism ocean
The method of recycled fiber element enzyme in Aspergillus fermentation broth, the present invention is using improved tannin precipitation method separation and Extraction through cellulase-producing
The cellulase that microbial fermentation is produced, and powdery cellulase product is prepared into using vacuum freeze-drying method, make fiber
The rate of recovery of plain enzyme can reach 90-95%, and short with the used time, energy consumption is small, simple to operate, the advantage of cellulase high income,
Overcome separation and Extraction cellulase stage high cost in whole lignocellulosic ethanol！The difficulties such as complex operation
Topic, prepares suitable for cellulase industrialization is extensive.
Present invention method of recycled fiber element enzyme from the fermentation of Aspergillus niger liquid of ocean is realized by following steps：
1st, zymotic fluid clarification：The solid state fermentation liquor produced after marine microorganism solid state fermentation 7-8 days is filtered, from
The heart, obtains fermented clear liquid；Described marine microorganism is ocean black-koji mould, and Aspergillus sp.ZJUBE2010 are protected
It is hidden in China typical culture collection center, preservation address：Wuhan, China Wuhan University, deposit number：CCTCC
NO.M2010132, preservation date：On June 2nd, 2010；
2nd, tannin precipitation：By in above-mentioned fermented clear liquid addition agitator tank, stirring certain hour is turned with certain, will be certain
The tanning solution of amount is added in the fermented clear liquid in agitator tank, and continues to stir certain hour, then stands 1-2h, carries out fibre
The precipitation of the plain enzyme of dimension；
3rd, precipitation is collected：The zymotic fluid precipitated by tannin is centrifuged with certain carrying out, supernatant removes, collects heavy
Form sediment；
4th, precipitation is redissolved：Concentration by above-mentioned precipitation certain volume is that the polyglycol solution of 20mg/ml redissolves, and is held
Continuous stirring certain hour, then stands 1-2h, is redissolved；
5th, product is collected：By above-mentioned solution, it is placed in a centrifuge and a period of time is centrifuged with certain rotating speed, supernatant is
Cellulase solution after concentration, concentrate is placed in vacuum freeze drier and dries prepared powdery cellulase product.
In the method for the described element enzyme of the recycled fiber from the fermentation of Aspergillus niger liquid of ocean, to the hair obtained in step 1
The cellulase solution being collected into after being parsed through tannin precipitation polyethylene glycol in ferment clarified solution and step 4 uses filter paper enzyme activity respectively
Assay method determines its cellulase activity.
In the method for the described element of the recycled fiber from the fermentation of Aspergillus niger liquid of ocean enzyme, in step 2 consumption of tannin according to
The consumption of 10g/L is added in fermented clear liquid.
In the method for the described element of the recycled fiber from the fermentation of Aspergillus niger liquid of ocean enzyme in step 2 and step 4, stirring turns
Speed is 80r/min, and mixing time is 10min.
In the method for the described element enzyme of the recycled fiber from the fermentation of Aspergillus niger liquid of ocean, in step 3 and step 5, centrifugation
Rotating speed is 4000r/min, and centrifugation time is 20min.
In the method for the described element enzyme of the recycled fiber from the fermentation of Aspergillus niger liquid of ocean, in step 4, polyglycol solution
Concentration is 20g/L, and solute is the PEG that molecular weight is 6000, and solvent is Natural Water.
In the method for the described element enzyme of the recycled fiber from the fermentation of Aspergillus niger liquid of ocean, in step 4, polyglycol solution
Volume is redissolved according to the 1/10 of its original fermentation liquor volume to determine.
In the fermentation of Aspergillus niger liquid from ocean in the method for recycled fiber element enzyme, the bar of vacuum freeze drying in step 5
Part is that vacuum is 9-19Pa, and temperature is -50 DEG C~-80 DEG C, untill vacuumizing 12-36h when powdered no moisture.
It is a further object to provide methods described in recycled fiber element enzyme in the fermentation of Aspergillus niger liquid of ocean
Using.
The invention has the advantages that：By using improved tannin-polyethylene glycol method separation and Extraction cellulase,
And powdery cellulase product is prepared into using vacuum freeze-drying method, the rate of recovery of cellulase is reached 90-
95%, have the advantages that the used time is short, energy consumption is small, simple to operate, cellulase high income, overcome separation and Extraction cellulase rank
Section problem such as high cost, complex operation in whole lignocellulosic ethanol.The ocean aspergillus niger that the present invention is selected
There is the characteristics of its salt-resistance, enzyme system are abundant, expression quantity is high, secretion capacity is strong.The inventive method is reasonable in design, simple to operate
Feasible, used device low cost, energy consumption is small, high income, it is adaptable to the industrialized production of extensive separation and Extraction cellulase.
