CN101412991B

CN101412991B - Method for preparing mannanase by using glycerol as carbon source and fedbatch of substrate for induction

Info

Publication number: CN101412991B
Application number: CN2008102291970A
Authority: CN
Inventors: 韩国宝; 王武良
Original assignee: LIAONING HUAXING BIOTECHOLOGY CO Ltd
Current assignee: LIAONING HUAXING BIOTECHOLOGY CO Ltd
Priority date: 2008-12-02
Filing date: 2008-12-02
Publication date: 2011-07-27
Anticipated expiration: 2028-12-02
Also published as: CN101412991A

Abstract

The invention relates to a preparation method for mannanase by induction of a substrate batch fed into a carbon source of glycerol, in particular to a fermentation production process for genetically engineered strain of the mannanase with Pichia stipitis as a carrier. The invention is characterized in that a seed culture solution is used for culture in a first-stage seed culture fermentor; a basic culture solution is used for culture in a second-stage seed fermentor; part of the basic culture solution is poured into a production fermentor for culture at initial time, and the glycerol, methanol and mannan substrate are respectively fed into a fermentation solution to finish the fermentation at a certain proportion and speed in the fermentation process. The yield of the mannanase can be obviously increased by 15 to 24 percent.

Description

With glycerine is that carbon source and current adding substrate are induced the method for preparing mannase

Technical field

The present invention relates to the fermentation technique of microbial product, is that carbon source and current adding substrate are induced the method for preparing mannase with glycerine.

Background technology

The method of traditional mode of production mannase is to utilize glucose to make carbon source.Glucose in the high-temperature sterilization process, produces caramelization as carbon source, makes the fermentation culture based sols be brown or color of soy sauce.Its drawback is: the first, and caramelization produces nuisance, is unfavorable for saccharomycetic growth; The second, the aftertreatment need of work decolouring of dark fermented liquid increases production process and expense.The 3rd, do not add the substrate mannosans and induce, productive rate is low.

Summary of the invention

The purpose of this invention is to provide a kind of is that carbon source and current adding substrate are induced the method for preparing mannase with glycerine, can significantly increase mannosans production of enzyme 15% ~ 24%.

Of the present invention is that carbon source and current adding substrate are induced the method for preparing mannase with glycerine, employing is done the fermentation manufacturing technique that carrier is produced the genetic engineering bacterium of mannase with pichia spp, with glucose is the yeast base culture base bacterial classification of carbon source, it is characterized in that: cultivate with the seed culture fluid that contains glucose, peptone and yeast powder in first order seed cultivation and fermentation jar; In secondary seed cultivation and fermentation jar, cultivate with glycerinated basic culture solution; In producing fermentor tank, the initial stage drops into the glycerinated basic medium of part and cultivates, and in each stage of fermenting process, flows glycerol adding, methyl alcohol and substrate mannosans respectively to fermented liquid, and adjusts flow velocity, to finish fermentation.

Effect after the process modification:

Substrate is the target of zymin effect, and mannosans is the substrate of mannase.Add the substrate mannosans during the fermentation and mainly play the effect of stimulation inductive.It is carbon source and current adding substrate mannosans that the inventive method replaces traditional glucose with glycerine, is being that the positively effect that is produced in the genetic engineering bacterium production technique of carrier production mannase is with the pichia spp:

A, being carbon source with glycerine aborning, and is that carbon source is compared with glucose, can significantly reduce the generation of metabolism repressor alcohol, acetic acid, thereby increase the amount of the mannase of final production.

B, use glycerine and produce as carbon source, because glycerine can not produce caramelization as carbon source in the high-temperature sterilization process, thus can improve the color of fermented liquid greatly, thus make the yeast can normal growth.

C, because glycerine be the color that carbon source has been improved fermented liquid greatly, the fermented liquid processing of need not decolouring of light color has reduced the fermentation liquor treatment expense, can reduce by 10% productive expense.

D, current adding substrate mannosans, can play stimulates the inductive effect, thereby has improved the final output of mannase.

E, utilize present method to produce mannase, finally producing the mannase amount is carbon source and the not production technique increase by 15～24% of current adding substrate mannosans than with glucose, and production cost only is the less than 90% of traditional technology cost.

