CN102424803A - High-yield heat resistant type neutral phytase bacterial strain as well as fermentation culture medium and enzyme production method thereof - Google Patents
High-yield heat resistant type neutral phytase bacterial strain as well as fermentation culture medium and enzyme production method thereof Download PDFInfo
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Abstract
The invention discloses a high-yield heat resistant type neutral phytase bacterial strain. The classification naming of the bacterial strain is Bacillusnealsonii, the preservation number is China General Microbiological Center Culture Collection Center (CGMCC) No.5396, the preservation date is October 28th, 2011, and the preservation unit is CGMCC. The phytoenzyme produced by the bacterial strain has better high-temperature heat resistance, 41 percent of activity is also reserved after 30min under warm bath at 80 DEG C, under the condition of 90 DEG C, 73 percent of activity is also reserved after 10min, and 21 percent of activity is reserved after 30min. The invention also provides a bacterial strain culture medium and a method for producing heat resistant type neutral phytase by the bacterial strain.
Description
Technical field
The invention belongs to microorganism field, the method for particularly a kind of bacterial strain of high yield heat resistant type neutral phytase and fermention medium thereof and production heat resistant type neutral phytase.
Background technology
Phytic acid is one type of IP6 material, and chemical name is hexamethylene 6 alcohol-1,2,3,4,5, and 6-six dihydrogen phosphate are a kind of faint yellow viscous liquids, extensively are present in cereal, in leguminous plants and the oil crops, is the main storage form of phosphoric.Phytic acid has extremely strong sequestering power, shows very strong anti-oxidant action, can with multiple metals ion such as Zn
2+, Ca
2+, Cu
2+, Fe
2+, Mg
2+, Co
2+Isochela synthesizes corresponding insoluble complex, thereby influences animal digesting and assimilating mineral element; Phytic acid can with chelatings such as protein, amino acid, VITAMINs, its solvability is reduced, influence animal digesting and assimilating to these nutritive substances; Phytic acid can also combine with stomach en-, glycase, lypase, trypsinase etc. in the animal digestive tract to reduce its activity, thereby influences animal digesting and assimilating protein, glucide.The phosphorus of 60%-80% is that form with phytate phosphorus exists in the most plants forage; And monogastric animal (pig, poultry and fish etc.) lacks the Sumizyme PHY system of endogenous type; Therefore effectively metabolism is phytic acid and the phytate that is rich in the feed of staple with cereal, leguminous plants and oil crops; Its phosphorus utilization is merely 0 ~ 40%, is not excreted by a large amount of phytate phosphorus major parts of animal body utilization, causes the huge waste of phosphorus on the one hand; The phosphorus that excretes on the other hand gets in soil and the water, causes and discharges the pollution of phosphorus to environment in the aquaculture.And, must in feed, add inorganic phosphorus and other mineral salts in order to satisfy the nutritional need in the growth of animal process, the raising that this not only causes ample resources waste and feed cost has also produced great pollution to environment.And China's phosphor resource is relatively deficienter, costs an arm and a leg, and can not and have to add this contradiction of inorganic phosphorus increase feed cost by the simple stomach animal use thereby how to resolve phytate phosphorus a large amount of in the feed, and people have turned to enzyme-Sumizyme PHY edman degradation Edman to sight.
Sumizyme PHY (phytase) is the phytinic acid lytic enzyme; It is the general name of the enzyme of one type of ability catalysis phytic acid and the phytate inositol derivative and the phosphorus (or phosphoric acid salt) that are hydrolyzed into low phosphorylation; Be described as green feed additive; Extensively be present in plant, animal and the mikrobe, wherein microbe-derived Sumizyme PHY is the focus of Recent study.In feed, add Sumizyme PHY, improve phosphatic bioavailability, eliminate the anti-oxidant action of phytic acid; Reduce the drainage of phosphorus in environment, reduced agroecological burden thus, and slowed down the eutrophication of aquatic environment; Simultaneously also reduced the interpolation of inorganic phosphorus in the animal-feed, the latter is a kind of non-renewable resource.In addition, in food, use the absorption that Sumizyme PHY can improve iron and zinc, this crowd's nutrition for developing country has significant meaning.At present, Sumizyme PHY is as the monogastric animal feed additive, and its feeding effect is worldwide extensively proved conclusively.
