CN103805627A - Method for preparing high-purity lysine sulfate through once fermentation - Google Patents

Method for preparing high-purity lysine sulfate through once fermentation Download PDF

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CN103805627A
CN103805627A CN201310438355.4A CN201310438355A CN103805627A CN 103805627 A CN103805627 A CN 103805627A CN 201310438355 A CN201310438355 A CN 201310438355A CN 103805627 A CN103805627 A CN 103805627A
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parts
acid
lysine
product
lysine sulfate
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CN103805627B (en
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马吉银
陈崇安
孟刚
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NINGXIA EPPEN BIOTECH CO Ltd
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NINGXIA EPPEN BIOTECH CO Ltd
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Abstract

The invention provides a method for preparing a product containing L-lysine sulfate through fermentation. The method comprises a step of guiding a gene regarded unrelated to lysine metabolism into L-lysine generating bacteria. Moreover, the lysine content in the product reaches the purity of a commercially available product. In addition, the invention also provides a product produced by the method; compared with the existing products containing L-lysine sulfate, the product can remarkably improve the moisture absorption resistance so as to be suitable for long-time storage.

Description

The method of high purity lysine sulfate is prepared in disposable fermentation
Technical field
The invention belongs to amino acid fermentation field, particularly, the present invention relates to the method for fermentation preparation containing the product of L-lysine sulfate, it comprises innovation and imports one to the bacterium that produces 1B and is conventionally considered to the gene irrelevant with Methionin metabolism, and in product, lysine content has reached the purity of commercially available prod.In addition, the present invention also provides product and the application etc. that described method is produced.
Background technology
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, medicine, be widely used in grocery trade, feed industry, pharmacy industry and other chemical industry, especially contain impurity lysine salt (as, vitriol, hydrochloride), generally also can directly use as fodder additives.Current, the production of 1B is mainly by the fermentative production of microorganism, as utilized coryneform bacteria to produce.
In the various product of lysine salt, especially lysine sulfate, there is very large shortcoming, there is larger water absorbability, make thus water content wherein too high (as, be greater than 3%), too high like this product humidity easily causes product caking, is difficult for preserving, easily mouldy while preservation especially for a long time, even if this,, in the time using as the lower feed of safety requirements, does not also allow.Especially the inventor finds, China's wide farmers, penkeeping is small, lysine salt is little as fodder additives consumption, therefore whole bag is bought the lysine salt product of (often unit price is more cheap), therefore have more retention, while preservation for a long time, the water absorbability of product is larger on their impact.
For this reason, aginomoto company of Japan adopts and regulates the equivalent of acid radical anion and Methionin recently to overcome this defect, the equivalence ratio of acid radical anion and Methionin is adjusted between 0.68 ~ 0.95, be the equivalent that the equivalent of acid radical anion is less than Methionin, can reduce to a certain extent like this equilibrium moisture (referring to No. 03120099th, Chinese patent) of product.Aforesaid method, for lysine hydrochloride, can bring better resistance to water soak, under the routine preservation environment that is 33 ~ 58%, substantially can make the equilibrium moisture of product be controlled at below 3% in relative humidity; But, for lysine sulfate, to preserve under environment at this, equilibrium moisture substantially all can exceed 3%, even can exceed 5%.
Study for a long period of time and test through the inventor, find unexpectedly, in the poor lysine sulfate of resistance to water soak, add the amino acid of a certain proportion of other type, although can reduce the purity of lysine sulfate in product, but can make the resistance to water soak of lysine sulfate product be significantly improved, and the product of this purity does not affect its effectiveness as lysine additives for forage.
And, the inventor has also worked out a kind of new fermentation process, the thinking of pathways metabolism that generates Methionin with the continuous reinforcement of prior art is contrary, open other bypasses of non-Methionin metabolism, especially in very complicated metabolic pathway, searched out unexpectedly a kind of enzyme, the degree of opening is very moderate, thereby can disposable fermentation obtain Methionin and other type amino acid ligand than suitable product, without too much step of preparation process, the product obtaining still can be served as lysine additives for forage, and resistance to water soak is significantly improved.
