CN102776168A

CN102776168A - Method for improving enzymatic activity by fed-batch fermentation and batch culture

Info

Publication number: CN102776168A
Application number: CN2012102516222A
Authority: CN
Inventors: 熊光辉; 沈瑞华; 杨涛; 顾稀平; 曹艳; 钱小方; 丁建华
Original assignee: JIANGSU NANTIAN AGRICULTURAL TECHNOLOGY CHEMICAL CO LTD
Current assignee: JIANGSU NANTIAN AGRICULTURAL TECHNOLOGY CHEMICAL CO LTD
Priority date: 2012-07-20
Filing date: 2012-07-20
Publication date: 2012-11-14

Abstract

The invention relates to a method for improving enzymatic activity by fed-batch fermentation and batch culture. During fermentation, the requirements for cell growth and nitrilre hydratase synthesis nutrition are met by supplementing alkali liquor and glucose into fermentation medium. Growth time of cells can be prolonged, and enzyme production level of Nocardia is also increased greatly. The enzymatic activity reaches 2934.6U/ml 9.3 times of that of batch fermentation, and the feeding way is simple, easy and convenient to operate.

Description

A kind of method of cultivating batch culture raising enzyme activity through feeding medium during fermentation

Technical field

The present invention relates to a kind of method of cultivating batch culture raising enzyme activity through feeding medium during fermentation.

Background technology

In 19 end of the centurys, synthesized acrylic amide first from acrylate chloride and ammonia.1954, American Cyanamid Co. adopted vinyl cyanide sulphuric acid hydrolysis technology to carry out industrial production.1972, Mitsui east pressed chemical company at first to set up the full scale plant of skeleton copper catalyzing propone nitrile hydration system acrylic amide, and after this various countries have developed dissimilar catalyzer in succession, adopt this technology to carry out industrial production.The eighties, Japanese Ri Dong chemical industrial company has realized with the industrial production of biological catalyst by vinyl cyanide system acrylic amide.Microbial method is to utilize cell paste that biotechnology turns out as enzyme catalyst, catalyzing propone nitrile synthesis of acrylamide under suitable condition.The key that the microorganism catalysis method is produced acrylic amide is the Nitrile hydratase of the high vigor of preparation.The raising of Nitrile hydratase vigor helps vinyl cyanide and carries out to acrylic amide the Direction of Reaction, reduces the acrylic amide production cost, enhances productivity, and microbial method is produced the acrylic amide industrial expansion has important meaning.

Summary of the invention

The object of the invention is providing a kind of method of cultivating batch culture raising enzyme activity through feeding medium during fermentation, the activity of the Nitrile hydratase that improving ferments obtains, thus promote post-hydration production acrylic amide.

In order to solve the problems of the technologies described above, technical scheme of the present invention is: a kind of method of cultivating batch culture raising enzyme activity through feeding medium during fermentation is characterized in that may further comprise the steps:

1) seed culture: with Nocardia bacteria with solid medium activation 90-120 h in 28 ℃ of incubators; Choose and connect bacterium colony and in triangular flask, carry out seed culture, placing temperature to be set at 28 ± 2 ℃, rotating speed triangular flask is that the shaking table of 150-200 r/min is cultivated 48 h;

2) fed-batch fermentation: 5% seed liquor is inoculated in carries out fed-batch fermentation in the fermention medium and cultivate, fermentation condition is: fermentation culture is used the automatic control fermentor tank of 2.5L, and fermentation volume is 1.2 L; 28 ± 2 ℃ of leavening temperatures, mixing speed 150-200 r/min, air flow are 35-40 L/min; PH value and dissolved oxygen during the fermentation, are added alkali lye and glucose by the control of fermentor tank control unit in fermention medium; Satisfy the needs of the synthetic nutrition of thalli growth and Nitrile hydratase; Prolong the thalli growth time, cultivate the 40-50h secondary fermentation and finish, survey bacterial enzyme and live.

Further, described solid culture based component is: glucose 20 g/L, potassium hydrogenphosphate 0.5 g/L, yeast extract paste 5g/L, peptone 5 g/L, sal epsom 0.1g/L, monosodium glutamate 1g/L, potassium hydrogenphosphate 0.5g/L, urea 7g/L, agar 20 g/L.

Further, described fermentation culture based component is: glucose 25 g/L, yeast extract paste 5 g/L, urea 8g/L, monosodium glutamate 1.5 g/L, potassium primary phosphate 0.8/L, potassium hydrogenphosphate 0.8 g/L, sal epsom 1.0g/L, skimmer 150ppm.

Further, described alkali lye is added technology and is: when the pH of fermented liquid less than 6 the time, make fermented liquid pH value maintain 6.0-7.0 through adding alkali lye.

Further, described alkali lye is sodium hydroxide or ammoniacal liquor, and described pH value is 6.5.

Further, described alkali lye is ammoniacal liquor.