But Sumizyme PHY also exists a lot of deficiencies in practical application, at first be Sumizyme PHY production cost with hold at high price, influenced actual popularization.The enzyme work of China's Sumizyme PHY product is merely 500 U/g.The work of product enzyme increases substantially; Will reduce the cost of Sumizyme PHY significantly; Do not put into effect as yet under the situation that relevant rules limit at current livestock and poultry farm movement phosphorus content; The cost and price that has only further reduction Sumizyme PHY, and significantly be lower than the price in phosphorus source, Sumizyme PHY could obtain large-scale promotion and application in China.Next is that the thermotolerance of Sumizyme PHY is generally lower, does not have better heat-resisting property, just can't resist the enzyme deactivation that when the feed high temperature granulating, causes, has further improved its cost again.China is particularly considerable through the amount that animal excrement enter natural phosphor resource every year, and this has not only caused the waste of huge phosphor resource, but also has caused serious water body environment pollution.How effective subdues owing to feed absorbs the phosphor resource that runs off, and making it turns waste into wealth, and eliminates its pollution to environment, becomes the major issue of present urgent need solution.
Summary of the invention
The object of the present invention is to provide a kind of bacterial strain of high yield heat resistant type neutral phytase, the classification called after of bacterial strain: genus bacillus
Bacillus nealsonii, its deposit number is CGMCC No.5396.Preservation date is on October 28th, 2011, and depositary institution is Chinese microorganism strains preservation management committee common micro-organisms center.
Plant enzymes molecular weight by this bacterial strain production is 43kDa, and the optimum temperuture of reaction is 55 ℃, and optimum pH is 7.5; This enzyme is better to the pyritous thermotolerance; Under 80 ℃ of temperature are bathed, also keep 41% activity behind the 30min, and under 90 ℃ of conditions; Also keep 73% activity behind the 10min, keep 21% activity behind the 30min.Alkali is had tolerance preferably, and not acidproof, most of metals ion all has restraining effect in various degree to the activity of this Sumizyme PHY, and strengthens its restraining effect along with the rising of its concentration, and Ca
2+To this enzyme unrestraint effect, slight promoter action is arranged on the contrary.Has very strong substrate specificity.This enzyme will be higher than in thermotolerance and separate the similar Sumizyme PHY that obtains at present, has good commercial applications and is worth.
The present invention adopts present biochemical technology, from soil, separates and has screened a Bacillus strain strain safety, can high yield heat resistant type neutral phytase
Bacillus nealsonii, the product enzyme process of this bacterial strain is optimized, and it is produced the enzyme characteristic estimate.The present invention can not and have to add inorganic phosphorus by the simple stomach animal use thereby to increase this problem of feed cost significant for solving phytate phosphorus a large amount of in the feed.
The separation method of bacterial strain of the present invention is following: take from Zhejiang, Yunnan plant rhizosphere soil, the high-temperature zone pedotheque is through killing common assorted bacterium behind 80 ℃ of water-bath 10min; Obtain the purpose bacillus spore; Getting bacterium liquid again, to coat screening dull and stereotyped, utilizes its hydrolysis phytic acid ca to produce the characteristic screening purpose bacterial strain of transparent circle, the bacterial strain that the picking transparent circle the is bigger separation of ruling; Obtain its single bacterium colony, accomplish first run primary dcreening operation.Bacterial strain behind the primary dcreening operation also will be measured enzyme activity through the ammonium meta-vanadate method and carry out multiple sieve to determine whether to exist the height of false positive bacterial strain and strain enzyme-producing vigor, selects the highest bacterial strain of vigor, and called after ZJ0902 preserves with glycerine.
