CN1272430C - Method or improving alkaline pectase enzyme activity in preparing alkaline pectase process by fermenting method - Google Patents
Method or improving alkaline pectase enzyme activity in preparing alkaline pectase process by fermenting method Download PDFInfo
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- CN1272430C CN1272430C CN 200410065807 CN200410065807A CN1272430C CN 1272430 C CN1272430 C CN 1272430C CN 200410065807 CN200410065807 CN 200410065807 CN 200410065807 A CN200410065807 A CN 200410065807A CN 1272430 C CN1272430 C CN 1272430C
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Abstract
The present invention provides a method of improving the enzyme activation of an alkaline pectic enzyme during the process of preparing the alkaline pectic enzyme by a fermentation method, which belongs to the technical field of preparing biological enzymes. In the present invention, the alkaline pectic enzyme of high enzyme activity is obtained by controlling the transfer speed and the coefficient (K<L>a) of volume dissolved oxygen of oxygen in fermentation liquid; the proper coefficient of volume dissolved oxygen is attained by controlling the rotation speed of stirring; thus, the alkaline pectic enzyme of high enzyme activity are prepared and obtained by submerged fermentation of liquid, and the enzyme activity reaches 40 u/ml (which does not reported domestically). The method of the present invention has the advantages of high activity and low cost of the prepared alkaline pectic enzyme, short fermentation period, convenient operation method and easy industrialization. The fermented and prepared alkaline pectic enzyme uses lyase of polygalacturonase as a main ingredient, and can be used for refined processing of cotton textiles. The present invention effectively changes the existing traditional refined processing technology of cotton textiles and improves environments and the quality of products.
Description
Technical field
Fermentation method prepares the method that raising alkaline pectase enzyme is lived in the alkaline pectin enzyme process, belongs to the biological enzyme preparing technical field.The present invention relates to a kind of by the transmission speed-volume dissolved oxygen coefficient K of oxygen in fermented liquid in the control fermenting process
LThe a value improves the method that the alkaline pectase enzyme is lived, and obtains the alkaline pectase that high enzyme is lived.
Background technology
Polygalacturonase (pectinases) is that a class can be decomposed pectin substance in the plant tissue (by the D-galacturonic acid with α-1, the straight catenate polymkeric substance that 4 glycosidic links are connected to form) enzyme system, alkaline pectase then is meant the polygalacturonase that has greater activity in alkaline range, much more general finger polygalacturonic acid lyase (Polygalacturonate lyase, PGL).
At present, acid pectase has been widely used in the clarification of the extraction of pectin and drinks, and alkaline pectase has become vital biotechnological formulation in the cotton fabric treating processes especially except that being applied at aspects such as the purifying of plant virus, association with pulp bleaching.Because the natural impurity that exists in the cotton fibre, in traditional technology, the general high temperature highly basic that uses is handled cotton fabric, cotton fibre can be damaged because of forming SURGICEL on the one hand, the cotton fibre surface loses due feel because of the wax of having removed a large amount of lubricates, it will produce a large amount of trade effluents what is more important, and to the negative impact that environment produced, so traditional technology no longer adapts to the development of entire society.Solution to this problem just is the friendly type dyeing and finishing technology of development environment, promptly green dyeing and finishing technology, and wherein Applied Biotechnology is one of important channel.
At present, the 3E system that advocate in Europe (usefulness Efficient, economic Economy, ecological Ecology), 3R production mechanism (a secondary accuracy Right first time, rapid reaction Rapid responsible, reproduction Reproducibility), 4R principle (save Reduction, reclaim Recovery, reuse Reuse, circulation Recycle) have become the main flow of world textile dyeing and finishing industrial technology development, Future Society except the content of this invariability of quality product, is more emphasized the eubiosis and to the affinity of environment to the requirement of textile industry.At present, the application of zymin be the most ripe in the green dyeing and finishing processing also be the most universal technology.Enzyme is being brought into play important effect in a plurality of technical process of dyeing and finishing processing, and its utilisation technology is still improving constantly, and Application Areas continues expansion.Can believe that along with environmental protection consciousness and the continuous reinforcement that requires, in the weaving processing industry in future, enzyme will have its more wide development space and more positive development prospect.
Since Japanese scholar Koki Horikoshi in 1972 makes alkaline pectase with alkaliphile first, so far isolation identification goes out tens kinds of alkaline pectases, but wherein can be applied to the few in number of suitability for industrialized production, only Novo Nordisk company once developed the brand of producing with the genetic engineering modified bacterial classification alkaline pectase for ' BioPrep ' in 1999, released a kind of alkaline pectate lyase ScourzymeL subsequently again in 2003.But because the cost problem is not widely used so far.Therefore improving production of enzyme by certain method and reduce production costs is the key of exploitation, using basic polygalacturonase
Summary of the invention
The purpose of this invention is to provide a kind of fermentation method and prepare the method that raising alkaline pectase enzyme is lived in the alkaline pectin enzyme process, by control volume dissolved oxygen coefficient (K
LA) improve the alkaline pectase enzyme and live, alkaline pectin enzymic activity height, cost that this method makes are low, and fermentation period is short simultaneously, working method is easy, be easy to industrialization.The alkaline pectase of preparation is a main component with the polygalacturonic acid lyase, can be used for the refining treatment of cotton fabric.
