CN102660527B - Method for producing high-enzyme-activity pectate lyase by means of raw material pretreatment and feed fermentation - Google Patents

Method for producing high-enzyme-activity pectate lyase by means of raw material pretreatment and feed fermentation Download PDF

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CN102660527B
CN102660527B CN 201210172315 CN201210172315A CN102660527B CN 102660527 B CN102660527 B CN 102660527B CN 201210172315 CN201210172315 CN 201210172315 CN 201210172315 A CN201210172315 A CN 201210172315A CN 102660527 B CN102660527 B CN 102660527B
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alkaline pectase
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CN102660527A (en
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邹谋勇
李雪芝
赵建
曲音波
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Shandong University
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Abstract

The invention discloses a method for producing high-enzyme-activity pectate lyase by means of raw material pretreatment and feed fermentation. The method is implemented by liquefying treatment and feed-batch fermentation for starch in a feed culture medium. The method obviously increases unit volume enzyme activity and volume production efficiency of the pectate lyase. Due to enzyme liquefaction treatment to the starch in the feed culture medium, viscosity coefficients of liquid are greatly reduced, and stirring efficiency is improved. Logarithmic growth phase of fungi is prolonged by means of feed fermentation, and biomass liveweight is increased. Experiments show that fermentation coarse enzyme fluid of the pectate lyase with the poly galacturonic acid cracking enzyme activity of 531UmL-1 is obtained by the method, and the highest volume production efficiency reaches 13U(mL*h)-1. The method further has the advantages of low cost, short fermenting period, high equipment utilization rate, simple process and suitability for large-scale industrial production.

Description

Utilize raw materials pretreatment and fed-batch fermentation to produce the method for high enzyme alkaline pectase alive
Technical field
The present invention relates to a kind of live method of alkaline pectase of high enzyme of producing, relate in particular to and a kind ofly produce the live method of alkaline pectase of high enzyme by the starch in the supplemented medium being carried out liquefaction processing and fed-batch fermentation, belong to the biological enzyme preparing technical field.
Background technology
Polygalacturonase (pectinases) is that the enzyme that a class can be decomposed vegetable cell wall fraction pectin substance is general name, polygalacturonase can be divided into pectin hydrolase and pectin lyase from is olation, the former mainly comprises protopectinase (PPase), Rohapect MPE (PE), oligomeric galactose aldehydic acid lytic enzyme, poly-methyl galacturonate enzyme (PMG), polygalacturonase (PG); The latter mainly comprises polygalacturonic acid lyase (PGL) and poly-methyl galacturonate lyase (PMGL).The normal alkaline pectase that claims of people refers generally to the polygalacturonic acid lyase in the pectin lyase.Pectin lyase disconnects glycosidic link by trans-elimination cracking pectin polymer in the C-4 position, simultaneously at H atom of C-5 place cancellation.Thereby produce a Δ 4:5 unsaturated link(age).
At present for the existing a large amount of bibliographical information of the research of polygalacturonase, wherein the long acid pectase of search time is mainly used in the extraction of pectin fraction and the clarification of fruit wine fruit juice, alkaline pectase is at pulping bleaching, application in the cotton fabric treatment process has obtained certain progress, and particularly the applied research on the coming unstuck of bast-fibre increases day by day.Raw ramie is a kind of sheet bar that is bonded together by various colloids, and ultimate fibre is difficult for separately, therefore must be to the raw ramie processing of coming unstuck before the combing spinning.The mode of coming unstuck for a long time mainly is chemical Degumming, and its ultimate principle is to utilize stable different to remove the colloid composition in the raw ramie to alkali, mineral acid and oxygenant of Mierocrystalline cellulose and colloid composition in the raw ramie.The shortcoming of this method is that violent chemical treatment may cause flaxen fiber impaired, influences the fine quality of linen, consumes a large amount of industrial chemicals and heat energy simultaneously, causes serious environmental to pollute.In recent years, biological degumming has received more concern, and it mainly comprises microbial degumming, and enzyme comes unstuck and the biological-chemical combination degumming.Wherein enzyme comes unstuck by force because having flexibility of operation, and process is controlled easily, is convenient to add advantages such as boil-off assistant and the emphasis that becomes research.
