CN102763771B - Fermentation and coating of lysine and xylanase - Google Patents
Fermentation and coating of lysine and xylanase Download PDFInfo
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- CN102763771B CN102763771B CN2012101858839A CN201210185883A CN102763771B CN 102763771 B CN102763771 B CN 102763771B CN 2012101858839 A CN2012101858839 A CN 2012101858839A CN 201210185883 A CN201210185883 A CN 201210185883A CN 102763771 B CN102763771 B CN 102763771B
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- 230000004151 fermentation Effects 0.000 title claims abstract description 32
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title abstract description 14
- 239000004472 Lysine Substances 0.000 title abstract description 7
- 239000011248 coating agent Substances 0.000 title abstract description 5
- 238000000576 coating method Methods 0.000 title abstract description 5
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- 238000000034 method Methods 0.000 claims abstract description 56
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- 241000283690 Bos taurus Species 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention provides a fermentation method for preparing an L-lysine product. The method comprises steps of: producing an L-lysine fermentation broth through fermentation; mixing the fermentation broth with high temperature dehydration resistant xylanase which can be obtained by fermentation; and drying the mixture at high temperature, and coating with fat. In addition, the present invention also provides a product produced by the method and high temperature dehydration resistant xylanase, etc.
Description
Technical field
The invention belongs to amino acid whose fermentation field, particularly, the present invention relates to ferment prepares the method for 1B goods, and it comprises that fermentation produces the 1B fermented liquid, this fermented liquid can mix with the zytase of the high temperature resistant dehydration that obtains of can fermenting, and is coated with fat after the high temperature drying.In addition, the present invention also provides the product of described method production and the zytase of high temperature resistant dehydration etc.
Background technology
Therefore the zytase material that contains xylogen (such as, feed) of can effectively degrading adds the absorption rate that zytase can strengthen feed, thereby saves feeding cost in feed.For example, Chinese patent 96196760.9 discloses the enzyme additive of ruminant feed, and it comprises zytase.
1B is important amino acid starting material, can be used as seasonings, food, fodder additives use, also can be used as the effective or adjunct ingredient in healthcare products, the medicine, is widely used in grocery trade, feed industry, pharmacy industry and other chemical industry.Current, the production of 1B mainly is by the fermentative production of microorganism, as utilizing coryneform bacteria production.
Yet, when Methionin as feed the time, because it belongs to the small molecules nutritive substance, can be discharged into soon in the animal body, especially when huge uptake, affected the efficient that animal absorbs.Usually solution is a small amount of feeding animals repeatedly, but this will increase the workload of animal rearing.Therefore, the present invention proposes direct method with the coated Methionin goods of fermenting lysine nutrient solution preparation, can effectively slow down the speed that Methionin discharges in animal body.
According to the thinking of routine, coated Methionin goods can be mixed together in the animal-feed when raising feeding with the enzyme addn that contains zytase.Cumbersome in using like this, and mix easily inhomogeneously, corresponding utilization ratio is low.
Inventor's imagination, in the process of the coated Methionin goods of preparation, zytase and/or cellulase are directly mixed with the fermenting lysine nutrient solution, can be mixedly very even, then be coated with, yet but find to be limited by high temperature and the drying process that will experience in being coated with, conventional enzymic activity will greatly weaken, even completely lose.Unexpectedly, the inventor has found the heat-resisting and drought-enduring fungal strain of a strain, through arduous intensive research, still can keep high xylanase activity after the zytase experience high temperature drying of wherein separating, therefore be suitable for using easily therein, reduced and fed the trouble of using when raising.
Summary of the invention
The technical problem to be solved in the present invention is that the fermentation that provides new prepares the method for 1B goods, it comprises that fermentation produces the 1B fermented liquid, this fermented liquid can mix with the zytase of the high temperature resistant dehydration that obtains of can fermenting, and is coated with fat after the high temperature drying.In addition, the present invention also provides product that described method produces and used material etc. wherein.
Particularly, in first aspect, the invention provides the method that fermentation prepares the 1B goods, comprise, fermentation produces the 1B fermented liquid, and this fermented liquid can mix with the zytase of high temperature resistant dehydration, and the particles with fat of collecting after high temperature drying is coated.Therefore, the 1B goods of the method for first aspect present invention fermentation preparation divide two-layer, outer wrapping inner layer, and its mesectoderm is fat, and internal layer contains the zytase of 1B and high temperature resistant dehydration.These 1B goods can slow down the speed that Methionin discharges in animal body, and can have xylanase activity.These goods are fodder additives preferably.
