CN104878051A

CN104878051A - Method for improving fermentation yield of L-isoleucine through adding choline

Info

Publication number: CN104878051A
Application number: CN201510274396.3A
Authority: CN
Inventors: 陈宁; 徐国栋; 谢希贤; 徐庆阳; 张成林; 李燕军; 范晓光
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2015-05-25
Filing date: 2015-05-25
Publication date: 2015-09-02

Abstract

The invention relates to a method for improving the fermentation productivity of L-isoleucine and conversion ratio of saccharic acid through adding choline to a fermentation medium. The invention relates to an improved method for preparing L-isoleucine by adopting a fermentation method. According to the principle that the choline has the effects of promoting synthesis of methyl, regulating osmotic pressure in thallus, regulating lipid metabolism of thallus and the protein synthesis in the fermentation process of L-isoleucine, promoting the synthesis of methyl of L-isoleucine, promoting the enzyme activity in the thallus, promoting the growth of thallus, and realizing the substantial accumulation of L-isoleucine in the fermentation liquor, so as to improve the productivity and the saccharic acid conversion ratio, and the method that choline or choline chloride (choline hydrochloride) is added to the fermentation medium is adopted, as a result, the fermentation production of L-isoleucine is effectively improved.

Description

A kind of method improving ILE fermentation yield by adding choline

[technical field]: the present invention relates to a kind of by adding the method that choline improves ILE fermentation production rate and glucose acid invert ratio in the fermentation medium, belonging to the amino acid whose technical field of fermentative Production.

[background technology]: in recent years, along with the applied research of ILE in medicines and health protection, food-processing and fodder industry deepens continuously, market to the demand of ILE in continuous growth.Though ILE realizes suitability for industrialized production, current production rate still can not be satisfied the demand.According to statistics, the demand in domestic branched chain market in 2014 is about 20,000 tons, and the throughput of domestic production producer is only 1.6 ten thousand tons at present, and insufficiency of supply-demand is comparatively large, and annual requirement is with the speed increase of 15%.Therefore, the production technique optimizing ILE is further significant.Compared with Foreign Advanced Lerel, China ILE produce have that fermentation and acid is low, low conversion rate and the problem such as by product is many.China's ILE acid yield is 25-30g/L, and extraction yield is 65-70%, and Japanese ILE acid yield is 30-35g/L, and extraction yield is 70-75%.The rise of the prices of raw and semifnished materials in recent years and the raising of labor cost bring challenge to ILE fermentative Production, and then limit the application of ILE.

At present, the production method of ILE mainly contains: proteolysis method, chemical synthesis, fermentation method and enzyme process.Proteolysis method requires simple to production unit, cost is low, but there is the shortcomings such as seriously polluted, product yield is low, impurity is more.Chemical synthesis is subject to the restriction of raw material sources, and synthesis step is complicated, is difficult to widespread use.ILE has four kinds of optically active isomers, and most of synthesis method can only obtain racemic modification, just can obtain ILE after then being split by racemize, so chemical synthesis is difficult to realize fairly large suitability for industrialized production.At present, the method for fermentable is only had can to realize large-scale industrial production ILE.Fermentation method raw materials cost is low, be easy to control, in the technique of industrial production ILE instantly, occupy huge advantage.At present, main employing brevibacterium flavum produces Isoleucine as industrial fermentation and produces bacterial strain.Bacterium biosynthetic pathways metabolism is produced clearly under prerequisite at Isoleucine, can show that the yield that ILE is produced is 0.486g ILE/g glucose by calculating in theory, and net yield only has 0.15-0.20g ILE/g glucose at present, larger gap is had, so still have very large room for promotion compared with theoretical value.Therefore the productive rate and the glucose acid invert ratio that how to improve Production by Microorganism Fermentation ILE further become the key issue promoting the Sustainable development of ILE industry.

At present, ILE fermentation and acid and transformation efficiency are still in lower level.One of reason is simultaneously for the synthesis of ILE and thalline at earlier fermentation substrate glucose.There is the competition to carbon source and the energy between Growth of Cells and ILE synthesis, too high biomass will inevitably cause glucose acid invert ratio to decline.Two of reason is that the phase occurs because specific cell growth rate declines " fermentation conversion " after fermentation, and cell synthesizes the by products such as 1B in a large number.Three of reason is that methyl donor is not enough, and synthesis ILE is restricted, cause producing acid and transformation efficiency lower.In addition, in the fermentation middle and later periods, bacterial enzyme is lived and is declined comparatively fast, causes later stage rate of producing acid to decline.

Choline is a kind of organic bases, and belong to vitamin B group, its molecular formula is (CH3) ₃n (CH ₂) ₂oH, in strong basicity, easily moisture absorption, has and promotes methyl synthesis, regulates osmotic pressure in somatic cells, reconciles the effect such as thalline metabolism of fat and protein synthesis, can improve the synthesis of methyl in somatic cells, improve thalline endoenzyme and live, promote thalli growth.Based on this principle, this technology improves ILE fermentation production rate and glucose acid invert ratio by the method for adding choline or Lipotril in the medium.

[summary of the invention]: the present invention adopts the method for adding a certain amount of choline or choline chloride 60 in the medium to improve Isoleucine Producing Mutant of Brevibacterium flavum ILE fermenting process glucose acid invert ratio and productive rate.