Aspect the production of alkaline pectase, but Japanese scholar Koki Horkoshi in reported first in 1972 alkaliphile secreted alkaline polygalacturonase, Junwei Cao, T.SaKai, Tohru Kobayashi etc. also in succession separation screening to the bacterial strain that produces alkaline pectase.China was studied alkaline pectase since the beginning of the nineties, and the generation bacterium of having reported comprises Erwinia, Alkaliphilic bacillus, subtilis, spiral shell spore bacterium etc., but research work mainly rests on bacterial screening and to the research of zymologic property; Aspect the fermentation research of alkaline pectase, rest on mostly and shake a bottle condition optimizing, in the initial optimization research of small-sized fermentation jar condition of enzyme production.Liu Xi etc. optimize the condition of enzyme production of high-production alkalescence pectinase subtilis, make enzyme activity bring up to 14.82U mL -1(optimization of alkaline pectase superior strain condition of enzyme production. biotechnology, 2008,18 (6): 71-74.).Jiang Hongju etc. optimize substratum and the fermentation culture conditions of Paenibacillus polymyxa fermentation product alkaline pectase, make enzyme work reach 311U mL -1(research of alkaline pectase fermentation condition is produced in Paenibacillus polymyxa 20185 liquid state fermentations. and China brewages, and 2011,235:111-114.).Dong Yunzhou etc. adopt the temperature control strategy that alkaline pectase is carried out fermentation optimization in the small-sized fermentation jar, and the enzyme work that makes genus bacillus WSH03-09 produce alkaline pectase reaches 5.99U mL -1(fermentation of bacillus produces alkaline pectase temperature control strategy. use and the environmental organism journal 2005,11 (3): 359-362.).Liu Huijuan etc. have studied the influence that differing temps (32-41 ℃) is produced the alkaline pectase kinetic parameter to genus bacillus WSHB04-02 batch fermentation, can make the enzyme work of alkaline pectase reach 53.0U mL behind the temperature optimization -1, production intensity is 3315U (hL) -1(fermentation of bacillus is produced the temperature control strategy of alkaline pectase. process engineering journal, 2007,7 (4): 786-789.).Chinese patent ZL 03131952.1 discloses the bacillus pumilus that alkaline pectase is produced in a strain, and it is 20UmL that liquid submerged fermentation obtains the alkaline pectase enzyme activity -1Chinese patent ZL 200410065807.X discloses a kind of by the transfer rate volume dissolved oxygen coefficient (K of oxygen in fermented liquid LA) method of raising alkaline pectin enzyme activity, its enzyme activity has reached 40U mL -1But alkaline pectin enzyme activity and the industrial requirement of above-mentioned report also have a segment distance.Li Zuming etc. to gram Lloyd's genus bacillus carried out ultraviolet mutagenesis breeding and solid state fermentation conditions optimization (breeding of high-production alkalescence pectinase bacterial strain and the research of fermentation condition thereof. industrial microorganism, 2008,38 (3): 27-31.); Chinese patent ZL 94105708.9, CN 101096650B, ZL 200410073818.2, CN 101235361A disclose the method that the different strains solid fermentation is produced alkaline pectase, because the enzyme activity determination method differs, enzymatic productivity can't direct visual comparison; Simultaneously also rarely seen its is applied to industrial production because solid fermentation apparatus requires high, complex process.ZL97109044 discloses the method for a strain Alkaliphilic bacillus and China grass degumming thereof, but since usually time long (16-24h), the low industrial applications that also is difficult to of pectinase activity.ZL01106844.2 discloses a strain alkaline pectin enzyme-producing bacteria CCTCC NO:M200038, and the gained enzyme liquid that will ferment is applied to China grass degumming, fails to be applied to suitability for industrialized production owing to its fermenting enzyme vigor is lower.Therefore further optimization production technology, by zymotechnique control and optimization and improvement alkaline pectase enzyme activity and volume production efficiency, reducing the production cost of alkaline pectase, is the key that technological development and engineering are amplified, and also is the prerequisite that promotes the alkaline pectase industrial applications.Particularly deep liquid fermentation process optimization and the control for high concentration substrate seems particularly important.The report of the alkaline pectase of high yield adopts solid fermentation (high concentration of substrate) more at present, but its follow-up separation and purification process complexity, the zymoprotein yield is lower, has increased production cost.And high concentration substrate liquid submerged fermentation production alkaline pectase exists stirring efficiency low, problems such as mass transfer effect difference.The fed-batch fermentation technology has obtained in the time using widely reducing just the fermented liquid concentration of substrate and prolong enzymatic production, and it has played vital role to fermentation middle and later periods cell growth division, keeping of cell viability, can effectively improve or the stabilized enzyme protein yield.In the methods involving that retrieves, about yet there are no report by the method for raw material being carried out pre-treatment and fed-batch fermentation production alkaline pectase.