More specifically, in first aspect, the invention provides the method that fermentation prepares the 1B goods, it comprises:
(1) under fermentation conditions cultivates the bacterium of the product 1B of the polynucleotide imported the coding pyridine nucleotide transhydrogenase, obtain fermented liquid, keep filtrate after filtering;
(2) under fermentation conditions cultivate the secreting, expressing bacterium of the polynucleotide of the zytase imported the high temperature resistant dehydration of encoding, keep supernatant liquor after filtering;
(3) supernatant liquor of the filtrate of mixing step (1) acquisition and step (2) acquisition obtains mixed solution;
(4) the mixed solution high temperature spray-drying that step (3) is obtained, and choose wantonly and sieve and/or fragmentation, collecting granules; With
The particles with fat of (5) step (4) being collected is coated.
The coated particle of fat can be sprayed on the particle surface by the fat that the fluidized granulation seed-coating machine will melt to be realized, thus preferred fat be under the room temperature state for solid-state fusing point a little more than room temperature (as, 45-80 ℃, preferably 50-65 ℃) fat.Preferably in the method for first aspect present invention, fat is fractionation palm fat preferably, is more preferably hundred Canons (BergaFat), and it can obtain easily by commercial sources.In the specific embodiment of the present invention, coated used fat is hundred HTL316 of Canon.
The inventor finds that lipid layer coated (spraying) must be thicker, and the anti-degradation capability of the 1B goods that prepare is stronger.Preferably in the method for first aspect present invention, in step (5), the weight ratio of the particle that step (4) obtains and fat is 2～8: 8～2, is preferably 3～7: 7～3, more preferably 5～6.5: 5～3.5.
In step (4), for wherein the dry size distribution that obtains is inhomogeneous, can sieve, collect the particle of certain particle size range; And/or for the larger particle of particle diameter dry and/or that screening obtains, can pulverize, then proceed screening, collect the particle of certain particle size range.Preferably in the method for first aspect present invention, the particle diameter of the particle of collection is less than 0.8mm, preferably less than 0.5mm.Particle greater than 0.5mm.Preferred in the method for first aspect present invention in addition, high temperature spray-drying can carry out in fluid bed dryer.For the shape that makes final particle is more even, fluid bed dryer inner exhaust gas temperature is unsuitable too high, usually is not higher than 100 ℃, preferably is not higher than 85 ℃, more preferably remains on 80 ± 3 ℃.Therefore, preferably in the method for first aspect present invention, in the step (4), high temperature is 60-100 ℃, is preferably 65-90 ℃, more preferably 75-85 ℃.
Methionin is as micromolecular compound, and the high temperature dehydration drying can not produce too much disadvantageous effect to it; And zytase through high temperature and/or dehydration, might make its degraded and/or sex change as macro-molecular protein, thereby causes remarkable reduction or the forfeiture of its enzymic activity, can't play respective action.Therefore the present invention can adopt the zytase of high temperature resistant dehydration, and wherein high temperature is 60-100 ℃, is preferably 65-90 ℃, more preferably 75-85 ℃.
Preferably when using as fodder additives, because the requirement of zytase is less than Methionin, therefore preferred lysine content is high in the 1B goods of the method for first aspect present invention fermentation preparation, and zytase content is low simultaneously, can be low to moderate the activity that satisfies the required zytase of feed.Preferably in the method for first aspect present invention, the volume ratio of the supernatant liquor that the filtrate that step (1) obtains and step (2) obtain is 10: 0.01-10 is preferably 10: 0.05-5, more preferably 10: 0.1～1.
Polynucleotide can be imported in the bacterium by various modes well-known to those skilled in the art, make this bacterium express the enzyme of described polynucleotide encoding, such as pyridine nucleotide transhydrogenase and/or zytase.Described polynucleotide can directly be imported into, such as utilizing the transfered cells such as microsome, particle gun; Also can indirectly be imported into, for example can be by being structured in transfered cell on the plasmid vector.The described polynucleotide that import can be incorporated on the genome of cell and express, and expression also can dissociate.In the method for preferred first aspect present invention, the polynucleotide of codase can utilize shuttle plasmid to import in the bacterium, as the polynucleotide of the pyridine nucleotide transhydrogenase of encoding can utilize intestinal bacteria-coryneform bacteria shuttle plasmid to import in the bacterium (such as coryneform bacteria) that produces 1B, and the polynucleotide of the zytase of and for example encoding can utilize E. coli-Yeast bacterium shuttle plasmid to import in the secreting, expressing bacterium (such as pichia yeast).