Further, the inventor finds, after having added sulfuric acid salify, lysine content in the product obtaining in this disposable fermentation is difficult to go beyond 55%, and in the market in product sold take high density product as main (general lysine content as 55% or more than), therefore lysine content in product is risen to at least 55%, could be than being easier to carry out marketing.For this reason, the inventor also provides the method for the fermentative production high purity lysine sulfate that a kind of step is easy.
Summary of the invention
The technical problem to be solved in the present invention is that the fermentation preparation that provides new contains the method for the product of L-lysine sulfate, and it comprises innovation and is conventionally considered to the gene irrelevant with Methionin metabolism to one of the bacterium importing of producing 1B.In addition, the present invention also provides product and the application etc. that the method is produced.
Particularly, in first aspect, the invention provides the method for fermentation preparation containing the product of L-lysine sulfate, it comprises:
(1) by encoding amino acid sequence, the polynucleotide of the albumen as shown in SEQ ID No:1 or its variant import the bacterium that produces 1B;
(2) bacterium that under fermentation conditions liquid culture step (1) obtains, collects the supernatant liquor of cultivating, and allows this supernatant liquor by filter membrane, retains filtrate, comprises the amino acid of following weight proportion in described filtrate:
54 ~ 60 parts of Methionins, are preferably 55 ~ 57 parts, more preferably 55.7 parts;
1.3 ~ 1.9 parts of aspartic acids, are preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts;
0.8 ~ 1.2 part of Threonine, is preferably 0.9 ~ 1.1 part, more preferably 1 part;
0.45 ~ 0.7 part of Serine, is preferably 0.55 ~ 0.65 part, more preferably 0.6 part;
2 ~ 3 parts, L-glutamic acid, is preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts;
0.8 ~ 1.1 part of glycine, is preferably 0.85 ~ 1.05 part, more preferably 0.96 part;
1.3 ~ 1.7 parts of L-Ala, are preferably 1.4 ~ 1.6 parts, more preferably 1.5 parts;
0.8 ~ 1.2 part of α-amino-isovaleric acid, is preferably 0.9 ~ 1.1 part, more preferably 0.99 part;
0.35 ~ 0.55 part of methionine(Met), is preferably 0.4 ~ 0.5 part, more preferably 0.44 part;
0.5 ~ 0.8 part of Isoleucine, is preferably 0.6 ~ 0.7 part, more preferably 0.63 part;
1.1 ~ 1.6 parts of leucines, are preferably 1.25 ~ 1.45 parts, more preferably 1.36 parts;
0.55 ~ 0.9 part, tyrosine, is preferably 0.65 ~ 0.8 part, more preferably 0.72 part;
0.5 ~ 0.85 part of phenylalanine, is preferably 0.6 ~ 0.75 part, more preferably 0.67 part;
0.8 ~ 1.2 part of Histidine, is preferably 0.9 ~ 1.1 part, more preferably 1.02 parts; With,
Arginine,, is preferably 1.1 ~ 1.3 parts, more preferably 1.18 parts by 1 ~ 1.5 part;
(3) filtrate obtaining to step (2), adds sulfuric acid concentrate drying to water ratio to be less than 3%(w/w).
In the specific embodiment of the present invention, the albumen of described polynucleotide encoding aminoacid sequence as shown in SEQ ID No:1.The thinking of pathways metabolism that generates Methionin with the continuous reinforcement of prior art is contrary, the method of first aspect present invention is carried out moderate other bypasses of opening non-Methionin metabolism by introducing the RNA polymerase sigma-32 factor, thereby can disposable fermentation obtain Methionin and other type amino acid ligand than suitable product, without too much step of preparation process.The RNA polymerase sigma-32 factor is by specifically heat-shocked promotor being worked, and participates in heat-shocked process, affect transcribing of heat-shocked associated protein, thereby the poisonous substance that the microorganism in glutamic acid fermentation overcomes metabolism generation is exerted an influence.Although the RNA polymerase sigma-32 factor is disclosed (can referring to NCBI(http: //www.ncbi.nlm.nih.gov) albumen and gene accession number AAB18436.1; Also can be referring to No. 96193336th, Chinese patent application), but RNA polymerase sigma-32 produces the impact distributing 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 described variant be to the aminoacid sequence as shown in SEQ ID No:1 add, disappearance or replace the aminoacid sequence that and several amino-acid residue obtain.
