CN101525604B - Method for increasing output of alkaline pectinase produced by fermentation by adding sorbitol - Google Patents
Method for increasing output of alkaline pectinase produced by fermentation by adding sorbitol Download PDFInfo
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- CN101525604B CN101525604B CN2009100308312A CN200910030831A CN101525604B CN 101525604 B CN101525604 B CN 101525604B CN 2009100308312 A CN2009100308312 A CN 2009100308312A CN 200910030831 A CN200910030831 A CN 200910030831A CN 101525604 B CN101525604 B CN 101525604B
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Abstract
The invention discloses a method for increasing the output of alkaline pectinase produced by fermentation by adding sorbitol, relating to the technical field of application of the carbon source adding policy in the optimization of fermentation. In the method, recombinant Pichia pastoris GS115 is adopted as fermentation strains, and sorbitol is added into the fermentation liquid at a constant speed or accelerated speed during fermentation induction phase to improve the activity of cells, weaken the toxicity of methanol, and increase the output of the alkaline pectinase. The lower adding amountof sorbitol can not inhibit the enzyme production effect of methanol to the strains, on the other hand, the toxicity to the strains from the methanol can be reduced to a certain extent, and the efficiency of enzyme production is further increased. When the sorbitol is added at a varying speed (0.9g/h-7.2g/h), the activity of enzyme is increased from 930U/ml to 1541U/ml. The invention is simple and effective in process, capable of being applied in the industrial production of pectinase in the future, and instructive to the optimization of fermentation of other Pichia pastoris.
Description
Technical field
Utilize recombinant yeast pichia pastoris to efficiently express alkaline pectase as the host, a kind of with the method for adding sorbyl alcohol raising fermentation production of alkaline pectic enzyme output, relate to carbon source and add the applied technical field of strategy in optimizing fermentation.
Background technology
Alkaline pectase is that a class can efficiently be decomposed pectin substance in the plant tissue (by the D-galacturonic acid with α-1 under alkaline condition, the general name of the enzyme straight catenate polymkeric substance that 4 glycosidic links are connected to form) is as a kind of bioscouring enzyme preparation of brand-new gentleness of the cleaner production that is used to weave and receive much concern.In early-stage Study, this research department obtains on the basis of a strain alkaline pectase superior strain WSHB04-02 at separation screening, its protokaryon alkaline pectin ester lyase gene PL is utilized the connection of shuttle vectors pPIC9K, and successful expression is in pichia spp GS115, and culture presevation number is CGMCC NO.2143.Research work afterwards all centers on the recombinant yeast pichia pastoris optimizing fermentation and launches.At the induction period of fermenting process, because for a long time stream adds methyl alcohol, the pair cell vigor has stronger toxic action, and therefore the vigor that how can keep cell again in abduction delivering stage efficiently is further to improve the key issue that enzyme is lived.Utilize the low-speed flow of sorbyl alcohol to add just and can address this problem, improve the output of polygalacturonase, do not see bibliographical information as yet.
Summary of the invention
The purpose of this invention is to provide a kind of method, utilize the present invention can significantly improve the output of alkaline pectase with the strategy raising fermentation production of alkaline pectic enzyme output of adding sorbyl alcohol.
Technical scheme of the present invention: a kind of with the method for adding sorbyl alcohol raising fermentation production of alkaline pectic enzyme output, with recombinant yeast pichia pastoris GS115 is fermentation strain, add sorbyl alcohol in the fermentation inducement phase in fermented liquid with constant speed or acceleration, to improve cell viability, weaken the toxic action of methyl alcohol, and then improve the output of alkaline pectase;
Flow acceleration is an optional constant speed in the Neosorb meter scope of 0.9g/h to 7.2g/h, is made into concentration expressed in percentage by volume 50% Sorbitol Solution USP and carries out stream and add;
Or flow acceleration improves flow velocity 0.6g/h with every 10h, progressively is increased to 7.2g/h from 0.9g/h, is made into concentration expressed in percentage by volume 50% Sorbitol Solution USP and carries out stream and add.
The output of alkaline pectase improves with the raising of sorbyl alcohol flow velocity, but when its flow velocity was too high, the output of alkaline pectase can reduce again.When the constant interpolation speed of sorbyl alcohol was 3.6g/h, the output of alkaline pectase was brought up to 1389U/mL by the 930U/mL of contrast (not adding sorbyl alcohol).When adopting gradient to increase the interpolation flow velocity of sorbyl alcohol, find that the output of alkaline pectase is brought up to 1541U/mL.
The mensuration that the alkaline pectase enzyme is lived: get a certain amount of fermented liquid in the centrifugal 10min of 10000r/min, supernatant liquor is as the sample of alkaline pectin enzyme assay.Comprise thick enzyme diluent (generally diluting 200-800 does not doubly wait) 20 μ L in the reaction system, 2mL contains glycine-NaOH damping fluid (buffer formulation: glycine 1.8775g, NaOH 0.35g, CaCl of 0.2% polygalacturonic acid
2-2H
2O 0.028g is settled to 500mL), with the enzyme liquid of non-activity as blank, with the adding starting enzymatic reaction of the buffered soln that contains substrate; Reaction conditions is 45 ℃ of reaction 15min, with the phosphoric acid termination reaction of 3mL 0.03mol/L, measures its absorbance at the 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 polyester galacturonic acid of 1 μ mol.