The fermentation of 1B has many prior aries to select for those skilled in the art, for example can utilize wild-type pyridine nucleotide transhydrogenase or its variant.The wild-type pyridine nucleotide transhydrogenase is that those skilled in the art know, it comprises α subunit and β subunit, and wherein the sequence of α subunit and β subunit is respectively shown in NCBI (http://www.ncbi.nlm.nih.gov) albumen and gene accession number NP416120.1 and AAC74674.1.Preferred variant can be Chinese patent 201110065683.5 disclosed pyridine nucleotide transhydrogenase variants, in actual production, increased the output (also can referring to Chinese patent 201110151495.4) of Methionin, it comprises α subunit variant and β subunit variant, wherein α subunit variant is replaced by other natural amino acids on the position of M102, L127 or Q322 with respect to wild-type α subunit, and preferred the replacement is M102L, L127R and Q322K; β subunit variant is replaced by other natural amino acids on the position of A398 or D400 with respect to the wild-type beta subunit, and preferred the replacement is A398S and D400H.In the specific embodiment of the present invention, adopt the pyridine nucleotide transhydrogenase variant, it is nucleotide sequence coded by shown in SEQ ID No:5.
In the method for first aspect present invention, the zytase of high temperature resistant dehydration is the zytase that produces of the bacterial strain found unexpectedly of the inventor preferably, this zytase is very low with existing known zytase homology, and has an advantage of high temperature resistant dehydration, even verified by after the high temperature evaporation drying, still can keep the enzymic activity more than 80%, thereby so that the method for first aspect present invention can be able to smooth enforcement.In the method for preferred first aspect present invention, the aminoacid sequence of the zytase of high temperature resistant dehydration:
(1) shown in SEQ ID No:2; Or
(2) be to SEQ ID No:2 disappearance, add and/or replace one or several (preferably be no more than 7, more preferably no more than 5, as be no more than 3) amino-acid residue and the sequence of the reservation SEQ ID No:2 activity that obtains.Those skilled in the art can prepare the misfolded proteins that obtains through disappearance, interpolation and/or substituted amino acid residue by recombinant DNA technology.In the specific embodiment of the present invention, zytase is nucleotide sequence coded by shown in SEQ ID No:1.
In step (2), remove by filter thalline and keep supernatant liquor, wherein preferably use 0.22 μ m filter membrane to filter.Supernatant liquor can be purified and/or be concentrated, also can be directly used in the method for first aspect present invention, preferably the latter.
In the method for preferred first aspect present invention, in step (2), the xylosidase enzymic activity of supernatant liquor is not less than 1000IU/L, preferably is not less than 5000IU/L, more preferably is not less than 10000IU/L, for example is not less than 15000IU/L.The zytase of high temperature resistant dehydration of the present invention is dry through high temperature dehydration, still can substantially keep original enzyme activity.The method of under fermentation conditions cultivating in the preferred step (2) comprises:
(i) with in the bacterium of the zytase of the high temperature resistant dehydration of secreting, expressing (such as, yeast) the access liquid nutrient medium, be cultured to OD600 in 28-33 ℃ and reach 3.0-8.0;
(ii) nutrient solution that step (i) is obtained is inoculated in the liquid nutrient medium with the inoculum size of 3-7% (volume), cultivates 20-28 hour in 28-33 ℃;
(iii) the access amount of methyl alcohol with 0.3-0.7% (volume) added in the nutrient solution of step (ii) acquisition, inducing culture 6-12 days, added the methyl alcohol of 0.3-0.7% (volume) access amount in per 24 hours during this time, ventilation control dissolved oxygen is greater than 20%, and control pH is 5.8-6.4.
In this article, inoculum size or access measurer capable field technique personnel institute can conventional understanding implication, specifically when representing with volume percent, refer to other liquid volumes of bacterial culture fluid (the bacterium liquid of access) or access with respect to the percentage of the culture volume that is access in.
Wherein, liquid nutrient medium is the liquid culture agent of the bacterium that can make the zytase of the high temperature resistant dehydration of secreting, expressing (such as, yeast) growth.Step (i) can be identical with liquid nutrient medium in the step (ii), also can make difference, preferably identical, as being the BMGY liquid nutrient medium, its prescription is in the 100mM potassium phosphate buffer (pH6.0), contain 1% (weight) yeast extract, 2% (weight) peptone, yeast nitrogenous base 1.34% (weight), vitamin H 4*10
-5% (weight), glucose 2% (weight), glycerine 1% (volume).