More preferably in the method for first aspect present invention, the aminoacid sequence of described variant be to aminoacid sequence as shown in SEQ ID No:1 add, disappearance or replace the aminoacid sequence that and several amino-acid residue obtain, and the polynucleotide of this variant of encoding import and produce after the bacterium of 1B under fermentation conditions liquid culture, the amino acid that the supernatant liquor of cultivation comprises weight proportion described in first aspect present invention.
Those skilled in the art can derive according to the aminoacid sequence of the RNA polymerase sigma-32 factor nucleotide sequence of its coding, and the preferably nucleotide sequence of codon modify, with the degree that regulates this pathways metabolism to open.In the specific embodiment of the present invention, the nucleotide sequence of described polynucleotide is as shown in SEQ ID NO:2.
Described polynucleotide can be imported into the bacterium that produces 1B by various modes well-known to those skilled in the art, as long as can make the bacterium that produces 1B express the described RNA polymerase sigma-32 factor.Described polynucleotide can directly be imported into, for example, utilize the transfered cell such as microsome, particle gun; Also can indirectly be imported into, for example can be by being structured in transfered cell on the carriers such as plasmid.The described polynucleotide 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, engineering bacteria is intestinal bacteria, being more preferably the intestinal bacteria to 1B feedback inhibition desensitization, is most preferably the intestinal bacteria of expressing the dihydrodipicolinic acid synthase to the desensitization of 1B feedback inhibition (referring to No. 94194962nd, Chinese patent).Because intestinal bacteria itself are suitable as cloning host bacterium, therefore preferred described polynucleotide be import by calcium chloride transformation colibacillary.
In this article, representing " part " used when proportioning, be in order to be illustrated in described 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 to composition, can be regarded as the ratio of these compositions in said composition.
In this article, inoculum size have those skilled in the art can the conventional implication of understanding, specifically, in representing with volume percent, refer to the percentage of bacterial culture fluid (the bacterium liquid of access) volume with respect to the culture volume being access in.
Use filter membrane can remove impurity, especially high molecular impurity, enrichment Methionin, thus improve Methionin purity.Filter membrane can be ultra-filtration membrane, can be also nanofiltration membrane, preferably ultra-filtration membrane.The inventor finds, uses the filter membrane that molecular weight cut-off is 1000Da, can disposable lysine content in the finished product be brought up to more than 55%, reaches the needs that are applicable to marketing, and simple operating steps.Therefore, preferably, in the method for first aspect present invention, filter membrane is that molecular weight cut-off is the filter membrane of 500 ~ 5000Da, the filter membrane that preferably molecular weight cut-off is 800 ~ 3000Da, being more preferably the filter membrane that molecular weight cut-off is 900 ~ 2000Da, is most preferably that molecular weight cut-off is the filter membrane of 1000Da.
In the method for first aspect present invention, add sulfuric acid, 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, and more preferably 1.5 ~ 2.5:0.75 ~ 1.25, most preferably are 2:1.
Liquid concentrated and dry can be by the conventional equipment in this area, as, vaporizer, drying machine and/or baking oven, carry out.Preferably, in the method for first aspect present invention, supernatant liquor is concentrated through vaporizer successively, sprays into granulation in fluid bed dryer, and in oven drying.
In second aspect, the invention provides the product (it is fodder additives preferably) containing L-lysine sulfate, wherein lysine content is greater than 54%(w/w), be preferably greater than 55%(w/w), also preferred content is 54 ~ 60%(w/w), more preferably 55 ~ 57%(w/w), most preferably be 55.7%(w/w).Such lysine content is convenient to marketing.
Preferably the product of second aspect present invention comprises 72.1 ~ 80.1 parts of lysine sulfate, is preferably 73.5 ~ 76.1 parts, more preferably 74.4 parts; 1.3 ~ 1.9 parts of aspartic acids, are preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts; With, 2 ~ 3 parts, L-glutamic acid, is preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts.
