CN103484493B - Method of preparing lysine through fermentation salt and products thereof - Google Patents
Method of preparing lysine through fermentation salt and products thereof Download PDFInfo
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- CN103484493B CN103484493B CN201310403725.0A CN201310403725A CN103484493B CN 103484493 B CN103484493 B CN 103484493B CN 201310403725 A CN201310403725 A CN 201310403725A CN 103484493 B CN103484493 B CN 103484493B
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Abstract
The invention provides the method for the standby product containing 1B salt of fermentation, it bacterium comprised to product 1B imports a gene being considered to irrelevant with lysine metabolism.In addition, present invention also offers the product that described method is produced, this product, relative to the existing product containing 1B salt, can significantly improve resistance to water soak, thus suitable long-term preservation.
Description
Technical field
The invention belongs to field of amino acid fermentation, specifically, the present invention relates to the method for the standby product containing L-lysine sulfate of fermentation, it comprises and innovatively imports a gene being usually considered to irrelevant with lysine metabolism to the bacterium producing 1B.In addition, present invention also offers product and application etc. that described method produces.
Background technology
1B is important amino acid starting material, can use as seasonings, food, fodder additives, also can as the effective or adjunct ingredient in healthcare products, medicine, be widely used in grocery trade, feed industry, pharmacy industry and other chemical industry, especially containing impurity lysine salt (as, vitriol, hydrochloride), generally can directly use as fodder additives.Current, the production of 1B mainly by the fermentative production of microorganism, is produced as utilized coryneform bacteria.
In the various product of lysine salt, especially lysine sulfate, there is very large shortcoming, namely there is larger water absorbability, make thus water content wherein too high (as, be greater than 3%), product humidity too high so easily causes product to lump, and not easily preserves, easily mouldy when preserving especially for a long time, even if this, when the feed lower as safety requirements uses, does not also allow.Especially the present inventor finds, China's wide farmers, penkeeping is small, lysine salt is little as fodder additives consumption, therefore the lysine salt product of (often unit price is more cheap) bought by whole bag, therefore have more retention, during long-term preservation, the impact of water absorbability on them of product is larger.
For this reason, aginomoto company of Japan adopts and regulates the equivalence ratio of acid radical anion and Methionin to overcome this defect, the equivalence ratio of acid radical anion and Methionin is adjusted between 0.68 ~ 0.95, namely the equivalent of acid radical anion is less than the equivalent of Methionin, can reduce the equilibrium moisture (see No. 03120099th, Chinese patent) of product so to a certain extent.Aforesaid method, for lysine hydrochloride, can bring better resistance to water soak, under the routine preservation environment that relative humidity is 33 ~ 58%, the equilibrium moisture of product substantially can be made to control below 3%; But for lysine sulfate, under this Conservation environment, equilibrium moisture, even can more than 5% substantially all more than 3%.
Study for a long period of time through the present inventor and test, surprisingly, the amino acid of other type a certain proportion of is added in the lysine sulfate of resistance to water soak difference, although the purity of lysine sulfate in product can be reduced, but the resistance to water soak of lysine sulfate product can be made to be significantly improved, and the product of this purity does not affect its effectiveness as lysine additives for forage.
And, the present inventor also investigated a kind of new fermentation process, contrary with the thinking that prior art continuous strengthens the pathways metabolism generating Methionin, open other bypasses of non-lysine metabolism, especially in very complicated metabolic pathway, a kind of enzyme has been searched out unexpectedly, the degree opened is very moderate, thus disposable fermentation can obtain Methionin with other type amino acid ligand than suitable product, without the need to too much step of preparation process, the product obtained still can as lysine additives for forage, and resistance to water soak is significantly improved.
Summary of the invention
The technical problem to be solved in the present invention is to provide the method for the standby product containing L-lysine sulfate of new fermentation, and it comprises and innovatively imports a gene being usually considered to irrelevant with lysine metabolism to the bacterium producing 1B.In addition, present invention also offers product and application etc. that the method produces.
