CN102719502A - Method for producing L-alanine by mutating lactate-production bacteria - Google Patents
Method for producing L-alanine by mutating lactate-production bacteria Download PDFInfo
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Abstract
The invention discloses a method for producing L-alanine by mutating lactate-production bacteria, which belongs to the microbe technical field, according to the invention, lactobacillus delbruckii bulgaricus subspecies can be taken as a starting bacterial classification, the method comprises the following steps: activating the starting strain, mutating processed cells by low energy ion implantation, screening the mutant strain capable of producing L-alanine by a reversible inhibition test, amplifying and culturing seed liquid, producing L-alanine by anaerobic fermentation, and purifying and processing a broth to prepare the finished product. The invention has the beneficial effects that the low energy ion implantation mutation method enables wide mutation spectrum, low mortality, high direct mutation rate and stable property; the raw material glucose belongs to the renewable natural resource, the source is wide, the price is low, the anaerobic fermentation reduces the power consumption, and the production cost can be reduced; an ultrafilter membrane is used for filtering and a nanofiltration membrane is used for filtering and removing the impurities during the purification process, the product quality can be ensured, and the method provided by the invention is suitable for large scale industrial production.
Description
Technical field
The present invention relates to a kind of mutagenesis lactic acid and produce the method that bacterium produces the L-L-Ala, belong to microbial technology field.
Background technology
The L-L-Ala is one of amino acid of constitutive protein matter, though be the nonessential amino acid of human body, is the highest amino acid of content in the blood of human body, has purposes widely in fields such as medicine, macromolecular material, food.At field of medicaments: the L-L-Ala is a main raw material of producing Y factor and L-aminopropanol as medicine intermediate; L-L-Ala and carbohydrate metabolism are closely related, are main amino donor in transamination reaction, have the important physical function, often add in the transfusion clinically; In addition, the L-L-Ala still is a kind of good hydragog(ue).At polymeric material field: discovered in recent years adds the L-L-Ala to the performance that can effectively improve POLYACTIC ACID in the POLYACTIC ACID.At field of food: the L-L-Ala has unique effect of improving local flavor, cooperates use can strengthen the local flavor of food and drink with other amino acid; The interpolation of L-L-Ala can obviously improve proteinic utilization ratio in the food and drink, can also improve organic acid tart flavour, more and more receives people's welcome and attention.
At present, the major industry working method of L-L-Ala is an enzymatic conversion method, promptly obtains the L-L-Ala through the microorganism cells catalysis L-aspartic acid that is rich in L-aspartic acid-beta-decarboxylase activity.The characteristics of enzymatic conversion method technology are that enzyme activity is high, and facility investment is little, and extraction process is simple, but its main raw material(s) L-aspartic acid price is more expensive, causes its production cost higher.Pascal Hols etc. has reported and has utilized genetic engineering means to transform milk-acid bacteria; With glucose, sucrose or lactose etc. is the method (US 6 627 420 B1) that fermenting raw materials is produced the L-L-Ala; It is complicated that but gene engineering method makes up the process of bacterial classification; Technical requirements is high, and the engineering strain that obtains is difficult to genetic stability and preservation.High reason waits superior strain (Anhui University of Engineering Science & Technology's journal, 2008,23 (1): 19-24) of having reported the method screening L-L-Ala that utilizes complex mutations such as ultraviolet ray and laser; But because ultraviolet ray is different to the mutagenic effect of various mikrobes; Add that mikrobe absorbs very difficult assurance of size of roentgen dose X, and be prone to the repair of generation light after the sudden change, so the poor effect of ultraviolet mutagenesis; Positive mutation rate is lower, and mortality ratio is higher.
Ion implantation organism selection by mutation is a kind of new direction of artificial mutation, is that collection chemomorphosis, physical mutagenesis are the comprehensive mutafacient system of one.Compare with traditional mutation source such as X ray, gamma-rays, ultraviolet ray and various chemical mutagen, ion implantation not only has energy deposition, also has effects such as momentum transfer, quality deposition and charge-exchange.Low energy ion beam is the common result of directly effect and indirect action to the mutagenesis of mikrobe; Its direct effect mainly is to inject sputter effect that the ionic momentum transfer produces in cell surface and be deposited on the etching injury that cell interior produces, and indirect action mainly is to inject the effect that radical caused that energy of ions is deposited on the cell interior generation.It can cause chromosomal distortion, causes damage, the fracture of DNA chain base, thereby genetic material is changed on gene level or molecular level or lacks, and significantly improves the frequency of variation.