The mode that adopts traditional identification of strains to combine with molecular biology identification is identified the bacterial strain that screening obtains.With reference to " uncle's Jie Shi Bacteria Identification handbook (the 8th edition); ZJ0902 is carried out gramstaining and spore staining; Observe its morphological structure and gemma position, combine some special physiological reactions of bacillus again, designed corresponding substratum and a series of biochemical reaction pipe.Biochemical reactions phenomenon by this bacterium tentatively is located the tyrothricin in bacillus.The Physiology and biochemistry qualification result of bacterial strain is as shown in the table.
| Certified variety | The result | Certified variety | The result |
| Gramstaining | ＋ | Catalase | ＋ |
| Whether produce gemma | Square column, middle life, no expansion external capsule | Urea decomposition | ＋ |
| Mobility | － | The phenylalanine(Phe) deamination | ＋ |
| Optimum temperuture | 37℃ | Glucose response | ＋ |
| 5%NaCl is growth down | ＋ | The SANMALT-S reaction | ＋ |
| 7%NaCl is growth down | － | The lactose reaction | ＋ |
| The starch hydrolysis | － | The sucrose reaction | － |
| Cellulose hydrolysis | － | The fructose reaction | － |
| Gelatine liquefication | － | The wood sugar reaction | － |
| Reduction nitrate salt | ＋ | The raffinose reaction | － |
| Nagler's reaction | － | The pectinose reaction | － |
| Indoles produces reaction | － | The rhamnosyl reaction | － |
| Citric acid reactions | － | Deep sugar reaction | － |
| H 2S produces reaction | － | The N.F,USP MANNITOL reaction | － |
| The V-P reaction | ＋ | The sorbyl alcohol reaction | － |
| The MR reaction | － | The Gluconic Acid Ammonium salt reaction | ＋ |
| Casein decomposition test | － | The ONPG reaction | － |
| The litmus milk reaction | Product acid reddens, and solidifies | The sodium hippurate reaction | － |
| The two hydrolysis reaction of l-arginine | － | Aerogenesis | － |
Annotate: "+" is positive, and "-" is negative.
The present invention also provides the fermention medium of above-mentioned bacterial strains, it is characterized in that, is made up of following components in weight percentage: 3.7% wheat bran, 2.27% peptone, 0.5% an ammonium nitrate, 0.00863% inorganic phosphorus, 0.2%CaCl
2, 0.05% KCl, 0.03%MgSO
4, 0.003%FeSO
4, 0.003% MnSO
4, 0.03% NaCl, surplus is a water.
Further, the pH value of said substratum is 7.0.
The present invention produces the enzyme substratum by common genus bacillus and sets out; Select for use and be easy to obtain and inexpensive raw material; Progressively replace carbon source, nitrogenous source and inorganic salt in the substratum, again to the temperature of its cultivation, inoculum size; Culture condition such as pH and liquid amount is optimized, and the culture condition that filters out the righttest medium component and the best is to improve the output of Sumizyme PHY.Utilize part factorial analysis experiment that each factor is carried out analysis of science to the influence degree of producing enzyme again; Confirm main affecting factors; Confirm the experimental center point through steepest climbing experiment again; Through the response surface experiment its mutual interactive relation is carried out research and analysis at last, confirm the final proportioning of substratum, to improve the ability that strain fermentation produces enzyme greatly.
A further object of the invention is to provide a kind of method of producing the heat resistant type neutral phytase; It is characterized in that may further comprise the steps: bacterial classification CGMCC No.5396 is inoculated into fermention medium; Said fermention medium is made up of following components in weight percentage: 3.7% wheat bran, 2.27% peptone, 0.5% an ammonium nitrate; 0.00863% inorganic phosphorus, 0.2%CaCl
2, 0.05% KCl, 0.03%MgSO
4, 0.003%FeSO
4, 0.003% MnSO
4, 0.03% NaCl, surplus is a water, pH7.0; 7%, 34 ℃ of fermentation of inoculum size 84 hours; The gained fermented liquid is centrifugal, concentrate through PEG20000 successively, the preliminary column chromatography of DEAE-Sepharose FF, the method for G-100 polydextran gel secondary column chromatography is carried out separation and purification to Sumizyme PHY, gets heat resistant type neutral phytase product.
