CN106434827A - Production of phenylalanine through co-fermentation of immobilized mixed bacteria - Google Patents
Production of phenylalanine through co-fermentation of immobilized mixed bacteria Download PDFInfo
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- CN106434827A CN106434827A CN201611056284.1A CN201611056284A CN106434827A CN 106434827 A CN106434827 A CN 106434827A CN 201611056284 A CN201611056284 A CN 201611056284A CN 106434827 A CN106434827 A CN 106434827A
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- fermentation
- phenylalanine
- immobilized
- thalline
- ammonia
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- 238000000855 fermentation Methods 0.000 title claims abstract description 51
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 title claims abstract description 40
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 12
- 241000894006 Bacteria Species 0.000 title claims abstract description 9
- 230000004151 fermentation Effects 0.000 claims abstract description 48
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 28
- 241000186145 Corynebacterium ammoniagenes Species 0.000 claims abstract description 15
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 14
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 11
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 claims abstract description 10
- 241000223254 Rhodotorula mucilaginosa Species 0.000 claims abstract description 9
- WBYWAXJHAXSJNI-VOTSOKGWSA-N trans-cinnamic acid Chemical compound OC(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-N 0.000 claims abstract description 7
- 239000004202 carbamide Substances 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 23
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000012530 fluid Substances 0.000 claims description 8
- 239000012535 impurity Substances 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 7
- 238000010521 absorption reaction Methods 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 235000013877 carbamide Nutrition 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000003729 cation exchange resin Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 239000010419 fine particle Substances 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 238000009413 insulation Methods 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000010902 straw Substances 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 229920000936 Agarose Polymers 0.000 claims description 2
- 238000010168 coupling process Methods 0.000 claims description 2
- 238000004132 cross linking Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000007921 spray Substances 0.000 claims description 2
- 241000186146 Brevibacterium Species 0.000 claims 1
- 239000004372 Polyvinyl alcohol Substances 0.000 claims 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 1
- 239000007791 liquid phase Substances 0.000 claims 1
- 239000012071 phase Substances 0.000 claims 1
- 229920002451 polyvinyl alcohol Polymers 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 4
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 abstract description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 abstract description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 abstract description 2
- 239000001099 ammonium carbonate Substances 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract description 2
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 abstract 1
- 108700023158 Phenylalanine ammonia-lyases Proteins 0.000 abstract 1
- 235000011114 ammonium hydroxide Nutrition 0.000 abstract 1
- 238000003912 environmental pollution Methods 0.000 abstract 1
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- 229960005190 phenylalanine Drugs 0.000 description 22
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 238000012239 gene modification Methods 0.000 description 4
- 230000005017 genetic modification Effects 0.000 description 4
- 235000013617 genetically modified food Nutrition 0.000 description 4
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229930016911 cinnamic acid Natural products 0.000 description 3
- 235000013985 cinnamic acid Nutrition 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 210000001822 immobilized cell Anatomy 0.000 description 3
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- -1 ammonium hydrogen carbonate Phenylalanine Chemical compound 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000012262 fermentative production Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- BTNMPGBKDVTSJY-UHFFFAOYSA-N keto-phenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=CC=C1 BTNMPGBKDVTSJY-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 239000001903 2-oxo-3-phenylpropanoic acid Substances 0.000 description 1
- VAJVDSVGBWFCLW-UHFFFAOYSA-N 3-Phenyl-1-propanol Chemical compound OCCCC1=CC=CC=C1 VAJVDSVGBWFCLW-UHFFFAOYSA-N 0.000 description 1