Summary of the invention
To above-mentioned existing technical problem, the present invention provides a kind of mutagenesis lactic acid to produce the method that bacterium produces the L-L-Ala, with lactobacillus delbruockii subspecies bulgaricus
Lactobacillus delbrueckii subsp. bulgaricusBe the bacterial classification that sets out, adopt low energy ion injection method mutagenic treatment cell, but and filter out the mutant strain of producing the L-L-Ala through the retroactive inhibition assay method, using this mutant strain is that substrate, anaerobic condition bottom fermentation are produced the L-L-Ala with glucose.
The present invention is for realizing above-mentioned purpose, and realize through following technical scheme: present method produces the bacterium lactobacillus delbruockii subspecies bulgaricus with lactic acid
Lactobacillus delbrueckii subsp. bulgaricusBe the bacterial classification that sets out; Mutant strain → amplification cultivation seed liquor → anaerobically fermenting production L-L-Ala → fermented liquid purifying treatment of the bacterium but activation is set out → low energy ion injection method mutagenic treatment cell → retroactive inhibition assay method screening production L-L-Ala → make finished product, concrete steps are following.
A, the activation bacterium that sets out: will set out the bacterium lactobacillus delbruockii subspecies bulgaricus on slant medium 42 ℃ be cultured to logarithmic phase, process bacteria suspension with the saline water of 0.85 %, be diluted to the 1mL bacteria suspension and contain the bacterium number and be about 10
7, behind the assorted bacterium of microscopy bacterial classification stalwartness and nothing, draw the 0.1mL bacteria suspension and evenly coat on the aseptic blank petridish the drying one-tenth mycoderm of aseptic wind.
B, low energy ion inject mutagenesis mutagenic treatment cell: microscopy is acellular overlapping after, under the vacuum condition somatic cells is carried out ion implantation processing, injecting ion is the N ion, dosage is 1.5 * 10
14-5 * 10
16Ions/cm
2, energy is 15-20keV, pulse is injected, and injects 5s at every turn, pitch time 15-40s; The mycoderm that will pass through after the ion implantation processing carries out wash-out with the 1mL SPSS, gets the 0.1mL elutriant then and coats on the solid plate, places 37 ℃ of incubators to cultivate 48h.
But the mutant strain of L-L-Ala is produced in C retroactive inhibition assay method screening: single bacterium colony of selecting after ion implantation is numbered, and is inoculated in the fermention medium of reference numeral, and fermented liquid pH value is at 6.5-7.5,37 ℃ of anaerobically fermenting 48h; Filtering fermentation liquor is removed thalline, and filtrating adds 1.5% agar through 121 ℃ of steam sterilizing 20min, behind 115 ℃ of steam sterilizing 20min, adds the 0.01-0.05% suppressor factor, and is dull and stereotyped, and the coating indicator, places 35-40 ℃ of incubator to cultivate; The mutant strain of choosing the dull and stereotyped pairing numbering of indicator growth carries out continuous passage to be cultivated, and through stability test, finally obtains two strains and is numbered
Lds.0108 with
Lds.1109 L-L-Ala superior strain.
D, amplification cultivation seed liquor:
Lds.0108 with
Lds.1109 pass through slant culture and seed tank culture, 37 ℃ of culture temperature, slant culture time 24h, seed tank culture time 18h respectively successively.
E, anaerobically fermenting are produced the L-L-Ala: the seed liquor after will increasing is inoculated in the fermention medium, and the substrate glucose concn is 6-12%, and leavening temperature is 35-40 ℃; The medium pH value is 6.5-7.5; Keep anaerobic environment, treat that remaining sugar concentration is less than 0.1% fermentation completion, fermentation time 30-52h.
