CN102911976A - Method for preparing medium-purity lysine sulphate through fermentation - Google Patents
Method for preparing medium-purity lysine sulphate through fermentation Download PDFInfo
- Publication number
- CN102911976A CN102911976A CN2012104400818A CN201210440081A CN102911976A CN 102911976 A CN102911976 A CN 102911976A CN 2012104400818 A CN2012104400818 A CN 2012104400818A CN 201210440081 A CN201210440081 A CN 201210440081A CN 102911976 A CN102911976 A CN 102911976A
- Authority
- CN
- China
- Prior art keywords
- parts
- product
- acid
- lysine sulfate
- lysine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000004472 Lysine Substances 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 31
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 238000000855 fermentation Methods 0.000 title claims abstract description 20
- 230000004151 fermentation Effects 0.000 title claims abstract description 20
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 title claims description 22
- 229910021653 sulphate ion Inorganic materials 0.000 title 1
- 235000018977 lysine Nutrition 0.000 claims abstract description 27
- VJDRZAPMMKFJEA-JEDNCBNOSA-N (2s)-2,6-diaminohexanoic acid;sulfuric acid Chemical compound OS(O)(=O)=O.NCCCC[C@H](N)C(O)=O VJDRZAPMMKFJEA-JEDNCBNOSA-N 0.000 claims abstract description 18
- 239000000047 product Substances 0.000 claims description 56
- 150000001413 amino acids Chemical group 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 31
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 25
- 229940024606 amino acid Drugs 0.000 claims description 24
- 235000001014 amino acid Nutrition 0.000 claims description 24
- 229930182817 methionine Natural products 0.000 claims description 24
- 241000894006 Bacteria Species 0.000 claims description 21
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 15
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 14
- 239000000654 additive Substances 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- 229960002989 glutamic acid Drugs 0.000 claims description 13
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 12
- 235000003704 aspartic acid Nutrition 0.000 claims description 12
- 108091033319 polynucleotide Proteins 0.000 claims description 11
- 102000040430 polynucleotide Human genes 0.000 claims description 11
- 239000002157 polynucleotide Substances 0.000 claims description 11
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 7
- 230000000968 intestinal effect Effects 0.000 claims description 7
- 239000004475 Arginine Substances 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 6
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 6
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 6
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 6
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 6
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 6
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 6
- 239000004473 Threonine Substances 0.000 claims description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 6
- 150000001510 aspartic acids Chemical class 0.000 claims description 6
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 6
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 6
- 229960000310 isoleucine Drugs 0.000 claims description 6
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 230000008676 import Effects 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 238000009630 liquid culture Methods 0.000 claims description 4
- 125000000539 amino acid group Chemical group 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 238000000586 desensitisation Methods 0.000 claims description 3
- 230000005764 inhibitory process Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 abstract description 12
- 230000004060 metabolic process Effects 0.000 abstract description 9
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 238000010521 absorption reaction Methods 0.000 abstract description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 abstract 3
- 241000233866 Fungi Species 0.000 abstract 1
- 235000019766 L-Lysine Nutrition 0.000 abstract 1
- 230000007774 longterm Effects 0.000 abstract 1
- 229960003646 lysine Drugs 0.000 description 25
- 239000012535 impurity Substances 0.000 description 11
- 239000000203 mixture Substances 0.000 description 8
- 108010045235 heat-shock sigma factor 32 Proteins 0.000 description 7
- 230000002035 prolonged effect Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 150000007523 nucleic acids Chemical group 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 150000005838 radical anions Chemical class 0.000 description 3
- 239000006200 vaporizer Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000004459 forage Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000005469 granulation Methods 0.000 description 2
- 230000003179 granulation Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 230000002787 reinforcement Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 108091000044 4-hydroxy-tetrahydrodipicolinate synthase Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000186031 Corynebacteriaceae Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229960005337 lysine hydrochloride Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Landscapes
- Fodder In General (AREA)
Abstract
The invention provides a method for preparing an L lysine sulphate product through fermentation. The method comprises the step of introducing a gene, thought to be irrelevant to metabolism of lysine, into a fungus producing L-lysine. In addition, the invention further provides a product produced through the method. The product has remarkably improved moisture absorption resistance and is suitable for long-term preservation as compared with the present product containing L- lysine sulphate.
