CN1544622A - Method for preparing phytase by the fermentation of methanol nutrient type recombinant yeast - Google Patents
Method for preparing phytase by the fermentation of methanol nutrient type recombinant yeast Download PDFInfo
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- CN1544622A CN1544622A CNA2003101088064A CN200310108806A CN1544622A CN 1544622 A CN1544622 A CN 1544622A CN A2003101088064 A CNA2003101088064 A CN A2003101088064A CN 200310108806 A CN200310108806 A CN 200310108806A CN 1544622 A CN1544622 A CN 1544622A
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- phytase
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- methyl alcohol
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Abstract
The invention relates to an improved method of preparing phytase by recombined methanol culture yeast fermenting, adopting low-price carbon source and nitrogen source to make cell growth in the course of engineering phytase fermenting, adopting variable-pH fermenting technique, thus implementing high-density expression of engineering phytase. Compared with existing techniques, it has low cost and the made phytase has higher enzyme activity.
Description
Technical field
The present invention relates to a kind ofly prepare improving one's methods of phytase, belong to bioengineering field with reorganization methyl alcohol nutritional type yeast fermentation.
Background technology
Phytic acid (claiming phytinic acid again) with the form of phytate, extensively exists in the plant materials usually, can not be utilized by the simple stomach animal.Phytase, be that phytinic acid lytic enzyme (Myo-inositol-hexaphosphatephosphohydrolase) can make phytate phosphorus be degraded into inositol and phosphoric acid, it is the general name that a class catalysis inositol hexaphosphate is taken off the phosphate group reaction enzymes, there are three types: phytinic acid 3-phosphohydrolase (Myo-inositol-hexaphosphate 3-phosphohydrolase, EC 3.1.3.8), (Myo-inositol-hexaphosphate 6-phosphohydrolase is EC3.1.3.26) with nonspecific orthophosphoric acid salt monoesters phosphohydrolase for phytinic acid 6-phosphohydrolase.The hydrolytic action of phytic acid is from the inositol six-ring on the 3rd and the 6th the O-P key, hydrolysis one by one then.The phytase that acts on the 6th O-P of phytic acid is called 6-phytase (phytinic acid 6-phosphohydrolase), and is called 3-phytase (phytinic acid 3-phosphohydrolase) since the 3rd phytase that O-P degrades.Phytase is mainly produced by plant (phytinic acid 6-phosphohydrolase) and microorganism (phytinic acid 3-phosphohydrolase), it can make the utilization ratio of phosphorus in the plant feed improve 60%, the excretion of phosphorus reduces 40% in the ight soil, reduces the anti-oxidant action of phytate and reduces the effects such as pollution of phytate phosphorus to environment.
The existing method that adopts fermentation method to prepare phytase has US 5,436,156, and three kinds of being reported of CN 1405303A and CN1184156A are said all to exist the low defective such as preparation cost height that reaches of phytase activity (FTU) on the whole.
Summary of the invention
The object of the invention is, provides a kind of and prepares the method for phytase with reorganization methyl alcohol nutritional type yeast fermentation, overcomes the deficiencies in the prior art with this.
Design of the present invention is such:
Reorganization methyl alcohol nutritional type yeast fermentation prepares phytase and is divided into two stages: one is the cell growth phase; Two is the protein expression stage of foreign gene.At the cell growth phase, its target is to realize high-cell density, owing to the too high often cell growth inhibiting of concentration of substrate (carbon source), therefore exists again and criticizes (fermentation) stage of cultivation and feed supplement cultivation (fermentation) stage.For this genetic engineering bacterium is to express the phytase that added value is lower and fermentation scale needs are very big, the carbon source that the cell growth phase is required and the price of nitrogenous source are the keys of the cost of decision whole process of production, adopting cheap syrup, sucrose, molasses, lactose, starch hydrolyzate or cerelose among the present invention is that (carbon source is higher for relatively at high price glycerine or used glucose concn in the prior art for carbon source, reach 100g/L, the low only 5g/L of the present invention has avoided the insufficient shortcoming of generation glucose effect and oxygen supply); Being easy to transportation and storage ammonium salt, corn steep liquor, analysis for soybean powder or cottonseed meal is nitrogenous source (nitrogenous source is for being difficult for the ammoniacal liquor of storage and transportation in the prior art).In addition, the present invention has realized the purpose of low-cost high-density fermentative preparation phytase by suitable feed supplement mode and to the control of the pH value of whole fermentation process.