F, fermented liquid purifying treatment: adopt ceramic super-filtering film or organic tubular ultra-filtration membrane to filter earlier, further filter through nf membrane then.
G, make finished product: nanofiltration liquid is through the vacuum concentration evaporation, and the L-L-Ala is separated out in crystallization, through centrifugal, and dry finished product.
The invention has the beneficial effects as follows: lactic acid produces the bacterium lactobacillus delbruockii subspecies bulgaricus as setting out bacterial classification; Under anaerobic has higher glycolysis-ability; In actual application, can practice thrift a large amount of power costs; And this bacterial classification is as the food grade bacterial classification, and lactic acid and L-Ala that its fermentation produces are analogs, can improve the efficient of gain mutant greatly.The low energy ion that adopts injects mutagenesis and has advantages such as mutation spectrum is wide, mortality ratio is low, positive mutation rate is high, proterties is stable.Used starting material glucose belongs to renewable pure natural resource, and wide material sources are cheap, and anaerobically fermenting reduced power consumption, has reduced production cost.Use ultrafiltration membrance filter and nf membrane to filter in the purification process and remove impurity, guaranteed quality product, be fit to large-scale industrial production.
Embodiment
Below in conjunction with embodiment the present invention is described further.
The present invention with lactic acid produce the bacterium lactobacillus delbruockii subspecies bulgaricus (
Lactobacillus delbrueckii subsp. bulgaricus) be the bacterial classification that sets out; But the screening of the activated bacterium that sets out → low energy ion injection method mutagenic treatment cell → retroactive inhibition assay method is produced mutant strain → amplification cultivation seed liquor → anaerobically fermenting of L-L-Ala and is produced steps such as L-L-Ala → fermented liquid purifying treatment → make finished product; Can know that implementation process mainly is divided into two stages; At first be the mutagenesis of producing the lactobacillus delbruockii subspecies bulgaricus mutant strain of L-L-Ala, utilize the lactobacillus delbruockii subspecies bulgaricus mutant strain that screens then
Lds.0108 with
Lds.1109 fermentative prodn L-L-Ala.
Embodiment 1-1: the mutagenesis of producing the lactobacillus delbruockii subspecies bulgaricus mutant strain of L-L-Ala.
1, nutrient media components.
1.1 the composition of slant medium: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, agar 1.5%, sodium hydroxide is transferred pH to 7.0,121 ℃ of steam sterilizing 20min.
1.2 the composition of fermention medium: glucose 6%, potassium primary phosphate 0.2%, potassium hydrogenphosphate 1.2%, anhydrous magnesium sulfate 0.02%, ammoniacal liquor is transferred pH to 7.0,115 ℃ of steam sterilizing 20min.
For avoiding causing interference to going on foot screening L-L-Ala generation bacterium down, in the fermention medium process for preparation, no any organism that contains L-Ala mixes.
2, operation steps.
The bacterium 2.1 activation is set out: will set out the bacterium lactobacillus delbruockii subspecies bulgaricus on the slant medium 42 ℃ cultivate about 18h, to logarithmic phase, process bacteria suspension with 0.85% saline water, be diluted to the 1mL bacteria suspension and contain the bacterium number and be about 10
7, behind the assorted bacterium of microscopy bacterial classification stalwartness and nothing, draw the 0.1mL bacteria suspension and evenly coat on the aseptic blank petridish that diameter is 9cm the drying one-tenth mycoderm of aseptic wind.
2.2 low energy ion injection method mutagenic treatment cell: microscopy is acellular overlapping after, under vacuum condition, carry out ion implantation processing.The ion that injects is the N ion, ion energy 15keV, implantation dosage 1.5 * 10
14Ions/cm
2, injection length 5s, pitch time 15s.Ion implanter is provided by Hefei Inst. of Plasma Physics, Chinese Academy of Sciences.The mycoderm that will pass through after the ion implantation processing carries out wash-out with the 1mL SPSS, gets the 0.1mL elutriant then and coats (the same slant medium of composition) on the solid plate, places 37 ℃ of incubators to cultivate 48h.