Description
Technical field
The invention belongs to the amino acid fermentation field, particularly, the present invention relates to ferment prepares the method for the product that contains L-lysine sulfate, and it comprises that innovation ground is considered to the gene that has nothing to do with the Methionin metabolism usually to one of the bacterium importing of producing 1B.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, the medicine, be widely used in grocery trade, feed industry, pharmacy industry and other chemical industry, especially contain impurity lysine salt (as, vitriol, hydrochloride), generally can directly use as fodder additives.Current, the production of 1B mainly is 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, thus so that water content wherein 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, the water absorbability of product is larger on their impact during prolonged preservation.
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, is under 33～58% the routine preservation environment in relative humidity, substantially can so that the equilibrium moisture of product be controlled at below 3%; Yet, for lysine sulfate, to preserve under the environment at this, equilibrium moisture substantially all surpasses 3%, even can surpass 5%.
Study for a long period of time and test through the inventor, found 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 the product, but meeting is so that the resistance to water soak of lysine sulfate product is 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 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, 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.
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 that the fermentation preparation contains the product of L-lysine sulfate, it comprises:
(1) albumen or the polynucleotide of its variant of encoding amino acid sequence shown in SEQ ID No:l is imported the bacterium that produces 1B;
(2) bacterium of under fermentation conditions liquid culture step (1) acquisition, and collect the supernatant liquor of cultivating, comprise the amino acid of following weight proportion in the described supernatant liquor:
51～55 parts of Methionins are preferably 51.5～53.5 parts, more preferably 52.3 parts;
1.2～1.8 parts of aspartic acids are preferably 1.45～1.6 parts, more preferably 1.54 parts;
0.8～1.1 part of Threonine is preferably 0.9～1 part, more preferably 0.94 part;
0.4～0.65 part of Serine is preferably 0.5～0.6 part, more preferably 0.56 part;
2～3 parts in L-glutamic acid is preferably 2.2～2.6 parts, more preferably 2.4 parts;
0.75～1.1 part of glycine is preferably 0.85～1 part, more preferably 0.92 part;
1.2～1.6 parts of L-Ala are preferably 1.3～1.5 parts, more preferably 1.4 parts;
0.75～1.1 part of α-amino-isovaleric acid is preferably 0.85～1 part, more preferably 0.93 part;
0.3～0.5 part of methionine(Met) is preferably 0.35～0.48 part, more preferably 0.42 part;
0.45～0.75 part of Isoleucine is preferably 0.55～0.65 part, more preferably 0.61 part;
1～1.5 part of leucine is preferably 1.2～1.35 parts, more preferably 1.27 parts;
0.5～0.8 part in tyrosine is preferably 0.6～0.75 part, more preferably 0.67 part;
0.5～0.8 part of phenylalanine is preferably 0.6～0.7 part, more preferably 0.64 part;
0.8～1.2 part of Histidine is preferably 0.9～1.05 part, more preferably 0.97 part; With,
Arginine, is preferably 1～1.2 part, more preferably 1.1 parts by 0.9～1.3 part;
(3) supernatant liquor 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 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 the 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 AABl8436.1 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 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 and replace the aminoacid sequence that one or several amino-acid residue obtains to the aminoacid sequence shown in SEQ IDNo:1, and the polynucleotide of this variant of encoding import to produce behind 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 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, most preferably is 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 are to import colibacillary by calcium chloride transformation.
In this article, expression used " part " during proportioning, be in order to be illustrated in the 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.
Add sulfuric acid in the method for first aspect present invention, thus can with this basic aminoacids salify of Methionin, be easier in drying process, condense into particle.The ratio of Methionin can be constant in the add-on of sulfuric acid and the supernatant liquor.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.
Liquid concentrated and dry can be by this area equipment commonly used, 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 the fluid bed dryer, and in oven drying.