- ULGJWNIHLSLQPZ-UHFFFAOYSA-N 7-[(6,8-dichloro-1,2,3,4-tetrahydroacridin-9-yl)amino]-n-[2-(1h-indol-3-yl)ethyl]heptanamide Chemical compound C1CCCC2=NC3=CC(Cl)=CC(Cl)=C3C(NCCCCCCC(=O)NCCC=3C4=CC=CC=C4NC=3)=C21 ULGJWNIHLSLQPZ-UHFFFAOYSA-N 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- ZGBSOTLWHZQNLH-UHFFFAOYSA-N [Mg].S(O)(O)(=O)=O Chemical compound [Mg].S(O)(O)(=O)=O ZGBSOTLWHZQNLH-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- DEDGUGJNLNLJSR-UHFFFAOYSA-N alpha-hydroxycinnamic acid Natural products OC(=O)C(O)=CC1=CC=CC=C1 DEDGUGJNLNLJSR-UHFFFAOYSA-N 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 108010003977 aminoacylase I Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000013051 drainage agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 210000004709 eyebrow Anatomy 0.000 description 1
- 210000000720 eyelash Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/222—Phenylalanine
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a method for producing phenylalanine through co-fermentation of immobilized mixed bacteria, comprising the steps of culturing Brevibacterium ammoniagenes, culturing Rhodotorula rubra, immobilizing mixed bacteria, preparing a mixed medium, fermenting with a fluidized bed, and purifying phenylalanine. The defects that fermentation enzymatic activity of a single bacterium is low and the bacterium is easily inactivated under high ammonia and high pH are overcome through the production of phenylalanine through co-fermentation of immobilized mixed bacteria; immobilized fermentation also provides a simplified purifying step of fermented phenylalanine. Ammonia produced by urea fermentation through Brevibacterium ammoniagenes is used as an ammonia source, Rhodotorula rubra is fermented through the ammonia and added trans-cinnamic acid, and phenylalanine is produced by fermenting the trans-cinnamic acid and ammonia through phenylalanine ammonialyase produced by Rhodotorula rubra; the fermentation process is free of ammonia water and ammonium bicarbonate, zero environmental pollution is caused, and product post-extraction process is simpler and more feasible in free cell fermentation than the prior art.
Description
Technical field
The invention belongs to field of microbial fermentation, more particularly to produces phenylpropyl alcohol ammonia using immobilized mixed strain combined ferment
The method of acid.
Background technology
Phenylalanine all has application in food processing and the industry such as medicine, cosmetics.As food nutrition hardening agent and
Food and drink additive and high sweet health sweeting agent（APM）Main synthesis material, particularly APM heat is low, is suitable to obesity
Crowd and patients with diabetes mellitus, are approved sale now with multinational.It is particularly which and has been proved to the work(such as refreshment and fat-reducing
Effect, is also used as the use of amino acidses cancer therapy drug.
The synthetic method of phenylalanine mainly has four kinds at present：Extraction method, chemical synthesiss, microbe fermentation method, enzyme process,
Wherein enzyme process includes phenylpyruvic acid enzyme process and cinnamic acid enzymatic again.Most domestic medium-sized and small enterprises are carried out using fermentation method at present
Production, but through production in a few years, its defect gradually comes out：Acid production rate is low, and the production cycle is longer, and wastewater flow rate is big, energy consumption
High, domestic cinnamic acid price also persistently drops in addition, reaches ten thousand yuan/ton of 1.8-2.0, and especially Guangxi cinnamic acid resource is richer
Richness, further increases the applicability of fermentation method.But as fermentative Production is using the artificial side for adding ammonia and ammonium hydrogen carbonate
Method provides ammonia source and high pH condition, causes growth of microorganism and is restricted, and the PAL of inside microbes exists
Just inactivate in the very short time, the fermentative Production of phenylalanine is seriously constrained, replaced type ammonia source is provided and improves production
Environment becomes urgent needss.
Immobilization technology is modern biotechnology and its a kind of core technology in industrialization link.Thousand rent lands, one youth of Japan
First enzyme immobilization technology is applied to industrialized production, he began one's study immobilized aminoacylase from nineteen sixty, 1969 years
Industrial applications are entered, this is a breakthrough in the world.Immobilized cell technique is referred to will be free using various physico-chemical methods
Cellular localization in limited space and makes a kind of technology which maintains vigour and can recycle, it be in immobilized enzyme base
Grow up on plinth.Immobilized cell has many advantages, can keep cytoactive for a long time, anti-shearing force, can profit repeatedly
With, low cost, optimize industrial flow, simplify equipment, the synthesis and accumulation for secondary substance is improved.Immobilized cell technique
Key be the property of carrier material and immobilized method.