Summary of the invention
In prior art, the alkaline pectin enzyme activity that liquid fermenting produces is low, and the cost height is not suitable for the deficiency of suitability for industrialized production, the purpose of this invention is to provide a kind of raw materials pretreatment and fed-batch fermentation of utilizing and produces the live method of alkaline pectase of high enzyme.
The method of utilizing raw materials pretreatment and fed-batch fermentation to produce high enzyme alkaline pectase alive of the present invention, be to improve alkaline pectase enzyme activity and production efficiency thereof by the starch in the supplemented medium being carried out liquefaction processing and fed-batch fermentation, its step is as follows:
(1) seed culture: under aseptic condition, activated inclined plane bacterial classification subtilis CCTCC NO:M200038, and be seeded to and carry out seed culture in the 50ml seed culture medium, 7-9h is cultivated in 36-37 ℃ of shaking table concussion;
(2) fermentation just: seed culture fluid is seeded to the 7.5L fermentor tank that fermention medium is housed, carries out fermentation culture, fermentor tank liquid amount 3-4L, inoculum size 10%, temperature 33-34 ℃, air flow 2.5-4.5L min -1
(3) the starch pre-treatment in the supplemented medium: warm amylase carries out liquefaction processing to the starch in the supplemented medium in using, and enzyme concentration is 20-45U g -1, liquefaction temperature is 45-65 ℃, liquefying time is 20-40min;
(4) fed-batch fermentation: just ferment to 13-19h, add pretreated supplemented medium by first fermention medium volume 10-25%, continue fermentation, temperature is 33-34 ℃, air flow 2.5-4.5L min -1, fermentation period is 36-48h;
(5) crude enzyme liquid preparation: after fermentation finished, with the centrifugal 10min of fermented liquid 12000rpm, supernatant liquor was the crude enzyme liquid that contains alkaline pectase;
Above-mentioned seed culture medium is formed: glucose 8-13g L -1, yeast powder 3-6g L -1, peptone 3-6g L -1, NaCl 3-6g L -1, K 2HPO 48-12g L -1, pH 8.0;
Above-mentioned fermention medium is formed: starch 15-25g L -1, wheat bran 35-45g L -1, (NH 4) 2SO 41-2g L -1, MgSO 47H 2O 0.5-1.5g L -1
Above-mentioned supplemented medium is formed: starch 110-135g L -1, wheat bran 8-13g L -1, (NH 4) 2SO 46-14g L -1, MgSO 47H 2O 5-8g L -1
Above-mentioned utilization utilizes raw materials pretreatment and fed-batch fermentation to produce in the method for high enzyme alkaline pectase alive:
The preferred 115-130g L of starch concentration in the described supplemented medium -1
The preferred 25-40U g of warm amylase enzyme concentration during step (3) is described -1, the preferred 50-60 of liquefaction temperature ℃, the preferred 25-35min of liquefying time.
Step (4) is described to be added after the pre-treatment time of supplemented medium and is preferably and just ferments to 14-18h; Additional amount is preferably the 10-20% of fermention medium volume just, the optimum 20% disposable supplemented medium of adding by first fermention medium volume.
