CN103444983A - Lysine and cellulase fermented and coated feed - Google Patents
Lysine and cellulase fermented and coated feed Download PDFInfo
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- CN103444983A CN103444983A CN2013104037227A CN201310403722A CN103444983A CN 103444983 A CN103444983 A CN 103444983A CN 2013104037227 A CN2013104037227 A CN 2013104037227A CN 201310403722 A CN201310403722 A CN 201310403722A CN 103444983 A CN103444983 A CN 103444983A
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- cellulase
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- fat
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Classifications
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- 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
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to lysine and cellulase fermented and a coated feed, and provides a method for preparing an L-lysine product by fermentation. The method comprises the steps: fermenting to produce an L-lysine fermented liquor, mixing the L-lysine fermented liquor with high-temperature-resisting dewatered cellulase obtained through the fermentation, and coating by using fat after high temperature drying. In addition, the invention also provides a product produced by using the method, the high-temperature-resisting dewatered cellulase and the like.
Description
Technical field
The invention belongs to amino acid whose fermentation field, particularly, the present invention relates to the method that fermentation prepares the 1B goods, it comprises that fermentation produces the 1B zymotic fluid, this zymotic fluid can mix with the cellulase of the high temperature resistant dehydration that obtains of can fermenting, and after high temperature drying, with fat, is coated with.In addition, the present invention also provides the product of described method production and the cellulase of high temperature resistant dehydration etc.
Background technology
Can effectively the degrade material (as, feed) of containing cellulose of cellulase therefore adds the absorption rate that cellulase 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 cellulase.
1B is important amino acid starting material, can be used as flavouring, food, feed addictive use, also can be used as the effective or adjunct ingredient in health products, medicine, is widely used in grocery trade, feed industry, pharmacy industry and other chemical industry.Current, the production of 1B is mainly by the fermenting and producing of microorganism, as utilized corynebacteria production.
Yet, when lysine, as feed the time, because it belongs to little molecular nutrition material, can be discharged into soon in animal body, especially, when huge uptake, affected the efficiency that animal absorbs.Usually the method solved is feeding animals in multiple times on a small quantity, but this will increase the workload of animal feeding.Therefore, the present invention proposes the direct method for preparing coated lysine goods with the fermenting lysine nutrient solution, can effectively slow down the speed that lysine discharges in animal body.
Routinely, coated lysine goods can be with the enzyme additive of cellulase in feeding while raising and being mixed together animal feed.Cumbersome in using like this, and easily mix inhomogeneously, corresponding utilization rate reduces.
Inventor's imagination, in preparation, in the process of coated lysine goods, cellulase is directly mixed with the fermenting lysine nutrient solution, can be mixedly very even, then be coated with, yet but find to be limited by being coated with high temperature and the dry run that will experience, conventional enzymatic activity will greatly weaken, and 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 the high-cellulose enzymatic activity after the cellulase experience high temperature drying of wherein separating, therefore be suitable for using easily therein, reduced and fed the trouble of using while 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 zymotic fluid, this zymotic fluid can mix with the cellulase of the high temperature resistant dehydration that obtains of can fermenting, and after high temperature drying, with fat, is coated with.In addition, the present invention also provides product that described method produces and material used etc. wherein.
Particularly, in first aspect, the invention provides the method that fermentation prepares the 1B goods, comprise, fermentation produces the 1B zymotic fluid, and this zymotic fluid can mix with the cellulase 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 ectomesoderm is fat, and the cellulase that internal layer contains 1B and high temperature resistant dehydration.These 1B goods can slow down the speed that lysine discharges in animal body, and can have cellulase activity.These goods are feed addictive preferably.
More specifically, in first aspect, the invention provides the method that fermentation prepares the 1B goods, it comprises:
(1) under fermentation conditions cultivate the bacterium of the product 1B of the polynucleotides that imported the coding pyridine nucleotide transhydrogenase, obtain zymotic fluid, after filtering, retain filtrate;
(2) under fermentation conditions cultivate the secreting, expressing bacterium of the polynucleotides of the cellulase that has imported the high temperature resistant dehydration of encoding, after filtering, retain supernatant;
(3) supernatant that the filtrate that blend step (1) obtains and step (2) obtain, obtain mixed liquor;
(4) mixed liquor high temperature spray-drying step (3) obtained, and optionally sieved and/or fragmentation collecting granules; With
(5) particles with fat of step (4) being collected is coated.