More preferably the amino acid that the product of second aspect present invention comprises following weight proportion:
72.1 ~ 80.1 parts of lysine sulfate, are preferably 73.5 ~ 76.1 parts, more preferably 74.4 parts;
1.3 ~ 1.9 parts of aspartic acids, are preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts;
0.8 ~ 1.2 part of Threonine, is preferably 0.9 ~ 1.1 part, more preferably 1 part;
0.45 ~ 0.7 part of Serine, is preferably 0.55 ~ 0.65 part, more preferably 0.6 part;
2 ~ 3 parts, L-glutamic acid, is preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts;
0.8 ~ 1.1 part of glycine, is preferably 0.85 ~ 1.05 part, more preferably 0.96 part;
1.3 ~ 1.7 parts of L-Ala, are preferably 1.4 ~ 1.6 parts, more preferably 1.5 parts;
0.8 ~ 1.2 part of α-amino-isovaleric acid, is preferably 0.9 ~ 1.1 part, more preferably 0.99 part;
0.35 ~ 0.55 part of methionine(Met), is preferably 0.4 ~ 0.5 part, more preferably 0.44 part;
0.5 ~ 0.8 part of Isoleucine, is preferably 0.6 ~ 0.7 part, more preferably 0.63 part;
1.1 ~ 1.6 parts of leucines, are preferably 1.25 ~ 1.45 parts, more preferably 1.36 parts;
0.55 ~ 0.9 part, tyrosine, is preferably 0.65 ~ 0.8 part, more preferably 0.72 part;
0.5 ~ 0.85 part of phenylalanine, is preferably 0.6 ~ 0.75 part, more preferably 0.67 part;
0.8 ~ 1.2 part of Histidine, is preferably 0.9 ~ 1.1 part, more preferably 1.02 parts; With,
Arginine,, is preferably 1.1 ~ 1.3 parts, more preferably 1.18 parts by 1 ~ 1.5 part.
Confirm through animal, even if further comprise fermentation impurity, to raise middle use be still safe to the product of second aspect present invention animal is fed, and more because the amino acid whose of other kinds adds, can also improve to a certain extent effect of fodder additives.Therefore, preferably the product of second aspect present invention is fodder additives.
Study for a long period of time and test through the inventor, find unexpectedly, in the poor lysine sulfate of resistance to water soak, add the amino acid of a certain proportion of other type, can make the resistance to water soak of lysine sulfate product be significantly improved, therefore suitable long-term preservation.In this article, equilibrium moisture content can be exchanged and use with equilibrium moisture, is the conventional water absorbability indexs of those skilled in the art, refer under certain relative humidity environment, water ratio when product water ratio reaches balance (that is, water ratio neither can increase, and also can not reduce).Preferably can under routine preservation condition, (relative humidity is 33 ~ 58%) preserve for a long time, (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 further comprise impurity, as fermentation impurity.Through inventor research, adopt other impurity in product prepared by the method for first aspect present invention, the amino acid product that can not make aforementioned proportion under routine preservation condition water ratio higher than 3%(w/w).Therefore preferably the product of second aspect present invention is to be prepared by the method for first aspect present invention.
In the third aspect, the invention provides lysine sulfate, aspartic acid and L-glutamic acid and combine the application in the product of preparation second aspect present invention, 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 to combine the application in the product of preparation second aspect present invention.Wherein, this product water ratio is little, and preferably (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).
The present invention has following beneficial effect: product resistance to water soak of the present invention is good, and the water ratio in product is reduced more than 50%, is suitable in the southern and northern wide geographic area standing storage of China, transportation and use; Product of the present invention is as 1B feed, safe and reliable, and effect promotes to some extent; The lysine content of product of the present invention has existing commercially available Methionin nicotinate product correspondence, and institute is so that business promotion; Fermentation process implementation step of the present invention is simple, only needs one time fermentation, has saved production cost, without potential safety hazard; The purification step of fermentation process of the present invention, has facilitated operation, has also saved production cost.