Specifically, in first aspect, the invention provides the method for the standby product containing L-lysine sulfate of fermentation, it comprises:
(1) polynucleotide of the albumen of encoding amino acid sequence as shown in SEQIDNo:1 or its variant are imported the bacterium producing 1B;
(2) the under fermentation conditions bacterium that obtains of liquid culture step (1), and collect the supernatant liquor cultivated, comprise the amino acid of following weight proportion in described supernatant liquor:
Methionin 51 ~ 55 parts, is preferably 51.5 ~ 53.5 parts, is more preferably 52.3 parts;
Aspartic acid 1.2 ~ 1.8 parts, is preferably 1.45 ~ 1.6 parts, is more preferably 1.54 parts;
Threonine 0.8 ~ 1.1 part, is preferably 0.9 ~ 1 part, is more preferably 0.94 part;
Serine 0.4 ~ 0.65 part, is preferably 0.5 ~ 0.6 part, is more preferably 0.56 part;
2 ~ 3 parts, L-glutamic acid, is preferably 2.2 ~ 2.6 parts, is more preferably 2.4 parts;
Glycine 0.75 ~ 1.1 part, is preferably 0.85 ~ 1 part, is more preferably 0.92 part;
L-Ala 1.2 ~ 1.6 parts, is preferably 1.3 ~ 1.5 parts, is more preferably 1.4 parts;
α-amino-isovaleric acid 0.75 ~ 1.1 part, is preferably 0.85 ~ 1 part, is more preferably 0.93 part;
Methionine(Met) 0.3 ~ 0.5 part, is preferably 0.35 ~ 0.48 part, is more preferably 0.42 part;
Isoleucine 0.45 ~ 0.75 part, is preferably 0.55 ~ 0.65 part, is more preferably 0.61 part;
Leucine 1 ~ 1.5 part, is preferably 1.2 ~ 1.35 parts, is more preferably 1.27 parts;
0.5 ~ 0.8 part, tyrosine, is preferably 0.6 ~ 0.75 part, is more preferably 0.67 part;
Phenylalanine 0.5 ~ 0.8 part, is preferably 0.6 ~ 0.7 part, is more preferably 0.64 part;
Histidine 0.8 ~ 1.2 part, is preferably 0.9 ~ 1.05 part, is more preferably 0.97 part; With,
Arginine, is preferably 1 ~ 1.2 part, is more preferably 1.1 parts by 0.9 ~ 1.3 part;
(3) to the supernatant liquor that step (2) obtains, sulfuric acid is added and concentrate drying is less than 3%(w/w to water ratio).
In the specific embodiment of the present invention, the albumen of described polynucleotide encoding aminoacid sequence as shown in SEQIDNo:1.Contrary with the thinking that prior art continuous strengthens the pathways metabolism generating Methionin, the method of first aspect present invention carrys out moderate other bypasses of opening non-lysine metabolism by introducing the RNA polymerase sigma-32 factor, thus disposable fermentation can obtain Methionin with other type amino acid ligand than suitable product, without the need to too much step of preparation process.The RNA polymerase sigma-32 factor by working to heat-shock promoters specifically, and participates in heat shock protocol, affects transcribing of heat-shocked associated protein, thus to the microorganism in glutamic acid fermentation overcome metabolism produce poisonous substance have an impact.(can see NCBI(http: //www.ncbi.nlm.nih.gov) albumen and gene accession number AAB18436.1 although the RNA polymerase sigma-32 factor is disclosed; Also can see No. 96193336th, Chinese patent application), but RNA polymerase sigma-32 produces the impact of distribution but without any enlightenment on fermenting lysine and on amino acid wherein.Therefore, preferably in the method for first aspect present invention, the aminoacid sequence of aminoacid sequence for obtaining the aminoacid sequence interpolation such as shown in SEQIDNo:1, disappearance or replacement one and several amino-acid residue of described variant.
More preferably in the method for first aspect present invention, the aminoacid sequence of aminoacid sequence for adding the aminoacid sequence such as shown in SEQIDNo:1, lacking and replace one or several amino-acid residue and obtain of described variant, and the polynucleotide of this variant of encoding import under fermentation conditions liquid culture after the bacterium producing 1B, the supernatant liquor of cultivation comprises the amino acid of weight proportion as described in first aspect present invention.
Those skilled in the art can derive the nucleotide sequence of its coding according to the aminoacid sequence of the RNA polymerase sigma-32 factor, the preferably nucleotide sequence of codon modify, with the degree regulating this pathways metabolism to open.In the specific embodiment of the present invention, the nucleotide sequence of described polynucleotide is as shown in SEQIDNO:2.
Described polynucleotide can be imported into the bacterium producing 1B, as long as the bacterium of product 1B can be made to express the described RNA polymerase sigma-32 factor by various mode well-known to those skilled in the art.Described polynucleotide can directly be imported into, such as, utilize the transfered cell such as microsome, particle gun; Also can indirectly be imported into, such as can by being structured in transfered cell on the carriers such as plasmid.The genome that the described polynucleotide imported can be incorporated into cell is expressed, and also can dissociate expression.In the method for preferred first aspect present invention, engineering bacteria is intestinal bacteria, being more preferably the intestinal bacteria to the desensitization of 1B feedback inhibition, is most preferably the intestinal bacteria (see No. 94194962nd, Chinese patent) of the dihydrodipicolinic acid synthase expressed the desensitization of 1B feedback inhibition.Because intestinal bacteria itself are suitable as cloning host bacterium, therefore preferred described polynucleotide be imported by calcium chloride transformation colibacillary.
In this article, " part " used when representing proportioning, be to represent in the composition, the weight proportion of each composition, this can understand to those skilled in the art.Weight " part " when more than one composition limits in composition, can be regarded as these compositions ratio in the composition.