The coated particle of fat can be sprayed on the fat of fusing on particle surface and realize by the fluidized granulation seed-coating machine, thus preferred fat be under 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 fat used is hundred HTL316 of Canon.
Inventor's discovery, fat deposit 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 particle that step (4) obtains and fatty weight ratio are 2 ~ 8:8 ~ 2, are preferably 3 ~ 7:7 ~ 3, more preferably 5 ~ 6.5:5 ~ 3.5.
In step (4), the particle diameter skewness for wherein drying obtains, can be sieved, and collects the particle of certain particle size range; And/or, for the larger particle of particle diameter dry and/or that screening obtains, can be pulverized, 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 is less than 0.5mm.Be greater than the particle of 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 not higher than 100 ℃, preferably, not higher than 85 ℃, more preferably remains on 80
+3 ℃.Therefore, preferably, in the method for first aspect present invention, in step (4), high temperature is 60-100 ℃, is preferably 65-90 ℃, more preferably 75-85 ℃.
Lysine is as micromolecular compound, and the high temperature dehydration drying can not produce too much adverse effect to it; And cellulase through high temperature and/or dehydration, likely makes its degraded and/or sex change as macro-molecular protein, thereby cause remarkable reduction or the forfeiture of its enzymatic activity, can't play respective action.Therefore the present invention can adopt the cellulase of high temperature resistant dehydration, and wherein high temperature is 60-100 ℃, is preferably 65-90 ℃, more preferably 75-85 ℃.
Preferably when using as feed addictive, because the requirement of cellulase is less than lysine, therefore in the 1B goods of the method for first aspect present invention fermentation preparation, preferably lysine content is high, and cellulase content is low simultaneously, can be low to moderate the activity that meets the required cellulase of feed.Preferably, in the method for first aspect present invention, the volume ratio of the supernatant that the filtrate that step (1) obtains and step (2) obtain is 10:0.01-10, is preferably 10:0.1-5, more preferably 10:0.5 ~ 2.
Polynucleotides can be imported in bacterium by various modes well-known to those skilled in the art, make this bacterium express the enzyme of described polynucleotide encoding, as pyridine nucleotide transhydrogenase and/or cellulase.Described polynucleotides 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 plasmid vector.The described polynucleotides that import can be incorporated on the genome of cell and express, and expression also can dissociate.Preferably in the method for first aspect present invention, the polynucleotides of codase can utilize shuttle plasmid to import in bacterium, as the polynucleotides of the pyridine nucleotide transhydrogenase of encoding can utilize Escherichia coli-corynebacteria shuttle plasmid to import in the bacterium (as corynebacteria) that produces 1B, the polynucleotides of the cellulase of and for example encoding can utilize E. coli-Yeast bacterium shuttle plasmid to import in secreting, expressing bacterium (as pichia yeast).
Pyridine nucleotide transhydrogenase is that pyridine nucleotide transhydrogenase is 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 as NCBI(http: //www.ncbi.nlm.nih.gov) as shown in albumen and gene accession number NP416120.1 and AAC74674.1.Preferred variant can be the disclosed pyridine nucleotide transhydrogenase variant of Chinese patent 201110065683.5, increased the output (also can referring to Chinese patent 201110151495.4) of lysine in actual production, 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 preferably replacing 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 preferably replacing is A398S and D400H.In the specific embodiment of the present invention, adopt the pyridine nucleotide transhydrogenase variant, it is nucleotide sequence coded by as shown in SEQ ID No:5.
In the method for first aspect present invention, the cellulase that the cellulase of the high temperature resistant dehydration bacterial strain that preferably inventor finds unexpectedly produces, this cellulase is very low with existing known cellulase homology, and there is the advantage of high temperature resistant dehydration, even verified by after the high temperature evaporation drying, still can retain the enzymatic activity more than 90%, thereby make the method for first aspect present invention can be able to smooth enforcement.In the method for preferred first aspect present invention, the amino acid sequence of the cellulase of high temperature resistant dehydration:
(1) as 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, by recombinant DNA technology, can prepare the misfolded proteins obtained through disappearance, interpolation and/or substituted amino acid residue.In the specific embodiment of the present invention, cellulase is nucleotide sequence coded by as shown in SEQ ID No:1.
In step (2), remove by filter thalline and retain supernatant, wherein preferably use 0.22 μ m filter membrane to be filtered.Supernatant can be purified and/or be concentrated, also can be directly used in the method for first aspect present invention, preferably the latter.