2.3 but the mutant strain of L-L-Ala is produced in retroactive inhibition assay method screening: select the single bacterium colony that grows after ion implantation and number, each single bacterium colony is chosen in the fermention medium of reference numeral 37 ℃ of anaerobically fermenting 48h respectively with transfering loop.The pH value can descend in the fermenting process, and then stream adds the ammoniacal liquor or the liquefied ammonia of concentration 25%, keeps fermented liquid pH value at 6.5-7.5.Filtering fermentation liquor is removed thalline, and its filtrating is through 121 ℃ of steam sterilizing 20min.After sterilization, do not add 1.5% agar in the fermented liquid of mycetome, behind 115 ℃ of steam sterilizing 20min,, fall dull and stereotyped adding wherein after 0.01% the suppressor factor L-amino-ethyl sulfonic acid filtration sterilization.To coat on this flat board as the proteus vulgaris of indicator, and place 37 ℃ of incubators to cultivate 48h.
If indicator grows containing on the flat board of suppressor factor, explain and contain the L-L-Ala in the fermented liquid, then the bacterial strain of reference numeral is the mutant strain that produces the L-L-Ala, and the bacterial strain of this numbering of picking is being temporary in 4 ℃ of refrigerators behind the cultivation 18h on the slant medium.
Embodiment 1-2: the mutagenesis of producing the lactobacillus delbruockii subspecies bulgaricus mutant strain of L-L-Ala.
1, nutrient media components: the composition of slant medium and fermention medium is with embodiment 1.
2, operation steps.
The bacterium 2.1 activation is set out: the method by embodiment 1 is processed mycoderm with starting strain.
2.2 low energy ion injection method mutagenic treatment cell: microscopy is acellular overlapping after, under vacuum condition, carry out ion implantation processing.Injecting ion is the N ion, ion energy 20keV, implantation dosage 6.0 * 10
15Ions/cm
2, injection length 5s, pitch time 30s.The mycoderm that will pass through after the ion implantation processing carries out wash-out with the 1mL SPSS, gets the 0.1mL elutriant then and coats on the slant medium, places 37 ℃ of incubators to cultivate 72h.
2.3 but the mutant strain of L-L-Ala is produced in the screening of retroactive inhibition assay method: single bacterium colony that will grow is numbered, and each single bacterium colony is chosen in the fermention medium of reference numeral 37 ℃ of anaerobically fermenting 48h respectively with transfering loop.The pH value descends in the fermenting process, and stream adds the ammoniacal liquor of concentration 25%, keeps fermented liquid pH value 6.5 to 7.5.Filtering fermentation liquor is removed thalline, and its filtrating is through 121 ℃ of steam sterilizing 20min.After sterilization, do not add 1.5% agar in the fermented liquid of mycetome, behind 115 ℃ of steam sterilizing 20min,, fall dull and stereotyped adding wherein after 0.03% the suppressor factor L-amino-ethyl sulfonic acid filtration sterilization.To coat on this flat board as the proteus vulgaris of indicator, and place 37 ℃ of incubators to cultivate 72h.
If indicator grows containing on the flat board of suppressor factor, explain and contain the L-L-Ala in the fermented liquid, then the bacterial strain of reference numeral is the mutant strain that produces the L-L-Ala, and the bacterial strain of this numbering of picking is being temporary in 4 ℃ of refrigerators behind the cultivation 18h on the slant medium.
Embodiment 1-3: the mutagenesis of producing the lactobacillus delbruockii subspecies bulgaricus mutant strain of L-L-Ala.
1, nutrient media components: the composition of slant medium and fermention medium is with embodiment 1.
2, operation steps.
The bacterium 2.1 activation is set out: the method by embodiment 1 is processed mycoderm with starting strain.
2.2 low energy ion injection method mutagenic treatment cell: microscopy is acellular overlapping after, under vacuum condition, carry out ion implantation processing, injecting ion is the N ion, ion energy 20keV, implantation dosage 5.0 * 10
16Ions/cm
2, injection length 5s, pitch time 40s.The mycoderm that will pass through after the ion implantation processing carries out wash-out with 0.5 mL SPSS, gets the 0.1mL elutriant then and coats on the slant medium, places 37 ℃ of incubators to cultivate 96h.