For the ease of understanding, below will be described in detail the present invention 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 sheets, 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 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 by the following examples content of the present invention.As do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art and commercially available common instrument, reagent, can be referring to references such as the manufacturers instructions 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 embodiment 1 RNA polymerase sigma-32 factor gene construct
According to NCBI(http: //www.ncbi.nlm.nih.gov) the aminoacid sequence of albumen accession number AAB18436.1, the inventor has designed appropriate expression type codon (non-expression amount optimizing codon), and entrust the gene of the composite coding RNA polymerase sigma-32 of the Shanghai Sheng Gong Bioisystech Co., Ltd factor and be built in colibacillus expression plasmid pET-20b (+) (can be purchased from Novagen company of the U.S., goods number Cat. No. 69739-3) by commercial sources.Clone's process is carried out with reference to the operational guidance of " molecular cloning experiment guide " and commercialization reagent used, and concise and to the point process is as follows:
Pass through automatic dna synthesizer, the nucleic acid fragment of synthetic RNA polymerase sigma-32 factor gene, 5 ' end of these nucleic acid fragments is carried out to phosphorylation with T4 polynucleotide kinase (purchased from TaKaRa company), then equimolar ratio is mixed after these 5 nucleic acid fragments in 65 ℃ of sex change 5 minutes, annealing is cooled to 16 ℃, adds T4 DNA ligase (purchased from TaKaRa company) to connect 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 (has been introduced EcoR I restriction enzyme site) as shown in the SEQ ID No:3 of sequence table, reverse primer (introduced Xho I restriction enzyme site) as shown in the SEQ ID No:4 of sequence table, reaction conditions was: with 94 ° of C sex change 4 minutes, then extend and within 35 seconds, carry out 35 circulations with 94 ° of C sex change 30 seconds, 63 ° of C annealing 60 seconds 72 ° of C, finally extend 4 minutes and be cooled to 4 ° of C with 72 ° of C.
The above-mentioned PCR product of agarose gel electrophoresis, reclaim the fragment of about 900bp size, by EcoR I and this fragment of XhoI double digestion, and be connected with T4 DNA ligase with pET-20b (+) plasmid through these two endonuclease digestions, be transformed in intestinal bacteria Top10 F '.Choose positive colony, extract plasmid wherein, through sequence verification, corresponding nucleotide sequence is as shown in the SEQ ID No:2 of inventor's design, the RNA polymerase sigma-32 factor of having encoded as shown in SEQ ID No:1, is returned the plasmid building (called after pET-sigma) by company.
the preparation of the L-lysine sulfate product of embodiment 2 Escherichia coli fermentations
Coli strain W3110 (the tyrA)/pCABD2 of the product 1B that the method described in No. 94194962nd, Chinese patent is built, transform pET-sigma plasmid, transform after positive colony (called after W3110 (tyrA)/pCABD2-sigma) through identifying to obtain, bacterial strain W3110 (tyrA)/pCABD2 and W3110 (tyrA)/pCABD2-sigma are carried out to fermenting lysine experiment No. 03120099 with reference to Chinese patent respectively.In brief, bacterial strain is accessed in liquid LB substratum to shaking culture and reach 0.35 to OD500, (every liter of culture medium prescription is 100g glucose, 60g (NH for inoculum size access fermenting lysine substratum take 5% 4) 2sO 4, 50g CaCO 3, 35mL peptone-B(Soy Protein Enzymatic Hydrolysate, purchased from the true Bioisystech Co., Ltd in Shanghai, total nitrogen content is not less than 3%), 1g KH 2pO 4, 400mg MgSO 47H 2o, 200mg METHIONINE, 10mg FeSO 47H 2o, 8.1mg MnSO 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 ℃ of vibrations (250rpm).Then, centrifugal collection medium supernatant (fermented liquid).Then the ultra-filtration membrane by molecular weight cut-off 1000Da (can purchased from German Sartorius AG, goods number: 