In this article, inoculum size have those skilled in the art can the conventional implication understood, specifically when representing with volume percent, refer to the percentage of bacterial culture fluid (the bacterium liquid of access) volume relative to the culture volume be access in.
Add sulfuric acid in the method for first aspect present invention, thus can with this basic aminoacids salify of Methionin, be easier to condense into particle in drying process.In the add-on of sulfuric acid and supernatant liquor, the ratio of Methionin can be constant.Preferably in the method for first aspect present invention, the mol ratio of Methionin and sulfuric acid is 1 ~ 3:0.5 ~ 1.5, is more preferably 1.5 ~ 2.5:0.75 ~ 1.25, most preferably is 2:1.
Concentrated and the dry equipment can commonly used by this area of liquid, e.g., vaporizer, drying machine and/or baking oven, carry out.Preferably in the method for first aspect present invention, supernatant liquor concentrates through vaporizer successively, sprays into granulation in fluid bed dryer, and in oven drying.
In second aspect, the invention provides the product containing L-lysine sulfate, it comprises lysine sulfate 68.1 ~ 73.5 parts, is preferably 68.8 ~ 71.5 parts, is more preferably 69.9 parts; Aspartic acid 1.2 ~ 1.8 parts, is preferably 1.45 ~ 1.6 parts, is more preferably 1.54 parts; With, 2 ~ 3 parts, L-glutamic acid, is preferably 2.2 ~ 2.6 parts, is more preferably 2.4 parts.
The product of preferred second aspect present invention comprises the amino acid of following weight proportion:
Lysine sulfate 68.1 ~ 73.5 parts, is preferably 68.8 ~ 71.5 parts, is more preferably 69.9 parts;
Aspartic acid 1.2 ~ 1.8 parts, is preferably 1.45 ~ 1.6 parts, is more preferably 1.54 parts;
Threonine 0.8 ~ 1.1 part, is preferably 0.9 ~ 1 part, is more preferably 0.94 part;
Serine 0.4 ~ 0.65 part, is preferably 0.5 ~ 0.6 part, is more preferably 0.56 part;
2 ~ 3 parts, L-glutamic acid, is preferably 2.2 ~ 2.6 parts, is more preferably 2.4 parts;
Glycine 0.75 ~ 1.1 part, is preferably 0.85 ~ 1 part, is more preferably 0.92 part;
L-Ala 1.2 ~ 1.6 parts, is preferably 1.3 ~ 1.5 parts, is more preferably 1.4 parts;
α-amino-isovaleric acid 0.75 ~ 1.1 part, is preferably 0.85 ~ 1 part, is more preferably 0.93 part;
Methionine(Met) 0.3 ~ 0.5 part, is preferably 0.35 ~ 0.48 part, is more preferably 0.42 part;
Isoleucine 0.45 ~ 0.75 part, is preferably 0.55 ~ 0.65 part, is more preferably 0.61 part;
Leucine 1 ~ 1.5 part, is preferably 1.2 ~ 1.35 parts, is more preferably 1.27 parts;
0.5 ~ 0.8 part, tyrosine, is preferably 0.6 ~ 0.75 part, is more preferably 0.67 part;
Phenylalanine 0.5 ~ 0.8 part, is preferably 0.6 ~ 0.7 part, is more preferably 0.64 part;
Histidine 0.8 ~ 1.2 part, is preferably 0.9 ~ 1.05 part, is more preferably 0.97 part; With,
Arginine, is preferably 1 ~ 1.2 part, is more preferably 1.1 parts by 0.9 ~ 1.3 part.
Confirm through animal, even if comprise fermentation impurities further, the product of second aspect present invention animal is fed raise in use be still safe, more because the amino acid whose of other kinds adds, effect of fodder additives can also be improved further.Therefore, the product of preferred second aspect present invention is fodder additives.
Study for a long period of time through the present inventor and test, surprisingly, in the lysine sulfate of resistance to water soak difference, add the amino acid of other type a certain proportion of, the resistance to water soak of lysine sulfate product can be made to be significantly improved, therefore suitable long-term preservation.In this article, equilibrium moisture content can be exchanged with equilibrium moisture and be used, be those skilled in the art the water absorbability index commonly used, refer under certain relative humidity conditions, product water ratio reaches water ratio time balance (that is, water ratio both can not increase, and also can not reduce).Preferably (relative humidity is 33 ~ 58%) can preserve for a long time under conventional storage conditions, (balance) water ratio of the product of second aspect present invention is less than 5%(w/w), be preferably less than 3%(w/w), be more preferably less than 2.5%(w/w).