Through aforesaid method, inoculum size 7% is shaken bottled liquid measure 75 mL/250 mL; Reach behind 34 ℃ of fermentation 84h and produce the enzyme mxm.; The Sumizyme PHY vigor reaches 13625.8U/ mL, the 8251U/mL of contrast original strain, and enzyme work has improved 65%; And the time of producing enzyme also extended to 84h by 72h, improved the enzymatic productivity of this bacterium greatly.
Description of drawings
Fig. 1 is the plate screening figure as a result of high yield heat resistant type neutral phytase bacterial strain of the present invention.
Fig. 2 A is the gramstaining qualification result figure of bacterial strain of the present invention.
Fig. 2 B is the gemma gramstaining qualification result figure of bacterial strain of the present invention.
Fig. 3 is the molecular biology identification figure as a result of bacterial strain of the present invention.
Fig. 4 is growing state and the product enzyme process synoptic diagram of bacterial strain of the present invention on fermentor tank.
Fig. 5 is that bacterial strain of the present invention produces heat-resistance phytase characteristic synoptic diagram.
Embodiment
Embodiment 1.High yield heat resistant type neutral phytase bacterial strain screening
Get Zhejiang, various places, Yunnan plant rhizosphere soil, the high-temperature zone pedotheque through behind 80 ℃ of water-bath 10min, killing common assorted bacterium, obtains the purpose bacillus spore, is diluted to 10 with saline water again
-3, 10
-4, 10
-5, get 1mL bacterium liquid and coat screening dull and stereotyped (2% glucose, 0.2%CaCl
2, 0.5%NH
4NO
3, 0.05% KCl, 0.05%MgSO
4.7H
2O, 0.001% FeSO
4.7H
2O, 0.001% MnSO
4.4H
2O, 0.3% phytic acid ca, 2% agar pH7.0), utilizes its hydrolysis phytic acid ca to produce the characteristic screening purpose bacterial strain of transparent circle, and the bacterial strain that the picking transparent circle is bigger separations of ruling obtained its single bacterium colony, completion first run primary dcreening operation.Plate screening figure as a result is as shown in Figure 1.
Consider that but the material in some bacterium metabolism substratum produces a large amount of acidic metabolites; This metabolite also possibly decompose phytic acid ca, produces transparent hydrolysis circle, causes false positive; So bacterial strain behind the primary dcreening operation; Also will measure enzyme activity through the ammonium meta-vanadate method and carry out multiple sieve to determine whether to exist the height of false positive bacterial strain and strain enzyme-producing vigor, select the highest bacterial strain of vigor, called after ZJ0902 preserves with glycerine.
The ammonium meta-vanadate method is a kind of phytase activity measuring method commonly used, has good sensitivity and stability, and its concrete steps are: get fermented liquid 10 mL; 4000 r/min are centrifugal, and 15 min remove thalline, and 0.1 mL diluent+1.9 mL Tris-HCl (pH7.5)+4 mL sodium phytates (2 mmol/L) are got in 10 times of dilutions; 55 ℃ of reaction 30 min add 4 mL reaction terminating liquids (pure nitric acid 165 mL of 100 g/L ammonium molybdates, 250 mL+2.35 g/L ammonium vanadate, 250 mL+65%, water is supplemented to 1000 mL) again; 10 min develop the color; Behind centrifugal 10 min of 4000 r/min, survey the OD value in 415 nm places, live according to deciding phosphorus typical curve calculating enzyme again.Blank adds reaction substrate again for adding reaction terminating liquid earlier.The enzyme unit (U/mL) that lives: at 55 ℃, under the condition of pH7.5, PM discharges 1 nmol from concentration is the substrate sodium phytate of 2 mmol/L the needed enzyme amount of inorganic phosphorus is 1 enzyme unit that lives.