In step (1), remove by filter thalline and keep filtrate, wherein preferably use ultra-filtration membrane to filter.Filtrate can be purified and/or is concentrated, also can be directly used in the method for first aspect present invention, preferably the latter.
In the method for preferred first aspect present invention, in step (1), the content of 1B is higher than 50g/L in the filtrate, preferably is higher than 70g/L, more preferably is higher than 90g/L.This can utilize many prior aries to realize, comprises the improvement of bacterial classification and/or the improvement of fermentation flow process.The method of under fermentation conditions cultivating in the exemplary step (1) comprises:
The bacterium that (a) will produce 1B is accessed the first fermentor tank in 31-38 ℃ of cultivation 10-20 hour;
(b) nutrient solution that step (a) is obtained is inoculated in the second fermentor tank with the inoculum size of 3-7% (volume), cultivates 3-8 hour in 36-40 ℃;
(c) to the second fermentor tank continuous flow sugaring, wherein the dosage that per hour flows of sugar is the 0.2-0.35% (weight) that cultivates liquid measure in the second fermentor tank, carries out 10-18 hour;
(d) to the second sugaring of fermentor tank continuous flow and nitrogenous source, wherein the dosage that per hour flows of sugar is the 0.3-0.5% (weight) that cultivates liquid measure in the second fermentor tank, and the dosage that per hour flows of nitrogenous source is the 0.1-0.25% (weight) that cultivates liquid measure in the second fermentor tank, carried out preferred 50-55 hour 30-65 hour.
In this article, " first " and " second " is the fermentor tank of modifying for the district office when modifying fermentor tank, and namely the first fermentor tank is different with the second fermentor tank.Wherein, the culture medium prescription in preferred the first fermentor tank is: contain glucose 500-750 kilogram, cane molasses 50-300 kilogram, corn steep liquor 400-600 kilogram, KH in per 18 cubic metres of substratum
2PO
4The 30-80 kilogram, MgSO
47H
2O 3-15 kilogram, FeSO
47H
2The O0.1-1 kilogram, MnSO
47H
2O 0.1-1 kilogram, vitamin H 5-30 gram, and folic acid 3-10 gram, and/or the culture medium prescription of preferred the second fermentor tank is: contain glucose 10000-15000 kilogram in per 315 cubic metres of substratum, cane molasses 50-3000 kilogram, corn steep liquor 10000-15000 kilogram, KH
2PO
4The 700-1200 kilogram, MgSO
47H
2O 5-220 kilogram, FeSO
47H
2O 5-25 kilogram, MnSO
47H
2O 5-220 kilogram, vitamin H 150-300 gram, and folic acid 50-120 gram.
Step (c) and part culture condition (d) (as, temperature, pH etc.) with step (b) can be identical, also can be different.In the step (b), do not flow add operation; And in step (c) with (d), pH maintains between 6.5 to 7.8, and this can add alkali by stream simply or acid realizes.
In this article, the stream dosage has those skilled in the art institute can the conventional implication of understanding, and specifically the time as expressed in weight percent, the weight that refers to the adding material accounts for the percentage of the weight that is added into material (such as, nutrient solution).Preferred steps (c) and (d) in sugar be glucose.In step (c), the dosage that per hour flows of glucose is the 0.2-0.35% (weight) that cultivates liquid measure in the second fermentor tank, is preferably 0.27-0.33% (weight).In step (d), the dosage that per hour flows of glucose is the 0.3-0.5% (weight) that cultivates liquid measure in the second fermentor tank, is preferably 0.42-0.48% (weight).
Nitrogenous source in the preferred steps (d) is inorganic nitrogen-sourced, and preferably sulfate of ammoniac or ammonium chloride are such as sulfate of ammoniac.In step (d), the dosage that per hour flows of sulfate of ammoniac is the 0.1-0.25% (weight) that cultivates liquid measure in the second fermentor tank, is preferably 0.17-0.23% (weight).
In second aspect, the invention provides the 1B goods (as, fodder additives), it comprises the particle with the fat parcel, wherein particle comprises the zytase of 1B and high temperature resistant dehydration, and the zytase of preferred high temperature resistant dehydration is the zytase that the bacterial strain found unexpectedly of the inventor produces.Preferably the goods in second aspect present invention are to be fermented by the method for first aspect present invention to prepare.