Embodiment

A kind of is that carbon source and current adding substrate mannosans are induced the method for preparing mannase with glycerine, employing is done the genetic engineering bacterium fermentation manufacturing technique that carrier is produced mannase with pichia spp, it is characterized in that with glycerine being carbon source and current adding substrate mannosans, concrete operation is as follows:

One, substratum

1. seed culture medium: containing weight percent is glucose 3%, peptone 2%, and yeast powder 2%, surplus is a water;

2. basic medium: containing weight percent is glycerine 2%～10%, potassium primary phosphate 1%～5%, and vitriolate of tartar 1%～5%, purity is 85% phosphatase 11 %～5%, magnesium sulfate heptahydrate 1%～5%, surplus is a water.It is more than 99.8% that used all glycerine all require purity.

Two, zymotechnique

1, culture of strains

Adopt seed culture medium when spawn culture and first order seed fermentation.

A. get an amount of production with producing mannase spawn culture inclined-plane, insert in 3～4 1000ml triangular flasks interior dress 500ml seed culture medium respectively;

B. cultivate: 30～32 ℃, 250～350 rev/mins, grew 20～30 hours.

2, one grade fermemtation

A, packing in the stainless steel seeding tank accounts for 70% seed culture medium of tank volume, 121 ℃ of steam sterilizings, and 15min is cooled to 30 ℃;

B, get the cultured bottle bacterial classification that shakes of above-mentioned steps 1 and insert in the seed culture fluid;

C, cultivation: 30 ℃, 200～300 rev/mins, pH keeps a certain steady state value in 4.0～6.0 scopes, and air flow 0.5～2vvm grew 20～30 hours.

3, second order fermentation

A. packing in the stainless steel seeding tank accounts for the basic culture solution of tank volume 70%～75%, 121 ℃ of steam sterilizings, and 15min is cooled to 30 ℃;

B. when the secondary seed cultivation and fermentation and the starting stage of fermentative production all adopt basic culture solution.Under sterile state, insert above-mentioned steps 2 cultured strain liquids in the secondary seed jar;

C. cultivate: 30 ℃, 200～300 rev/mins, pH keeps a certain steady state value in 4.0～6.0 scopes, and air flow 0.5～2vvm grew 12～30 hours.

4, produce fermentation

Drop into the basic culture solution that is equivalent to fermentor tank volume 35～45% in a, the stainless steel fermentor tank, 121 ℃ of steam sterilizings, 15min is cooled to 30 ℃;

B, with above-mentioned steps 3 cultured strain liquids in sterile state inserts fermentor tank;

C, cultivation and fermentation:

(1) culture condition: 30 ℃, 180～250 rev/mins, constant ph 4.0～6.0 these scopes, air flow 0.5～2vvm;

(2) compound concentration 50% aqueous glycerin solution, the sterilization cooling is equipped with stream and adds usefulness;

(3) after glycerine consumption in the basic culture solution fully, (measuring residual sugar content with the glycerine meter is 0), in the flow velocity (with initial fermentating liquid volume is benchmark, down with omiting) of 10～40ml/h/l, stream adds the ready glycerine solution of above-mentioned step (2) to fermention medium;

(4) begin after stream adds methyl alcohol, reduce flow velocity to the 1～4ml/h/l of glycerine;

(5) whole glycerine stream added process lasting about 50～80 hours.

D. induce

(1) pichia spp produces gene induced dose of enzyme: methyl alcohol

(2) glycerine at the uniform velocity stream add 4h or after the longer time, to the bacterium turnout be 200～250g weight in wet base/L, and fermented liquid begins stream and adds methyl alcohol when not having the visible recombinant protein and producing, methyl alcohol stream adds process and lasts till that fermentation finished preceding 10～15 hours.

(3) methyl alcohol stream added-time employing concentration is the methyl alcohol more than 99.8%, and initial flow acceleration is 4～7ml/h/L.

(4) yeast is the carbon source continued growth with methyl alcohol, and methyl alcohol induces it to produce mannase as the inductor of Pichia yeast engineering simultaneously;

(5) along with the quick accumulation that continues to reach culture of cultivating, answer the stream dosage 8～10ml/h/l of corresponding increase methyl alcohol, and need to increase stirring and air flow and pressure boost, so that oxygen more is dissolved in the fermented liquid;

(6) methyl alcohol begins after stream adds 3～5h, when Pichia yeast engineering begins to produce mannase, beginning and according to the vigor of mannase, with the speed of 0.5～1ml/h/l simultaneously stream add 0.002～0.5% mannan solution, stream adds lasting 30～50 hours, up to finishing fermentation.