Embodiment 2.Identification of strains
The mode that adopts traditional identification of strains to combine with molecular biology identification is identified the bacterial strain that screening obtains.With reference to " uncle's Jie Shi Bacteria Identification handbook (the 8th edition); ZJ0902 is carried out gramstaining and spore staining (coloration result is shown in Fig. 2 A, 2B); Observe its morphological structure and gemma position; Combine some special physiological reactions of bacillus again, designed corresponding substratum and a series of biochemical reaction pipe.Biochemical reactions phenomenon by this bacterium tentatively is located the tyrothricin in bacillus.The Physiology and biochemistry qualification result of bacterial strain is as shown in the table:
| Certified variety | The result | Certified variety | The result |
| Gramstaining | ＋ | Catalase | ＋ |
| Whether produce gemma | Square column, middle life, no expansion external capsule | Urea decomposition | ＋ |
| Mobility | － | The phenylalanine(Phe) deamination | ＋ |
| Optimum temperuture | 37℃ | Glucose response | ＋ |
| 5%NaCl is growth down | ＋ | The SANMALT-S reaction | ＋ |
| 7%NaCl is growth down | － | The lactose reaction | ＋ |
| The starch hydrolysis | － | The sucrose reaction | － |
| Cellulose hydrolysis | － | The fructose reaction | － |
| Gelatine liquefication | － | The wood sugar reaction | － |
| Reduction nitrate salt | ＋ | The raffinose reaction | － |
| Nagler's reaction | － | The pectinose reaction | － |
| Indoles produces reaction | － | The rhamnosyl reaction | － |
| Citric acid reactions | － | Deep sugar reaction | － |
| H 2S produces reaction | － | The N.F,USP MANNITOL reaction | － |
| The V-P reaction | ＋ | The sorbyl alcohol reaction | － |
| The MR reaction | － | The Gluconic Acid Ammonium salt reaction | ＋ |
| Casein decomposition test | － | The ONPG reaction | － |
| The litmus milk reaction | Product acid reddens, and solidifies | The sodium hippurate reaction | － |
| The two hydrolysis reaction of l-arginine | － | Aerogenesis | － |
Annotate: "+" is positive, and "-" is negative.
Extract the genomic dna that test kit extracts this bacterium with DNA of bacteria, as template with primer P1, P2 and archaeal dna polymerase through the increased 16SrDNA gene order of this bacterium of round pcr.Pcr amplification product is identified with 0.8% agarose gel electrophoresis and is checked order.Wherein primer P1, P2 sequence are:
Primer P1 sequence: 5 '-AGAGTTTGATCMTGGCTCAG-3 '
Primer P2 sequence: 5 '-AAGGAGGTGWTCCARCC-3 '
The result shows that the sequence length of the 16S rDNA that obtains of amplification is 1469bp, in the Genbank DB, carries out the Blast sequence alignment, selects the bacterial strain of each close kind of homology, and constructing system is grown tree, can confirm this bacterial strain with
Bacillus nealsoniiBelong to a branch together.In conjunction with morphological specificity, the physiological and biochemical property qualification result of front, can the bacterial strain ZJ0902 of screening be positioned in the bacillus
Bacillus nealsoniiThe molecular biology identification of bacterial strain of the present invention figure as a result is as shown in Figure 3.This bacterial classification was in preservation on October 28 in 2011, and its deposit number is CGMCC No.5396, and depositary institution is Chinese microorganism strains preservation management committee common micro-organisms center.
Embodiment 3.ZJ0702 strain enzyme-producing performance study and fermentation condition optimization
Fermention medium to the ZJ0702 bacterial strain is further optimized, to improve the ability that it produces enzyme.The present invention produces the enzyme substratum by common genus bacillus and sets out; Select for use and be easy to obtain and inexpensive raw material; Progressively replace carbon source, nitrogenous source and inorganic salt in the substratum, again to the temperature of its cultivation, inoculum size; Culture condition such as pH and liquid amount is optimized, and the culture condition that filters out optimum medium component and the best is to improve the output of Sumizyme PHY.Utilize part factorial analysis experiment that each factor is carried out analysis of science to the influence degree of producing enzyme; Confirm main affecting factors; Confirm the experimental center point through steepest climbing experiment again; Through the response surface experiment its mutual interactive relation is carried out research and analysis at last, confirm the final proportioning of substratum, to improve the ability that the ZJ0902 strain fermentation produces enzyme.