3051460901E-SG) respectively by supernatant liquor, with the 1B content in the quantitative filtrate of HPLC and other compositions.Result is as shown in table 1, with respect to the filtrate of W3110 (tyrA)/pCABD2, the lysine content of W3110 (tyrA)/pCABD2-sigma slightly declines, but other amino acid whose kinds and ratio obviously improve, other impurity (are mainly the impurity (inorganic salt) lower than amino acid molecular amount, and estimate most ofly to be removed by membrane retention higher than the impurity (sugar, polysaccharide, peptide and albumen) of amino acid molecular amount) content 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 filtrate
Fermentation strain W3110(tyrA)/pCABD2 W3110(tyrA)/pCABD2-sigma
Lysine content 11.93g/L 11.36g/L
Other aminoacids contents 1.15g/L 3.11g/L
Each amino acid and content ratio thereof Methionin, 78.6; Threonine, 2.66; Serine, 1.36; L-glutamic acid, 1.01; Glycine, 0.93; L-Ala, 0.75; Methionine(Met), 0.61; 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, 55.7; Aspartic acid 1.61; Threonine, 1.00; Serine, 0.60; L-glutamic acid, 2.56; Glycine, 0.96; L-Ala, 1.50; α-amino-isovaleric acid, 0.99; Methionine(Met), 0.44; Isoleucine, 0.63; Leucine, 1.36; Tyrosine, 0.72; Phenylalanine, 0.67; Histidine, 1.02; Arginine, all the other amino acid of 1.18(do not detect (< 0.01))
Higher than the impurity of amino acid molecular amount 1.10g/L 1.06g/L
Lower than the impurity of amino acid molecular amount 0.99g/L 1.02g/L
Add the vitriol oil to the above-mentioned filtrate being obtained by different strains respectively, the mol ratio that makes the Methionin in sulfuric acid and supernatant liquor is 1:2, be then placed on vaporizer concentrated after, spray into granulation in fluid bed dryer with vaporific.Particle is placed in to 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 3 1B products
Get the L-lysine sulfate product (being designated as sigma) of being prepared by W3110 (tyrA)/pCABD2-sigma of embodiment 2 and the L-lysine sulfate product of being prepared by W3110 (tyrA)/pCABD2 (being designated as contrast 1).
In addition, the chemically pure reagent of 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 of getting following weight proportion mixes (being designated as contrast 3): lysine sulfate, 69.9; Aspartic acid 1.54; With, L-glutamic acid, 2.4.
Above-mentioned sample is placed under the environment with different relative humidity 7 days respectively at 25 ℃, then measure the equilibrium moisture content in product at that time, result is as shown in table 2, improve the easy moisture absorption of lysine sulfate product of production based on prior art, under routine preservation environment, (relative humidity 33 ~ 58%) is difficult to control water ratio below 3% and preserves for a long time, if and mixed with other amino acid of some particular types and ratio, can significantly strengthen its resistance to water soak, make it long-term preservation, wherein aspartic acid and L-glutamic acid are larger to the resistance to water soak gain effects of lysine sulfate, but can not strengthen its resistance to water soak completely, impurity in tunning can reduce the resistance to water soak of lysine sulfate to a certain extent, as long as still can keep wherein having other amino acid of 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% 3.94% 1.01% 0.78% 1.61%
Relative humidity 43% 5.25% 2.43% 1.35% 2.18%
Relative humidity 58% 7.43% 3.74% 1.62% 2.79%
the effectiveness of embodiment 4 L-lysine sulfate products to calf growth effect
Entrust herding institute of Ningxia academy of agricultural sciences to carry out simultaneous test with the L-lysine sulfate product of being prepared by W3110 (tyrA)/pCABD2-sigma and the commercially available 85%L-lysine hydrochloride fodder additives of embodiment 2, feed Deng addition (in 1B wherein) calf of raising 4 ~ 6 week age, take the feed that do not add Methionin as contrast, the product of embodiment 2 on average makes calf adding weight (ADG) improve 17.0%, 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 that also contains more other kinds in the product of embodiment 2, duration of test, does not have untoward reaction report.