Choline has raising thalline endoenzyme and lives in ILE fermenting process, promote thalli growth, regulate osmotic pressure in somatic cells, reconcile thalline metabolism of fat and protein synthesis, promote the effects such as ILE methyl synthesis, therefore, by adding the method for choline or choline chloride 60 in the fermentation medium, ILE fermentation production rate and glucose acid invert ratio is effectively improved.

This method, when not increasing extras and human input, achieves the shortening of whole fermentation period and the significantly raising of ILE output and transformation efficiency, is suitable for suitability for industrialized production.

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

A kind of method improving ILE fermentation production rate and glucose acid invert ratio provided by the invention, it is characterized in that: add choline or choline chloride 60 in the fermentation medium, its concentration is in the medium made to be 0.01 ~ 3g/L, preferably 0.05 ~ 2g/L, more preferably 0.1 ~ 1g/L.Wherein said choline salt is choline chloride 60.

The present invention has the synthesis of raising methyl according to choline to ILE production bacterium, enhancing enzyme is lived, the principle of raising cell permeability proposes and is applicable to the method that raising Isoleucine produces bacterium ILE fermentation production rate and glucose acid invert ratio, the method, by adding a certain amount of choline in the medium, reaches the object of high yield ILE.

[embodiment]:

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

Embodiment 1:

The bacterial strain brevibacterium flavum adopted; Substratum is at the existing fermention medium [glucose 100g/L (divide and disappear, 0.075MPa moist heat sterilization 15min), (NH that generally adopt ₄) ₂sO ₄3g/L, MgSO ₄7H ₂o 0.6g/L, MnSO ₄7H ₂o 15mg/L, FeSO ₄7H ₂o 15mg/L, KH ₂pO ₄1.3g/L, V _b10.1mg/L, Dried Corn Steep Liquor Powder 15g/L (0.01MPa moist heat sterilization 20min) NaOH and hydrochloric acid adjust pH to 7.0.Sterilising conditions: 0.1MPa moist heat sterilization 20min] middle interpolation 0.2g/L choline chloride 60, cultural method: bacterial classification is accessed seed culture medium [glucose 30g/L, yeast powder 5g/L, ammonium sulfate 5g/L, Dried Corn Steep Liquor Powder 20g/L, KH ₂pO ₄2g/L, MgSO ₄7H ₂o 0.8g/L], inoculum size is 10%, at 31 DEG C, pH be 7.0 and dissolved oxygen be automatically control to cultivate 15h in fermentor tank to the logarithm middle and later periods in 5L under 20% condition, automatically control in fermentor tank by the 5L of inoculum size access containing fermention medium of 10%, pass into suitable air, regulate agitation as appropriate rotating speed, adopt different oxygen supply Schema control dissolved oxygen: 0-20h is 30-50%, 20-50h is 10-20%, by the ammoniacal liquor control pH of auto-feeding 25% at 7.0-7.2, appropriate bubble enemy froth breaking is added by stream, and to add concentration by stream be that residual sugar controls about 1.5% by the glucose solution of 800g/L, fermentation 50h terminates.When putting tank, the output of ILE is 35.6g/L, and glucose acid invert ratio is 17.0%, improves 7.23% and 7.60% respectively than control experiment (ILE output is 33.2g/L, and glucose acid invert ratio is 15.8%).

Embodiment 2:

The bacterial strain adopted is brevibacterium flavum; Substratum is add 1.0g/L choline chloride 60 in the existing fermention medium (with embodiment 1) generally adopted; Cultural method is with embodiment 1.When putting tank, the output of ILE is 40.5g/L, and glucose acid invert ratio is 18.7%, improves 22.0% and 18.4% respectively than control experiment (ILE output is 33.2g/L, and glucose acid invert ratio is 15.8%).

Embodiment 3:

The bacterial strain adopted is brevibacterium flavum; Substratum is add 2.0g/L choline chloride 60 in the existing fermention medium (with embodiment 1) generally adopted; Cultural method is with embodiment 1.When putting tank, the output of ILE is 36.9g/L, and glucose acid invert ratio is 17.9%, improves 11.1% and 13.3% respectively than control experiment (ILE output is 33.2g/L, and glucose acid invert ratio is 15.8%).

Embodiment 4:

The bacterial strain adopted is brevibacterium flavum; Substratum is add 3.0g/L choline chloride 60 in the existing fermention medium (with embodiment 1) generally adopted; Cultural method is with embodiment 1.When putting tank, the output of ILE is 36.2g/L, and glucose acid invert ratio is 17.6%, improves 9.0% and 11.4% respectively than control experiment (ILE output is 33.2g/L, and glucose acid invert ratio is 15.8%).

Claims

1. one kind is improved the method for ILE fermenting process productive rate and glucose acid invert ratio, its key step is: adopt Isoleucine to produce bacterium and obtain ILE through fermentation, it is characterized in that, add choline or its salt in the medium, make its concentration in the medium be 0.01 ~ 3g/L.

2. method according to claim 1, preferably content is: wherein the concentration of said choline or its salt is 0.05 ~ 2g/L.

3. method according to claim 1, the content of its best is: wherein the concentration of said choline or its salt is 0.1 ~ 1g/L.

4. any one method according to claims 1 to 3, is characterized in that: wherein said choline salt is choline chloride 60.