The product of second aspect present invention can not contain other impurity (that is, the product of second aspect present invention is made up of above-mentioned amino acid), also can comprise impurity further, as fermentation impurities.Through the present inventor's research, other impurity in the product adopting the method for first aspect present invention to prepare, can not make the amino acid products of aforementioned proportion under conventional storage conditions water ratio higher than 3%(w/w).Therefore preferably the product of second aspect present invention is prepared by the method for first aspect present invention.
In the third aspect, the invention provides lysine sulfate, aspartic acid and L-glutamic acid combine preparing water ratio little containing L-lysine sulfate fodder additives in application, preferably provide lysine sulfate, aspartic acid, Threonine, Serine, L-glutamic acid, glycine, L-Ala, α-amino-isovaleric acid, methionine(Met), Isoleucine, leucine, tyrosine, phenylalanine, Histidine and arginine combine preparing water ratio little containing L-lysine sulfate fodder additives in application.Preferably wherein, (balance) water ratio is less than 5%(w/w), be more preferably less than 3%(w/w), be most preferably less than 2.5%(w/w).
Preferably in the application of third aspect present invention, the fodder additives containing L-lysine sulfate is the product of second aspect present invention.
The present invention has following beneficial effect: product resistance to water soak of the present invention is good, makes the water ratio in product reduce by more than 50%, is suitable in the southern and northern wide geographic area standing storage of China, transport and use; Product of the present invention is as 1B feed, safe and reliable, and effect promotes to some extent; Fermentation process implementation step of the present invention is simple, and one time fermentation can obtain the product of corresponding amino acid ligand ratio, has saved production cost, without potential safety hazard.
For the ease of understanding, below will be described in detail the present invention by specific embodiment.It is important to note that these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification sheets, many changes of the present invention, to change concerning one of ordinary skill in the art be all apparent.
In addition, the present invention refer to open source literature, and these documents are to more clearly describe the present invention, and their entire contents is all included in and carried out reference herein, just looks like that repeated description is excessively the same in this article for their full text.
Embodiment
Further illustrate content of the present invention by the following examples.As do not specialized, the conventional means that technique means used in embodiment is well known to those skilled in the art and commercially available common instrument, reagent, can see references such as the manufacturers instructions of " Molecular Cloning: A Laboratory guide (the 3rd edition) " (Science Press), " Microbiology Experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and reagent.
the preparation of embodiment 1RNA polysaccharase sigma-32 factor gene construct
According to NCBI(http: //www.ncbi.nlm.nih.gov) the aminoacid sequence of Protein Accession AAB18436.1, the present inventor devises appropriate expression type codon (non-express amount optimizing codon), and entrust the gene of the Shanghai Sheng Gong Bioisystech Co., Ltd composite coding RNA polymerase sigma-32 factor by commercial sources and be built in colibacillus expression plasmid pET-20b (+) (can purchased from American Novagen company, goods number Cat.No.69739-3).Cloning procedure carries out with reference to the operational guidance of " Molecular Cloning: A Laboratory guide " and commercial reagents used, and simplified process is as follows:
Pass through automatic dna synthesizer, the nucleic acid fragment of synthesis RNA polymerase sigma-32 factor gene, with T4 polynucleotide kinase (purchased from TaKaRa company), 5 ' of these nucleic acid fragments end is carried out phosphorylation, then equimolar ratio mixes after these 5 nucleic acid fragments in 65 DEG C of sex change 5 minutes, annealing is cooled to 16 DEG C, adds T4DNA ligase enzyme (purchased from TaKaRa company) and connects 12 hours.Then, get the above-mentioned connection product of 1 μ L and carry out pcr amplification in 50 μ L reaction volumes, wherein forward primer (introduces EcoRI restriction enzyme site) as shown in the SEQIDNo:3 of sequence table, reverse primer (introduces XhoI restriction enzyme site) as shown in the SEQIDNo:4 of sequence table, reaction conditions was: with 94 ° of C sex change 4 minutes, then to anneal 60 seconds with 94 ° of C sex change 30 seconds, 63 ° of C and 72 ° of C extend 35 seconds carries out 35 circulations, finally extend 4 minutes with 72 ° of C and be cooled to 4 ° of C.
The above-mentioned PCR primer of agarose gel electrophoresis, reclaim the fragment of about 900bp size, by this fragment of EcoRI and XhoI double digestion, and be connected with T4DNA ligase enzyme with pET-20b (+) plasmid through these two endonuclease digestions, be transformed in intestinal bacteria Top10F '.Choose positive colony, extract plasmid wherein, through sequence verification, shown in the SEQIDNo:2 that corresponding nucleotide sequence designs as the present inventor, encode the RNA polymerase sigma-32 factor as shown in SEQIDNo:1, by company, the plasmid built (called after pET-sigma) is returned.
