CN102925505B - Method for preparing highly-purified L-Lysine sulphate through one-time fermentation - Google Patents
Method for preparing highly-purified L-Lysine sulphate through one-time fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims description 21
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Abstract
The invention provides a method for preparing products containing L-Lysine sulphate by fermenting, wherein a gene which is known to have nothing to do with lysine metabolism is lead into bacteria which produce L-Lysine, and the content of lysine in the L-Lysine sulphate achieves purity of market products. Furthermore, the invention further provides the L-Lysine sulphate produced by the method, and the L-Lysine sulphate can obviously increase moisture absorption resistance ability relative to existing products containing the L-Lysine sulphate, thereby being suitable for long-term preservation.
Description
Technical field
The invention belongs to the amino acid fermentation field, particularly, the preparation that the present invention relates to ferment contains the method for 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 use as fodder additives.Current, the production of 1B is mainly by the fermentative production of microorganism, as utilizing coryneform bacteria production.
In the various product of lysine salt, especially lysine sulfate, have very large shortcoming, namely have larger water absorbability, make thus wherein water content too high (as, greater than 3%), too high like this product humidity easily causes product caking, is difficult for preserving, and is especially easily mouldy during prolonged preservation, even this does not allow when using as the lower feed of safety requirements 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 defective, the equivalence ratio of acid radical anion and Methionin is adjusted between 0.68～0.95, be the equivalent of acid radical anion less than the equivalent of Methionin, can reduce to a certain extent like this equilibrium moisture (referring to No. the 03120099th, Chinese patent) of product.Aforesaid method can bring better resistance to water soak for lysine hydrochloride, in relative humidity is under 33～58% routine preservation environment, substantially can make the equilibrium moisture of product be controlled at below 3%; Yet, for lysine sulfate, to preserve under environment at this, 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 the poor lysine sulfate of 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 it as the effectiveness of 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 opposite, 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, need not too much step of preparation process, the product that obtains 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 in the market in product sold take the high density product as main (general lysine content as 55% or more than), therefore lysine content in product is risen at least 55%, could be than being easier to carry out marketing.For this reason, the inventor also provides the method for the easy fermentative production high purity lysine sulfate of a kind of step.
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 imports one to the bacterium that produces 1B with comprising innovation 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 that the fermentation preparation contains the product of L-lysine sulfate, it comprises:
(1) polynucleotide of the albumen as shown in SEQ ID No:1 or its variant import the bacterium that produces 1B with encoding amino acid sequence;
(2) bacterium of under fermentation conditions liquid culture step (1) acquisition, collect the supernatant liquor of cultivating, and allow this supernatant liquor by filter membrane, keeps 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 that obtains to step (2), add sulfuric acid and concentrate drying to water ratio 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 opposite, the method of first aspect present invention is opened other bypasses of non-Methionin metabolism moderately by introducing the RNA polymerase sigma-32 factor, thereby can disposable fermentation obtain Methionin and other type amino acid ligand than suitable product, need not too much step of preparation process.The RNA polymerase sigma-32 factor is by working to the heat-shocked promotor specifically, 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.(can be referring to 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 be referring to No. the 96193336th, Chinese patent application), but RNA polymerase sigma-32 produces the impact that distributes but without any enlightenment on fermenting lysine and on wherein amino acid.Therefore, preferably in the method for first aspect present invention, the aminoacid sequence that the aminoacid sequence of described variant obtains for the aminoacid sequence as shown in SEQ ID No:1 being added, lack or replaces and several amino-acid residue.
More preferably in the method for first aspect present invention, the aminoacid sequence of described variant is for adding, lack the aminoacid sequence as shown in SEQ IDNo:1 or replacing 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, and the supernatant liquor of cultivation comprises the amino acid of 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.In the method for preferred 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. the 94194962nd, Chinese patent).Because intestinal bacteria itself are suitable as the cloning host bacterium, therefore preferred described polynucleotide be import by calcium chloride transformation colibacillary.
In this article, expression " part " used during 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 " can be regarded as the ratio of these compositions in said composition when more than one composition limits in to composition.