The on-line parameter that relates in the aforesaid method is measured
Dissolved oxygen (DO), pH, temperature adopt the on-line automatic control of electrode, and DO is relative dissolved oxygen level, that is: the 30min that ventilated before substratum is not inoculated makes the substratum dissolved oxygen level that reaches capacity, this moment DO to demarcate be 100%, it is 0% that the saturated sodium sulfite dissolved oxygen is demarcated.
The offline parameter that relates in the aforesaid method is measured
Biomass: get fermented liquid 1mL(with the centrifugal back of fermented liquid supernatant in contrast) the perchloric acid 1mL of adding 1M in colorimetric cylinder, behind the boiling water bath 20min, go to the centrifugal 10min of 10000rpm in the centrifuge tube, get supernatant 1ml constant volume to 10mL, measure its light absorption value in ultraviolet spectrophotometer 260nm place and measure light absorption value, reference standard curve calculation dry cell weight.The standard curve making method is: dilution obtains the concentration gradient thalline, measures OD respectively 260, and corresponding weighing dry cell weight, be X-axis with the dry cell weight, OD 260Be Y-axis drawing standard curve.
Alkaline pectase (PGL) activity: get the 5mL fermented liquid, with 1200rpm, 4 ℃ of centrifugal 10min get supernatant and get 20 μ L after with pH 9.6Gly-NaOH damping fluid dilution certain multiple and add 2mL 0.2%(w/v) the polygalacturonic acid solution (with pH 9.6, contain 0.44mM CaCl 2, the configuration of 0.05M Gly-NaOH damping fluid), 45 ℃ of reaction 15min with 3mL0.03M phosphoric acid solution termination reaction, measure its light absorption value in ultraviolet spectrophotometer 235nm place.
A standard enzyme unit of activity (IU) is defined as: the unit time (1min) makes the polygalacturonic acid cleavage produce the enzyme amount of 1 μ mol unsaturated polyester galacturonic acid.Wherein the molar absorptivity of unsaturated galacturonic acid at wavelength 235nm place is 4600(M).
Beneficial effect of the present invention:
By adopting amylase that the high density starch in the supplemented medium is carried out liquefaction processing, effectively reduce the viscosity factor of feed supplement liquid, improved flowability and the oxygen carrying capacity of feed liquid, reduced fermentor tank and stirred resistance, for pump flow feeding substratum provides possibility; Improve dissolving sugar concentration in the substrate simultaneously, make cell obtain nutrition rapidly, division growth has improved the growth velocity of subtilis fast.Prolong the thalli growth time by fed-batch fermentation, improve biomass, prolong effective integration time of alkaline pectase; Make the unit enzyme activity of alkaline pectase reach 531U mL -1, volume production efficiency reaches 13U (mlh) -1(not appearing in the newspapers both at home and abroad).This method has shortened fermentation period greatly, thereby has improved the fermentor tank utilising efficiency, has reduced production cost; Operation is simple for this method, for suitability for industrialized production has been established solid basis.The alkaline pectase of fermentative production just enzyme liquid can be used for processings of coming unstuck of fibrilia raw material, can effectively shorten even saves the chemical Degumming operation, improves the quality of flaxen fiber product, and the minimizing waste liquid is to the pollution of environment.
Description of drawings
Fig. 1 is that fed-batch fermentation is to the influence of alkaline pectin enzyme activity.
Fig. 2 is that influence to the alkaline pectin enzyme activity is handled in starch material amylase liquefaction in the supplemented medium.