2.3 but the mutant strain of L-L-Ala is produced in the screening of retroactive inhibition assay method: single bacterium colony that will grow is numbered, and each single bacterium colony is chosen in the fermention medium of reference numeral 37 ℃ of anaerobically fermenting 48h respectively with transfering loop.The pH value descends in the fermenting process, and stream adds the ammoniacal liquor of concentration 25%, keeps fermented liquid pH value 6.5 to 7.5.Filtering fermentation liquor is removed thalline, and its filtrating is through 121 ℃ of steam sterilizing 20min.After sterilization, do not add 1.5% agar in the fermented liquid of mycetome, behind 115 ℃ of steam sterilizing 20min,, fall dull and stereotyped adding wherein after 0.05% the suppressor factor L-amino-ethyl sulfonic acid filtration sterilization.To coat on this flat board as the proteus vulgaris of indicator, and place 37 ℃ of incubators to cultivate 96h.
If indicator grows containing on the flat board of suppressor factor, explain and contain the L-L-Ala in the fermented liquid, then the bacterial strain of reference numeral is the mutant strain that produces the L-L-Ala, and the bacterial strain of this numbering of picking is being temporary in 4 ℃ of refrigerators behind the cultivation 18h on the slant medium.
Repeat embodiment 1-1 to 1-3, the mutant strain that obtains is carried out continuous passage cultivation, fermenting stability test, finally obtain 2 strains and produce higher, the genetic stability mutant strain preferably of L-L-Ala concentration, be numbered
Lds.0108 with
Lds.1109.
Embodiment 2-1: utilize mutant strain
Lds.0108 produce the L-L-Ala in the 100L fermentation cylinder for fermentation.
1, nutrient media components.
1.1 the composition of slant medium: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, agar 1.5%, sodium hydroxide is transferred pH to 7.0,121 ℃ of steam sterilizing 20min.
1.2 the composition of seed culture medium: peptone 0.8%, yeast extract paste 0.5%, glycerine 0.5%, potassium primary phosphate 0.2%, potassium hydrogenphosphate 1.2%, anhydrous magnesium sulfate 0.02%, sodium hydroxide is transferred pH to 7.0,121 ℃ of steam sterilizing 20min.
1.3 the composition of fermention medium: glucose 6%, potassium primary phosphate 0.2%, potassium hydrogenphosphate 1.2%, anhydrous magnesium sulfate 0.02%, sodium hydroxide is transferred pH to 7.0,115 ℃ of steam sterilizing 20min.
2, operation steps.
2.1 amplification cultivation seed liquor: with mutant strain
Lds.0108 culture transferring is cultivated 24h down at 37 ℃ to slant medium.Bacterial strain on the inclined-plane is processed bacteria suspension with sterilized water.Inoculum size by 0.5% inserts in the shake-flask seed substratum, 1000mL Erlenmeyer flask liquid amount 300mL, and 37 ℃ of culture temperature are cultivated 18h on the shaking table of rotating speed 120rpm.
2.2 anaerobically fermenting is produced the L-L-Ala: by in 0.5% the inoculum size access 100L fermentor tank, leavening temperature is trained 35 ℃, rotating speed 200rpm with cultured seed liquid; Tank pressure 0.05Mpa; The pH value descends in the fermenting process, and stream adds the ammoniacal liquor of concentration 25%, keeps fermented liquid pH value 6.5 to 7.5.Whole fermentation process does not need bubbling air, keeps anaerobic state.Fermentation 30h, fermentating liquid PH value no longer reduces, and the ammoniacal liquor spending rate is zero, surveys remaining sugar concentration less than 0.05%, stops fermentation, and L-L-Ala concentration is 5.63% in the performance liquid detection fermented liquid.
2.3 fermented liquid purifying treatment: above-mentioned fermented liquid is removed macromolecular substance such as somatic cells through aperture 20 nanometer tubular type organic membrane membrane ultrafiltration, with 20L pure water dialysis washing and filtering mother liquor, L-L-Ala composition is wherein fully collected in the ultrafiltration clear liquid.The ultrafiltration clear liquid filters through 600 molecular weight nf membrane, sloughs pigment, further removes larger molecular weight impurity, phosphoric acid salt, divalent-metal ion etc.The washing of fully dialysing of nanofiltration mother liquor, the about altogether 185L of nanofiltration liquid.
