Cultivation method for saccharomycete excreting expression proteolytic enzyme

A technology of secretion expression and culture method, which is applied in the field of yeast culture for secretion and expression of protease, can solve the problems of cumbersome operation, difficulty in real-time monitoring, lack of technology, etc., and achieve low cost of culture, simple and accurate reaction control method, protease expression highly active effect

Inactive Publication Date: 2008-09-24
广州伯凯生物技术有限公司 +1
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  • Application Information

AI Technical Summary

Problems solved by technology

These methods, in the actual fermentation operation, are difficult to achieve real-time monitoring, there is a long delay between sampling and process adjustment, and the operation is cumbersome and the operating cost is relatively high, so it is difficult to achieve the best effect of the fermentation process
[0009] For the low-cost and efficient secretory expression of xylanase or mannanase, there is still a lack of corresponding technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 Pichia pastoris fermentation prepares mannanase

[0037] The strain used was P. pastoris GS115 / poly-man-B3, which was constructed by Guangzhou Bokai Biotechnology Co., Ltd.

[0038] Pick 2-3 colonies from YPD and culture them in the primary seed medium of BMGY (30mL in 300mL triangular flask) for about 16h, the OD of the bacteria 600 When it reaches 1.0, take 2mL of the above-mentioned culture solution and culture it in BMGY secondary seed medium (10 500mL triangular flasks with 50mL) for 16h, and the cells are 0D 600 Reached 7.0.

[0039] Calculated on the basis of 1L fermentation broth, first weigh a small amount of inorganic salt components, including copper sulfate 0.08mg, sodium iodide 0.12mg, sodium molybdate 0.45mg, manganese sulfate 8.0mg, cobalt chloride 1.5mg, boric acid 0.08mg, zinc sulfate 10mg , add water to dissolve and make up to 150mL, then add concentrated sulfuric acid 0.05mL; then weigh the main inorganic salt components, including magn...

Embodiment 2

[0047] Embodiment 2 Pichia pastoris fermentation prepares xylanase

[0048] The strain used is P. pastoris GS115 / poly-xyl-B5, constructed by Guangzhou Bokai Biotechnology Co., Ltd.

[0049] After the seed culture is completed with the medium of BMGY, inoculate the fermenter according to the inoculum amount of 3-5%. The fermentation medium is the same as before. Sterilize, connect the defoamer and the ammonia and methanol to be fed, and calibrate the dissolved oxygen electrode. Ferment at 3L / min, 330r / min, 30°C. During the growth period, the bacteria use glucose as a carbon source. After cultivating for 18-24 hours, the dissolved oxygen value of the fermentation broth rises to above 80%. At this time, start to feed methanol, and use the dissolved oxygen value for real-time regulation. The set control range is: when the dissolved oxygen value of the fermentation broth is lower than 10%. , stop feeding methanol; when the dissolved oxygen value rises above 60%, start feeding m...

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Abstract

The invention provides a method for culturing microzyme of secretory expression proteolytic enzyme. The method is characterized in that an inorganic salt culture medium is adopted for fermenting and culturing a recombinant methylotrophic yeast. Fermentation and culture comprise the growth period of the microzyme and the secretory expression period of the proteolytic enzyme. In the secretory expression period of the proteolytic enzyme, the feed flow of methanol can be controlled in real time according to the variation of oxygen dissolved in fermentation liquid, and the PH value of the fermentation liquid is adjusted with ammonia, thus expressing the proteolytic enzyme at high efficiency. Compared with the prior art, the method for culturing the microzyme of the secretory expression proteolytic enzyme has the characteristics of low culture cost, convenient and accurate reaction control mode and high expression activity of the proteolytic enzyme.

Description

Technical field: [0001] The invention relates to a yeast culture method for secreting and expressing protease. Background technique: [0002] Mannan-oligosaccharides and xylo-oligosaccharides are considered to be high-end products in functional oligosaccharides because of their good bifidus factor effects. Xylanase and mannanase are necessary biological enzymes for large-scale production of these two oligosaccharides. [0003] In order to obtain highly active xylanase and mannanase, people have tried various methods, especially applying genetic engineering means to obtain various recombinant strains to express secretase protein. In the published literature reports, Bacillus is more widely used. In recent years, some people have used Pichia pastoris (P. pastoris) to express mannanase and xylanase. For example, literature 1 uses the recombinant bacteria P. pastoris GS115-Atx to prepare xylanase. It is believed that the organic medium composed of bran and beef extract can be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N1/32C12N9/60C12N9/24
Inventor 张雷王玉亭吴道贫
Owner 广州伯凯生物技术有限公司
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