In second aspect, the invention provides the product that contains L-lysine sulfate, it comprises 68.1～73.5 parts of lysine sulfate, is preferably 68.8～71.5 parts, more preferably 69.9 parts; 1.2～1.8 parts of aspartic acids are preferably 1.45～1.6 parts, more preferably 1.54 parts; With, 2～3 parts in L-glutamic acid is preferably 2.2～2.6 parts, more preferably 2.4 parts.
The product of preferred second aspect present invention comprises the amino acid of following weight proportion:
68.1～73.5 parts of lysine sulfate are preferably 68.8～71.5 parts, more preferably 69.9 parts;
1.2～1.8 parts of aspartic acids are preferably 1.45～1.6 parts, more preferably 1.54 parts;
0.8～1.1 part of Threonine is preferably 0.9～1 part, more preferably 0.94 part;
0.4～0.65 part of Serine is preferably 0.5～0.6 part, more preferably 0.56 part;
2～3 parts in L-glutamic acid is preferably 2.2～2.6 parts, more preferably 2.4 parts;
0.75～1.1 part of glycine is preferably 0.85～1 part, more preferably 0.92 part;
1.2～1.6 parts of L-Ala are preferably 1.3～1.5 parts, more preferably 1.4 parts;
0.75～1.1 part of α-amino-isovaleric acid is preferably 0.85～1 part, more preferably 0.93 part;
0.3～0.5 part of methionine(Met) is preferably 0.35～0.48 part, more preferably 0.42 part;
0.45～0.75 part of Isoleucine is preferably 0.55～0.65 part, more preferably 0.61 part;
1～1.5 part of leucine is preferably 1.2～1.35 parts, more preferably 1.27 parts;
0.5～0.8 part in tyrosine is preferably 0.6～0.75 part, more preferably 0.67 part;
0.5～0.8 part of phenylalanine is preferably 0.6～0.7 part, more preferably 0.64 part;
0.8～1.2 part of Histidine is preferably 0.9～1.05 part, more preferably 0.97 part; With,
Arginine, is preferably 1～1.2 part, more preferably 1.1 parts by 0.9～1.3 part.
Confirm through animal, even further comprise fermentation impurity, to raise middle use still be safe to the product of second aspect present invention animal is fed, and more because the amino acid whose adding of other kinds, can also further improve 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, meeting is so that the resistance to water soak of lysine sulfate product is 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, such as fermentation impurity.Through inventor's research, other impurity in the product of the method preparation of employing first aspect present invention can not make amino acid product water ratio under the routine preservation condition of aforementioned proportion be 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 and unite application in the little fodder additives that contains L-lysine sulfate of preparation water ratio, 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 unite application in the little fodder additives that contains L-lysine sulfate of preparation water ratio.Preferably wherein, (balance) water ratio is more preferably less than 3% (w/w) less than 5% (w/w), most preferably less than 2.5% (w/w).
Preferably in the application of third aspect present invention, the fodder additives that contains L-lysine sulfate is the product of second aspect present invention.
The present invention has following beneficial effect: product resistance to water soak of the present invention is good, so that the water ratio in the product reduces 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; Fermentation process implementation step of the present invention is simple, and one time fermentation can obtain the product of corresponding amino acid ligand ratio, has saved production cost, without potential safety hazard.