Further, the sugared technology of described benefit is: in the fermentation middle and later periods, make its concentration remain on 6-15g/L through the glucose solution of adding 150-180g/L.

Beneficial effect of the present invention: adopt the present invention during the fermentation; Through in fermention medium, adding alkali lye and glucose, satisfy the needs of the synthetic nutrition of thalli growth and Nitrile hydratase, can not only prolong the thalli growth time; And Nocardia bacteria product enzyme level is enhanced; It is 9.3 times of batch fermentation that the enzyme work of this technology reaches 2934.6U/ml, and the feed supplement mode is simple, is convenient to operation.

Embodiment

Embodiment 1 batch fermentation controlled trial

(1) solid medium

Glucose 20 g/L, potassium hydrogenphosphate 0.5 g/L, yeast extract paste 5g/L, peptone 5 g/L, sal epsom 0.1g/L, monosodium glutamate 1g/L, potassium hydrogenphosphate 0.5g/L, urea 7g/L, agar 20 g/L.

(2) fermention medium

Glucose 25 g/L, yeast extract paste 5 g/L, urea 8g/L, monosodium glutamate 1.5 g/L, potassium primary phosphate 0.8/L, potassium hydrogenphosphate 0.8 g/L, sal epsom 1.0g/L, skimmer 150ppm.

(3) seed culture

Nocardia bacteria with solid medium activation 90-120 h in 28 ℃ of incubators, is chosen and connect bacterium colony and in triangular flask, carry out seed culture, and placing temperature to be set at 28 ± 2 ℃, rotating speed triangular flask is that the shaking table of 150-200 r/min is cultivated 48 h;

(4) batch fermentation is cultivated

The Nitrile hydratase fermentative prepn is controlled automatically in the fermentor tank at 2.5 L and carried out, and fermentation condition is: fermentation volume is 1.2 L, 28 ± 2 ℃ of leavening temperatures, and mixing speed 150-200 r/min, air flow are 35-40 L/min.

(5) fermentation result

Fermentation is carried out finishing behind the 40h, and enzyme is lived and is 314.56U/ml.

Embodiment 2 fed-batch fermentations

(1) (2) (3) step is with embodiment 1

(4) fed-batch fermentation is cultivated

Inoculum size with 5% inserts fermention medium, and fermentation culture is used the 2.0L fermentor tank, liquid amount 1.2L, and 28 ± 2 ℃ of leavening temperatures, mixing speed 150-200 r/min, air flow are 35-40 L/min.When pH value of solution in the fermention medium less than 6.5 the time, add 2% ammoniacal liquor and keep fermented liquid pH 6.5, in the fermentation middle and later periods, the glucose solution of adding 180g/L makes its concentration remain on 6-15g/L.

(5) fermentation result

Fermentation is carried out finishing behind the 50h, has prolonged 10h with the contrast batch fermentation, and the bacterial enzyme work that the fed-batch formula is cultivated reaches 2934.6 U/ml, is 9.3 times of batch fermentation.

Claims

1. one kind is passed through the method that feeding medium during fermentation is cultivated batch culture raising enzyme activity, it is characterized in that may further comprise the steps:

2. according to claims 1 seed culture medium; It is characterized in that: the solid culture based component is: glucose 20 g/L, potassium hydrogenphosphate 0.5 g/L, yeast extract paste 5g/L, peptone 5 g/L, sal epsom 0.1g/L, monosodium glutamate 1g/L, potassium hydrogenphosphate 0.5g/L; Urea 7g/L, agar 20 g/L.

3. according to claims 1 described a kind of method of cultivating batch culture raising enzyme activity through feeding medium during fermentation, it is characterized in that: described fermentation culture based component is: glucose 25 g/L, yeast extract paste 5 g/L, urea 8g/L, monosodium glutamate 1.5 g/L, potassium primary phosphate 0.8/L, potassium hydrogenphosphate 0.8 g/L, sal epsom 1.0g/L, skimmer 150ppm.

4. according to claims 1 described a kind of method of cultivating batch culture raising enzyme activity through feeding medium during fermentation; It is characterized in that: described alkali lye is added technology and is: when the pH of fermented liquid less than 6 the time, make fermented liquid pH value maintain 6.0-7.0 through adding alkali lye.

5. according to claims 4 described a kind of methods of cultivating batch culture raising enzyme activity through feeding medium during fermentation, it is characterized in that: described alkali lye is sodium hydroxide or ammoniacal liquor, and described pH value is 6.5.

6. according to claims 5 described a kind of methods of cultivating batch culture raising enzyme activity through feeding medium during fermentation, it is characterized in that: described alkali lye is ammoniacal liquor.

7. according to claims 1 described a kind of method of cultivating batch culture raising enzyme activity through feeding medium during fermentation; It is characterized in that: the sugared technology of described benefit is: in the fermentation middle and later periods, make its concentration remain on 6-15g/L through the glucose solution of adding 150-180g/L.