Nutrient media components after optimizing is 3.7% wheat bran, 2.27% peptone, 0.5% an ammonium nitrate, 0.00863% inorganic phosphorus, 0.2%CaCl
2, 0.05% KCl, 0.03%MgSO
4, 0.003%FeSO
4, 0.003% MnSO
4, 0.03% NaCl; Culture condition is: 34 ℃, and inoculum size 7%, pH7.0; Shake bottled liquid measure 75 mL/250 mL, reach behind the fermentation 84h and produce the enzyme mxm., the Sumizyme PHY vigor reaches 13625.8U/ mL; The 8251U/mL of contrast original strain; Enzyme work has improved 65%, and the time of producing enzyme also extended to 84h by 72h, improved the enzymatic productivity of this bacterium greatly.
Embodiment 4.Jar fermentation research and process control on the bacterial strain
Adopt 5L small-sized fermentation jar simulation industry to produce enzyme, adopt two kinds of different modes of common enlarged culturing and high-density culture to study the technology that it produces enzyme, wherein common enlarged culturing is: the 3L fermention medium; Inoculum size inoculation by 7%; 34 ℃ of temperature, rotating speed 400r/min, every fermentation 12h get once its enzyme work of appearance mensuration, protein content, reducing sugar content; And on-line determination produces the pH in the enzyme process; Fermentation Process of Parameter such as dissolved oxygen, culture temperature is studied strain growth state and product enzyme, the relation of utilization of carbon source etc. in its process.High-density culture is: by the common fermentation mode 72h that ferments, when the carbon source approach exhaustion, mend the bran water solution 80mL into 3.7%.The comparison of the dual mode through common enlarged culturing and high-density culture, the variation of parameter and to producing the influence of enzyme in the research process, thus finally confirm fermentation parameter.Growing state and the product enzyme process synoptic diagram of bacterial strain of the present invention on fermentor tank is as shown in Figure 4.
The result shows: the product enzyme of high-density culture will be apparently higher than common enlarged culturing; Confirm that high-density culture is the mode of its enzymatic production; Specific strategy is enzymatic production 72h, mends the bran water solution 80mL into 3.7% when carbon source is about to exhaust, and this moment, fermentation time prolonged 24h; Enzyme work has improved 10%, and enzyme work has reached 22551.8U/mL.Its vigor will be much higher than the present Sumizyme PHY of reporting of the same type, has reached the level of suitability for industrialized production.
Embodiment 5. the separation and purification of heat-resistance phytase and the property research of enzyme
Under the best medium condition, strain fermentation produces enzyme 72h, and the centrifugal 20min of fermented liquid 12000 r/min is obtained crude enzyme liquid; Pack in the dialysis tubing, spill PEG20000, draw the moisture in the crude enzyme liquid on the surface; Every 3h mends and spills PEG20000 one time, and it is carried out directly concentrating, and gets fermented liquid 2 mL after PEG20000 concentrates; Last DEAE-Sepharose FF ion exchange column; Tris-HCl damping fluid with the pH 7.0 that contains 0-1.0 mol/L NaCl under the control of computer constant flow pump carries out gradient elution, and flow velocity is 1 mL/min, and every pipe is collected 5 mL.Measure every pipe and collect the enzyme work of liquid and the light absorption value at 280 nm places; Be associated with active chromatographic solution; Concentrated chromatographic solution 2 mL that got DEAE-Sepharose FF pillar at last cross the G-100 polydextran gel and carry out the secondary separation purifying; Tris-HCl damping fluid with pH 7.0 under the control of computer constant flow pump carries out wash-out, and flow velocity is 15mL/h, and every pipe is collected 5 mL.Measure every pipe and collect the enzyme work of liquid and the light absorption value at 280 nm places.Be associated with the component that enzyme is lived, get appearance on each chromatographic solution 15 μ L, carry out the SDS-PAGE electrophoretic analysis; Proof obtains pure Sumizyme PHY, again to its optimum reaction condition, thermotolerance; The tolerance of pH, each item zymologic properties such as the influence of metals ion and substrate specificity are studied.