Specific embodiment
The present invention is elaborated with reference to embodiment, to make those of ordinary skill in the art refering to after this specification
Can implement according to this.
Described marine microorganism is ocean black-koji mould, and Aspergillus sp.ZJUBE2010 are preserved in Chinese allusion quotation
Type culture collection, preservation address：Wuhan, China Wuhan University, deposit number：CCTCC NO.M2010132, preservation day
Phase：On June 2nd, 2010.
Embodiment 1
The method of recycled fiber element enzyme, is realized by following steps in a kind of aspergillus niger microbial fermentation solution from ocean：
1st, zymotic fluid clarification：By the zymotic fluid of ocean aspergillus niger solid state fermentation 7d-8d or so carry out 5 layers of filtered through gauze,
With 4000r/min in centrifuge, 20min is centrifuged, obtains fermented clear liquid；
2nd, tannin precipitation：By in fermented clear liquid addition agitator tank, a certain amount of tannin is added to the fermentation
In clarified solution, 10min is stirred with the rotating speed of 80r/min, stand 1h, carry out the precipitation of cellulase；
3rd, precipitation is collected：The zymotic fluid that will be precipitated by tannin, with 4000r/min, is centrifuged 20min, and supernatant is removed,
Collect precipitation；
4th, precipitation is redissolved：The polyglycol solution of the 1/10 of above-mentioned precipitation original volume 20mg/ml is redissolved, with 80r/
Min persistently stirs the 5min times, then stands 1h, is redissolved；
5th, product is collected, and the solution of above-mentioned redissolution is placed in a centrifuge with 4000r/min, and 20min, supernatant is centrifuged
Cellulase solution after as concentrating, concentrate is placed in vacuum freeze drier and dries prepared powdery cellulase product
Product.
The condition of the vacuum freeze drying is that vacuum is 9-19Pa, and temperature is -50 DEG C~-80 DEG C, vacuumizes 12-
Untill 36h is when powdered no moisture.
Filter paper enzyme activity (FPA) is determined:50mg (1x6cm) whatman filter paper is taken, enzyme liquid 0.5mL is added, pH4.8 is added
Sodium citrate one citrate buffer solution 2mL, 50 DEG C of reaction 1h, add 2.5mlDNS terminating reactions, boiling water bath 5min colour developings are surveyed
Determine A54O.
Enzyme activity is defined:Under the above-described reaction conditions, the enzyme needed for catalytic substrate hydrolysis generation 1umol glucose per minute
It is 1 enzyme activity unit U to measure.
In the method for the described element enzyme of the recycled fiber from the fermentation of Aspergillus niger liquid of ocean, to the hair obtained in step 1
Cellulase precipitation is collected into through tannin precipitation determine its fibre with filter paper enzyme activity assay method respectively in ferment clarified solution and step 3
The plain enzyme activity of dimension.
Tannin as cellulase precipitating reagent, be the original that can pass through Hydrogenbond with cellulase protein using tannin
Reason, and the combination of this hydrogen bond has invertibity, and polyethylene glycol can parse cellulase from enzyme-tannin complexes
Come, the enzyme preparation not saliferous prepared with the tannin precipitation method, purity is high, not only conforms to edible requirement, and can be as oral
Medicinal enzyme preparation, also can be used for textile process and fermentation industry etc., be determined by experiment, using tannin precipitated cellulose enzyme after,
Cellulase is parsed using polyethylene glycol, the cellulase in crude enzyme liquid can be efficiently extracted.Operating procedure of the present invention is simple
Feasible, used device low cost, energy consumption is small, high income, it is adaptable to the industrialized production of extensive separation and Extraction cellulase.
Embodiment 2
The zymotic fluid of ocean aspergillus niger solid state fermentation 7d is first carried out into single filter with 5 layers of gauze, then through 4000r/min's
Centrifuge carries out centrifugation 20min, obtains clarified broth.The zymotic fluid of the microbial fermentation 7d-8d of cellulase-producing or so enters
Row filtering, centrifugation, obtain fermented clear liquid.
By in fermented clear liquid addition agitator tank, it is stirred with the rotating speed of 80r/min, weighs tannin and be added to institute
In stating fermented clear liquid so that the concentration of tannin is 10g/ml, and continue to stir 10min, stand 1h, carry out the heavy of cellulase
Form sediment.