For the ease of understanding, below will describe in detail the present invention by specific embodiment.It needs to be noted, these descriptions only are 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 all are 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 specify by the following examples content of the present invention.As do not specialize, the conventional means that used technique means is well known to those skilled in the art among the embodiment 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 AAB 18436.1 of NCBI (http://www.nebi.nlm.nih.gov), the inventor has designed appropriate expression type codon (non-expression amount optimizing codon), and entrust Shanghai to give birth to the gene of the worker composite coding RNA polymerase sigma-32 of the Bioisystech Co., Ltd factor and be built among the 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 used commercialization reagent, 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 ' end of these nucleic acid fragments is carried out phosphorylation, then equimolar ratio is mixed behind these 5 nucleic acid fragments in 65 ℃ of sex change 5 minutes, annealing is cooled to 16 ℃, adds T4 dna ligase (available from TaKaRa company) and connects 12 hours.Then, get the above-mentioned connection product of 1 μ L and in 50 μ L reaction volumes, carry out pcr amplification, wherein forward primer (has been introduced EeoR I restriction enzyme site) shown in the SEQ ID No:3 of sequence table, reverse primer (introduced Xho I restriction enzyme site) shown in the SEQ ID No:4 of sequence table, reaction conditions was: with 94 ℃ of sex change 4 minutes, then extend with 94 ℃ of sex change 30 seconds, 63 ℃ of annealing 60 seconds and 72 ℃ and carried out 35 circulations in 35 seconds, extended 4 minutes with 72 ℃ at last and be cooled to 4 ℃.
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 among the intestinal bacteria Top10F '.Choose positive colony, extract plasmid wherein, through sequence verification, the corresponding nucleotide sequence is shown in the SEQ ID No:2 of inventor's design, encoded the RNA polymerase sigma-32 factor shown in SEQ IDNo:1 is returned the plasmid (called after pET-sigma) that builds by company.
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 made up/pCABD2, transform the pET-sigma plasmid, through identifying that obtaining to transform positive colony (behind the 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 the 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) is with the 1B content in the quantitative supernatant liquor of HPLC and other compositions.The result is as shown in table 1, fermented liquid 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, the content of other impurity (be higher than the impurity (sugar, polysaccharide, peptide and albumen) of amino acid molecular amount and be lower than the impurity (inorganic salt) of amino acid molecular amount) is substantially constant, if therefore the character of both tunnings has difference, estimation is owing to other amino acid whose kind and ratio.
Comparison of ingredients in table 1 fermented liquid
Add the vitriol oil to the above-mentioned supernatant liquor that is obtained by different strains respectively so that the mol ratio of the Methionin in sulfuric acid and the supernatant liquor is 1: 2, then place concentrate on the vaporizer after, spray into granulation in the fluid bed dryer with vaporific.Particle is placed the baking oven inner drying, until water ratio is no more than 1%, obtain 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 the product at that time, the result is as shown in table 2, the easy moisture absorption of lysine sulfate product that obtains according to 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, then 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 the 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
Embodiment 4L-lysine sulfate product is to the effectiveness of 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 the calf of raising for 4～6 ages in week Deng addition (in 1B wherein), take the feed that do not add Methionin as contrast, the product of embodiment 2 on average makes calf adding weight (ADG) improve 17.5%, and commercially available product on average improves 15.8%, wherein slightly is better than the commercially available prod and may gives 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 (10)
1. the fermentation preparation contains the method for the product of L-lysine sulfate, and it comprises:
(1) albumen or the polynucleotide of its variant of encoding amino acid sequence shown in SEQ ID No:l is imported the bacterium that produces 1B;
(2) bacterium of under fermentation conditions liquid culture step (1) acquisition, and collect the supernatant liquor of cultivating, comprise the amino acid of following weight proportion in the described supernatant liquor:
51～55 parts of Methionins are preferably 51.5～53.5 parts, more preferably 52.3 parts;
1.2～1.8 parts of aspartic acids are preferably 1.45～1.6 parts, more preferably 1.54 parts;
0.8～1.1 part of Threonine is preferably 0.9～1 part, more preferably 0.94 part;
0.4～0.65 part of Serine is preferably 0.5～0.6 part, more preferably 0.56 part;
2～3 parts in L-glutamic acid is preferably 2.2～2.6 parts, more preferably 2.4 parts;
0.75～1.1 part of glycine is preferably 0.85～1 part, more preferably 0.92 part;
1.2～1.6 parts of L-Ala are preferably 1.3～1.5 parts, more preferably 1.4 parts;
0.75～1.1 part of α-amino-isovaleric acid is preferably 0.85～1 part, more preferably 0.93 part;
0.3～0.5 part of methionine(Met) is preferably 0.35～0.48 part, more preferably 0.42 part;
0.45～0.75 part of Isoleucine is preferably 0.55～0.65 part, more preferably 0.61 part;
1～1.5 part of leucine is preferably 1.2～1.35 parts, more preferably 1.27 parts;
0.5～0.8 part in tyrosine is preferably 0.6～0.75 part, more preferably 0.67 part;
0.5～0.8 part of phenylalanine is preferably 0.6～0.7 part, more preferably 0.64 part;
0.8～1.2 part of Histidine is preferably 0.9～1.05 part, more preferably 0.97 part; With,
Arginine, is preferably 1～1.2 part, more preferably 1.1 parts by 0.9～1.3 part;
(3) supernatant liquor that obtains to step (2), add sulfuric acid and concentrate drying to water ratio less than 3% (w/w).
