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Method for preparing batroxobin through fermentation of recombinant methanol yeast

A technology of methanolic yeast and batroxobin, applied in the direction of microorganism-based methods, biochemical equipment and methods, hydrolytic enzymes, etc.

Inactive Publication Date: 2011-11-16
辽宁远大诺康医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above research results show that it is feasible to produce snake venom-like thrombin by genetic engineering, but it is necessary to use eukaryotic expression system

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Pick a single colony cultured on the YPD plate medium at 30°C and carry out shaking culture in a shaker flask for 18 hours, then transfer to 1.5 liters to continue culturing overnight, and directly inoculate into a 30 liter fermenter, 15 liters of the medium is a salt medium, dissolve Oxygen is kept at 30%, and the number of revolutions is 700-800rpm. After the carbon source was exhausted, the glycerol flow was fed.

[0029] When the OD value reaches 300-350, it enters the methanol induction stage, and methanol is added to 0.2% within 2 hours. When methanol starts to be used, the methanol concentration is gradually increased to 0.5%-1%, and the culture temperature is gradually reduced to 25°C, and the pH is adjusted to 6.8-7.0. During the fermentation process, the tank pressure should be maintained between 0.04-0.06MPa, and the air flow Between 0.2-0.25VVM, the speed is maintained between 700-800rpm.

[0030] When the OD is close to 600 and the activity reaches 90U, d...

Embodiment 2

[0033] Pick a single colony cultured on the YPD plate medium at 30°C and carry out shaking culture in a shaker flask for 18 hours, then transfer to 1.5 liters to continue culturing overnight, and directly inoculate into a 30 liter fermenter, 15 liters of the medium is a salt medium, dissolve Oxygen is kept at 30%, and the number of revolutions is 700-800rpm. After the carbon source was exhausted, the glycerol flow was fed.

[0034] According to the fermentation method of invitrogin, the pilot-scale fermentation was carried out. Others induced more than 200 hours. When the protein activity in the fermentation broth no longer increased, the fermentation broth was centrifuged to collect the supernatant, the protein activity reached 90KU / ml, and the output reached 2.34×10 6 KU. After microfiltration and sterilization, the active protein with a purity of more than 97% is obtained by using hydrophobic column chromatography, ion exchange column chromatography, gel column chromatog...

Embodiment 3

[0036] Seed liquid: After fermenting in 1.5L yeast nitrogen base medium at 28°C for 24 hours, transfer to 30L fermentation liquid for fermentation. 13.5 L salt medium, the composition is as follows: H 3 PO 4 , 27ml / L; CaSO 4 2H 2 O, 0.9g / L; K 2 SO 4 , 18g / L; MgSO 4 ·7H 2 O, 15g / L; KOH, 4.13g / L; glycerin 40g / L; 4.4ml / L trace mineral solution.

[0037] During fermentation, NH 4 Adjust the pH value with OH to maintain it at 5.0. Ferment with vigorous agitation (1000rpm) under oxygen at 30°C, and add an antifoaming agent. After 24 hours of fermentation, add 50% glycerin containing 12ml / L trace minerals, the dissolved oxygen concentration is 30%, and continue to cultivate for 10 hours. After carbonogen starvation for 30 min, methanol was added for induction. The rate of adding methanol in the first 5 hours is low, and does not provide oxygen, so that the yeast adapts to methanol. 100% methanol contains 12ml / L trace mineral solution, and its dissolved oxygen is more than 3...

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Abstract

The invention aims to provide a method for preparing batroxobin through fermentation of recombinant methanol yeast. The method comprises batch fermentation and fed-batch fermentation of yeast, and methanol induced expression of the batroxobin, wherein the yeast is recombinant site-directed mutated batroxobin that the 45-position arginine of natural batroxobin is mutated into lysine. The method is characterized in that: a carbon source for the batch fermentation and fed-batch fermentation of the yeast is glycerol, and the pH is 5.0-5.5; and the pH in the methanol induced expression of the batroxobin is 6.8-7.0, the temperature is 25DEG C and the methanol concentration is 0.5 percent. Research results indicate that: by the method, the fermentation efficiency can be greatly improved, and the expression level of a target product is obviously improved.

Description

technical field [0001] The invention relates to a fermentation process for expressing recombinant batroxobin, an antithrombotic drug, by methanolic yeast. Background technique: [0002] With the increase of patients with thromboembolic diseases, the development of antithrombotic drugs has become one of the hot spots. On the one hand, people use molecular biology techniques to modify the molecular structure of existing drugs in order to obtain better thrombolytic drugs. On the other hand, we are actively looking for some natural thrombolytic drugs with good safety, good curative effect and few side effects. [0003] Expressing certain proteins in E.coli is already a basic means of producing pharmaceutical proteins more routinely, but in the actual research on snake venom thrombin, it is found that the amount of snake venom thrombin expressed in E.coli is very small. Pan Hua et al. used the RT-PCR method to amplify the thrombin-like gene Pallas from Agkistrodon halys Pallas,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/60C12R1/645
Inventor 薛百忠王宏英薛雁徐梅王建华姜大威杨宇孟威
Owner 辽宁远大诺康医药有限公司
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