A lot of scholars are attempted by genetic engineering means at this stage, microorganism are carried out genetic modification, for fermenting and producing
Chinese invention patent " a kind of side of producing L-phenylalanine by microorganism fermentation of phenylalanine, such as publication number CN102399835A
Method ", and a kind of Chinese invention patent " method of fermenting and producing L-phenylalanine " of publication number CN102181503A all adopts
Genetic engineering means, carry out genetic modification to Corynebacterium glutamicum and escherichia coli, respectively in order to fermenting and producing phenylalanine.
But the phenylalanine yield for being produced by the method for genetic modification is relatively low, and there is potential genetic risk, needs numerous studies
As a result with experimental data support just can enter industrialized production, find natural strain carry out phenylalanine production also compel in eyebrow
Eyelash.If natural being fixed of strain can be processed using immobilization technology, in the bar without ammonia and ammonium hydrogen carbonate
Phenylalanine is produced under part, it will huge change is brought to phenylalanine industry.
Content of the invention
The invention aims to providing a kind of method that immobilized mixed strain combined ferment produces phenylalanine.Gram
Prior art has been taken and has manually added ammonia and caused enzyme inactivation very fast, separating step complexity is extracted after product, and genetic modification is deposited
Shortcoming in potential risk.
The purpose of the present invention is through the following steps that realizing：
（1）Brevibacterium ammoniagene is cultivated：Brevibacterium ammoniagene is accessed in culture medium, in fermentation tank according to the inoculum concentration of 1%-1.5%
Fermentation is stirred, centrifugation after fermentation is finished obtains brevibacterium ammoniagene thalline；
（2）Rhodothece rubra culture：Rhodothece rubra is accessed in culture medium according to the inoculum concentration of 1.5%-3%, is carried out in fermentation tank
Stirring fermentation, centrifugation after fermentation is finished obtains Rhodotorula rubra HMC thalline；
（3）Mixed bacteria immobilization：By step（1）And step（2）The brevibacterium ammoniagene thalline for obtaining and Rhodotorula rubra HMC thalline
According to 1:2 ratio is mixed, and is processed to mixing being fixed of thalline；
（4）The preparation of mixed culture medium：By Fructus Hordei Germinatus remove impurity, straw and weeds are removed, then fine particle is ground into pulverizer
Shape, with mass volume ratio as 1:4（KG/L）Ratio add pure water, be put in water-bath 65 DEG C of insulation saccharifying 7 hours constantly
Stirring, uniform to ensure saccharifying.Using 4 layers of filtered through gauze remove impurity, saccharified liquid is boiled the appropriate egg albumen of addition and is helped as absorption
Filtering agent, after cooling, then with 4 layers of filtered through gauze, that is, obtains the beerwort that clarifies, adds 2% carbamide, 0.2% magnesium sulfate, 2% Portugal
Grape sugar, 2% trans-cinnamic acid had both obtained mixed culture medium；
（5）Fluid bed ferments：Immobilized mixing thalline is put in fermentation fluid bed, is passed through mixed culture medium and is fluidized
Bed fermentation；
（6）Phenylalanine is purified：After the completion of fermentation, fermentation liquid is collected by cation exchange resin eluting, then carries out spray dried
Dry obtain the phenylalanine that purity is more than 97.8%.
Further, the step（1）And step（2）Used in culture medium be：5 grams of peptone, yeast extract powder 2
Gram, 20 grams of glucose, 1 gram of dipotassium hydrogen phosphate, 0.5 gram of magnesium sulfate, 1000 milliliters of distilled water.