the L-lysine sulfate product of embodiment 2 Escherichia coli fermentation
To coli strain W3110 (the tyrA)/pCABD2 of the product 1B that the method described in No. 94194962nd, Chinese patent builds, transform pET-sigma plasmid, obtain after conversion positive colony (called after W3110 (tyrA)/pCABD2-sigma) through qualification, bacterial strain W3110 (tyrA)/pCABD2 and W3110 (tyrA)/pCABD2-sigma is carried out fermenting lysine experiment with reference to No. 03120099th, Chinese patent respectively.In brief, bacterial strain is accessed shaking culture in LB liquid medium and reach 0.35 to OD500, (often liter of culture medium prescription is the inoculum size access fermenting lysine substratum with 5%: 100g glucose, 60g (NH
4)
2sO
4, 50gCaCO
3, 35mL peptone-B(SoyProteinEnzymaticHydrolysate, purchased from the true Bioisystech Co., Ltd in Shanghai, total nitrogen content is not less than 3%), 1gKH
2pO
4, 400mgMgSO
47H
2o, 200mgL-methionine(Met), 10mgFeSO
47H
2o, 8.1mgMnSO
44H
2o, 300 μ g vitamin Hs and 200 μ g VITMAIN B1, be adjusted to pH7 with Tris-HCl) in cultivate 72 hours with 36 DEG C of vibrations (250rpm).Then, collected by centrifugation medium supernatant (fermented liquid), with the 1B content in the quantitative supernatant liquor of HPLC and other compositions.Result is as shown in table 1, relative to the fermented liquid of W3110 (tyrA)/pCABD2, the lysine content of W3110 (tyrA)/pCABD2-sigma slightly declines, but other amino acid whose kinds and ratio significantly improve, the content of other impurity (higher than the impurity (sugar, polysaccharide, peptide and albumen) of amino acid molecular amount and the impurity (inorganic salt) lower than amino acid molecular amount) is substantially constant, if therefore the character of both tunnings has difference, estimation is owing to other amino acid whose kind and ratio.
Comparison of ingredients in table 1 fermented liquid
| Fermentation strain | W3110(tyrA)/pCABD2 | W3110(tyrA)/pCABD2-sigma |
| Lysine content | 12.58g/L | 11.96g/L |
| Other aminoacids contents | 1.23g/L | 3.29g/L |
| Each amino acid and content ratio thereof | Methionin, 72.1; Threonine, 2.48; Serine, 1.27; L-glutamic acid, 0.95; Glycine, 0.87; L-Ala, 0.70; Methionine(Met), 0.58; α-amino-isovaleric acid, Histidine and arginine, be all less than all the other amino acid of 0.1(and do not detect (< 0.01)) | Methionin, 52.3; Aspartic acid 1.54; Threonine, 0.94; Serine, 0.56; L-glutamic acid, 2.4; Glycine, 0.92; L-Ala, 1.4; α-amino-isovaleric acid, 0.93; Methionine(Met), 0.42; Isoleucine, 0.61; Leucine, 1.27; Tyrosine, 0.67; Phenylalanine, 0.64; Histidine, 0.97; Arginine, all the other amino acid of 1.1(do not detect (< 0.01)) |
| Higher than the impurity of amino acid molecular amount | 2.61g/L | 2.32g/L |
| Lower than the impurity of amino acid molecular amount | 1.03g/L | 1.05g/L |
Add the vitriol oil respectively to the above-mentioned supernatant liquor obtained by different strains, make the mol ratio of the Methionin in sulfuric acid and supernatant liquor be 1:2, be then placed in after vaporizer concentrates, spray into granulation in fluid bed dryer with vaporific.Particle is placed in baking oven inner drying, until water ratio is no more than 1%, obtains L-lysine sulfate product.
the resistance to water soak test of embodiment 3L-lysine product
The L-lysine sulfate product (being designated as sigma) prepared by W3110 (tyrA)/pCABD2-sigma of Example 2 and the L-lysine sulfate product (being designated as contrast 1) prepared by W3110 (tyrA)/pCABD2.
In addition, the chemically pure reagent getting following weight proportion mixes (being designated as contrast 2): lysine sulfate, 69.9; Aspartic acid 1.54; Threonine, 0.94; Serine, 0.56; L-glutamic acid, 2.4; Glycine, 0.92; L-Ala, 1.4; α-amino-isovaleric acid, 0.93; Methionine(Met), 0.42; Isoleucine, 0.61; Leucine, 1.27; Tyrosine, 0.67; Phenylalanine, 0.64; Histidine, 0.97; With, arginine, 1.1.
In addition, the chemically pure reagent getting following weight proportion mixes (being designated as contrast 3): lysine sulfate, 69.9; Aspartic acid 1.54; With, L-glutamic acid, 2.4.