Preferably, in the method for first aspect present invention, in step (2), the cellulase activity of supernatant is not less than 1000IU/L, preferably is not less than 5000IU/L, more preferably is not less than 8000IU/L, for example is not less than 12000IU/L.The cellulase of high temperature resistant dehydration of the present invention, through the high temperature dehydration drying, still can retain original enzyme activity substantially.The method of under fermentation conditions cultivating in preferred step (2) comprises:
(i) by the bacterium of the cellulase of the high temperature resistant dehydration of secreting, expressing (as, saccharomycete) access fluid nutrient medium, be cultured to OD600 in 28-33 ℃ and reach 3.0-8.0;
The nutrient solution (ii) step (i) obtained is with the 3-7%(volume) inoculum concentration be inoculated in fluid nutrient medium, cultivate 20-28 hour in 28-33 ℃;
(iii) by methyl alcohol with the 0.3-0.7%(volume) the access amount add step (ii) to obtain nutrient solution in, induce and cultivate 3-8 days, within every 24 hours during this time, add the 0.3-0.7%(volume) methyl alcohol of access amount, ventilation is controlled dissolved oxygen and is greater than 20%, and to control pH be 5.8-6.4.
In this article, inoculum concentration or access measurer capable field technique personnel institute can conventional understanding implication, specifically, when meaning with percent by volume, refer to the percentage of other liquid volumes of bacterial culture fluid (the bacterium liquid of access) or access with respect to the culture volume be access in.
Wherein, fluid nutrient medium is the Liquid Culture agent of the bacterium that can make the cellulase of the high temperature resistant dehydration of secreting, expressing (as, saccharomycete) growth.Step (i) and the step fluid nutrient medium in (ii) can be identical, also can make difference, preferably identical, as be the BMGY fluid nutrient medium, its formula is in 100mM kaliumphosphate buffer (pH6.0), containing 1%(weight) yeast extract, 2%(weight) peptone, yeast nitrogenous base 1.34%(weight), biotin 4*10
-5%(weight), glucose 2%(weight), glycerine 1%(volume).
In step (1), remove by filter thalline and retain filtrate, wherein preferably use milipore filter to be filtered.Filtrate can be purified and/or is concentrated, also can be directly used in the method for first aspect present invention, preferably the latter.
Preferably, in the method for first aspect present invention, in step (1), in filtrate, the content of 1B is higher than 50g/L, preferably higher than 70g/L, more preferably 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 exemplary step (1) comprises:
(a) bacterium that will produce 1B is accessed the first fermentation tank in 31-38 ℃ of cultivation 10-20 hour;
(b) nutrient solution step (a) obtained is with the 3-7%(volume) inoculum concentration be inoculated in the second fermentation tank, cultivate 3-8 hour in 36-40 ℃;
(c), to the second fermentation tank Continuous-flow sugaring, wherein the dosage that per hour flows of sugar is to cultivate the 0.2-0.35%(weight of liquid measure in the second fermentation tank), carry out 10-18 hour;
(d) to the second sugaring of fermentation tank Continuous-flow and nitrogenous source, wherein the dosage that per hour flows of sugar is to cultivate the 0.3-0.5%(weight of liquid measure in the second fermentation tank), and the dosage that per hour flows of nitrogenous source is to cultivate the 0.1-0.25%(weight of liquid measure in the second fermentation tank), carry out 30-65 hour, preferably 50-55 hour.
In this article, " first " and " second ", when modifying fermentation tank, is the fermentation tank of modifying for district office, and the first fermentation tank and the second fermentation tank are different.Wherein, preferably the culture medium prescription in the first fermentation tank is: contain glucose 500-750 kilogram, cane molasses 50-300 kilogram, corn steep liquor 400-600 kilogram, KH in every 18 cubic metres of culture mediums
2pO
4the 30-80 kilogram, MgSO
47H
2o 3-15 kilogram, FeSO
47H
2o 0.1-1 kilogram, MnSO
47H
2o 0.1-1 kilogram, biotin 5-30 gram, and folic acid 3-10 gram, and/or preferably the culture medium prescription of the second fermentation tank is: contain glucose 10000-15000 kilogram in every 315 cubic metres of culture mediums, 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, biotin 150-300 gram, and folic acid 50-120 gram.
Step (c) and part condition of culture (d) (as, temperature, pH etc.) with step (b) can be identical, also can be different.In step (b), do not flowed add operation; And, in step (c) with (d), pH maintains between 6.5 to 7.8, this can add alkali by stream simply or acid realizes.