In the third aspect, the invention provides the zytase of high temperature resistant dehydration or its encoding gene, its carrier, bacterial classification.Particularly, the present invention:
At the zytase that high temperature resistant dehydration is provided aspect the I, its aminoacid sequence:
(1) shown in SEQ ID No:2; Or
(2) be to SEQ ID No:2 disappearance, add and/or replace one or several (preferably be no more than 7, more preferably no more than 5, as be no more than 3) amino-acid residue and the sequence of the reservation SEQ ID No:2 activity that obtains.
At the gene that the zytase of the I aspect of encoding is provided aspect the II, preferably its nucleotide sequence is shown in SEQ ID No:1.
The carrier that comprises the gene of II aspect is being provided aspect the III.
Bacterial classification is being provided aspect the IV, secreting, expressing bacterium (such as, yeast) especially, it comprises the gene of II aspect, or transforms or transfection with the carrier of III aspect.
In the method that the fermentative production zytase is provided aspect the V, it comprises the bacterial classification of under fermentation conditions cultivating the IV aspect, isolates the zytase of I aspect from culture.
In the purposes of the zytase that the I aspect is provided aspect the VI in preparation feed or fodder additives.
The present invention has following beneficial effect: goods of the present invention are safe and reliable, can effectively reduce the release rate of Methionin in the animal body, have xylanase activity, can further improve the utilization ratio of feed; Zytase of the present invention has the advantage of high temperature resistant dehydration.
For the ease of understanding, below will describe in detail the present invention by specific embodiment.It needs to be noted that these descriptions only are exemplary descriptions, do not consist of limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change all are apparent concerning one of ordinary skill in the art.
In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that their full text repeats in this article and clearly put down in writing.
Embodiment
Further specify by the following examples content of the present invention.As do not specialize, the conventional means that used technique means is well known to those skilled in the art among the embodiment and commercially available common instrument, reagent can be referring to the references such as manufacturers instruction of " molecular cloning experiment guide (the 3rd edition) " (Science Press), " Microbiology Experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and reagent.
The preparation of the expression construct of the xylanase gene of the heat-resisting and anti-dehydration of embodiment 1
Bacterial strain according to our discovery, entrust Institute of Microorganism, Academia Sinica, carry out sequential analysis, found new xylanase gene, its nucleotide sequence is shown in the SEQ ID No:1 of sequence table, and the aminoacid sequence of coded zytase is shown in SEQ ID No:2.Then, make up the yeast of secreting, expressing according to the operational guidance of " molecular cloning experiment guide " and used commercialization reagent, in brief, the gene of this zytase of being about to encode (has been introduced EcoR I restriction enzyme site) with forward primer shown in the SEQ ID No:3 of sequence table, use EcoR I and Not I double digestion after reverse primer (has been introduced Not I restriction enzyme site) shown in the SEQ ID No:4 of the sequence table amplification, and be connected with the T4DNA ligase enzyme through the pPIC3.5 of these two endonuclease digestions plasmid (can available from Invitrogen company), be transformed in the bacillus coli DH 5 alpha.Choose positive colony, extract plasmid wherein, after sequence verification is correct, behind positive plasmid usefulness Sal I linearization for enzyme restriction, be transformed into pichia yeast GS115 strain by electrotransformation, choose positive Yeast transformant at the flat board that contains G418, return.
Fermentation preparation and the determination of activity of the zytase of the heat-resisting and anti-dehydration of embodiment 2
The positive Yeast transformant that embodiment 1 is obtained is inoculated into 50ml BMGY liquid nutrient medium, and (100mM potassium phosphate buffer (pH6.0) wherein contains 1% yeast extract, 2% peptone, yeast nitrogenous base 1.34%, vitamin H 4*10
-5%, glucose 2%, glycerine 1%) in, be cultured to OD600 and reach 5.0 in 30 ℃, 200rpm.
Above-mentioned culture is transferred in the 1L BMGY liquid nutrient medium, cultivated 24 hours in 30 ℃, 200rpm, add 5ml methyl alcohol, then continuing at 30 ℃, 200rpm cultivated 8 days, added 5ml methyl alcohol in per 24 hours during this time, ventilation control dissolved oxygen (DO) is greater than 20%, and control pH is 6.0 (regulating with ammoniacal liquor).After cultivation was finished, with 0.22 μ m membrane filtration and keep supernatant liquor, this supernatant liquor detected through SDS-PAGE and wherein is rich in protein corresponding to zytase, and fermentation obtains the zytase supernatant liquor thus.