2. method claimed in claim 1, wherein the aminoacid sequence of variant is for adding, lack and replace the aminoacid sequence that one or several amino-acid residue obtains to the aminoacid sequence shown in SEQ ID No:1, the polynucleotide of this variant of optimized encoding import behind the bacterium that produces 1B under fermentation conditions liquid culture, and the supernatant liquor of cultivation comprises the as claimed in claim 1 amino acid of weight proportion.
3. 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.
4. method claimed in claim 1, wherein bacterium is intestinal bacteria, preferably to the intestinal bacteria of 1B feedback inhibition desensitization.
5. method claimed in claim 1, wherein the nucleotide sequence of polynucleotide is shown in SEQ ID NO:2.
6. contain the product (it is fodder additives preferably) of L-lysine sulfate, it comprises 68.1～73.5 parts of lysine sulfate, is preferably 68.8～71.5 parts, more preferably 69.9 parts; 1.2～1.8 parts of aspartic acids are preferably 1.45～1.6 parts, more preferably 1.54 parts; With, 2～3 parts in L-glutamic acid is preferably 2.2～2.6 parts, and more preferably 2.4 parts, preferably it comprises:
68.1～73.5 parts of lysine sulfate are preferably 68.8～71.5 parts, more preferably 69.9 parts;
1.2～1.8 parts of aspartic acids are preferably 1.45～1.6 parts, more preferably 1.54 parts;
0.8～1.1 part of Threonine is preferably 0.9～1 part, more preferably 0.94 part;
0.4～0.65 part of Serine is preferably 0.5～0.6 part, more preferably 0.56 part;
2～3 parts in L-glutamic acid is preferably 2.2～2.6 parts, more preferably 2.4 parts;
0.75～1.1 part of glycine is preferably 0.85～1 part, more preferably 0.92 part;
1.2～1.6 parts of L-Ala are preferably 1.3～1.5 parts, more preferably 1.4 parts;
0.75～1.1 part of α-amino-isovaleric acid is preferably 0.85～1 part, more preferably 0.93 part;
0.3～0.5 part of methionine(Met) is preferably 0.35～0.48 part, more preferably 0.42 part;
0.45～0.75 part of Isoleucine is preferably 0.55～0.65 part, more preferably 0.61 part;
1～1.5 part of leucine is preferably 1.2～1.35 parts, more preferably 1.27 parts;
0.5～0.8 part in tyrosine is preferably 0.6～0.75 part, more preferably 0.67 part;
0.5～0.8 part of phenylalanine is preferably 0.6～0.7 part, more preferably 0.64 part;
0.8～1.2 part of Histidine is preferably 0.9～1.05 part, more preferably 0.97 part; With,
Arginine, is preferably 1～1.2 part, more preferably 1.1 parts by 0.9～1.3 part.
7. product claimed in claim 6, its water ratio preferably less than 3% (w/w), is more preferably less than 2.5% (w/w) less than 5% (w/w).
8. product claimed in claim 6, it is by arbitrary described method preparation of claim 1～5.
9. lysine sulfate, aspartic acid and L-glutamic acid are united at the preparation water ratio less than the application in the fodder additives that contains L-lysine sulfate of 5% (w/w) (preferably less than 3% (w/w), being more preferably less than 2.5% (w/w)).