Further, the step（1）And step（2）Cultivation temperature be 28 DEG C, respectively ferment 24 hours and 36 hours.
Further, the step（3）Middle immobilization mixing thalline adopts absorption method, covalent coupling method, cross-linking method, embedding
One kind in method, microencapsulation, preferably polyethylene alcohol and agarose mixing investment.
Further, the step（5）Middle fluid bed fermentation time is 56 hours, is detected by high performance liquid chromatography,
In fermentation liquid, concentration of phenylalanine is not further added by stopping fermentation.
Further, the high performance liquid chromatography testing conditions are, chromatographic column：Alltech C18 post；Mobile phase：First
Alcohol/water, 10/90 (V/V)；Column temperature：25℃；Flow velocity：0.65 mL/min;Sample size：10 µL；Detector：SHIMADZU company
LC-10AT HPLC system, UV210 nm.
Further, the brevibacterium ammoniagene and rhodothece rubra are purchased from the management of Chinese industrial Microbiological Culture Collection
The heart, preserving number is CICC 20101, CICC 32621.
Compared with prior art, the beneficial effects of the present invention is：Mixing by brevibacterium ammoniagene and Rhodotorula rubra HMC
Strain immobilization fermentation, brevibacterium ammoniagene fermentation carbamide produces ammonia, and outside cooperation plus trans-cinnamic acid enters one using rhodothece rubra
Step fermentation, produces phenylalanine, has reached the purpose of not additional ammonia, and using be all that natural industrial metaplasia produces strain, no
There is transgenic risk, immobilization fermentation also simplify the rear extraction process of product phenylalanine.Whole fermentative process conditions temperature
With controlled, technological process is simple, also not using strong acid and highly basic, while ensure that working condition is gentle, increased enzyme activity,
The use time of enzyme is extended, rear extraction step is simplified, and increased the yield of phenylalanine.
Specific embodiment
Below by specific embodiment, the present invention is further detailed.
Embodiment 1：
By 100 grams of peptone, 40 grams of yeast extract powder, 400 grams of glucose, 20 grams of dipotassium hydrogen phosphate, the dissolving of 10 grams of magnesium sulfate with
In 20KG distilled water, brevibacterium ammoniagene is accessed, in 28 DEG C of bottom fermentations 24 hours, centrifugation obtained thalline 4.3KG.By Rhodotorula rubra HMC
Same access etc. in the culture medium of quality, in 28 DEG C of bottom fermentations 36 hours, centrifugation obtained thalline 8.8KG.Two kinds of thalline are mixed
Processed using being fixed of trehalose investment after uniform.By Fructus Hordei Germinatus 100KG remove impurity, straw and weeds are removed, then uses powder
Broken machine is ground into fine particle shape, adds pure water 400KG, is put in water-bath 65 DEG C of insulation saccharifying 7 hours and is stirred continuously, with
Ensure that saccharifying is uniform.Using 4 layers of filtered through gauze remove impurity, saccharified liquid is boiled addition 1KG egg albumen as absorption filter aid, treat cold
But after, then with 4 layers of filtered through gauze, that is, obtain the beerwort 410KG for clarifying, add 8.2KG carbamide, 0.82KG magnesium sulfate,
8.2KG glucose, 8.6KG trans-cinnamic acid had both obtained mixed culture medium.Immobilization mixing thalline is put in fluid bed, is passed through
Mixed culture medium is fermented, and after fermenting 56 hours, using high performance liquid chromatography, testing conditions are：Chromatographic column：Alltech
C18 post；Mobile phase：Methanol/water, 10/90 (V/V)；Column temperature：25℃；Flow velocity：0.65 mL/min;Sample size：10 µL；Detection
Device：The LC-10AT HPLC system of SHIMADZU company, UV210 nm, the yield of phenylalanine in fermentation liquid is detected, concentration is not
It is further added by, stops fermentation.By cation exchange resin eluting collect after, then carry out spray drying obtain purity for 97.8% with
On phenylalanine 4.5KG.