Beneficial effect of the present invention: utilize the present invention can significantly improve the output of alkaline pectase.Lower sorbyl alcohol addition not only can not suppress the product enzyme effect of methyl alcohol to thalline, can also weaken the methyl alcohol toxic action that thalline is subjected to a certain extent, produces enzyme efficient thereby further improve.In addition, provided by the present inventionly improve the strategy that produces foreign protein the expression system of other pichia spp is also had certain experiences and directive function by adding sorbyl alcohol.
Embodiment
Comparative examples: the bacterial classification that the present invention is used: recombinant yeast pichia pastoris P.pastoris GS115 is the host, integrates the encoding gene from the alkaline pectase in the Bacillus sp.WSHB04-02 bacterial strain, has His
+And Mut
+Phenotype, copy number are 2-3.Recombinant yeast pichia pastoris P.pastoris GS115 is open at [biotechnology journal 2008.24 (4) .p635-639].
Substratum:
Seed culture medium: glucose 20g/L, peptone 20g/L, yeast powder 10g/L.
The batch fermentation substratum is: 85% phosphoric acid 26.7mL/L, CaSO
40.93g/L, K
2SO
418.2g/L, MgSO
47H
2O 14.9g/L, KOH 4.13g/L, glycerine 40.0g/L, PTM1 4.35mL/L, 25% ammoniacal liquor is transferred pH5.5.
Feed supplement growth medium: 50% (W/V) glycerine (containing 12mL/L PTM1).
Fermentation inducement substratum: 100% methyl alcohol (containing 12mL/L PTM1).
Culture condition: first glycerine pipe from the preservation bacterial classification goes to and shakes bottle at 30 ℃, cultivated 24 hours in the shaking table of 200rpm, in the 3L fermentor tank, ferment then, fs is about 36h in vegetative period, and subordinate phase is about 100h inductive phase, and fermentation condition is: pH 5.5, regulate mixing speed and air flow and keep dissolved oxygen more than 30%, Ventilation Rate 2.0vvm, vegetative period temperature maintenance at 30 ℃, inductive phase, temperature was reduced to 22 ℃.
Based on the fermentative Production alkaline pectase under this technology, output can reach about 930U/mL.
Embodiment 1: ferment tank adopts the sorbyl alcohol constant speed to add strategy, the same comparative examples of all the other conditions.When entering inductive phase, fermentation adds sorbyl alcohol (50%, v/v, the Neosorb of 1.8g/h).The result: the output of alkaline pectase is brought up to 1176U/mL by the 930U/mL of contrast.
Embodiment 2: ferment tank adopts the sorbyl alcohol constant speed to add strategy, the same comparative examples of all the other conditions.When entering inductive phase, fermentation adds sorbyl alcohol (50%, v/v, the Neosorb of 3.6g/h).The result: the output of alkaline pectase is brought up to 1389U/mL by the 930U/mL of contrast.
Embodiment 3: ferment tank adopts the sorbyl alcohol constant speed to add strategy, the same comparative examples of all the other conditions.When entering inductive phase, fermentation adds sorbyl alcohol (50%, v/v, the Neosorb of 5.4g/h).The result: the output of alkaline pectase is brought up to 1211U/mL by the 930U/mL of contrast.
Embodiment 4: ferment tank adopts the sorbyl alcohol speed change to add strategy, the same comparative examples of all the other conditions.Interpolation sorbyl alcohol when fermentation enters inductive phase (50%, v/v, flow acceleration progressively is increased to 7.2g/h from 0.9g/h, every 10h improves flow velocity 0.6g/h).The result: the output of alkaline pectase is brought up to 1541U/mL by the 930U/mL of contrast.
Claims (1)
1. one kind is improved the method for fermentation production of alkaline pectic enzyme output with adding sorbyl alcohol, it is characterized in that with recombinant yeast pichia pastoris GS115 be fermentation strain, add sorbyl alcohol in the fermentation inducement phase in fermented liquid with constant speed or acceleration, to improve cell viability, weaken the toxic action of methyl alcohol, and then improve the output of alkaline pectase;
Used bacterial classification: recombinant yeast pichia pastoris P.pastoris GS115 is the host, integrates the encoding gene from the alkaline pectase in the Bacillus sp.WSHB04-02 bacterial strain, has His
+And Mut
+Phenotype, copy number are 2-3;
Flow acceleration is an optional constant speed in the Neosorb meter scope of 0.9g/h to 7.2g/h, is made into concentration expressed in percentage by volume 50% Sorbitol Solution USP and carries out stream and add;
Or flow acceleration improves flow velocity 0.6g/h with every 10h, progressively is increased to 7.2g/h from 0.9g/h, is made into concentration expressed in percentage by volume 50% Sorbitol Solution USP and carries out stream and add.
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| CN101525604B true CN101525604B (en) | 2011-03-30 |
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| CN102367437A (en) * | 2011-10-25 | 2012-03-07 | 江南大学 | Method for producing pectinase by fermentation of recombinant Escherichia coli |
| CN106282278B (en) * | 2015-06-29 | 2021-06-08 | 广东东阳光药业有限公司 | Fermentation method of human papilloma virus L1 protein |
| CN105713850A (en) * | 2016-03-23 | 2016-06-29 | 江南大学 | Pichia pastoris strain capable of constitutively expressing alkaline pectinase and application thereof |
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