CN103451223A

CN103451223A - High-purity lysine sulfate preparation method based on one-time fermentation

Info

Publication number: CN103451223A
Application number: CN2013104388806A
Authority: CN
Inventors: 马吉银; 陈崇安; 孟刚
Original assignee: NINGXIA EPPEN BIOTECH CO Ltd
Current assignee: NINGXIA EPPEN BIOTECH CO Ltd
Priority date: 2012-11-07
Filing date: 2012-11-07
Publication date: 2013-12-18
Anticipated expiration: 2032-11-07
Also published as: CN103451223B

Abstract

The invention provides an L-lysine sulfate product preparation method based on fermentation. The L-lysine sulfate product preparation method based on fermentation comprises the steps that a gene which is considered to be irrelevant to lysine metabolism is led into fungi which can produce L-lysine, and the lysine content in a product can reach the level of the purity of products in the market. In addition, the invention further provides the product prepared with the method. Compared with an existing product containing the L-lysine sulfate, the product prepared with the method is capable of obviously improving hygroscopicity resistance and suitable for being stored for a long time.

Description

Disposable fermentation prepares the method for high purity lysine sulfate

Technical field

The invention belongs to the amino acid fermentation field, particularly, the present invention relates to the method for fermentation preparation containing the product of L-lysine sulfate, it comprises that innovation ground imports one to the bacterium that produces 1B and usually is considered to the gene irrelevant with the Methionin metabolism, and in product, lysine content has reached the purity of commercially available prod.In addition, the present invention also provides product that described method produces and application etc.

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 as fodder additives, use.Current, the production of 1B is mainly by the fermentative production of microorganism, as utilized coryneform bacteria production.

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, especially easily mouldy during prolonged preservation, even, when this uses at the feed lower as safety requirements, do not allow yet.Especially the inventor finds, China's wide farmers, penkeeping is small, lysine salt is little as the fodder additives consumption, therefore whole bag is bought the lysine salt product of (often unit price is more cheap), therefore have more retention, during prolonged preservation, the water absorbability of product is larger on their impact.

For this reason, Japan aginomoto company adopts the equivalent of regulating 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% in relative humidity, substantially can make the equilibrium moisture of product be controlled at below 3%; Yet, for lysine sulfate, at this, to preserve under environment, equilibrium moisture substantially all can surpass 3%, even can surpass 5%.

Study for a long period of time and test through the inventor, found unexpectedly, add the amino acid of a certain proportion of other type in poor lysine sulfate at resistance to water soak, 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 searched out unexpectedly a kind of enzyme in very complicated metabolic pathway, 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 obtained still can be as lysine additives for forage, and resistance to water soak is significantly improved.

Further, the inventor finds, after having added the sulfuric acid salify, lysine content in the resulting product of this disposable fermentation is difficult to go beyond 55%, and take in the market the high density product in product sold 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 that innovation ground imports one to the bacterium that produces 1B and usually is considered to the gene irrelevant with the Methionin metabolism.In addition, the present invention also provides product that the method produces and application etc.

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, collect the supernatant liquor of cultivating, and allow this supernatant liquor by filter membrane, retains filtrate, comprises the amino acid of following weight proportion in described filtrate:

54 ~ 60 parts of Methionins, be preferably 55 ~ 57 parts, more preferably 55.7 parts;

1.3 ~ 1.9 parts of aspartic acids, be preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts;

0.8 ~ 1.2 part of Threonine, be preferably 0.9 ~ 1.1 part, more preferably 1 part;

0.45 ~ 0.7 part of Serine, be preferably 0.55 ~ 0.65 part, more preferably 0.6 part;

2 ~ 3 parts, L-glutamic acid, be preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts;

0.8 ~ 1.1 part of glycine, be preferably 0.85 ~ 1.05 part, more preferably 0.96 part;

1.3 ~ 1.7 parts of L-Ala, be preferably 1.4 ~ 1.6 parts, more preferably 1.5 parts;

0.8 ~ 1.2 part of α-amino-isovaleric acid, be preferably 0.9 ~ 1.1 part, more preferably 0.99 part;

0.35 ~ 0.55 part of methionine(Met), be preferably 0.4 ~ 0.5 part, more preferably 0.44 part;

0.5 ~ 0.8 part of Isoleucine, be preferably 0.6 ~ 0.7 part, more preferably 0.63 part;

1.1 ~ 1.6 parts of leucines, be preferably 1.25 ~ 1.45 parts, more preferably 1.36 parts;

0.55 ~ 0.9 part, tyrosine, be preferably 0.65 ~ 0.8 part, more preferably 0.72 part;

0.5 ~ 0.85 part of phenylalanine, be preferably 0.6 ~ 0.75 part, more preferably 0.67 part;

0.8 ~ 1.2 part of Histidine, be preferably 0.9 ~ 1.1 part, more preferably 1.02 parts; With,