The result is following; Concentrate through PEG20000, the preliminary column chromatography of DEAE-Sepharose FF, the method for G-100 polydextran gel secondary column chromatography is carried out separation and purification to the bacillus category Sumizyme PHY; Obtained single Sumizyme PHY pure protein; The purifying multiple is 44.14, and the recovery is 5.72%, explains that this separation method is carrying out purifying or highly effective to such Sumizyme PHY.And the zymologic property of this Sumizyme PHY is: molecular weight is 43kDa, and the optimum temperuture of reaction is 55 ℃, and optimum pH is 7.5.Fig. 5 is that bacterial strain of the present invention produces heat-resistance phytase its activity characteristic synoptic diagram under different temperature and pH value.This enzyme is better to the pyritous thermotolerance, under 80 ℃ of temperature are bathed, also keeps 41% activity behind the 30min, and under 90 ℃ of conditions, also keeps 73% activity behind the 10min, keeps 21% activity behind the 30min.Alkali is had tolerance preferably, and not acidproof, most of metals ion all has restraining effect in various degree to the activity of this Sumizyme PHY, and strengthens its restraining effect along with the rising of its concentration, and Ca
2+To this enzyme unrestraint effect, slight promoter action is arranged on the contrary.Has very strong substrate specificity.Hence one can see that, and this enzyme will be higher than in thermotolerance has separated the similar Sumizyme PHY that obtains at present, has good commercial applications and is worth.
Those of ordinary skill in the art will be appreciated that; Above embodiment is used for explaining the present invention; And be not that conduct is to qualification of the present invention; As long as in essential scope of the present invention, all will drop in the scope of claims of the present invention variation, the modification of the above embodiment.
Claims (4)
1. the bacterial strain of a high yield heat resistant type neutral phytase, the classification called after of bacterial strain:
Bacillus nealsonii, its deposit number is CGMCC No.5396.
2. the fermention medium of the said bacterial strain of claim 1 is characterized in that, is made up of following components in weight percentage: 3.7% wheat bran, 2.27% peptone, 0.5% an ammonium nitrate, 0.00863% inorganic phosphorus, 0.2%CaCl
2, 0.05% KCl, 0.03%MgSO
4, 0.003%FeSO
4, 0.003% MnSO
4, 0.03% NaCl, surplus is a water.
3. fermention medium as claimed in claim 2 is characterized in that, the pH value of said substratum is 7.0.
4. method of producing the heat resistant type neutral phytase; It is characterized in that may further comprise the steps: bacterial classification CGMCC No.5396 is inoculated into fermention medium; Said fermention medium is made up of following components in weight percentage: 3.7% wheat bran, 2.27% peptone, 0.5% an ammonium nitrate; 0.00863% inorganic phosphorus, 0.2%CaCl
2, 0.05% KCl, 0.03%MgSO
4, 0.003%FeSO
4, 0.003% MnSO
4, 0.03% NaCl, surplus is a water, pH7.0; 7%, 34 ℃ of fermentation of inoculum size 84 hours; The gained fermented liquid is centrifugal, concentrate through PEG20000 successively, the preliminary column chromatography of DEAE-Sepharose FF, the method for G-100 polydextran gel secondary column chromatography is carried out separation and purification to Sumizyme PHY, gets heat resistant type neutral phytase product.
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| CN104732084A (en) * | 2015-03-20 | 2015-06-24 | 上海交通大学 | Model construction method for reducing rapidly digestible starch content on basis of response surface method |
| CN107893045A (en) * | 2017-12-28 | 2018-04-10 | 云南中烟工业有限责任公司 | A kind of bacterial strain Bacillus nealsoniiYFM3 and its preparation method and application |
| CN115074281A (en) * | 2022-06-27 | 2022-09-20 | 华中农业大学 | Bacillus ginseng strain capable of highly producing organic acid and application thereof in microecological preparation |
| CN115725635A (en) * | 2022-07-28 | 2023-03-03 | 青岛蔚蓝生物集团有限公司 | Pichia pastoris mutant strain and application thereof in production of neutral phytase |
| CN116337791A (en) * | 2023-05-31 | 2023-06-27 | 北京挑战生物技术有限公司 | In-vitro detection method for release rate of phosphorus phytate in feed raw material |
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