Technical scheme:
The present invention is said to prepare the method for phytase with reorganization methyl alcohol nutritional type yeast fermentation, comprises the protein expression stage of cell batch fermentation, fed-batch fermentation and foreign gene, and the promotor that relates to has alcohol oxidase promotor (P
AOX) and methyl alcohol promotor (P
MOX) etc., it is characterized in that the carbon source in the stage is syrup, sucrose, molasses, lactose, starch hydrolyzate or cerelose at said cell batch fermentation and fed-batch fermentation; Nitrogenous source is ammonium salt, corn steep liquor, analysis for soybean powder or cottonseed meal; At the said cell batch fermentation and the pH=5.0-5.5 in fed-batch fermentation stage, at the pH=4.0-4.8 in protein expression stage of said foreign gene;
Wherein said reorganization methyl alcohol nutritional type yeast comprises pichia spp (Pichia) or debaryomyces hansenii (P.hansenesis) etc., and preferred yeast is pichia spp (Pichia).
The preferred ammonium salt of said nitrogenous source, more preferably ammonium sulfate.
The whole process pH that ferments in the prior art is 5.0-5.5, be permanent pH fermentation, and the present invention adopts and becomes pH control fermentation, and the mode that becomes pH not to be that prior art is said realize by adding acid or adding alkali, realize but adopt by control carbon source feed supplement mode.
The advantage of invention is, adopted cheap carbon source and nitrogenous source to carry out the method for the cell growth of engineering phytase fermenting process, and the technology that has adopted change pH fermentation, thereby the high-density that realizes the engineering phytase is expressed, with prior art (Van Gorcom R.F.M.et al., Patent No.US 5436156,1995; Yao Bin etc., CN 1405303A and CN 1184156A) to compare, the cheaper and prepared phytase activity of its cost of the said preparation method of the present invention is higher.
Specific implementation method
Be further described bright below by case study on implementation, its objective is to be better understanding content of the present invention, illustrated embodiment does not limit protection scope of the present invention:
Embodiment 1
Used bacterial strain is SP8847 or YY113 (Mut
sHis
+), by Shanghai forever industry Nong Ke Bioisystech Co., Ltd make up.
Will be from flat board picking one single bacterium colony insert that (1L contains: yeast powder 10g, peptone 20g, glucose 10g) 30 ℃ of following 220r/min cultivated 20 hours the YPD seed culture medium (500mL Erlenmeyer flask liquid amount 50mL).Be connected to by 10% inoculum size then that (1L contains: cerelose 5.0g, ammonium sulfate [(NH in the 50L fermentor tank of dress 20L batch culture base
4)
2SO
4] 20g, PTM1 12mL, phosphoric acid 26mL), transfer pH to 5.2 with 2mol/LKOH before the inoculation.Keep dissolved oxygen concentration more than 25% by control air flow and rotating speed.Batch culture finishes the back to the greatest extent in the carbon source metabolism, and (1L contains: cerelose 500g, ammonium sulfate [(NH to mend supplemented medium again
4)
2SO
4] 5.0g, PTM1 12mL) make the cell continued growth, reached high-density, i.e. cell optical density(OD) (OD at 40-50 hour that ferments
600) more than 400.Closing to an end in cell growth was set at 4.8 with the pH set(ting)value by 5.2 in preceding 1 hour, made pH because the organic acid of metabolism carbon source generation drops to set(ting)value, stopped to mend carbon source (cerelose) afterwards and entered the phytase expression phase.After stopping to mend cerelose, after cerelose metabolism to the greatest extent, dissolved oxygen concentration (DO) will rise to more than 70% in 1 minute, begin to mend the expression that methyl alcohol carries out the engineering phytase then.
Embodiment 2
Remove and use the cane sugar substitution cerelose, the pH=5.5 of cell growth phase, outside the pH=4.8 of expression phase, other conditions are identical with embodiment 1.
Embodiment 3
Remove with syrup and substitute cerelose, the pH=5.0 of cell growth phase, outside the pH=4.0 of expression phase, other conditions are identical with embodiment 1.
Embodiment 4
Remove with molasses and substitute cerelose, the pH=5.2 of cell growth phase, outside the pH=4.4 of expression phase, other conditions are identical with embodiment 1.