Yi Pin biotech inc, <110> Ningxia
The method of high purity lysine sulfate is prepared in the disposable fermentation of <120>
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<400> 2
atgaccgata aaatgcagag cctggcgctg gcgccggtgg gcaacctgga tagctatatt 60
cgcgcggcga acgcgtggcc gatgctgagc gcggatgaag aacgcgcgct ggcggaaaaa 120
ctgcattatc atggcgatct ggaagcggcg aaaaccctga ttctgagcca tctgcgcttt 180
gtggtgcata ttgcgcgcaa ctatgcgggc tatggcctgc cgcaggcgga tctgattcag 240
gaaggcaaca ttggcctgat gaaagcggtg cgccgcttta acccggaagt gggcgtgcgc 300
ctggtgagct ttgcggtgca ttggattaaa gcggaaattc atgaatatgt gctgcgcaac 360
tggcgcattg tgaaagtggc gaccaccaaa gcgcagcgca aactgttttt taacctgcgc 420
aaaaccaaac agcgcctggg ctggtttaac caggatgaag tggaaatggt ggcgcgcgaa 480
ctgggcgtga ccagcaaaga tgtgcgcgaa atggaaagcc gcatggcggc gcaggatatg 540
acctttgatc tgagcagcga tgatgatagc gatagccagc cgatggcgcc ggtgctgtat 600
ctgcaggata aaagcagcaa ctttgcggat ggcattgaag atgataactg ggaagaacag 660
gcggcgaacc gcctgaccga tgcgatgcag ggcctggatg aacgcagcca ggatattatt 720
cgcgcgcgct ggctggatga agataacaaa agcaccctgc aggaactggc ggatcgctat 780
ggcgtgagcg cggaacgcgt gcgccagctg gaaaaaaacg cgatgaaaaa actgcgcgcg 840
gcgattgaag cgtaa 855
<210> 3
<211> 24
<212> DNA
<213> artificial sequence
<400> 3
cgaattcgat gaccgataaa atgc 24
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<400> 4
gctcgagtta cgcttcaatc gc 22

Claims (10)

1. fermentation preparation is containing the method for the product of L-lysine sulfate, and it comprises:
(1) by encoding amino acid sequence, the polynucleotide of the albumen as shown in SEQ ID No:1 import the bacterium that produces 1B;
(2) bacterium that under fermentation conditions liquid culture step (1) obtains, collects the supernatant liquor of cultivating, and allows the filter membrane that this supernatant liquor is 1000Da by molecular weight cut-off, retains filtrate, comprises the amino acid of following weight proportion in described filtrate:
54 ~ 60 parts of Methionins, are preferably 55 ~ 57 parts;
1.3 ~ 1.9 parts of aspartic acids, are preferably 1.5 ~ 1.65 parts;
0.8 ~ 1.2 part of Threonine, is preferably 0.9 ~ 1.1 part;
0.45 ~ 0.7 part of Serine, is preferably 0.55 ~ 0.65 part;
2 ~ 3 parts, L-glutamic acid, is preferably 2.3 ~ 2.8 parts;
0.8 ~ 1.1 part of glycine, is preferably 0.85 ~ 1.05 part;
1.3 ~ 1.7 parts of L-Ala, are preferably 1.4 ~ 1.6 parts;
0.8 ~ 1.2 part of α-amino-isovaleric acid, is preferably 0.9 ~ 1.1 part;
0.35 ~ 0.55 part of methionine(Met), is preferably 0.4 ~ 0.5 part;
0.5 ~ 0.8 part of Isoleucine, is preferably 0.6 ~ 0.7 part;
1.1 ~ 1.6 parts of leucines, are preferably 1.25 ~ 1.45 parts;
0.55 ~ 0.9 part, tyrosine, is preferably 0.65 ~ 0.8 part;
0.5 ~ 0.85 part of phenylalanine, is preferably 0.6 ~ 0.75 part;
0.8 ~ 1.2 part of Histidine, is preferably 0.9 ~ 1.1 part; With,
Arginine,, is preferably 1.1 ~ 1.3 parts by 1 ~ 1.5 part;
(3) filtrate obtaining to step (2), adds sulfuric acid concentrate drying to water ratio to be less than 3%(w/w).
2. method claimed in claim 1, wherein the mol ratio of Methionin and sulfuric acid is 1 ~ 3:0.5 ~ 1.5, is preferably 1.5 ~ 2.5:0.75 ~ 1.25, more preferably 2:1.
3. method claimed in claim 1, wherein bacterium is intestinal bacteria, preferably the intestinal bacteria to the desensitization of 1B feedback inhibition.