In this article, the stream dosage has the implication that those skilled in the art institute can conventional understanding, specifically the time as expressed in weight percent, refers to the percentage that the weight that adds material accounts for the weight that is added into material (as, nutrient solution).Preferred steps (c) and (d) in sugar be glucose.In step (c), the dosage that per hour flows of glucose is to cultivate the 0.2-0.35%(weight of liquid measure in the second fermentation tank), be preferably 0.27-0.33%(weight).In step (d), the dosage that per hour flows of glucose is to cultivate the 0.3-0.5%(weight of liquid measure in the second fermentation tank), be preferably 0.42-0.48%(weight).
Nitrogenous source in preferred steps (d) is inorganic nitrogen-sourced, and preferably sulfate of ammoniac or ammonium chloride, as sulfate of ammoniac.In step (d), the dosage that per hour flows of sulfate of ammoniac is to cultivate the 0.1-0.25%(weight of liquid measure in the second fermentation tank), be preferably 0.17-0.23%(weight).
In second aspect, the invention provides the 1B goods (as, feed addictive), it comprises the particle with the fat parcel, the cellulase that wherein particle comprises 1B and high temperature resistant dehydration, the cellulase of preferred high temperature resistant dehydration is the cellulase that the bacterial strain found unexpectedly of the inventor produces.Preferably the goods in second aspect present invention are to be fermented and prepared by the method for first aspect present invention.
In the third aspect, the invention provides the cellulase of high temperature resistant dehydration or its encoding gene, its carrier, bacterial classification.Particularly, the present invention:
Providing the cellulase of high temperature resistant dehydration aspect I, its amino acid sequence:
(1) as 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.
The gene of the cellulase of the I aspect of encoding is being provided aspect II, and preferably its nucleotide sequence is as shown in SEQ ID No:1.
The carrier of the gene that comprises the II aspect is being provided aspect III.
Bacterial classification is being provided aspect IV, secreting, expressing bacterium (as, yeast) especially, the gene that it comprises the II aspect, or transform or transfection with the carrier of III aspect.
In the method that the fermenting and producing cellulase is provided aspect V, it comprises the bacterial classification of under fermentation conditions cultivating the IV aspect, isolates the cellulase of I aspect from culture.
Purposes in the cellulase that the I aspect is provided aspect VI is preparing feed or feed addictive.
The present invention has following beneficial effect: goods of the present invention are safe and reliable, can effectively reduce the rate of release of lysine in animal body, have cellulase activity, can further improve the utilization rate of feed; Cellulase of the present invention has the advantage of high temperature resistant dehydration.
For the ease of understanding, below will to the present invention, be described in detail by specific embodiment.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification, many variations of the present invention, change are all 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, and the full text that just looks like them repeats in this article and clearly put down in writing the same.
The specific embodiment
Further illustrate by the following examples content of the present invention.As do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art 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 cellulose enzyme gene of the heat-resisting and anti-dehydration of embodiment 1
The bacterial strain of finding according to us, entrust Institute of Microorganism, Academia Sinica, carry out sequence analysis, found new cellulose enzyme gene, its nucleotide sequence is as shown in the SEQ ID No:1 of sequence table, and the amino acid sequence of coded cellulase is as shown in SEQ ID No:2.Then, build the yeast of secreting, expressing according to the operating guidance of " molecular cloning experiment guide " and commercialization reagent used, in brief, the gene of this cellulase of being about to encode (has been introduced EcoR I restriction enzyme site) with forward primer as shown in the SEQ ID No:3 of sequence table, use EcoR I and Not I double digestion after amplification that reverse primer (has been introduced Not I restriction enzyme site) as shown in the SEQ ID No:4 of sequence table, and be connected with the T4 DNA ligase with pPIC3.5 plasmid through these two endonuclease digestions (can purchased from Invitrogen company), be transformed in bacillus coli DH 5 alpha.Choose positive colony, extract plasmid wherein, after sequence verification is correct, positive plasmid, with after Sal I linearization for enzyme restriction, is transformed into to pichia yeast GS115 strain by electrotransformation, containing on the flat board of G418, choosing positive Yeast transformant, return.
