CN101962662A

CN101962662A - Method for reducing by-product acid in fermentation course of L-leucine

Info

Publication number: CN101962662A
Application number: CN 201010527450
Authority: CN
Inventors: 陈宁; 史建明; 徐庆阳; 谢希贤; 刘淑云
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2010-11-02
Filing date: 2010-11-02
Publication date: 2011-02-02

Abstract

The invention relates to a method for reducing by-product acid in the fermentation course of L-leucine, in particular to a method for reducing by-product acid in the course of preparing L-leucine by utilizing a fermentation method. The invention adopts a method of adding citric acid or salt thereof to a culture medium to effectively reduce the formation of the by-product acid of HAc, L-valine, L-alanine and the like in the fermentation course of the L-leucine according to a principle of regulating cell metabolism flow distribution.

Description

A kind of method that reduces secondary acid in the L-leucine fermenting process

[technical field]: the present invention relates to the method for secondary acid in a kind of L-of minimizing leucine fermenting process, the invention belongs to the biochemical engineering field.

[background technology]: brevibacterium flavum is that industrial fermentation is produced the leucic bacterial strain commonly used of L-.HAc, L-Xie Ansuan and L-L-Ala are main secondary acid in L-leucine production process, a large amount of accumulation severe inhibition thalline vigor of these secondary acid, leucine output is descended, and the existence of these secondary acid simultaneously also causes disadvantageous effect to leucic separation and Extraction.

In recent years, the result of study in metabolic engineering field shows that pyruvic acid and acetyl-CoA are the key nodes of the synthetic L-leucine metabolism network of brevibacterium flavum, and HAc, L-Xie Ansuan and L-L-Ala are the main by products of L-leucine batch fermentation process in addition.When the EMP flow exceeded the metabolic capacity of TCA and L-leucine biosynthetic pathway, the pyruvic acid metabolism was obstructed, thereby by the further metabolism of other approach, caused the generation of by product.Therefore batch fermentation is produced in the leucic process of L-, weakens the EMP Embden Meyerbof Parnas pathway metabolic flux, and the generation and the control TCA circular flow that reduce by product HAc, L-Xie Ansuan and L-L-Ala are the keys that improves L-leucine yield.

[summary of the invention]: the present invention adopts the method for adding a certain amount of citric acid or its salt in substratum to reduce the generation that brevibacterium flavum produces by product HAc, L-Xie Ansuan and L-L-Ala in the L-leucine process.

Citric acid or its salt can suppress the pyruvate kinase activity, cause the phosphoenolpyruvic acid accumulation, and phosphoenolpyruvic acid can the strongly inhibited phosphofructokinase, and citric acid can suppress the vigor of phosphofructokinase by the retarding effect of strengthening ATP simultaneously.Because pyruvate kinase and phosphofructokinase are the key enzymes of EMP Embden Meyerbof Parnas pathway, can be so add an amount of Trisodium Citrate in the fermention medium so that the EMP Embden Meyerbof Parnas pathway metabolic flux weaken, thus reduce the generation of by product.The middle and later periods thalline that ferments in addition is in stationary phase, the main effect of HMP approach is that the carbon skeleton that therefore enters the HMP approach finally reenters EMP Embden Meyerbof Parnas pathway at the GAP node for the catalytic reaction of the acetohydroxy acid of L-leucine route of synthesis reduction isomerase provides reducing power NADPH.HMP approach flow increases, thereby reducing power NADPH growing amount increases, and helps the leucic generation of L-.Suitably control TCA circular flow is necessary for increasing leucic synthesizing of L-.Because citrate synthase is a TCA round-robin rate-limiting enzyme, add the activity of Trisodium Citrate meeting competitive inhibition citrate synthase.So adding an amount of Trisodium Citrate can restricted T CA metabolic flux, reduce the waste of carbon skeleton, enter the biosynthetic metabolism stream of L-leucine thereby improve acetyl-CoA, improved the leucic output of L-.

This method has realized the minimizing of secondary acid in the fermenting process and the raising of L-leucine output under the situation that does not increase extras and human input, be suitable for suitability for industrialized production.

The objective of the invention is to be achieved through the following technical solutions:

A kind of method that reduces secondary acid in the L-leucine fermenting process provided by the invention is characterized in that: add citric acid or its salt in fermention medium, make its concentration in substratum be 0.01～16g/L, preferred 0.05～10g/L, more preferably 0.1～5g/L.Wherein said Citrate trianion is Trisodium Citrate or Tripotassium Citrate.