Get fresh supernatant liquor, be divided into three parts, a refrigeration stand-by (A), a lyophilize is dissolved (B) again with potassium phosphate buffer (pH6.0) to solid, portion evaporation drying in 80 ℃ of baking ovens is dissolved (C) again with potassium phosphate buffer (pH6.0) to solid; The commercially available zytase (can available from Jiangsu Province's paddy bio tech ltd difficult to understand) of 5000IU/g specification is adopted in contrast, with being divided into three parts after potassium phosphate buffer (pH6.0) dissolving, a refrigeration stand-by (CA), the lyophilize (CB) of two parts of difference and 80 ℃ of baking oven evaporation dryings (CC) then heavily are dissolved in potassium phosphate buffer (pH6.0) in addition.Measure respectively these xylanase activities, the enzyme that is converted into original unit's volume supernatant liquor or unit weight is lived, calculate the rate of loss because the enzyme that high temperature dehydration causes is lived, the result is as shown in table 1, show with respect to the commercially available prod and almost lost the situation that enzyme is lived, even if the high temperature resistant dehydration of zytase of the present invention is through high temperature dehydration, still can keep the activity more than 80%, substantially keep protoenzyme function alive.
The high temperature dehydration enzyme of table 1 the present invention and the commercially available zytase rate of loss of living
| Sample | Enzyme is lived | Rate of loss | Sample | Enzyme is lived | Rate of loss |
| A | 2.38*10 4IU/L | CA | 5.21*10 3IU/g | ||
| B | 2.20*10 4IU/L | 6.8％ | CB | 5.05*10 3IU/g | 3.1％ |
| C | 1.96*10 4IU/L | 17.6％ | CC | 0.87*10 3IU/g | 83.3％ |
The fermentative production of embodiment 3L-Methionin
As described in Chinese patent 201110065683.5, pyridine nucleotide transhydrogenase variant gene (its nucleotide sequence is shown in the SEQ ID No:5 of sequence table) is cloned into intestinal bacteria-coryneform bacteria shuttle plasmid pMS2 according to a conventional method (can be available from the U.S. representative microbial preservation center (ATCC), goods number ATCC 67189) between EcoR I and the Xba I restriction enzyme site, electricity is transformed into the coryneform bacteria engineering bacteria of 1B fermentation (can be available from the U.S. representative microbial preservation center (ATCC), goods number ATCC 31269) in, obtains the coryneform bacteria engineering bacteria.
(culture medium prescription wherein is 600 kilograms of glucose, 200 kilograms of cane molasses, 520 kilograms of corn steep liquors, KH with this coryneform bacteria engineering bacteria 20 cubic metres of fermentor tanks of inoculum size access take 0.5%
2PO
445 kilograms, MgSO
47H
27 kilograms of O, FeSO
47H
20.5 kilogram of O, MnSO
47H
20.5 kilogram of O, vitamin H 12 gram, and folic acid 5 grams, water is settled to 18 cubic metres), in 35 ℃ of saturated aerated culture 15 hours, improve cell density.
Then, (culture medium prescription wherein is: 12000 kilograms of glucose, 1000 kilograms of cane molasses, 12000 kilograms of corn steep liquors, KH with 350 cubic metres of fermentor tanks of the direct injection of the nutrient solution of 20 cubic metres of fermentor tanks
2PO
41000 kilograms, MgSO
47H
2100 kilograms of O, FeSO
47H
212 kilograms of O, MnSO
47H
2The O 120 kg, vitamin H 200 gram, and folic acid 85 grams, water is settled to 315 cubic metres), in 38 ℃ of saturated aerated culture 5 hours.Then, per hour stream adds 1000 kilograms of glucose, continues 15 hours, during exhaust vapour to keep volume; Afterwards, per hour stream adds 1500 kilograms of glucose and 650 kilograms of ammonium sulfate, continuing fermentation 50 hours, during exhaust vapour to keep volume.During stream adds, add NaOH and concentrated hydrochloric acid pH is maintained between 6.5 to 7.8, add alkali when namely being lower than lower bound, be higher than high acid adding in limited time.It is complete to ferment, and thin-layer chromatography detects and wherein produces 1B 97g/L, reaches the standard of industrial application.