10. application claimed in claim 9, the fodder additives that wherein contains L-lysine sulfate are the arbitrary described products of claim 6～8.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210440081 CN102911976B (en) | 2012-11-07 | 2012-11-07 | Method for preparing medium-purity lysine sulphate through fermentation |
| CN201310403725.0A CN103484493B (en) | 2012-11-07 | 2012-11-07 | Method of preparing lysine through fermentation salt and products thereof |
| CN201310403491.XA CN103805626B (en) | 2012-11-07 | 2012-11-07 | Method of preparing lysine through fermentation salt and products thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210440081 CN102911976B (en) | 2012-11-07 | 2012-11-07 | Method for preparing medium-purity lysine sulphate through fermentation |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310403491.XA Division CN103805626B (en) | 2012-11-07 | 2012-11-07 | Method of preparing lysine through fermentation salt and products thereof |
| CN201310403725.0A Division CN103484493B (en) | 2012-11-07 | 2012-11-07 | Method of preparing lysine through fermentation salt and products thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102911976A true CN102911976A (en) | 2013-02-06 |
| CN102911976B CN102911976B (en) | 2013-10-16 |
Family
ID=47610555
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310403725.0A Active CN103484493B (en) | 2012-11-07 | 2012-11-07 | Method of preparing lysine through fermentation salt and products thereof |
| CN 201210440081 Active CN102911976B (en) | 2012-11-07 | 2012-11-07 | Method for preparing medium-purity lysine sulphate through fermentation |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310403725.0A Active CN103484493B (en) | 2012-11-07 | 2012-11-07 | Method of preparing lysine through fermentation salt and products thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN103484493B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4393135A (en) * | 1979-12-10 | 1983-07-12 | Ajinomoto Company Incorporated | Method for producing L-glutamic acid by fermentation |
| CN1142856A (en) * | 1993-12-08 | 1997-02-12 | 味之素株式会社 | Process for producing L-Lysine by fermentation |
| CN1181785A (en) * | 1995-02-20 | 1998-05-13 | 味之素株式会社 | Stress-tolerant microorganism and method of the prodn. of fermentation product |
| EP1064106B1 (en) * | 1998-03-18 | 2003-07-16 | Technologies Inc. Biosphere | Method for bio-refining organic waste material to produce denatured and sterile nutrient products |
| CN1943392A (en) * | 2005-10-08 | 2007-04-11 | 德古萨股份公司 | Feedstuff additive containing l-lysine |
-
2012
- 2012-11-07 CN CN201310403725.0A patent/CN103484493B/en active Active
- 2012-11-07 CN CN 201210440081 patent/CN102911976B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4393135A (en) * | 1979-12-10 | 1983-07-12 | Ajinomoto Company Incorporated | Method for producing L-glutamic acid by fermentation |
| CN1142856A (en) * | 1993-12-08 | 1997-02-12 | 味之素株式会社 | Process for producing L-Lysine by fermentation |
| CN1181785A (en) * | 1995-02-20 | 1998-05-13 | 味之素株式会社 | Stress-tolerant microorganism and method of the prodn. of fermentation product |
| EP1064106B1 (en) * | 1998-03-18 | 2003-07-16 | Technologies Inc. Biosphere | Method for bio-refining organic waste material to produce denatured and sterile nutrient products |
| CN1943392A (en) * | 2005-10-08 | 2007-04-11 | 德古萨股份公司 | Feedstuff additive containing l-lysine |
Non-Patent Citations (3)
| Title |
|---|
| MANFRED KIRCHER ET AL: "The fermentative production of l-lysine as an animal feed additive", 《CHEMOSPHERE》, 31 December 2001 (2001-12-31), pages 27 - 31 * |
| 石现瑞 等: "65%赖氨酸硫酸盐物理性状的研究", 《新研究》, no. 7, 31 December 2006 (2006-12-31), pages 18 - 20 * |
| 邝声耀 等: "新型赖氨酸应用推广的研究进展", 《四川畜牧兽医》, no. 11, 31 December 2005 (2005-12-31) * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103484493A (en) | 2014-01-01 |
| CN103484493B (en) | 2016-02-17 |
| CN102911976B (en) | 2013-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6341936B2 (en) | High production strain of 5-aminolevulinic acid and its production method and use | |
| CN102021154B (en) | Method for improving yield of arginine by mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase | |
| CN102342370B (en) | Production process of fermented soya bean meal rich in limiting amino acids | |
| CN103555779B (en) | A kind of method of fermentative production γ-aminobutyric acid | |