Embodiment
Embodiment 1
Subtilis CCTCC NO:M200038(applicant is preserved in Chinese typical culture center (CCTCC) on November 20th, 2000, relevant patent is seen ZL01106844.2) through slant activation, provoke lawn be seeded to be equipped with seed culture medium shake the bottle cultivate, culture condition is: 300ml triangular flask liquid amount is 50ml, 37 ℃ of leavening temperatures, 8h is cultivated in the 200rpm concussion.With the first fermention medium of inoculum size 10% inoculation, temperature 33-34 ℃, air flow 2.5-4.5L min -1With middle temperature amylase the starch in the supplemented medium is carried out liquefaction processing, enzyme concentration is 25U g -1, liquefaction temperature is 60 ℃, liquefying time is 35min.Just ferment to 14h, add the later supplemented medium of pre-treatment by 20% of first fermention medium volume, 33-34 ℃ of control temperature, air flow 2.5-4.5L min -1Fermentation period is 36-48h.After fermentation finished, with the centrifugal 10min of fermented liquid 12000rpm, supernatant liquor was the crude enzyme liquid that contains alkaline pectase; Through measuring, the high alkalinity pectinase activity of described crude enzyme liquid is 465U mL -1Fermenting process is seen Fig. 1.Wherein culture medium prescription is as follows:
Seed culture medium is formed: glucose 8g L -1, yeast powder 3g L -1, peptone 3g L -1, NaCl 3g L -1, K 2HPO 48g L -1, pH 8.0;
Fermention medium is formed: starch 15g L -1, wheat bran 35g L -1, (NHz 4) 2SO 41g L -1, MgSO 47H 2O 0.5gL -1
Supplemented medium is formed: starch 115g L -1, wheat bran 8g L -1, (NH 4) 2SO 46g L -1, MgSO 47H 2O 5g L -1
Embodiment 2
Fermentation process, condition are except following change, and all the other are with embodiment 1.In the preprocessing process, middle temperature amylase enzyme concentration is 32.5U g -1, liquefaction temperature is 55 ℃, liquefying time is 30min.Just ferment to the 16h feed supplement.Fermentation period is 36-48h, and the high alkalinity pectinase activity of the crude enzyme liquid of acquisition is 531U mL -1Fermenting process is seen Fig. 1.Wherein culture medium prescription is as follows:
Seed culture medium is formed: glucose 11g L -1, yeast powder 4,5g L -1, peptone 4.5g L -1, NaCl 4.5g L -1, K 2HPO 410g L -1, pH 8.0;
Fermention medium is formed: starch 20g L -1, wheat bran 40g L -1, (NH 4) 2SO 41.5g L -1, MgSO 47H 2O 1gL -1
Supplemented medium is formed: starch 122.5g L -1, wheat bran 10.5g L -1, (NH 4) 2SO 410g L -1, MgSO 47H 2O6.5g L -1
Embodiment 3
Fermentation process, condition are except following change, and all the other are with embodiment 1.In the preprocessing process, middle temperature amylase enzyme concentration is 40U g -1, liquefaction temperature is 50 ℃, liquefying time is 25min.Just ferment to the 18h feed supplement.Fermentation period is 36-48h, and the final alkaline pectin enzyme activity of the crude enzyme liquid of acquisition is 479UmL -1Fermenting process is seen Fig. 1.Wherein culture medium prescription is as follows:
Seed culture medium is formed: glucose 13g L -1, yeast powder 6g L -1, peptone 6g L -1, NaCl 6g L -1, K 2HPO 412g L -1, pH 8.0;
Above-mentioned fermention medium is formed: starch 25g L -1, wheat bran 45g L -1, (NH 4) 2SO 42g L -1, MgSO 47H 2O 1.5g L -1
Above-mentioned supplemented medium is formed: starch 130g L -1, wheat bran 13g L -1, (NH 4) 2SO 414g L -1, MgSO 47H 2O8g L -1
Embodiment 4
Fermention medium and supplemented medium are mixed, carry out batch fermentation (being not carry out feed supplement in the process), seed activation, cultivation, inoculum size, leavening temperature and air flow are identical with embodiment 1.Fermentation 48h, the final alkaline pectin enzyme activity of the crude enzyme liquid of acquisition is 203U mL -1Fermenting process is seen Fig. 1.Compare with embodiment 1,2,3, the crude enzyme liquid alkaline pectase enzyme activity that present embodiment obtains is obviously lower.
Embodiment 5
Seed activation, cultivation and inoculum size are carried out fed-batch fermentation experiment, 122.5g L in the experimental group supplemented medium with embodiment 1 at the 500mL triangular flask -1High density starch with in warm amylase carry out liquefaction processing, enzyme concentration is 45U g -1, liquefaction temperature is 65 ℃, liquefying time is 40min.Handle in contrast not carry out amylase.This condition bottom fermentation 60h, the crude enzyme liquid alkaline pectase enzyme activity of acquisition reaches 347U ml -1Fermenting process is seen Fig. 2.