4. containing the fodder additives of L-lysine sulfate, wherein lysine content is 54 ~ 60%(w/w), more preferably 55 ~ 57%(w/w), most preferably be 55.7%(w/w); And described product comprises 72.1 ~ 80.1 parts of lysine sulfate, be preferably 73.5 ~ 76.1 parts, more preferably 74.4 parts; 1.3 ~ 1.9 parts of aspartic acids, are preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts; With, 2 ~ 3 parts, L-glutamic acid, is preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts.
5. fodder additives claimed in claim 4, it comprises:
72.1 ~ 80.1 parts of lysine sulfate, are preferably 73.5 ~ 76.1 parts, more preferably 74.4 parts;
1.3 ~ 1.9 parts of aspartic acids, are preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts;
0.8 ~ 1.2 part of Threonine, is preferably 0.9 ~ 1.1 part, more preferably 1 part;
0.45 ~ 0.7 part of Serine, is preferably 0.55 ~ 0.65 part, more preferably 0.6 part;
2 ~ 3 parts, L-glutamic acid, is preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts;
0.8 ~ 1.1 part of glycine, is preferably 0.85 ~ 1.05 part, more preferably 0.96 part;
1.3 ~ 1.7 parts of L-Ala, are preferably 1.4 ~ 1.6 parts, more preferably 1.5 parts;
0.8 ~ 1.2 part of α-amino-isovaleric acid, is preferably 0.9 ~ 1.1 part, more preferably 0.99 part;
0.35 ~ 0.55 part of methionine(Met), is preferably 0.4 ~ 0.5 part, more preferably 0.44 part;
0.5 ~ 0.8 part of Isoleucine, is preferably 0.6 ~ 0.7 part, more preferably 0.63 part;
1.1 ~ 1.6 parts of leucines, are preferably 1.25 ~ 1.45 parts, more preferably 1.36 parts;
0.55 ~ 0.9 part, tyrosine, is preferably 0.65 ~ 0.8 part, more preferably 0.72 part;
0.5 ~ 0.85 part of phenylalanine, is preferably 0.6 ~ 0.75 part, more preferably 0.67 part;
0.8 ~ 1.2 part of Histidine, is preferably 0.9 ~ 1.1 part, more preferably 1.02 parts; With,
Arginine,, is preferably 1.1 ~ 1.3 parts, more preferably 1.18 parts by 1 ~ 1.5 part.
6. the fodder additives described in claim 4 or 5, its water ratio is less than 5%(w/w), be preferably less than 3%(w/w), be more preferably less than 2.5%(w/w).
7. the fodder additives described in claim 4 or 5, it is to be prepared by arbitrary described method of claim 1 ~ 3.
8. lysine sulfate, aspartic acid and L-glutamic acid are combined in the application of preparing in fodder additives, and wherein, amino acid whose weight proportion is as follows:
72.1 ~ 80.1 parts of lysine sulfate, are preferably 73.5 ~ 76.1 parts, more preferably 74.4 parts;
1.3 ~ 1.9 parts of aspartic acids, are preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts; With,
2 ~ 3 parts, L-glutamic acid, is preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts.
9. 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 are combined in the application of preparing in fodder additives.
10. the application described in claim 8 or 9, wherein fodder additives is arbitrary described fodder additives of claim 4 ~ 7.
CN201310438355.4A 2012-11-07 2012-11-07 The method that disposable fermentation prepares high-purity lysine sulphate Active CN103805627B (en)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
冯占雨 等: "70%含量预混料专用型赖氨酸硫酸盐物理性状的研究", 《国家饲料工程技术研究中心（养殖技术）四川畜牧信息网》 *
冯占雨 等: "70%赖氨酸硫酸盐和98%赖氨酸盐酸盐在饲料中混合均匀度的比较研究", 《饲料博览》 *
谯仕彦: "新型氨基酸的应用研究", 《北方牧业》 *
邝声耀 等: "新型赖氨酸应用推广的研究进展", 《四川畜牧兽医》 *
陈林书 等: "赖氨酸硫酸盐类产品应用连续喷雾流化床造粒技术的生产线配置", 《食品与发酵工业》 *

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