fermentation preparation and the determination of activity of the cellulase of the heat-resisting and anti-dehydration of embodiment 2
The positive Yeast transformant that embodiment 1 is obtained is inoculated into 50ml BMGY fluid nutrient medium, and (100mM kaliumphosphate buffer (pH6.0), wherein containing 1% yeast extract, 2% peptone, yeast nitrogenous base 1.34%, biotin 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 1L BMGY fluid nutrient medium, in 30 ℃, 200rpm, cultivate 24 hours, add 5ml methyl alcohol, then continuing at 30 ℃, 200rpm cultivates 6 days, within every 24 hours during this time, add 5ml methyl alcohol, ventilation is controlled dissolved oxygen (DO) and is greater than 20%, and control pH is that 6.0(regulates with ammoniacal liquor).After cultivation completes, with 0.22 μ m membrane filtration and retain supernatant, this supernatant detects and wherein is rich in the protein that cellulase is corresponding through SDS-PAGE, and fermentation obtains the cellulase supernatant thus.
Get fresh supernatant, be divided into three parts, a refrigeration salt adding acid for adjusting pH to 4.8 stand-by (A), a freeze drying is again dissolved (B) with citrate buffer solution (pH4.8) to solid, and portion evaporation drying in 80 ℃ of baking ovens is again dissolved (C) with citrate buffer solution (pH4.8) to solid; Contrast adopts the commercially available cellulase (can purchased from Hubei emerging milky way chemical industry Co., Ltd) of 30,000 U/g specifications, be divided into three parts after dissolving with citrate buffer solution (pH4.8), a refrigeration stand-by (CA), the freeze drying (CB) of another two parts of difference and 80 ℃ of baking oven evaporation dryings (CC), then heavily be dissolved in citrate buffer solution (pH4.8).Measure respectively these cellulase activities, the enzyme that is converted into original unit's volume supernatant or Unit Weight is lived, the enzyme loss late alive that calculating causes due to high temperature dehydration, result is as shown in table 1, show almost to have lost with respect to commercially available prod the situation that enzyme is lived, the high temperature resistant dehydration of cellulase of the present invention, even if through high temperature dehydration, still can retain the activity of 93% left and right, substantially retain protoenzyme function alive.
The high temperature dehydration enzyme of table 1 the present invention and the commercially available cellulase loss late of living
| Sample | Enzyme is lived | Loss late | Sample | Enzyme is lived | Loss late |
| A | 1.59*10 4U/L | CA | 2.72*10 4U/g | ||
| B | 1.46*10 4U/L | 8.2% | CB | 2.45*10 3IU/g | 9.9% |
| C | 1.48*10 4U/L | 6.9% | CC | 0.23*10 2U/g | 91.5% |
the fermenting and producing of embodiment 3 1Bs
As described in Chinese patent 201110065683.5, pyridine nucleotide transhydrogenase variant gene (its nucleotide sequence is as shown in the SEQ ID No:5 of sequence table) is cloned into to Escherichia coli-corynebacteria shuttle plasmid pMS2(according to a conventional method can be purchased from U.S.'s representative microbial preservation center (ATCC), goods number ATCC 67189) between EcoR I and Xba I restriction enzyme site, electricity is transformed into the corynebacteria engineering bacteria of 1B fermentation (can be purchased from U.S.'s representative microbial preservation center (ATCC), goods number ATCC 31269), in, obtain the corynebacteria engineering bacteria.
This corynebacteria engineering bacteria be take to 0.5% inoculum concentration access 20 cubic metres of fermentation tanks, and (culture medium prescription wherein is 600 kilograms of glucose, 200 kilograms of cane molasses, 520 kilograms of corn steep liquors, KH
2pO
445 kilograms, MgSO
47H
27 kilograms of O, FeSO
47H
20.5 kilogram of O, MnSO
47H
20.5 kilogram of O, biotin 12 grams, and folic acid 5 grams, water is settled to 18 cubic metres), in 35 ℃ of saturated ventilations, cultivate 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 the nutrient solution of 20 cubic metres of fermentation tanks directly to be injected to 350 cubic metres of fermentation tanks
2pO
41000 kilograms, MgSO
47H
2100 kilograms of O, FeSO
47H
212 kilograms of O, MnSO
47H
2the O 120 kg, biotin 200 grams, and folic acid 85 grams, water is settled to 315 cubic metres), in 38 ℃ of saturated ventilations, cultivate 5 hours.Then, per hour stream adds 1000 kilograms of glucose, continues 15 hours, during exhaust steam to keep volume; Afterwards, per hour stream adds 1500 kilograms of glucose and 650 kilograms of ammonium sulfate, continuing fermentation 50 hours, during exhaust steam to keep volume.During stream adds, add NaOH and concentrated hydrochloric acid that pH is maintained between 6.5 to 7.8, add alkali during lower than lower bound, higher than the high acid adding of prescribing a time limit.Ferment complete, thin-layer chromatography detects and wherein produces 1B 97g/L, reaches the standard of commercial Application.