Arginine,, be preferably 1.1 ~ 1.3 parts, more preferably 1.18 parts by 1 ~ 1.5 part;

(3) filtrate obtained to step (2), add 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 the heat-shocked promotor being worked, and participates in the heat-shocked process, affect transcribing of heat-shocked associated protein, thereby the poisonous substance that the microorganism in glutamic acid fermentation overcomes the metabolism generation is exerted an influence.Although the RNA polymerase sigma-32 factor is disclosed (can be 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 that distributes 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 adds, lacks or replaces for the aminoacid sequence to as shown in SEQ ID No:1 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 adds, lacks for the aminoacid sequence to as shown in SEQ ID No:1 or replaces the aminoacid sequence that and several amino-acid residue obtain, and the polynucleotide of this variant of encoding import to 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, the nucleotide sequence of codon modify preferably, the degree of opening to regulate this pathways metabolism.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, such as utilizing the transfered cells such as microsome, particle gun; Also can indirectly be imported into, such as 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 to express intestinal bacteria to the dihydrodipicolinic acid synthase of 1B feedback inhibition desensitization (referring to No. 94194962nd, Chinese patent).Because intestinal bacteria itself are suitable as the cloning host bacterium, therefore preferred described polynucleotide be by calcium chloride transformation, import colibacillary.

In this article, " part " used when meaning proportioning, be in order to be illustrated in described composition, the weight proportion of each composition, and this can understand to those skilled in the art.Weight " part " when more than one composition is limited 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, when meaning with volume percent, refer to the percentage of bacterial culture fluid (the bacterium liquid of access) volume with respect to the culture volume be access in.

Use filter membrane can remove impurity, high molecular impurity especially, 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 the filter membrane that molecular weight cut-off is 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 the filter membrane that molecular weight cut-off is 1000Da.

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, and more preferably 1.5 ~ 2.5:0.75 ~ 1.25, most preferably be 2:1.

Concentrated and the dry equipment that can be commonly used by this area of liquid, 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), 55 ~ 57%(w/w more preferably), 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, be preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts; With, 2 ~ 3 parts, L-glutamic acid, be preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts.

The amino acid that more preferably product of second aspect present invention comprises following weight proportion:

72.1 ~ 80.1 parts of lysine sulfate, be preferably 73.5 ~ 76.1 parts, more preferably 74.4 parts;

Arginine,, be preferably 1.1 ~ 1.3 parts, more preferably 1.18 parts by 1 ~ 1.5 part.

Through animal, confirm, even 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 the 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, found unexpectedly, add the amino acid of a certain proportion of other type at resistance to water soak in poor lysine sulfate, can make the resistance to water soak of lysine sulfate product be significantly improved, therefore suitable prolonged preservation.In this article, equilibrium moisture content can with the equilibrium moisture Alternate, be those skilled in the art's water absorbability indexs commonly used, refer under certain relative humidity environment, water ratio when the product water ratio reaches balance (that is, water ratio neither can increase, and also can not reduce).Preferably can be under the routine preservation condition (relative humidity is 33 ~ 58%) prolonged preservation, (balance) water ratio of the product of second aspect present invention is less than 5%(w/w), preferably be 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 comprised of above-mentioned amino acid), also can further comprise impurity, as fermentation impurity.Through inventor research, other impurity in the product that adopts the method for first aspect present invention to prepare, the amino acid product that can not make aforementioned proportion under the routine preservation condition water ratio higher than 3%(w/w).Therefore preferably the product of second aspect present invention is prepared by method by 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), most preferably be less than 2.5%(w/w).

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 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 the 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, facilitated operation, also saved production cost.

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 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 this paper in and carried out reference, 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 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 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 U.S. Novagen company, 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, with T4 polynucleotide kinase (purchased from TaKaRa company), 5 ' end of these nucleic acid fragments is carried out to phosphorylation, 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 with 72 ° of C, extend 4 minutes and be cooled to 4 ° 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 the 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, the 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, returned by company the plasmid (called after pET-sigma) built.

the preparation of the L-lysine sulfate product of embodiment 2 Escherichia coli fermentations