Embodiment 2：
By 300 grams of peptone, 120 grams of yeast extract powder, 1200 grams of glucose, 60 grams of dipotassium hydrogen phosphate, 30 grams of dissolvings of magnesium sulfate
In 60KG distilled water, brevibacterium ammoniagene is accessed, in 28 DEG C of bottom fermentations 24 hours, centrifugation obtained thalline 12.45KG.By dark red ferment
Female bacterium is equally accessed etc. in the culture medium of quality, and in 28 DEG C of bottom fermentations 36 hours, centrifugation obtained thalline 25.5KG.By two kinds of thalline
Processed using being fixed of trehalose investment after mix homogeneously.By Fructus Hordei Germinatus 200KG remove impurity, straw and weeds are removed, then
Fine particle shape being ground into pulverizer, add pure water 780KG, is put in water-bath 65 DEG C of insulation saccharifying 7 hours and constantly stirs
Mix, uniform to ensure saccharifying.Using 4 layers of filtered through gauze remove impurity, saccharified liquid is boiled addition 3KG egg albumen as absorption drainage
Agent, after cooling, then with 4 layers of filtered through gauze, that is, obtains the beerwort 800KG for clarifying, adds 16KG carbamide, 1.6KG sulphuric acid
Magnesium, 16KG glucose, 16.8KG trans-cinnamic acid had both obtained mixed culture medium.Immobilization mixing thalline is put in fluid bed,
It is passed through mixed culture medium to be fermented, after fermenting 56 hours, using high performance liquid chromatography, testing conditions are：Chromatographic column：
Alltech C18 post；Mobile phase：Methanol/water, 10/90 (V/V)；Column temperature：25℃；Flow velocity：0.65 mL/min;Sample size：10
µL；Detector：The LC-10AT HPLC system of SHIMADZU company, UV210 nm, the yield of phenylalanine in fermentation liquid is detected,
Concentration is not further added by, and stops fermentation.After cation exchange resin eluting is collected, then carry out spray drying and obtain purity be
More than 97.8% phenylalanine 10.3KG.
Claims (7)
1. immobilized mixed strain combined ferment production phenylalanine, it is characterised in that comprising having the following steps：
（1）Brevibacterium ammoniagene is cultivated：Brevibacterium ammoniagene is accessed in culture medium, in fermentation tank according to the inoculum concentration of 1%-1.5%
Fermentation is stirred, centrifugation after fermentation is finished obtains brevibacterium ammoniagene thalline；
（2）Rhodothece rubra culture：Rhodothece rubra is accessed in culture medium according to the inoculum concentration of 1.5%-3%, is carried out in fermentation tank
Stirring fermentation, centrifugation after fermentation is finished obtains Rhodotorula rubra HMC thalline；
（3）Mixed bacteria immobilization：By step（1）And step（2）The brevibacterium ammoniagene thalline for obtaining and Rhodotorula rubra HMC thalline
According to 1:2 ratio is mixed, and is processed to mixing being fixed of thalline；
（4）The preparation of mixed culture medium：By Fructus Hordei Germinatus remove impurity, straw and weeds are removed, then fine particle is ground into pulverizer
Shape, with mass volume ratio as 1:4（KG/L）Ratio add pure water, be put in water-bath 65 DEG C of insulation saccharifying 7 hours constantly
Stirring, uniform to ensure saccharifying；Using 4 layers of filtered through gauze remove impurity, saccharified liquid is boiled the appropriate egg albumen of addition and is helped as absorption
Filtering agent, after cooling, then with 4 layers of filtered through gauze, that is, obtains the beerwort that clarifies, adds 2% carbamide, 0.2% magnesium sulfate, 2% Portugal
Grape sugar, 2% trans-cinnamic acid had both obtained mixed culture medium；
（5）Fluid bed ferments：Immobilized mixing thalline is put in fermentation fluid bed, is passed through mixed culture medium and is fluidized
Bed fermentation；
（6）Phenylalanine is purified：After the completion of fermentation, fermentation liquid is collected by cation exchange resin eluting, then carries out spray dried
Dry obtain the phenylalanine that purity is more than 97.8%.