being coated with of embodiment 4 1B feeds
The zymotic fluid of embodiment 3 preparation is carried out to film separation, filtering solid insoluble by Suntar-III type milipore filter (can purchased from SanDa film Science Co., Ltd).Then, filtrate is mixed with the volume ratio of 10:1 with the cellulase supernatant that embodiment 2 fermentations obtain, directly with vaporific, spray in fluid bed dryer and keep exhaust temperature 80
+3 ℃ constant, collects the particle of discharging.Particle to discharging is sieved, and the particle that is more than or equal to 0.5mm for particle diameter is sent into crusher in crushing, and the particle that the particle diameter then particle after fragmentation obtained with screening is less than 0.5mm mixes and again sprays in fluid bed dryer and keep exhaust temperature 80
+3 ℃ constant, collects the particle of discharging and sieve out the particle that particle diameter is less than 0.5mm, is the 1B feed granules agent with cellulase activity.The particle that particle diameter is greater than 0.5mm is again after fragmentation, and the particle of the discharging obtained with the filtrate drying of next batch mixes.
Get 370 HTL316(of gram hundred Canons (BergaFat) and can converge new peak herding Co., Ltd purchased from Nanning) in 60 ℃ of baking ovens, melt, 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 gram and forms coated layer, obtain the coated 1B feed addictive with cellulase activity.
the degraded test of the 1B feed addictive with cellulase activity that embodiment 5 is coated
According to Zhao He etc. (research of different proportion sheep's hay and ensilage dry matter degradability in cow rumen. Chinese Cattle industry science, 34 (2): Nylon Bag in the body of 13-16) describing, to coated feed addictive (the coated 1B feed granules agent with cellulase activity of take the is contrast) test of degrading of embodiment 4 preparation, using wherein 1B as testing index in adult dairy cattle.Degradation rate after impinging upon 2 hours is reached to (being 1B conservation rate less than 5%) more than 95%, and the conservation rate of coated feed addictive is as shown in the table:
| The time of experience (hour) | Conservation rate (%) | The time of experience (hour) | Conservation rate (%) |
| 2 | 66.6 | 16 | 19.5 |
| 4 | 39.8 | 24 | 12.3 |
| 6 | 30.0 | 36 | 9.9 |
| 8 | 23.9 | 48 | 5.8 |
Reach one week thereafter during animal experiment, animal subject, with respect to common feeding animals, is not observed bad reaction and is occurred.As can be seen here, 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 feed addictive has cellulase activity, thereby significantly improved the utilization rate of feed, and animal has been had no adverse effect simultaneously.
<110 > Ningxia Yi Pin biotech inc
<120 > fermentation of lysine, cellulase and coated feed
<130 > electronic application
<160> 5
<170> PatentIn version 3.3
<210> 1
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atgaaaggca gcgcggcgag cagcgtgctg ctgacctttc tggcgggcat tagccgcacc 60
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ccgggcgaag atccgtataa cccggatccg ccgattggcg tgggctggga aaccccgaac 180
ctgggcaacg gctttgtgac cccgagcgaa gcgagcaccg atgcggtgat ttgccataaa 240
gaagcgaccc cggcgcgcgg ccatgtgagc gtgaaagcgg gcgataaaat ttatattcag 300
tggcagccga acccgtggcc ggatagccat catggcccgg tgctggatta tctggcgccg 360
tgcaacggcc cgtgcgaaag cgtggataaa accagcctgc gcttttttaa aattgatggc 420
gtgggcctga ttgatggcag cagctatccg ggctattggg cggatgatga actggcggtg 480
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cgccatgaaa ttattgcgct gcatagcgcg ggcaacccgg atggcgcgca gctgtatccg 600