The zymotic fluid that will be precipitated by tannin is centrifuged on centrifuge, 4000r/min, and 20min is centrifuged, and supernatant is gone
Fall, collect precipitation.
The above-mentioned precipitation polyglycol solution of 1/10 original fermentation liquor volume is redissolved, polyglycol solution concentration is 20g/
L, 5min is persistently stirred with 80r/min, then stands 1h.
The solution of above-mentioned redissolution is placed in a centrifuge with 4000r/min, 20min is centrifuged, supernatant is after concentration
Cellulase solution, the enzyme activity rate of recovery be 96.2%.
It is 10Pa in vacuum to use vacuum freeze drying agent, 30h is freezed under the conditions of -50 DEG C powdery cellulase is obtained
, with embodiment 1, method of adopting international standards determines enzyme activity for product, assay method and standard, and the enzyme activity rate of recovery is 75%.
Embodiment 3
The zymotic fluid of ocean aspergillus niger solid state fermentation 7d is first carried out into single filter with 5 layers of gauze, then through 4000r/min's
Centrifuge carries out centrifugation 20min, obtains clarified broth.
By in fermented clear liquid addition agitator tank, it is stirred with the rotating speed of 80r/min, weighs tannin and be added to institute
In stating fermented clear liquid so that the concentration of tannin is 10g/ml, and continue to stir 10min, stand 1h, carry out the heavy of cellulase
Form sediment.
The zymotic fluid that will be precipitated by tannin is centrifuged on centrifuge, 4000r/min, and 20min is centrifuged, and supernatant is gone
Fall, collect precipitation.
The above-mentioned precipitation polyglycol solution of 1/10 original fermentation liquor volume is redissolved, polyglycol solution concentration is 20g/
L, 5min is persistently stirred with 80r/min, then stands 1h.
The solution of above-mentioned redissolution is placed in a centrifuge with 4000r/min, 20min is centrifuged, supernatant is after concentration
Cellulase solution, it is 97.4% to determine enzyme activity rate.
It is 10Pa in vacuum to use vacuum freeze drying agent, 30h is freezed under the conditions of -50 DEG C powdery cellulase is obtained
, with embodiment 1, method of adopting international standards determines enzyme activity for product, assay method and standard, and it is 73% to measure enzyme activity yield.
Embodiment 4
The zymotic fluid of ocean aspergillus niger solid state fermentation 7d is first carried out into single filter with 5 layers of gauze, then through 4000r/min's
Centrifuge, is centrifuged 20min, obtains clarified broth.
By in fermented clear liquid addition agitator tank, it is stirred with the rotating speed of 80r/min, weighs tannin and be added to institute
In stating fermented clear liquid so that the concentration of tannin is 10g/ml, and continue to stir 10min, stand 1h, carry out the heavy of cellulase
Form sediment.
The zymotic fluid that will be precipitated by tannin is centrifuged on centrifuge, 4000r/min, and 20min is centrifuged, and supernatant is gone
Fall, collect precipitation.
The above-mentioned precipitation polyglycol solution of 1/10 original fermentation liquor volume is redissolved, polyglycol solution concentration is 20g/
L, 5min is persistently stirred with 80r/min, then stands 1h.
Liquid will be redissolved, will be placed in a centrifuge with 4000r/min, 20min will be centrifuged, supernatant will be the cellulose after concentration
Enzyme liquid, it is 97.9% to determine enzyme activity rate.
It is 10Pa in vacuum to use vacuum freeze drying agent, 30h is freezed under the conditions of -50 DEG C powdery cellulase is obtained
, with embodiment 1, method of adopting international standards determines enzyme activity for product, assay method and standard, and it is 78% to measure enzyme activity yield.
Reference and traditional ammonium sulfate precipitation method, under 60% saturation degree (390g/L), ammonium sulfate precipitation is more complete, will
It carries out freeze-drying recovery, and error is reduced by three experiments, determines the cellulase activity rate of recovery 31.25%, and ammonium sulfate
Addition is big, and enzyme activity loss is serious, is compared to tannin-polyethylene glycol method and causes the cost recovery to increase, and the enzyme activity rate of recovery is low.Than
Relatively the results are shown in Table 1.