Embodiment 6
Fermentation process, condition are except following change, and all the other are with embodiment 1, and starch concentration is 110g L in the supplemented medium -1, middle temperature amylase enzyme concentration is 20U g -1, liquefaction temperature is 60 ℃, liquefying time is 20min.Just ferment to the 16h feed supplement.Fermentation period 36-48h, the high alkalinity pectinase activity of the crude enzyme liquid of acquisition is 437U mL -1
Embodiment 7
Fermentation process, condition are except following change, and all the other are with embodiment 1, and starch concentration is 135g L in the supplemented medium -1, middle temperature amylase enzyme concentration is 45U g -1, liquefaction temperature is 45 ℃, liquefying time is 30min.Just ferment to the 16h feed supplement, add supplemented medium according to 25% first fermention medium volume.Fermentation period 36-48h, the high alkalinity pectinase activity of the crude enzyme liquid of acquisition is 421U mL -1
Embodiment 8
Fermentation process, condition are except following change, and all the other are with embodiment 1, just ferment to 14h to add supplemented medium by 10% of first fermention medium volume, and fermentation period 36-48h, the high alkalinity pectinase activity of the crude enzyme liquid of acquisition is 340U mL -1

Claims (4)

1. one kind is utilized raw materials pretreatment and fed-batch fermentation to produce the live method of alkaline pectase of high enzyme, and step is:
(1) seed culture: under aseptic condition, activated inclined plane bacterial classification subtilis CCTCC NO:M200038, and be seeded to and carry out seed culture in the 50ml seed culture medium, 7-9h is cultivated in 36-37 ℃ of shaking table concussion;
(2) fermentation just: seed culture fluid is seeded to the 7.5L fermentor tank that fermention medium is housed, carries out fermentation culture, fermentor tank liquid amount 3-4L, inoculum size 10%, temperature 33-34 ℃, air flow 2.5-4.5L min -1
(3) the starch pre-treatment in the supplemented medium: warm amylase carries out liquefaction processing to the starch in the supplemented medium in using, and enzyme concentration is 20-45U g -1, liquefaction temperature is 45-65 ℃, liquefying time is 20-40min;
(4) fed-batch fermentation: just ferment to 13-19h, add pretreated supplemented medium by first fermention medium volume 10-25%, continue fermentation, temperature is 33-34 ℃, air flow 2.5-4.5L min -1, fermentation period is 36-48h;
(5) crude enzyme liquid preparation: after fermentation finished, with the centrifugal 10min of fermented liquid 12000rpm, supernatant liquor was the crude enzyme liquid that contains alkaline pectase;
Above-mentioned seed culture medium is formed: glucose 8-13g L -1, yeast powder 3-6g L -1, peptone 3-6g L -1, NaCl 3-6g L -1, K 2HPO 48-12g L -1, pH 8.0;
Above-mentioned fermention medium is formed: starch 15-25g L -1, wheat bran 35-45g L -1, (NH 4) 2SO 41-2g L -1, MgSO 47H 2O 0.5-1.5g L -1
Above-mentioned supplemented medium is formed: starch 110-135g L -1, wheat bran 8-13g L -1, (NH 4) 2SO 46-14g L -1, MgSO 47H 2O 5-8g L -1
2. utilize raw materials pretreatment and fed-batch fermentation to produce the method for high enzyme alkaline pectase alive according to claim 1, it is characterized in that the starch concentration in the described supplemented medium is 115-130g L -1
3. utilize raw materials pretreatment and fed-batch fermentation to produce the method for high enzyme alkaline pectase alive according to claim 1, it is characterized in that warm diastatic enzyme concentration was 25-40U g during step (3) was described -1, liquefaction temperature is 50-60 ℃, liquefying time is 25-35min.
4. utilize raw materials pretreatment and fed-batch fermentation to produce the method for high enzyme alkaline pectase alive according to claim 1, it is characterized in that, the time that step (4) is described adds supplemented medium after the pre-treatment, additional amount was the 10-20% of first fermention medium volume in order just to ferment to 14-18h.
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