Technical scheme of the present invention: the present invention separates from fruit surface and obtains a strain and produce the alkaline pectase bacterial strain, classification called after genus bacillus (Bacillus sp) WSHB04-02, be deposited in Wuhan China typical culture collection center on November 27th, 2004, deposit number is CCTCC NO:M204082.
Adopting genus bacillus CCTCC NO:M204082 is starting strain, makes alkaline pectase through seed culture and 7L fermentor tank liquid submerged fermentation, by the volume dissolved oxygen coefficient K in the control fermented liquid
LA,, K
LA is 200h
-1-500h
-1, obtain the alkaline pectase that high enzyme is lived.Its fermenting process is:
Seed culture: get the bacteria suspension of 1ml-70 ℃ of refrigeration, be inoculated in the 500ml triangular flask that the 50ml seed culture medium is housed and carry out seed culture;
The fermentation of 7L jar: the inoculum size according to 8% inserts in the fermention medium, carries out fermentation culture in the 7L fermentor tank.
Seed culture medium (g/L): corn steep liquor 30, sucrose 20, peptone 20, dipotassium hydrogen phosphate 18.4, potassium primary phosphate 6, pH7.0; Culture condition: incubation time is 10h～18h, and 30 ℃～40 ℃ of temperature are shaken a bottle rotating speed 200rpm.
Fermention medium (g/L): W-Gum 6～l0, yeast extract paste 8～12, peptone 6～10, dipotassium hydrogen phosphate 8～10, potassium primary phosphate 2～4, pH7.0; Fermentation condition: liquid amount 3.5L～4.5L, inoculum size 8%, fermentation time 20h～30h, 30 ℃～40 ℃ of temperature, air flow 3.5L/min～4.5L/min, K
LA is 200h
-1～500h
-1, with K
LA is 350h
-1Best.
The application of genus bacillus CCTCC NO:M204082 fermentation gained alkaline pectase: prepared alkaline pectase enzyme work reaches 40U/ml, can be used for the refining processing of cotton textiles.
On-line parameter is measured
PH, dissolved oxygen (DO) adopt electrode to show that automatically DO is relative dissolved oxygen level, that is: the saturated dissolved oxygen level when substratum not being inoculated is decided to be 100%, and saturated sodium sulfite solution is decided to be 0%.
Offline parameter is measured
K
LA-is the volume dissolved oxygen coefficient (h of impellent with difference in oxygen concentration between in the solution
-1): in the clear water system, measure with sulphite process
Dry cell weight: get a certain amount of fermented liquid, under the 10000r/min condition centrifugal 5 minutes, remove supernatant liquor, place 105 ℃ of baking ovens to dry to constant weight thalline, weigh.
Reducing sugar: 3,5-dinitrosalicylic acid sodium (DNS) method, get a certain amount of fermented liquid, under the 10000r/min condition centrifugal 5 minutes, go precipitation, remove the supernatant liquor 2ml after ionized water dilutes, to wherein adding DNS reagent 1.5ml, reacting by heating 5 minutes in boiling water bath is cooled to room temperature with mobile cold water then immediately immediately, again to wherein adding distilled water 21.5ml, measure its absorbance behind the mixing at spectrophotometer 520nm place.
Alkaline pectase (PGGL) activity: get a certain amount of fermented liquid, under the 10000r/min condition centrifugal 5 minutes, go precipitation, the supernatant liquor 20 μ l that remove after ionized water dilutes join glycine-sodium hydrate buffer solution (pH9.4) that 2ml contains 0.2% polygalacturonic acid, 45 ℃ of reaction 15min, phosphoric acid solution with 3ml 0.03M stops enzyme reaction, measures its absorbance at spectrophotometer 235nm place.
A standard enzyme unit alive (1U) is defined as: per minute makes the polygalacturonic acid cleavage produce the enzyme amount of the unsaturated galacturonic acid of 1 μ mol.Adopt unsaturated galacturonic acid to determine in the characteristic absorbance at 235nm place.
Beneficial effect of the present invention: screening has obtained a kind of high yield bacterium of alkaline pectase, by changing different mixing speed, reach different volume dissolved oxygen coefficients, through liquid submerged fermentation, thereby prepare the alkaline pectase that high enzyme is lived, enzyme work reaches 40U/ml (domestic not appearing in the newspapers); By changing different mixing speed, make fermentation period shorten greatly simultaneously, saved production cost simultaneously from enzyme work and two aspects of fermentation period, working method is easy, is easy to industrialization, for suitability for industrialized production is laid a good foundation.The alkaline pectase of fermentative preparation can be used for the refining processing of cotton textiles, changes existing traditional cotton textiles refining processing technology effectively, improves environment and cotton textile quality.