cagtgcttta acctggaaat taccggcagc ggcaccgtgg aaccggaagg cgtgccggcg 660
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acctatgaag tgccgggccc gagcctgatt ccgcaggcgg tgcagattga acagagcagc 780
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Met Lys Gly Ser Ala Ala Ser Ser Val Leu Leu Thr Phe Leu Ala Gly
1 5 10 15
Ile Ser Arg Thr Ser Ala His Gly Tyr Val Ser Asn Leu Val Ile Asn
20 25 30
Ile Gly Tyr Tyr Arg Gly Trp Leu Pro Gly Glu Asp Pro Tyr Asn Pro
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Asp Pro Pro Ile Gly Val Gly Trp Glu Thr Pro Asn Leu Gly Asn Gly
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Phe Val Thr Pro Ser Glu Ala Ser Thr Asp Ala Val Ile Cys His Lys
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Glu Ala Thr Pro Ala Arg Gly His Val Ser Val Lys Ala Gly Asp Lys
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Ile Tyr Ile Gln Trp Gln Pro Asn Pro Trp Pro Asp Ser His His Gly
100 105 110
Pro Val Leu Asp Tyr Leu Ala Pro Cys Asn Gly Pro Cys Glu Ser Val
115 120 125
Asp Lys Thr Ser Leu Arg Phe Phe Lys Ile Asp Gly Val Gly Leu Ile
130 135 140
Asp Gly Ser Ser Tyr Pro Gly Tyr Trp Ala Asp Asp Glu Leu Ala Val
145 150 155 160
Asn Gly Asn Gly Trp Leu Val Gln Ile Pro Glu Asp Ile Lys Pro Gly
165 170 175
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180 185 190
Pro Asp Gly Ala Gln Leu Tyr Pro Gln Cys Phe Asn Leu Glu Ile Thr
195 200 205
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tcagggatta ttgttgtcgg agcactgttg cagattggcc agggcggctg ggttagcttc 1440
cttagtttta tcgcggtgct tatagccagc attaatattt tcggtggctt caccgtgact 1500
cagcgcatgc tgaaaatgtt ccgcaaaaat taaatgtctg gaggattagt tacagctgca 1560
tacattgttg ccgcgatcct gtttatcttc agtctggccg gtctttcgaa acatgaaacg 1620
tctcgccagg gtaacaactt cggtatcgcc gggatggcga ttgcgttaat cgcaaccatt 1680
tttggaccgg atacgggtaa tgttggctgg atcttgctgg cgatggtcat tggtggggca 1740
attggtatcc gtctggcgaa gaaagttgaa atgaccgaaa tgccagaact ggtggcgatc 1800
ctgcatagct tcgtgggtct ggcggcagtg ctggttggct ttaacagcta tctgcatcat 1860
gacgcgggaa tggcaccgat tctggtcaat attcacctga cggaagtgtt cctcggtatc 1920
ttcatcgggg cggtaacgtt cacgggttcg gtggtggcgt tcggcaaact gtgtggcaag 1980
atttcgtcta aaccattgat gctgccaaac cgtcacaaaa tgaacctggc ggctctggtc 2040
gtttccttcc tgctgctgat tgtatttgtt cgcacggaca gcgtcggcct gcaagtgctg 2100
gcattgctga taatgaccgc aattgcgctg gtattcggct ggcatttagt cgcctccatc 2160
ggtggtgcag atatgccagt ggtggtgtcg atgctgaact cgtactccgg ctgggcggct 2220
gcggctgcgg gctttatgct cagcaacgac ctgctgattg tgaccggtgc gctggtcggt 2280
tcttcggggg ctatcctttc ttacattatg tgtaaggcga tgaaccgttc ctttatcagc 2340
gttattgcgg gtggtttcgg caccgacggc tcttctactg gcgatgatca ggaagtgggt 2400
gagcaccgcg aaatcaccgc agaagagaca gcggaactgc tgaaaaactc ccattcagtg 2460
atcattactc cggggtacgg catggcagtc gcgcaggcgc aatatcctgt cgctgaaatt 2520
actgagaaat tgcgcgctcg tggtattaat gtgcgtttcg gtatccaccc ggtcgcgggg 2580
cgtttgcctg gacatatgaa cgtattgctg gctgaagcaa aagtaccgta tgacatcgtg 2640
ctggaaatgg acgagatcaa tgatgacttt gctgataccg ataccgtact ggtgattggt 2700
gctaacgata cggttaaccc ggcgtcgcag catgatccga agagtccgat tgctggtatg 2760
cctgtgctgg aagtgtggaa agcgcagaac gtgattgtct ttaaacgttc gatgaacact 2820
ggctatgctg gtgtgcaaaa cccgctgttc ttcaaggaaa acacccacat gctgtttggt 2880
gacgccaaag ccagcgtgga tgcaatcctg aaagctctgt aa 2922
Claims (10)
1. fermentation prepares the method for 1B goods, comprises, fermentation produces the 1B zymotic fluid, and this zymotic fluid mixes with the cellulase of high temperature resistant dehydration, and the particles with fat of collecting after high temperature drying is coated.