The present invention has proposed to reduce the method that brevibacterium flavum produces the generation of by product HAc, L-Xie Ansuan and L-L-Ala in the L-leucine process according to the principle of adjusting the metabolism flow distribution, in fermention medium, add the vigor that an amount of Trisodium Citrate can suppress EMP Embden Meyerbof Parnas pathway key enzyme pyruvate kinase and phosphofructokinase, make the EMP Embden Meyerbof Parnas pathway metabolic flux weaken, thereby reduce the generation of by product.

[embodiment]:

The present invention is further illustrated below by embodiment, and the cited case does not limit protection scope of the present invention:

Embodiment 1:

The bacterial strain that adopts is a brevibacterium flavum;

Seed culture medium (g/L): glucose 30, corn steep liquor 10mL, soya-bean cake hydrolyzed solution 20mL, urea 2.0, KH ₂PO ₄1.0, MgSO ₄0.4, MnSO ₄0.01 Met 0.4, VB ₁300 μ g, VH 200 μ g.pH?7.0～7.2，0.075MPa，20min

Fermention medium (g/L): glucose 80, corn steep liquor 43.5mL, NH ₄Ac 16.7, (NH ₄) ₂SO ₄17, KH ₂PO ₄1.25, MgSO ₄0.5, MnSO ₄0.01, FeSO ₄0.01 Met 0.7, Glu 0.42, and Ile 0.06, VB ₁160 μ g, VH 50 μ g, CaCO ₃20 (branch disappears), pH 7.0～7.2,0.075MPa, 20min

Cultural method: scrape one from well-grown activated inclined plane and completely encircle lawn and change the 500mL that the 40mL seed culture medium is housed over to and do not have the baffle flask, 9 layers of gauze seal, and put on the patrolling shaking table (180r/min) 28 ℃ of following shaking culture 20h.Inoculum size by 10% inserts the 5L that contains fermention medium (containing the 0.1g/L Trisodium Citrate) and controls in the fermentor tank automatically, control fermented liquid temperature is 28 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～30h is 30%, 30h is later on 20%, control pH 7.0 by auto-feeding liquefied ammonia, add an amount of bubble enemy froth breaking by stream, and to add concentration by stream be that the glucose solution of 800g/L is controlled at 1.0～2.0% with residual sugar, ferments to 64h to stop.Record L-L-Ala content at 44h and 64h and be respectively 0.76g/L, 3.94g/L, comparison is according to the facts tested (44hL-L-Ala content 0.86g/L, 64hL-L-Ala content 4.75g/L) and has been reduced by 11.6% and 17.1% respectively.

Embodiment 2:

The bacterial strain that adopts is a brevibacterium flavum; Seed, fermention medium (with embodiment 1), the middle 5.0g/L Trisodium Citrate that adds in the fermention medium; Cultural method is with embodiment 1.Record the L-valine content at 44h and 64h and be respectively 1.00g/L, 4.36g/L, comparison is according to the facts tested (44hL-valine content 1.58g/L, 64hL-valine content 7.46g/L) and has been reduced by 36.7% and 41.6% respectively.

Embodiment 3:

The bacterial strain that adopts is a brevibacterium flavum; Seed, fermention medium (with embodiment 1), the middle 5.0g/L Trisodium Citrate that adds in the fermention medium; Cultural method is with embodiment 1.Record HAc content at 44h and 64h and be respectively 0.84g/L, 2.62g/L, comparison is according to the facts tested (44hHAc content 2.16g/L, 64hHAc content 10.09g/L) and has been reduced by 61.1% and 74.0% respectively.

Claims

1. one kind is reduced secondary sour method in the L-leucine fermenting process, its key step is: add the generation that citric acid or its salt reduce secondary acid in the employing brevibacterium flavum fermentative production L-leucine process in substratum, it is characterized in that adding citric acid or its salt in substratum, to make its concentration in substratum be 0.01～16g/L.

2. method according to claim 1 is characterized in that: wherein said Citrate trianion is Trisodium Citrate or Tripotassium Citrate.

3. method according to claim 1 is characterized in that: the concentration of wherein said citric acid or its salt is 0.05～10g/L.

4. method according to claim 1 is characterized in that: the concentration of wherein said citric acid or its salt is 0.1～5g/L.