Description of drawings
Fig. 1 is volume dissolved oxygen coefficient K in the alkaline pectin enzymic fermentation
LA is to the influence of enzyme work and fermentation period
The biological material specimens preservation
Genus bacillus, preservation date on November 27th, 2004, depositary institution's title and abbreviation: Chinese typical culture collection center C CTCC, deposit number is NO:M204082.
Embodiment
Embodiment 1
With the bacterial suspension inoculation of 1ml-70 ℃ of preservation in seed culture medium, the seed culture medium liquid amount is the bottled 50ml seed culture medium of the triangle of 500ml, through 37 ℃, the rotary type shaking table of 200rpm was cultivated after 14 hours, inoculum size according to 8% is inoculated in the 7L fermentor tank, and the fermention medium liquid amount is the canned 4L fermention medium of the fermentation of 7L.Fermentation condition is: 37 ℃ of leavening temperatures, air flow 4L/min is with mixing speed 600rpm control K
LA is 350h
-1, fermentation unit adopts automatic antifoaming system.Whole process adopts the various parameters of the method off-line analysis fermented liquid of timing sampling, comprises the parameters such as amount of enzyme work, dry cell weight and reducing sugar.Under this condition, its fermentation period is 8 hours, and the final enzyme work of fermenting reaches 40U/ml, and fermenting process is seen Fig. 1.
Embodiment 2
Changing mixing speed is 500rpm control K
LA is 200h
-1, all the other conditions are with embodiment 1, and under this operational condition, fermentation period is 12 hours, and the final enzyme that ferments is lived and is 39U/ml, and fermenting process is seen Fig. 1.
Embodiment 3
Changing mixing speed is 700rpm control K
LA is 500h
-1, all the other conditions are with embodiment 1, and under this operational condition, fermentation period is 12 hours, and the final enzyme that ferments is lived and is 34U/ml, and fermenting process is seen Fig. 1.
The comparative example 4
Changing mixing speed is 400rpm control K
LA is 100h
-1, all the other conditions are with embodiment 1, and under this operational condition, fermentation period is 16-24 hour, and the final enzyme that ferments is lived and is 30U/ml, and fermenting process is seen Fig. 1.
Claims (3)
1. one kind is produced the alkaline pectase bacterial strain, its classification called after genus bacillus (Bacillus sp.) WSHB04-02, and deposit number is CCTCC NO:M204082.
2. prepare at fermentation method and improve the method that the alkaline pectase enzyme is lived in the alkaline pectin enzyme process, it is characterized in that by the volume dissolved oxygen coefficient K in the control fermented liquid
LA obtains the alkaline pectase that high enzyme is lived, K
LA is 200-h
-1-500h
-1, its fermenting process is:
Fermentation strain: genus bacillus CCTCC NO:M204082;
Seed culture: get the bacteria suspension of 1ml-70 ℃ of refrigeration, be inoculated in the 500ml triangular flask that the 50ml seed culture medium is housed and carry out seed culture;
Seed culture medium is formed in g/L: corn steep liquor 30, sucrose 20, peptone 20, dipotassium hydrogen phosphate 18.4, potassium primary phosphate 6, pH7.0; Culture condition: incubation time is 10h～18h, and 30 ℃～40 ℃ of temperature are shaken a bottle rotating speed 200rpm;
The fermentation of 7L jar: the inoculum size according to 8% inserts seed liquor in the fermention medium, carries out fermentation culture in the 7L fermentor tank;
Fermention medium is formed in g/L: W-Gum 6～10, yeast extract paste 8～12, peptone 6～10, dipotassium hydrogen phosphate 8～10, potassium primary phosphate 2～4, pH7.0; Fermentation condition: liquid amount 3.5L～4.5L, inoculum size 8%, fermentation time 20h～30h, 30 ℃～40 ℃ of temperature, air flow 3.5L/min～4.5L/min, K
LA200h
-1～500h
-1
3. the method that raising alkaline pectase enzyme according to claim 2 is lived, K wherein
LA is 350h
-1
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157900B (en) * | 2007-09-04 | 2011-07-27 | 江南大学 | Alkaline pectic enzyme producing engineering strain and its construction and method for producing alkaline pectic enzyme with the same |
| CN102911956B (en) * | 2012-08-07 | 2014-05-14 | 江南大学 | Optimized alkaline pectinase gene and application thereof |
| CN106047745A (en) * | 2016-05-10 | 2016-10-26 | 西北农林科技大学 | A microorganism and applications thereof |
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