2. method claimed in claim 1, it comprises:
(1) under fermentation conditions cultivate the bacterium of the product 1B of the polynucleotides that imported the coding pyridine nucleotide transhydrogenase, obtain zymotic fluid, after filtering, retain filtrate;
(2) under fermentation conditions cultivate the secreting, expressing bacterium of the polynucleotides of the cellulase that has imported the high temperature resistant dehydration of encoding, after filtering, retain supernatant;
(3) supernatant that the filtrate that blend step (1) obtains and step (2) obtain, obtain mixed liquor;
(4) mixed liquor high temperature spray-drying step (3) obtained, and optionally sieved and/or fragmentation collecting granules; With
(5) particles with fat of step (4) being collected is coated.
3. the described method of claim 1 or 2, the particle of wherein collecting and fatty weight ratio are 2-8:8-2, are preferably 3-7:7-3, more preferably 5-6.5:5-3.5; And/or fat is fractionation palm fat, is more preferably hundred Canons, be most preferably hundred HTL316 of Canon; And/or high temperature is 60-100 ℃, is preferably 65-90 ℃, more preferably 75-85 ℃; And/or the particle diameter of the particle of collection is less than 0.8mm, preferably be less than 0.5mm.
4. method claimed in claim 2, wherein, in step (3), the volume ratio of the supernatant that the filtrate that step (1) obtains and step (2) obtain is 10:0.01-10, is preferably 10:0.1-5, more preferably 10:0.5 ~ 2.
5. the described method of claim 1 or 2, the wherein amino acid sequence of the cellulase of high temperature resistant dehydration:
(1) as 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.
6. method claimed in claim 2, wherein, in step (1), in filtrate, the content of 1B is higher than 50g/L, preferably higher than 70g/L, more preferably higher than 90g/L.
7. the described method of claim 1 or 2, wherein goods are feed addictives.
8. goods, feed addictive preferably, it comprises the particle with the fat parcel, the cellulase that wherein particle comprises 1B and high temperature resistant dehydration, preferably prepared by its arbitrary described method by claim 1-8.
9. the cellulase of high temperature resistant dehydration, its amino acid sequence:
(1) as 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.
10. II of the present invention, III, IV, V or VI aspect.
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| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769439A (en) * | 2005-09-22 | 2006-05-10 | 李军训 | Solid state production technology for thermally-stabilised cellulase of feed use |
| CN101381713A (en) * | 2007-09-08 | 2009-03-11 | 宝鸡市星星协力生物有限公司 | Preparation technology of high temperature resistant feeding cellulase |
| CN102318739A (en) * | 2011-06-08 | 2012-01-18 | 宁夏伊品生物科技股份有限公司 | Three-level fermentation of lysine and coating products thereof |
| CN102771630B (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 cellulose |
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| CN1417339A (en) * | 2001-11-06 | 2003-05-14 | 杭州华大基因研发中心 | High temperature-resisting cellulase gene sequence and its encoded polypeptide and prepn process |
| KR100822041B1 (en) * | 2006-12-21 | 2008-04-15 | 씨제이제일제당 (주) | - Corynebacterium glutamicum enhanced expression of moaA gene encoding molybdenum cofactor biosynthesis enzyme A and method for producing L-lysine using the same |
| CN101372693A (en) * | 2008-07-01 | 2009-02-25 | 吉林大学 | Heat resisting cellulase gene, recombinant engineering bacterium, heat resisting cellulase and use |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769439A (en) * | 2005-09-22 | 2006-05-10 | 李军训 | Solid state production technology for thermally-stabilised cellulase of feed use |
| CN101381713A (en) * | 2007-09-08 | 2009-03-11 | 宝鸡市星星协力生物有限公司 | Preparation technology of high temperature resistant feeding cellulase |
| CN102318739A (en) * | 2011-06-08 | 2012-01-18 | 宁夏伊品生物科技股份有限公司 | Three-level fermentation of lysine and coating products thereof |
| CN102771630B (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 cellulose |
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| CN102771630A (en) | 2012-11-14 |
| CN103444983B (en) | 2015-11-18 |
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