By above-mentioned sample respectively at 25 DEG C with under the environment of different relative humidity place 7 days, then the equilibrium moisture content in product is at that time measured, result is as shown in table 2, the easy moisture absorption of lysine sulfate product conventionally obtained, under routine preservation environment (relative humidity 33 ~ 58%) be difficult to control water ratio preserve for a long time below 3%, if and mix with other amino acid of some particular types and ratio, then can significantly strengthen its resistance to water soak, make it long-term preservation, wherein aspartic acid and the resistance to water soak gain effects of L-glutamic acid to lysine sulfate larger, but its resistance to water soak can not be strengthened completely, impurity in tunning can reduce the resistance to water soak of lysine sulfate to a certain extent, but as long as can keep other amino acid wherein having some particular types and ratio, just can offset the disadvantageous effect that impurity produces, and make it long-term preservation.
The equilibrium moisture content test result of table 2 variant production
| Sample | Contrast 1 | Contrast 2 | Contrast 3 | sigma |
| Relative humidity 33% | 3.22% | 1.01% | 0.78% | 1.36% |
| Relative humidity 43% | 4.90% | 2.43% | 1.35% | 1.59% |
| Relative humidity 58% | 6.03% | 3.74% | 1.62% | 2.08% |
embodiment 4L-lysine sulfate product is to the effectiveness of calf growth effect
The L-lysine sulfate product prepared by W3110 (tyrA)/pCABD2-sigma of herding institute of Ningxia academy of agricultural sciences embodiment 2 and commercially available 85%L-lysine hydrochloride fodder additives is entrusted to carry out simultaneous test, the calf raising 4 ~ 6 week age is fed Deng addition (in wherein 1B), not add the feed of Methionin for contrast, the product of embodiment 2 on average makes calf adding weight (ADG) improve 17.5%, and commercially available product on average improves 15.8%, wherein slightly be better than commercially available prod and may give the credit to the amino acid also containing other kinds more in the product of embodiment 2, duration of test, does not have untoward reaction to report.
<110> Ningbo Eppen Biotech Co., Ltd.
Method of <120> preparing lysine through fermentation salt and products thereof
<130>CN
<160>4
<170>PatentInversion3.5
<210>1
<211>284
<212>PRT
<213>Escherichiacoli
<400>1
MetThrAspLysMetGlnSerLeuAlaLeuAlaProValGlyAsnLeu
151015
AspSerTyrIleArgAlaAlaAsnAlaTrpProMetLeuSerAlaAsp
202530
GluGluArgAlaLeuAlaGluLysLeuHisTyrHisGlyAspLeuGlu
354045
AlaAlaLysThrLeuIleLeuSerHisLeuArgPheValValHisIle
505560
AlaArgAsnTyrAlaGlyTyrGlyLeuProGlnAlaAspLeuIleGln
65707580
GluGlyAsnIleGlyLeuMetLysAlaValArgArgPheAsnProGlu
859095
ValGlyValArgLeuValSerPheAlaValHisTrpIleLysAlaGlu
100105110
IleHisGluTyrValLeuArgAsnTrpArgIleValLysValAlaThr
115120125
ThrLysAlaGlnArgLysLeuPhePheAsnLeuArgLysThrLysGln
130135140
ArgLeuGlyTrpPheAsnGlnAspGluValGluMetValAlaArgGlu
145150155160
LeuGlyValThrSerLysAspValArgGluMetGluSerArgMetAla
165170175
AlaGlnAspMetThrPheAspLeuSerSerAspAspAspSerAspSer
180185190
GlnProMetAlaProValLeuTyrLeuGlnAspLysSerSerAsnPhe
195200205
AlaAspGlyIleGluAspAspAsnTrpGluGluGlnAlaAlaAsnArg
210215220
LeuThrAspAlaMetGlnGlyLeuAspGluArgSerGlnAspIleIle
225230235240
ArgAlaArgTrpLeuAspGluAspAsnLysSerThrLeuGlnGluLeu
245250255
AlaAspArgTyrGlyValSerAlaGluArgValArgGlnLeuGluLys
260265270
AsnAlaMetLysLysLeuArgAlaAlaIleGluAla
275280
<210>2
<211>855
<212>DNA
<213>Escherichiacoli
<400>2
atgaccgataaaatgcagagcctggcgctggcgccggtgggcaacctggatagctatatt60