Table 1
Claims (9)
1. in a kind of fermentation of Aspergillus niger liquid from ocean recycled fiber element enzyme method, it is characterised in that by following steps reality
It is existing：
(1) zymotic fluid clarification：The solid state fermentation liquor produced after marine microorganism solid state fermentation 7-8 days is filtered, is centrifuged, obtained
To fermented clear liquid；The marine microorganism is ocean black-koji mould, and Aspergillus sp.ZJUBE2010 are preserved in China
Type Tissue Collection, preservation address：Wuhan, China Wuhan University, deposit number：CCTCC NO.M2010132, preservation
Date：On June 2nd, 2010；
(2) tannin precipitation：By in above-mentioned fermented clear liquid addition agitator tank, stirring certain hour is turned with certain, will be a certain amount of
Tanning solution add agitator tank in fermented clear liquid in, and continue stir certain hour, then stand 1-2h, carry out fiber
The precipitation of plain enzyme；
(3) precipitation is collected：The zymotic fluid precipitated by tannin is centrifuged with certain carrying out, supernatant removes, collects precipitation；
(4) precipitation is redissolved：Concentration by above-mentioned precipitation certain volume is that the polyglycol solution of 20mg/ml redissolves, and is continued
Stirring certain hour, then stands 1-2h, is redissolved；
(5) product is collected：By above-mentioned solution, it is placed in a centrifuge and a period of time is centrifuged with certain rotating speed, supernatant is as dense
Cellulase solution after contracting, concentrate is placed in vacuum freeze drier and dries prepared powdery cellulase product.
2. in described a kind of fermentation of Aspergillus niger liquid from ocean according to claim 1 recycled fiber element enzyme method, its
It is characterised by, after being parsed through tannin precipitation polyethylene glycol in the fermented clear liquid and step (4) that are obtained in step (1)
The cellulase solution being collected into determines its cellulase activity with filter paper enzyme activity assay method respectively.
3. in a kind of fermentation of Aspergillus niger liquid from ocean according to claim 1 recycled fiber element enzyme method, its feature exists
In the consumption of tannin is added according to the consumption of 10g/L in fermented clear liquid in the step (2).
4. in a kind of fermentation of Aspergillus niger liquid from ocean according to claim 1 recycled fiber element enzyme method, its feature exists
In in the step (2) and step (4), the rotating speed of stirring is 80r/min, and mixing time is 10min.
5. in a kind of fermentation of Aspergillus niger liquid from ocean according to claim 1 recycled fiber element enzyme method, its feature exists
In in the step (3) and step (5), the rotating speed of centrifugation is 4000r/min, and centrifugation time is 20min.
6. in a kind of fermentation of Aspergillus niger liquid from ocean according to claim 1 recycled fiber element enzyme method, its feature exists
In in the step (4), the concentration of polyglycol solution is 20g/L, and solute is the PEG that molecular weight is 6000, and solvent is nature
Water.
7. in a kind of fermentation of Aspergillus niger liquid from ocean according to claim 1 recycled fiber element enzyme method, its feature exists
In in the step (4), the redissolution volume of polyglycol solution is according to depending on the 1/10 of its original fermentation liquor volume.
8. in a kind of fermentation of Aspergillus niger liquid from ocean according to claim 1 recycled fiber element enzyme method, its feature exists
In the condition of vacuum freeze drying in the step (5)：Vacuum is 9-19Pa, and temperature is -50 DEG C~-80 DEG C, is vacuumized
Untill 12-36h is when powdered no moisture.
9. the application according to any described methods of claim 1-8 in recycled fiber element enzyme in the fermentation of Aspergillus niger liquid of ocean.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876648A (en) * | 2012-10-25 | 2013-01-16 | 天津工业生物技术研究所 | Method for extracting cellulase from microorganism fermentation broth |
| CN105838697A (en) * | 2016-01-18 | 2016-08-10 | 浙江大学舟山海洋研究中心 | Fermentation method for producing salt-tolerant cellulase from marine aspergillus niger |
-
2017
- 2017-02-15 CN CN201710080722.6A patent/CN106801045A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876648A (en) * | 2012-10-25 | 2013-01-16 | 天津工业生物技术研究所 | Method for extracting cellulase from microorganism fermentation broth |
| CN105838697A (en) * | 2016-01-18 | 2016-08-10 | 浙江大学舟山海洋研究中心 | Fermentation method for producing salt-tolerant cellulase from marine aspergillus niger |
Non-Patent Citations (1)
| Title |
|---|
| 吴慧清等: "丹宁-聚乙二醇法提取纤维素酶的研究", 《食品与发酵工业》 * |
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Application publication date: 20170606 |