In this article, inoculum size have those skilled in the art can the conventional implication of understanding, specifically when representing with volume percent, refer to bacterial culture fluid (the bacterium liquid of access) volume with respect to the percentage of the culture volume that is 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 molecular weight cut-off to be the filter membrane of 1000Da, can disposable lysine content in the finished product be brought up to more than 55%, reaches the needs that are fit to marketing, and simple operating steps.Therefore, in the method for preferred first aspect present invention, filter membrane is that molecular weight cut-off is the filter membrane of 500～5000Da, and preferably molecular weight cut-off is the filter membrane of 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.
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, more preferably 1.5～2.5: 0.75～1.25, most preferably be 2: 1.
Concentrated and the dry equipment that can commonly use 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) that contains 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.
The product of preferred 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 product of second aspect present invention comprises the amino acid of 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 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, the product of preferred 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 in the poor lysine sulfate of resistance to water soak, 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 the water absorbability index that those skilled in the art commonly use, 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 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, adopt other impurity in the product of method preparation of first aspect present invention, the amino acid product that can not make aforementioned proportion under the routine preservation condition water ratio higher than 3% (w/w).Therefore the product of preferred second aspect present invention is by the method preparation of first aspect present invention.
In the third aspect, the invention provides lysine sulfate, aspartic acid and L-glutamic acid unite the preparation second aspect present invention product 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 unite the preparation second aspect present invention product in application.Wherein, this product water ratio is little, and preferred (balance) water ratio is more preferably less than 3% (w/w), most preferably less than 2.5% (w/w) less than 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, and is 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 corresponding, 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 describe in detail the present invention by specific embodiment.It needs to be noted, these descriptions are only exemplary descriptions, do not consist of limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change 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 1RNA polysaccharase sigma-32 factor gene construct
Aminoacid sequence according to the albumen accession number AAB18436.1 of NCBI (http://www.ncbi.nlm.nih.gov), the inventor has designed appropriate expression type codon (non-expression amount optimizing codon), and entrust Shanghai give birth to the gene of the work composite coding RNA polymerase sigma-32 of the Bioisystech Co., Ltd factor and be built in colibacillus expression plasmid pET-20b (+) (can available 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 (available from TaKaRa company), 5 ' of these nucleic acid fragments are held and carried out 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 T4DNA ligase enzyme (available from TaKaRa company) to connect 12 hours.Then, get the 1 above-mentioned connection product of μ L and carry out pcr amplification in 50 μ L reaction volumes, wherein forward primer (has been introduced the EcoRI 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 sex change 4 minutes, then extended in 60 seconds and 72 ℃ with 94 ℃ of sex change 30 seconds, 63 ℃ of annealing and carried out 35 circulations in 35 seconds, extended 4 minutes and be cooled to 4 ℃ with 72 ℃ at last.
The above-mentioned PCR product of agarose gel electrophoresis, reclaim the approximately fragment of 900bp size, with EcoR I and this fragment of XhoI double digestion, and be connected with the T4DNA ligase enzyme through the pET-20b of these two endonuclease digestions (+) plasmid, be transformed in intestinal bacteria Top10F '.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 encoded RNA polymerase sigma-32 factor as shown in SEQ IDNo:1 is returned by company the plasmid (called after pET-sigma) that builds.
The preparation of the L-lysine sulfate product of embodiment 2 Escherichia coli fermentations
The coli strain W3110 (tyrA) of the product 1B that the described method of No. the 94194962nd, Chinese patent is built/pCABD2, transform the pET-sigma plasmid, through identifying that obtaining to transform positive colony (after called after W3110 (tyrA)/pCABD2-sigma), carries out the fermenting lysine experiment for No. 03120099 with reference to Chinese patent respectively with bacterial strain W3110 (tyrA)/pCABD2 and W3110 (tyrA)/pCABD2-sigma.In brief, bacterial strain is accessed in liquid LB substratum shaking culture reach 0.35 to OD500, (every liter of culture medium prescription is 100g glucose, 60g (NH to the inoculum size access fermenting lysine substratum take 5%
4)
2SO
4, 50g CaCO
3, 35mL peptone-B (Soy Protein Enzymatic Hydrolysate, available from the true Bioisystech Co., Ltd in Shanghai, total nitrogen content is not less than 3%), 1g KH
2PO
4, 400mgMgSO
47H
2O, 200mg METHIONINE, 10mg FeSO
47H
2O, 8.1mg MnSO
44H
2O, 300 μ g vitamin Hs and 200 μ g VITMAIN B1 are adjusted to pH7 with Tris-HCl) in cultivated 72 hours with 36 ℃ of vibrations (250rpm).Then, centrifugal collection medium supernatant (fermented liquid).Then with supernatant liquor respectively the ultra-filtration membrane by molecular weight cut-off 1000Da (can be available 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 of amino acid molecular amount, polysaccharide, peptide and albumen) estimate most ofly to be removed by membrane retention) 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
Add the vitriol oil to the above-mentioned filtrate that is obtained by different strains respectively, the mol ratio that makes the Methionin in sulfuric acid and supernatant liquor is 1: 2, then be placed in concentrate on vaporizer after, spray into granulation in fluid bed dryer with vaporific.Particle is placed in 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 3L-lysine product
Get embodiment 2 by the L-lysine sulfate product (being designated as sigma) of W3110 (tyrA)/pCABD2-sigma preparation with by the L-lysine sulfate product (being designated as contrast 1) of W3110 (tyrA)/pCABD2 preparation.