When carrying out the cell grown cultures with carbon source of the present invention and nitrogenous source, cell optical density(OD) (OD
600) can reach 400-700, dry cell weight is more than the 150g/L, reaches the requirement of high density fermentation.The present invention compared with prior art the results are shown in following table:
Show the comparison that phytase is produced in cell growth carbon source of the present invention and nitrogenous source and invention both at home and abroad
| The growth phase carbon nitrogen source | The present invention | ???US.5436156 | ?CN?1405303A | ??CN?1184156A |
| Carbon source | Syrup, sucrose, molasses, lactose, starch hydrolyzate or cerelose 5.0g/L | Sweet dew-macrodex g/L yeast extracts 12.5g/L | Glycerine or glucose 100g/L | Glycerine or glucose 100g/L |
| Nitrogenous source | Ammonium salt, corn steep liquor, analysis for soybean powder or cottonseed meal 20g/L | Caseinhydrolysate 25g/L | 25% ammoniacal liquor closed-loop control stream adds | 25% ammoniacal liquor closed-loop control stream adds |
| Cell density (dry weight, g/L) | ????＞150 | ????＞100 | ?????＞100 | |
| Phytase activity/FTU | ?????2250 | ??????280 | ?????500 | ??????500 |
Claims (4)
1, a kind of method for preparing phytase with reorganization methyl alcohol nutritional type yeast fermentation, the protein expression stage that comprises cell batch fermentation, fed-batch fermentation and foreign gene, it is characterized in that the carbon source in the stage is syrup, sucrose, molasses, lactose, starch hydrolyzate or cerelose at said cell batch fermentation and fed-batch fermentation; Nitrogenous source is ammonium salt, corn steep liquor, analysis for soybean powder or cottonseed meal; At the said cell batch fermentation and the pH=5.0-5.5 in fed-batch fermentation stage, at the pH=4.0-4.8 in protein expression stage of said foreign gene;
Wherein said reorganization methyl alcohol nutritional type yeast is pichia spp (Pichia) or debaryomyces hansenii (P.hansenesis).
As the said preparation method of claim 1, it is characterized in that 2, wherein said nitrogenous source is an ammonium salt.
As the said preparation method of claim 2, it is characterized in that 3, wherein said nitrogenous source is an ammonium sulfate.
As any one preparation method in the claim 1～3, it is characterized in that 4, wherein said reorganization methyl alcohol nutritional type yeast is pichia spp (Pichia).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101088064A CN1544622A (en) | 2003-11-24 | 2003-11-24 | Method for preparing phytase by the fermentation of methanol nutrient type recombinant yeast |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101088064A CN1544622A (en) | 2003-11-24 | 2003-11-24 | Method for preparing phytase by the fermentation of methanol nutrient type recombinant yeast |
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| Publication Number | Publication Date |
|---|---|
| CN1544622A true CN1544622A (en) | 2004-11-10 |
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824444A (en) * | 2010-04-09 | 2010-09-08 | 上海化工研究院 | Method for preparing <13>C-labelled glucose |
| CN102286503A (en) * | 2011-09-05 | 2011-12-21 | 中国农业科学院生物技术研究所 | Paenibacillus neutral phytase |
| CN105802936A (en) * | 2016-05-26 | 2016-07-27 | 湖南南北旺生物技术有限公司 | Preparation method of high-temperature resistant neutral phytase, high-temperature resistant neutral phytase and application of high-temperature resistant neutral phytase |
| CN107922914A (en) * | 2015-06-17 | 2018-04-17 | 波特研究公司 | Breed microorganism and correlation technique and system |
-
2003
- 2003-11-24 CN CNA2003101088064A patent/CN1544622A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824444A (en) * | 2010-04-09 | 2010-09-08 | 上海化工研究院 | Method for preparing <13>C-labelled glucose |
| CN101824444B (en) * | 2010-04-09 | 2012-07-18 | 上海化工研究院 | Method for preparing <13>C-labelled glucose |
| CN102286503A (en) * | 2011-09-05 | 2011-12-21 | 中国农业科学院生物技术研究所 | Paenibacillus neutral phytase |
| CN107922914A (en) * | 2015-06-17 | 2018-04-17 | 波特研究公司 | Breed microorganism and correlation technique and system |
| CN105802936A (en) * | 2016-05-26 | 2016-07-27 | 湖南南北旺生物技术有限公司 | Preparation method of high-temperature resistant neutral phytase, high-temperature resistant neutral phytase and application of high-temperature resistant neutral phytase |
| CN105802936B (en) * | 2016-05-26 | 2019-10-29 | 湖南南北旺生物技术有限公司 | The preparation method and high temperature resistant neutral phytase of a kind of high temperature resistant neutral phytase and its application |
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