Being coated with of embodiment 4L-lysine feed
The fermented liquid of embodiment 3 preparation is carried out membrane sepn, filtering solid insoluble by Suntar-III type ultra-filtration membrane (can available from SanDa film Science company limited).Then, the zytase supernatant liquor that filtrate and embodiment 2 fermentations are obtained mixes with 10: 0.5 volume ratio, directly with vaporific spray in the fluid bed dryer and keep exhaust temperature 80 ± 3 ℃ constant, the particle of collection discharging.Particle to discharging sieves; send into crusher in crushing for particle diameter more than or equal to the particle of 0.5mm; then the particle after the fragmentation and the particle diameter that obtains of screening are mixed less than the particle of 0.5mm and also again spray in the fluid bed dryer and keep exhaust temperature constant at 80 ± 3 ℃; collect the particle of discharging and sieve out particle diameter less than the particle of 0.5mm, be the 1B feed granules agent with xylanase activity.Particle diameter is greater than the particle of 0.5mm again after the fragmentation, and the particle of the discharging that obtains with the filtrate drying of next batch mixes.
Getting the 370 gram HTL316 of hundred Canons (BergaFat) (can available from Nanning converge new peak herding company limited) melts in 60 ℃ of baking ovens, then with the fluidized granulation seed-coating machine it is all evenly sprayed on the surface of 1B feed granules of the above-mentioned preparation of 630 grams and form coated layer, namely get the coated 1B fodder additives with xylanase activity.
The degraded test of the 1B fodder additives with xylanase activity that embodiment 5 is coated with
According to Zhao He etc. (research of different ratios sheep's hay and silage dry matter degradability in cow rumen. Chinese Cattle industry science, 34 (2): Nylon Bag in the body of 13-16) describing, in adult dairy cattle to coated fodder additives (take the not coated 1B feed granules agent with xylanase activity as the contrast) test of degrading of embodiment 4 preparation, with 1B wherein as testing index.Degradation rate after impinging upon 2 hours is reached (being 1B conservation rate less than 5%) more than 95%, and the conservation rate of coated fodder additives is as shown in the table:
Reach a thereafter week during the animal experiment, animal subject is not observed untoward reaction and is occured with respect to common feeding animals.This shows, coating technique of the present invention can the remaining time of significant prolongation 1B in the animal stomach, in 24 hours, can in the ox stomach, discharge more equably, and simultaneously can be so that fodder additives has xylanase activity, thereby significantly improved the utilization ratio of feed, and simultaneously animal has been had no adverse effect.
Claims (28)
1. fermentation prepares the method for 1B goods, comprise, fermentation produces the 1B fermented liquid, this fermented liquid can mix with the zytase of high temperature resistant dehydration, the particles with fat of collecting after high temperature drying is coated, and wherein the aminoacid sequence of the zytase of high temperature resistant dehydration is shown in SEQ ID No:2.
2. method claimed in claim 1, it comprises:
(1) under fermentation conditions cultivates the bacterium of the product 1B of the polynucleotide imported the coding pyridine nucleotide transhydrogenase, obtain fermented liquid, keep filtrate after filtering;
(2) under fermentation conditions cultivate the secreting, expressing bacterium of the polynucleotide of the zytase imported the high temperature resistant dehydration of encoding, keep supernatant liquor after filtering;
(3) supernatant liquor of the filtrate of mixing step (1) acquisition and step (2) acquisition obtains mixed solution;
(4) the mixed solution high temperature spray-drying that step (3) is obtained, and choose wantonly and sieve and/or fragmentation, collecting granules; With
The particles with fat of (5) step (4) being collected is coated.
3. method claimed in claim 2, wherein in the step (5), the weight ratio of the particle that step (4) is collected and fat is 2-8: 8-2.
4. method claimed in claim 2, wherein in the step (5), the weight ratio of the particle that step (4) is collected and fat is 3-7: 7-3.
5. method claimed in claim 2, wherein in the step (5), the weight ratio of the particle that step (4) is collected and fat is 5-6.5: 5-3.5.
6. method claimed in claim 2, wherein in the step (5), fat is fractionation palm fat.
7. method claimed in claim 2, wherein in the step (5), fat is hundred Canons.
8. method claimed in claim 2, wherein in the step (5), fat is hundred HTL316 of Canon.
9. method claimed in claim 2, wherein in the step (4), high temperature is 60-100 ℃.
10. method claimed in claim 2, wherein in the step (4), high temperature is 65-90 ℃.