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 was being placed 7 days under with the environment of different relative humidity under 25 ℃ respectively, 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, (relative humidity 33～58%) is difficult to control water ratio below 3% and prolonged preservation under the routine preservation environment, 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 4L-lysine sulfate product to the calf growth effect
entrust academy of agricultural sciences, Ningxia herding institute to compare test with L-lysine sulfate product and the commercially available 85%L-lysine hydrochloride fodder additives by W3110 (tyrA)/pCABD2-sigma preparation of embodiment 2, feed Deng addition (in 1B wherein) calf of raising for 4～6 ages in week, 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 the 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 the untoward reaction report.
Claims (5)
1. the fermentation preparation contains the method for the product of L-lysine sulfate, and it comprises:
(1) polynucleotide with the albumen of encoding amino acid sequence as shown in SEQ ID No:l are cloned into pET-20b (+) plasmid, then this recombinant plasmid is imported in coli strain W3110 (tryA)/pCABD2 the bacterial strain W3110 (tryA) of acquisition product 1B/pCABD2-sigma;
(2) bacterium of under fermentation conditions liquid culture step (1) acquisition, collect the supernatant liquor of cultivating, and allow this supernatant liquor by filter membrane, keeps filtrate;
Described fermentation condition is: bacterial strain is accessed in liquid LB substratum shaking culture to OD
500Reach 0.35, with in 5% inoculum size access fermenting lysine substratum with 36 ℃ of shaking culture 72 hours, every liter of fermenting lysine culture medium prescription is: 100g glucose, 60g (NH
4)
2SO
4, 50g CaCO
3, 35mL peptone-B, 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 are adjusted to pH 7 with Tris-HCl;
Described filter membrane is that molecular weight cut-off is the ultra-filtration membrane of 1000Da;
The amino acid that comprises following weight proportion in described filtrate: 54～60 parts of Methionins, 1.3～1.9 parts of aspartic acids, 0.8～1.2 part of Threonine, 0.45～0.7 part of Serine, 2～3 parts, L-glutamic acid, 0.8～1.1 part of glycine, 1.3～1.7 parts of L-Ala, 0.8～1.2 part of α-amino-isovaleric acid, 0.35～0.55 part of methionine(Met), 0.5～0.8 part of Isoleucine, 1.1～1.6 parts of leucines, 0.55～0.9 part, tyrosine, 0.5～0.85 part of phenylalanine, 0.8～1.2 part of Histidine, 1～1.5 part of arginine;
(3) filtrate that obtains to step (2) adds sulfuric acid and concentrated, sprays into granulation in fluid bed dryer with vaporific, and with particle drying to water ratio less than 3%.
2. method claimed in claim 1, wherein add sulfuric acid in step (3), and the mol ratio that makes Methionin and sulfuric acid is 1～3: 0.5～1.5.
3. method claimed in claim 2, the mol ratio of wherein said Methionin and sulfuric acid is 1.5～2.5: 0.75～1.25.
4. method claimed in claim 3, the mol ratio of wherein said Methionin and sulfuric acid is 2: 1.
5. the arbitrary described method of claim 1-4, wherein the polynucleotide sequence of encoding amino acid sequence albumen as shown in SEQ ID No:l is as shown in SEQ ID NO:2.
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| CN201310438880.6A CN103451223B (en) | 2012-11-07 | 2012-11-07 | The method of high-purity lysine sulphate is prepared in disposable fermentation |
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