| CN103114069B (en) | Bacterium for fermenting L-tryptophan from mixed saccharum and fermentation method thereof | |
| CN104928226A (en) | Recombined corynebacterium glutamicum and application of corynebacterium glutamicum to 5-aminolevulinic acid production | |
| CN111066963A (en) | Preparation method of protein peptide zinc chelate suitable for ruminants | |
| Li et al. | Effects of amino acids and overexpression of dapA gene on the production of ε-poly-L-lysine by Streptomyces diastatochromogenes strains | |
| CN102925505B (en) | Method for preparing highly-purified L-Lysine sulphate through one-time fermentation | |
| CA3193923C (en) | Nitrogen-enhanced yeast-based fertilizer | |
| CN112980758A (en) | Method for increasing yield of 5-aminolevulinic acid synthesized by corynebacterium glutamicum | |
| CN102911976B (en) | Method for preparing medium-purity lysine sulphate through fermentation | |
| JPS61212249A (en) | Composition for feed | |
| CN102925506B (en) | Method for preparing high purity lysine sulfate through fermenting | |
| CN105670982A (en) | Recombinant strain as well as construction method and application thereof | |
| CN105265765A (en) | Fermented feed with high-nutrition components and preparation method of fermented feed | |
| CN103805646B (en) | Fermentation is for the method and products thereof of high purity lysine salt | |
| CN103451223A (en) | High-purity lysine sulfate preparation method based on one-time fermentation | |
| CN105264082B (en) | Extraction of nitrogen from organic material by ammonification by mixed bacterial populations | |
| CN105603017A (en) | Method for producing trans-4-hydroxyl-L-proline by means of fermentation by aid of recombinant corynebacterium acetoacidophilum | |
| CN103805627A (en) | Method for preparing high-purity lysine sulfate through once fermentation | |
| JP2013208074A (en) | Method for preparing 5-aminolevulinic acid | |
| CN103805626A (en) | Method for preparing lysine salt through fermentation and product thereof | |
| CN103451221A (en) | Method for preparing high-purity L-lysine salt through fermentation and product produced by using method | |
| CN102191227B (en) | Dihydrodipicolinate synthase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method for preparing medium-purity lysine sulphate through fermentation Effective date of registration: 20190201 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd.|INNER MONGOLIA EPPEN BIOTECH Co.,Ltd. Registration number: 2019640000001 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20200116 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd. Registration number: 2019640000001 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method for preparing medium-purity lysine sulphate through fermentation Effective date of registration: 20200120 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd. Registration number: Y2020640000002 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20210126 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd. Registration number: Y2020640000002 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Preparation of medium purity lysine sulfate by fermentation Effective date of registration: 20210127 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd. Registration number: Y2021640000001 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20220129 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd. Registration number: Y2021640000001 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method for preparing medium purity lysine sulfate by fermentation Effective date of registration: 20220323 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd. Registration number: Y2022640000002 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230315 Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd.|INNER MONGOLIA EPPEN BIOTECH Co.,Ltd.|Heilongjiang Yipin Biotechnology Co.,Ltd. Registration number: Y2022640000002 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method for Preparation of Medium Purity Lysine Sulfate through Fermentation Granted publication date: 20131016 Pledgee: Yongning County Branch of China Agricultural Development Bank Pledgor: NINGXIA EPPEN BIOTECH Co.,Ltd.|INNER MONGOLIA EPPEN BIOTECH Co.,Ltd.|Heilongjiang Yipin Biotechnology Co.,Ltd. Registration number: Y2024640000005 |