E, in fermentative production, the final volume of fermented liquid is advisable to account for 70%～75% of fermentor tank cumulative volume.

Three, producing fermenting process finishes: find bacterium when bacterioscopy and stop fermentation when beginning self-dissolving.

Four, the effect comparison of the inventive method and traditional technology experiment:

The effect comparison table of traditional technology and the inventive method

Illustrate:

1, continuous as can be seen from the above table 6 batches of contrast fermentations, the glycerol type fermentation has bigger advantage than the fermentation of glucose type;

2, glycerine is that carbon source and current adding substrate stimulate the final enzyme activity of inducing fermentation will exceed 15～24% than the final enzyme activity of glucose type fermentation not wait;

3, in the last handling process of fermented liquid, the decolouring cost then more demonstrates the advantage of glycerol type fermentation, and processing cost has only about 1/3 of glucose type fermentation thereafter.

Claims

1. be that carbon source and current adding substrate are induced the method for preparing mannase with glycerine, employing is done the fermentation manufacturing technique that carrier is produced the genetic engineering bacterium of mannase with pichia spp, it is characterized in that: in first order seed cultivation and fermentation jar, utilize seed culture fluid to cultivate: adopt seed culture medium when spawn culture and first order seed fermentation, its weight percentages is a glucose 3%, peptone 2%, yeast powder 2%, surplus are water; In secondary seed cultivation and fermentation jar, cultivate with basic culture solution: when the secondary seed cultivation and fermentation and the starting stage of fermentative production all adopt basic culture solution, its weight percentages is a glycerine 2%～10%, potassium primary phosphate 1%～5%, vitriolate of tartar 1%～5%, purity is 85% phosphatase 11 %～5%, magnesium sulfate heptahydrate 1%～5%, surplus are water; In producing fermentor tank, the initial stage drops into the part basic culture solution and cultivates, along with the carrying out of fermenting process according to a certain percentage with speed flow respectively glycerol adding, methyl alcohol and substrate mannosans to the fermented liquid to finish fermentation; The concentration of the stream glycerol adding aqueous solution is 50%; The concentration that stream adds methyl alcohol is more than 99.8%; The concentration that stream adds mannan solution is 0.002～0.5%; Induce in the operation glycerine at the uniform velocity stream add 4h or after the longer time, to the bacterium turnout be 200～250g weight in wet base/L, and fermented liquid begins stream and adds methyl alcohol when not having the generation of visible recombinant protein, methyl alcohol stream adds process and lasts till that fermentation finished preceding 10～15 hours; Initial flow acceleration is 4～7ml/h/L, along with the quick accumulation that continues to reach culture of cultivating, needs stream dosage to the 8～10ml/h/l of corresponding increase methyl alcohol; Methyl alcohol begins after stream adds 3～5h, when Pichia yeast engineering begins to produce mannase, beginning and according to the vigor of mannase, stream adds 0.002～0.5% mannan solution simultaneously, velocity flow with 0.5～1ml/h/l adds lasting 30～50 hours, up to finishing fermentation.

2. according to claim 1 is that carbon source and current adding substrate are induced the method for preparing mannase with glycerine, and it is characterized in that: in seed culture and fermentative production, the final volume of fermented liquid all keeps accounting for 70%～75% of fermentor tank cubic capacity.

3. according to claim 1 is that carbon source and current adding substrate are induced the method for preparing mannase with glycerine, it is characterized in that: replaced glucose with glycerine as carbon source during the fermentation, and the current adding substrate mannosans stimulates and induces to produce more mannase.

4. according to claim 1 is that carbon source and current adding substrate are induced the method for preparing mannase with glycerine, it is characterized in that: after glycerine consumption in the basic culture solution fully, measuring residual sugar content with the glycerine meter is 0 o'clock, adds 50% glycerine solution to fermention medium with the flow velocity stream of 10～40ml/h/l; Beginning the flow acceleration of glycerine to be reduced to 1～4ml/h/l after stream adds methyl alcohol.

5. according to claim 1 is that carbon source and current adding substrate are induced the method for preparing mannase with glycerine, it is characterized in that it is more than 99.8% that used all glycerine all require purity.

6. according to claim 1 is that carbon source and current adding substrate are induced the method for preparing mannase with glycerine, it is characterized in that: the culture condition of seed fermentation and production fermentation is 30 ℃, 200～300 rev/mins, pH keeps a certain steady state value in 4.0～6.0 scopes, air flow 0.5～2vvm grew 12～30 hours.