2. immobilized mixed strain combined ferment according to claim 1 produces phenylalanine, it is characterised in that the step
Suddenly（1）And step（2）Used in culture medium be：5 grams of peptone, 2 grams of yeast extract powder, 20 grams of glucose, dipotassium hydrogen phosphate 1
Gram, 0.5 gram of magnesium sulfate, 1000 milliliters of distilled water.
3. immobilized mixed strain combined ferment according to claim 1 and 2 produces phenylalanine, it is characterised in that institute
State step（1）And step（2）Cultivation temperature be 28 DEG C, respectively ferment 24 hours and 36 hours.
4. immobilized mixed strain combined ferment according to claim 1 produces phenylalanine, it is characterised in that the step
Suddenly（3）Middle immobilization mixing thalline is using the one kind in absorption method, covalent coupling method, cross-linking method, investment, microencapsulation, excellent
Select polyvinyl alcohol and agarose mixing investment.
5. immobilized mixed strain combined ferment according to claim 1 produces phenylalanine, it is characterised in that the step
Suddenly（5）Middle fluid bed fermentation time is 56 hours, is detected by high performance liquid chromatography, and in fermentation liquid, concentration of phenylalanine is no longer
Increase i.e. and stop fermentation.
6. immobilized mixed strain combined ferment according to claim 5 produces phenylalanine, it is characterised in that the height
Effect liquid phase chromatogram method testing conditions are, chromatographic column：Alltech C18 post；Mobile phase：Methanol/water, 10/90 (V/V)；Column temperature：25
℃；Flow velocity：0.65 mL/min;Sample size：10 µL；Detector：The LC-10AT HPLC system of SHIMADZU company, UV210
nm.
7. immobilized mixed strain combined ferment according to claim 1 produces phenylalanine, it is characterised in that the product
Ammonia brevibacterium and rhodothece rubra are purchased from Chinese industrial Microbiological Culture Collection administrative center, preserving number be CICC 20101,
CICC 32621.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1288949A (en) * | 2000-10-20 | 2001-03-28 | 安徽省科苑应用技术开发(集团)股份有限公司 | Conditions of culturing deep red saccharomycete to produce phenylalanine deaminase |
| CN1962861A (en) * | 2006-11-10 | 2007-05-16 | 南京工业大学 | Combination immobilization method for use in bio-catalytic conversion |
| CN101671716A (en) * | 2009-10-15 | 2010-03-17 | 淮北新旗氨基酸有限公司 | Method for manufacturing D-phenylalanine by bio-enzyme asymmetric transformation |
-
2016
- 2016-11-26 CN CN201611056284.1A patent/CN106434827A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1288949A (en) * | 2000-10-20 | 2001-03-28 | 安徽省科苑应用技术开发(集团)股份有限公司 | Conditions of culturing deep red saccharomycete to produce phenylalanine deaminase |
| CN1962861A (en) * | 2006-11-10 | 2007-05-16 | 南京工业大学 | Combination immobilization method for use in bio-catalytic conversion |
| CN101671716A (en) * | 2009-10-15 | 2010-03-17 | 淮北新旗氨基酸有限公司 | Method for manufacturing D-phenylalanine by bio-enzyme asymmetric transformation |
Non-Patent Citations (4)
| Title |
|---|
| （德）纳尔蔡斯（NARZISS，LUDWIG）: "《啤酒厂麦芽汁制备工艺技术 第6版》", 31 December 1991 * |
| 丁明玉等: "《离子色谱原理与应用》", 31 March 2001 * |
| 金其荣等: "《有机酸发酵工艺学》", 30 June 1989 * |
| 魏金明编著: "《中国药学大辞典》", 30 June 2010 * |
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