cgcgcggcgaacgcgtggccgatgctgagcgcggatgaagaacgcgcgctggcggaaaaa120
ctgcattatcatggcgatctggaagcggcgaaaaccctgattctgagccatctgcgcttt180
gtggtgcatattgcgcgcaactatgcgggctatggcctgccgcaggcggatctgattcag240
gaaggcaacattggcctgatgaaagcggtgcgccgctttaacccggaagtgggcgtgcgc300
ctggtgagctttgcggtgcattggattaaagcggaaattcatgaatatgtgctgcgcaac360
tggcgcattgtgaaagtggcgaccaccaaagcgcagcgcaaactgttttttaacctgcgc420
aaaaccaaacagcgcctgggctggtttaaccaggatgaagtggaaatggtggcgcgcgaa480
ctgggcgtgaccagcaaagatgtgcgcgaaatggaaagccgcatggcggcgcaggatatg540
acctttgatctgagcagcgatgatgatagcgatagccagccgatggcgccggtgctgtat600
ctgcaggataaaagcagcaactttgcggatggcattgaagatgataactgggaagaacag660
gcggcgaaccgcctgaccgatgcgatgcagggcctggatgaacgcagccaggatattatt720
cgcgcgcgctggctggatgaagataacaaaagcaccctgcaggaactggcggatcgctat780
ggcgtgagcgcggaacgcgtgcgccagctggaaaaaaacgcgatgaaaaaactgcgcgcg840
gcgattgaagcgtaa855
<210>3
<211>24
<212>DNA
<213> artificial sequence
<400>3
cgaattcgatgaccgataaaatgc24
<210>4
<211>22
<212>DNA
<213> artificial sequence
<400>4
gctcgagttacgcttcaatcgc22
Claims (19)
1. the method for the standby product containing L-lysine sulfate of fermentation, it comprises:
(1) polynucleotide of the albumen of encoding amino acid sequence as shown in SEQIDNo:1 are imported the intestinal bacteria producing 1B, it is the intestinal bacteria to the desensitization of 1B feedback inhibition;
(2) the under fermentation conditions bacterium that obtains of liquid culture step (1), and collect the supernatant liquor cultivated, comprise the amino acid of following weight proportion in described supernatant liquor:
Methionin 51 ~ 55 parts;
Aspartic acid 1.2 ~ 1.8 parts;
Threonine 0.8 ~ 1.1 part;
Serine 0.4 ~ 0.65 part;
2 ~ 3 parts, L-glutamic acid;
Glycine 0.75 ~ 1.1 part;
L-Ala 1.2 ~ 1.6 parts;
α-amino-isovaleric acid 0.75 ~ 1.1 part;
Methionine(Met) 0.3 ~ 0.5 part;
Isoleucine 0.45 ~ 0.75 part;
Leucine 1 ~ 1.5 part;
0.5 ~ 0.8 part, tyrosine;
Phenylalanine 0.5 ~ 0.8 part;
Histidine 0.8 ~ 1.2 part; With,
Arginine 0.9 ~ 1.3 part;
(3) to the supernatant liquor that step (2) obtains, sulfuric acid is added and concentrate drying is less than 3%w/w to water ratio.
2. method according to claim 1, wherein, in step (2), comprises the amino acid of following weight proportion in described supernatant liquor:
Methionin 51.5 ~ 53.5 parts;
Aspartic acid 1.45 ~ 1.6 parts;
Threonine 0.9 ~ 1 part;
Serine 0.5 ~ 0.6 part;
2.2 ~ 2.6 parts, L-glutamic acid;
Glycine 0.85 ~ 1 part;
L-Ala 1.3 ~ 1.5 parts;
α-amino-isovaleric acid 0.85 ~ 1 part;
Methionine(Met) 0.35 ~ 0.48 part;
Isoleucine 0.55 ~ 0.65 part;
Leucine 1.2 ~ 1.35 parts;
0.6 ~ 0.75 part, tyrosine;
Phenylalanine 0.6 ~ 0.7 part;
Histidine 0.9 ~ 1.05 part; With,
Arginine 1 ~ 1.2 part.
3. method according to claim 1, wherein the mol ratio of Methionin and sulfuric acid is 1 ~ 3:0.5 ~ 1.5.
4. method according to claim 3, wherein the mol ratio of Methionin and sulfuric acid is 1.5 ~ 2.5:0.75 ~ 1.25.
5. method according to claim 4, wherein the mol ratio of Methionin and sulfuric acid is 2:1.
6. method according to claim 1, wherein the nucleotide sequence of polynucleotide is as shown in SEQIDNO:2.
7., containing the fodder additives of L-lysine sulfate, it comprises:
Lysine sulfate 72.1 ~ 80.1 parts;
Aspartic acid 1.3 ~ 1.9 parts;
Threonine 0.8 ~ 1.2 part;
Serine 0.45 ~ 0.7 part;
2 ~ 3 parts, L-glutamic acid;
Glycine 0.8 ~ 1.1 part;
L-Ala 1.3 ~ 1.7 parts;
α-amino-isovaleric acid 0.8 ~ 1.2 part;
Methionine(Met) 0.35 ~ 0.55 part;
Isoleucine 0.5 ~ 0.8 part;
Leucine 1.1 ~ 1.6 parts;
0.55 ~ 0.9 part, tyrosine;
Phenylalanine 0.5 ~ 0.85 part;
Histidine 0.8 ~ 1.2 part; With,
Arginine, 1 ~ 1.5 part.