11. method claimed in claim 2, wherein in the step (4), high temperature is 75-85 ℃.
12. method claimed in claim 2, wherein in the step (4), the particle diameter of the particle of collection is less than 0.8mm.
13. method claimed in claim 2, wherein in the step (4), the particle diameter of the particle of collection is less than 0.5mm.
14. method claimed in claim 2, wherein in the step (3), the volume ratio of the supernatant liquor that the filtrate that step (1) obtains and step (2) obtain is 10: 0.01-10.
15. method claimed in claim 2, wherein in the step (3), the volume ratio of the supernatant liquor that the filtrate that step (1) obtains and step (2) obtain is 10: 0.05-5.
16. method claimed in claim 2, wherein in the step (3), the volume ratio of the supernatant liquor that the filtrate that step (1) obtains and step (2) obtain is 10: 0.1～1.
17. method claimed in claim 2, wherein in the step (1), the content of 1B is higher than 50g/L in the filtrate.
18. method claimed in claim 2, wherein in the step (1), the content of 1B is higher than 70g/L in the filtrate.
19. method claimed in claim 2, wherein in the step (1), the content of 1B is higher than 90g/L in the filtrate.
20. claim 1 or 2 described methods, wherein goods are fodder additivess.
21. goods, it comprises that wherein particle comprises the zytase of 1B and high temperature resistant dehydration with the particle of fat parcel, and wherein the aminoacid sequence of the zytase of high temperature resistant dehydration is shown in SEQ ID No:2.
22. the described goods of claim 21, it is by the arbitrary described method preparation of claim 1-20.
23. claim 21 or 22 described goods, it is fodder additives.
24. the zytase of high temperature resistant dehydration, its aminoacid sequence is shown in SEQ ID No:2.
25. the encoding gene of the zytase of the described high temperature resistant dehydration of claim 24.
26. the described gene of claim 25, its nucleotide sequence is shown in SEQ ID No:1.
27. comprise the carrier of claim 25 or 26 described genes.
28. bacterial classification, it comprises claim 25 or 26 described genes, or transforms or transfection with the described carrier of claim 27.
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| CN2012101858839A CN102763771B (en) | 2012-06-07 | 2012-06-07 | Fermentation and coating of lysine and xylanase |
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| BR112014008493A2 (en) * | 2011-11-11 | 2019-09-24 | Novozymes Inc | isolated polypeptide, composition, isolated polynucleotide, recombinant host cell, methods for producing a polypeptide, a mutant or source cell, and a protein, transgenic plant, part of the plant or plant cell, double stranded inhibitor mRNA molecule, method for inhibiting expression of a polypeptide, processes for degrading a cellulosic or xylan-containing material, for producing a fermentation product, and for fermentation of a cellulosic or xylan-containing material, and, broth formulation or cell culture composition |
| CN104472877B (en) * | 2014-12-17 | 2017-07-18 | 宁夏伊品生物科技股份有限公司 | The preparation technology of L lysine products |
| CN104431369B (en) * | 2014-12-17 | 2017-05-24 | 宁夏伊品生物科技股份有限公司 | Rumen-protected lysine feed |
| CN104480089B (en) * | 2014-12-17 | 2017-07-18 | 宁夏伊品生物科技股份有限公司 | The zytase of mutation and its application in L lysine products |
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| CN102191287B (en) * | 2011-03-18 | 2012-08-15 | 宁夏伊品生物科技股份有限公司 | Cis-fermentation preparation method of lysine |
| CN102191288B (en) * | 2011-03-18 | 2012-08-15 | 宁夏伊品生物科技股份有限公司 | Trans-lysine fermentation preparation |
| CN102318739B (en) * | 2011-06-08 | 2012-08-15 | 宁夏伊品生物科技股份有限公司 | Three-level fermentation of lysine and coating products thereof |
| CN102234666B (en) * | 2011-06-08 | 2012-08-15 | 宁夏伊品生物科技股份有限公司 | Fed-batch fermentation preparation of lysine |
| CN102763771B (en) * | 2012-06-07 | 2013-10-16 | 宁夏伊品生物科技股份有限公司 | Fermentation and coating of lysine and xylanase |
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Denomination of invention: Fermentation and coating of lysine and xylanase Effective date of registration: 20210127 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: INNER MONGOLIA EPPEN BIOTECH Co.,Ltd.|NINGXIA EPPEN BIOTECH Co.,Ltd. Registration number: Y2021640000001 |
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