The coli strain W3110 (tyrA) of the product 1B that No. 94194962 described method of Chinese patent built/pCABD2, transform the pET-sigma plasmid, after identifying that acquisition transforms positive colony (called after W3110 (tyrA)/pCABD2-sigma), bacterial strain W3110 (tyrA)/pCABD2 and W3110 (tyrA)/pCABD2-sigma are carried out to the 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 to take 5% inoculum size access fermenting lysine substratum 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 36 ℃ of vibrations (250rpm), cultivate 72 hours.Then, centrifugal collection medium supernatant (fermented liquid).Then by supernatant liquor respectively the ultra-filtration membrane by molecular weight cut-off 1000Da (can be purchased from German Sartorius AG, goods number: 3051460901E-SG), with the 1B content in the quantitative filtrate of HPLC and other compositions.Result is as shown in table 1, filtrate with respect to W3110 (tyrA)/pCABD2, the lysine content of W3110 (tyrA)/pCABD2-sigma slightly descends, but other amino acid whose kinds and ratio obviously improve, other impurity (are mainly the impurity (inorganic salt) lower than the amino acid molecular amount, and higher than the impurity (sugar, polysaccharide, peptide and albumen) of amino acid molecular amount, estimate most ofly by membrane retention, to be removed) 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, all be 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))
Impurity higher than the amino acid molecular amount	1.10g/L	1.06g/L
			Impurity lower than the amino acid molecular amount	0.99g/L	1.02g/L

Add the vitriol oil to the above-mentioned filtrate obtained by different strains respectively, the mol ratio that makes the Methionin in sulfuric acid and supernatant liquor is 1:2, after then being placed on vaporizer and concentrating, with vaporific, sprays into granulation in fluid bed dryer.Particle is placed in to the baking oven inner drying, until water ratio is no more than 1%, obtains the 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) prepared by W3110 (tyrA)/pCABD2-sigma of embodiment 2 and the L-lysine sulfate product 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.

By above-mentioned sample respectively under 25 ℃ to place under the environment of different relative humidity 7 days, 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 the routine preservation environment, (relative humidity 33 ~ 58%) is difficult to control water ratio below 3% and prolonged preservation, if and mix with other amino acid of some particular types and ratio, can significantly strengthen its resistance to water soak, make it prolonged 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 fully, 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 prolonged 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 the calf growth effect

Entrust academy of agricultural sciences, Ningxia herding institute to carry out simultaneous test with the L-lysine sulfate product 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, the feed that does not add Methionin of take is 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, do not have the untoward reaction report.

<110 > Ningxia Yi Pin biotech inc

<120 > disposable fermentation prepares the method for high purity lysine sulfate

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Claims

1. the 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, collect the supernatant liquor of cultivating, and allow 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, be preferably 55 ~ 57 parts;

1.3 ~ 1.9 parts of aspartic acids, be preferably 1.5 ~ 1.65 parts;

0.8 ~ 1.2 part of Threonine, be preferably 0.9 ~ 1.1 part;

0.45 ~ 0.7 part of Serine, be preferably 0.55 ~ 0.65 part;

2 ~ 3 parts, L-glutamic acid, be preferably 2.3 ~ 2.8 parts;

0.8 ~ 1.1 part of glycine, be preferably 0.85 ~ 1.05 part;

1.3 ~ 1.7 parts of L-Ala, be preferably 1.4 ~ 1.6 parts;

0.8 ~ 1.2 part of α-amino-isovaleric acid, be preferably 0.9 ~ 1.1 part;

0.35 ~ 0.55 part of methionine(Met), be preferably 0.4 ~ 0.5 part;

0.5 ~ 0.8 part of Isoleucine, be preferably 0.6 ~ 0.7 part;

1.1 ~ 1.6 parts of leucines, be preferably 1.25 ~ 1.45 parts;

0.55 ~ 0.9 part, tyrosine, be preferably 0.65 ~ 0.8 part;

0.5 ~ 0.85 part of phenylalanine, be preferably 0.6 ~ 0.75 part;

0.8 ~ 1.2 part of Histidine, be preferably 0.9 ~ 1.1 part; With,

Arginine,, be preferably 1.1 ~ 1.3 parts by 1 ~ 1.5 part;

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 to the intestinal bacteria of 1B feedback inhibition desensitization.

4. containing the product (it is fodder additives preferably) of L-lysine sulfate, wherein lysine content is 54 ~ 60%(w/w), 55 ~ 57%(w/w more preferably), 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, be preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts; With, 2 ~ 3 parts, L-glutamic acid, be preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts.

5. product claimed in claim 4, it comprises:

6. the described product of claim 4 or 5, its water ratio is less than 5%(w/w), preferably be less than 3%(w/w), be more preferably less than 2.5%(w/w).

7. the described product of claim 4 or 5, prepared by its arbitrary described method by claim 1 ~ 3.

8. lysine sulfate, aspartic acid and L-glutamic acid are combined the application in preparing fodder additives, and wherein, amino acid whose weight proportion is as follows:

1.3 ~ 1.9 parts of aspartic acids, be preferably 1.5 ~ 1.65 parts, more preferably 1.61 parts; With,

2 ~ 3 parts, L-glutamic acid, be preferably 2.3 ~ 2.8 parts, more preferably 2.56 parts.

9. application claimed in claim 8, wherein fodder additives is the arbitrary described product of claim 4 ~ 7.

10. the application of arbitrary described product in preparing fodder additives of claim 4 ~ 7.