8. fodder additives according to claim 7, it comprises:
Lysine sulfate 73.5 ~ 76.1 parts;
Aspartic acid 1.5 ~ 1.65 parts;
Threonine 0.9 ~ 1.1 part;
Serine 0.55 ~ 0.65 part;
2.3 ~ 2.8 parts, L-glutamic acid;
Glycine 0.85 ~ 1.05 part;
L-Ala 1.4 ~ 1.6 parts;
α-amino-isovaleric acid 0.9 ~ 1.1 part;
Methionine(Met) 0.4 ~ 0.5 part;
Isoleucine 0.6 ~ 0.7 part;
Leucine 1.25 ~ 1.45 parts;
0.65 ~ 0.8 part, tyrosine;
Phenylalanine 0.6 ~ 0.75 part;
Histidine 0.9 ~ 1.1 part; With,
Arginine 1.1 ~ 1.3 parts.
9. fodder additives according to claim 8, it comprises:
Lysine sulfate 74.4 parts;
Aspartic acid 1.61 parts;
Threonine 1 part;
Serine 0.6 part;
2.56 parts, L-glutamic acid;
Glycine 0.96 part;
L-Ala 1.5 parts;
α-amino-isovaleric acid 0.99 part;
Methionine(Met) 0.44 part;
Isoleucine 0.63 part;
Leucine 1.36 parts;
0.72 part, tyrosine;
Phenylalanine 0.67 part;
Histidine 1.02 parts; With,
Arginine 1.18 parts.
10. fodder additives according to claim 7, its water ratio is less than 5%w/w.
11. fodder additivess according to claim 10, its water ratio is less than 3%w/w.
12. fodder additivess according to claim 11, its water ratio is less than 2.5%w/w.
13. fodder additivess according to claim 7, it is prepared by arbitrary described method of claim 1 ~ 6.
14. lysine sulfate, aspartic acid and L-glutamic acid combine prepare water ratio be less than 5%w/w containing L-lysine sulfate fodder additives in application, wherein, amino acid whose weight proportion is as follows:
Lysine sulfate 72.1 ~ 80.1 parts;
Aspartic acid 1.3 ~ 1.9 parts; With,
2 ~ 3 parts, L-glutamic acid.
15. application according to claim 14, wherein, amino acid whose weight proportion is as follows:
Lysine sulfate 73.5 ~ 76.1 parts;
Aspartic acid 1.5 ~ 1.65 parts; With,
2.3 ~ 2.8 parts, L-glutamic acid.
16. application according to claim 15, wherein, amino acid whose weight proportion is as follows:
Lysine sulfate 74.4 parts;
Aspartic acid 1.61 parts; With,
2.56 parts, L-glutamic acid.
17. application according to claim 14, wherein, the water ratio containing the fodder additives of L-lysine sulfate is less than 3%w/w.
18. application according to claim 17, wherein, the water ratio containing the fodder additives of L-lysine sulfate is less than 2.5%w/w.
19. application according to claim 14, the fodder additives wherein containing L-lysine sulfate is arbitrary described fodder additives of claim 7 ~ 13.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4393135A (en) * | 1979-12-10 | 1983-07-12 | Ajinomoto Company Incorporated | Method for producing L-glutamic acid by fermentation |
| CN1142856A (en) * | 1993-12-08 | 1997-02-12 | 味之素株式会社 | Process for producing L-Lysine by fermentation |
| CN1181785A (en) * | 1995-02-20 | 1998-05-13 | 味之素株式会社 | Stress-tolerant microorganism and method of the prodn. of fermentation product |
| CN1943392A (en) * | 2005-10-08 | 2007-04-11 | 德古萨股份公司 | Feedstuff additive containing l-lysine |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1064106B1 (en) * | 1998-03-18 | 2003-07-16 | Technologies Inc. Biosphere | Method for bio-refining organic waste material to produce denatured and sterile nutrient products |
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2012
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4393135A (en) * | 1979-12-10 | 1983-07-12 | Ajinomoto Company Incorporated | Method for producing L-glutamic acid by fermentation |
| CN1142856A (en) * | 1993-12-08 | 1997-02-12 | 味之素株式会社 | Process for producing L-Lysine by fermentation |
| CN1181785A (en) * | 1995-02-20 | 1998-05-13 | 味之素株式会社 | Stress-tolerant microorganism and method of the prodn. of fermentation product |
| CN1943392A (en) * | 2005-10-08 | 2007-04-11 | 德古萨股份公司 | Feedstuff additive containing l-lysine |
Non-Patent Citations (3)
| Title |
|---|
| 65%赖氨酸硫酸盐物理性状的研究;石现瑞 等;《新研究》;20061231;18-20 * |
| The fermentative production of l-lysine as an animal feed additive;Manfred Kircher等;《chemosphere》;20011231;27-31 * |
| 新型赖氨酸应用推广的研究进展;邝声耀 等;《四川畜牧兽医》;20051231(第11期);摘要、第31页中栏第1段、第33页左栏第1段及左栏表格 * |
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