2.4 make finished product: utilize vacuum rotating thin film evaporation instrument to above-mentioned nanofiltration liquid distillation condensing crystal; Liquid concentrator is cooled to sufficient crystallising below 20 ℃, and suction filtration gets mother liquor 20L, dried crystals; Get L-L-Ala 3980.6g; Reach 99.2% through the check product content, specific rotatory power 14.7, quality meet GB 25543-2010.
Embodiment 2-2: utilize mutant strain
Lds.1109 produce the L-L-Ala 10 cubic metres of fermentation cylinder for fermentation.
1, nutrient media components.
1.1 the composition of slant medium: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, agar 1.5%, sodium hydroxide is transferred pH to 7.0,121 ℃ of steam sterilizing 20min.
1.2 the composition of seed culture medium: peptone 0.8%, yeast extract paste 0.5%, glycerine 0.5%, potassium primary phosphate 0.2%, potassium hydrogenphosphate 1.2%, anhydrous magnesium sulfate 0.02%, sodium hydroxide is transferred pH to 7.0,121 ℃ of steam sterilizing 20min.
1.3 the composition of fermention medium: glucose 9%, potassium primary phosphate 0.2%, potassium hydrogenphosphate 1.2%, anhydrous magnesium sulfate 0.02%, sodium hydroxide is transferred pH to 7.0,115 ℃ of steam sterilizing 20min.
2, operation steps.
2.1 amplification cultivation seed liquor: with mutant strain
Lds.1109 culture transferring is cultivated 24h down at 37 ℃ to slant medium respectively.Bacterial strain on the inclined-plane is processed bacteria suspension with sterilized water.Inoculum size by 0.5% inserts in the shake-flask seed substratum, and 1000mL Erlenmeyer flask liquid amount 300mL, cultivates 18h by 37 ℃ on the shaking table of rotating speed 120rpm.
2.2 anaerobically fermenting is produced the L-L-Ala: cultured shake-flask seed liquid by in 0.5% the inoculum size access 500L seeding tank, is cultivated 37 ℃, rotating speed 200rpm, tank pressure 0.05Mpa, anaerobism is cultivated 18h.Above-mentioned seeding tank seed liquor, culture transferring to useful volume are 10 cubic metres fermentor tank, 37 ℃ of leavening temperatures, and rotating speed 100rpm, tank pressure 0.05Mpa fermenting process pH value descends, and stream adds liquefied ammonia, keeps fermented liquid pH value 6.5 to 7.5.Whole fermentation process does not need bubbling air, keeps anaerobic state.Fermentation 40h, fermentating liquid PH value no longer reduces, and the liquefied ammonia spending rate is zero, surveys remaining sugar concentration less than 0.04%, stops fermentation, and L-L-Ala concentration is 7.52% in the performance liquid detection fermented liquid.
2.3 fermented liquid purifying treatment: above-mentioned fermented liquid is removed macromolecular substance such as somatic cells through aperture 50 nano ceramics membrane ultrafiltration, and pure water dialysis washing and filtering mother liquor is fully collected in the ultrafiltration clear liquid L-L-Ala composition wherein.The ultrafiltration clear liquid filters through 800 molecular weight nf membrane, sloughs pigment, further removes larger molecular weight impurity, phosphoric acid salt, divalent-metal ion etc.The washing of fully dialysing of nanofiltration mother liquor, about altogether 14.5 cubes of nanofiltration liquid.
2.4 make finished product: utilize the single-action vacuum-evaporator to above-mentioned nanofiltration liquid distillation condensing crystal, liquid concentrator is cooled to sufficient crystallising below 20 ℃, 3.2 cubic metres of suction filtration centrifuge mother liquors.Dried crystals gets L-L-Ala 506kg, reaches 99.5% through the check product content, and specific rotatory power 14.5, quality meet GB 25543-2010.
Embodiment 2-3: utilize mutant strain
Lds.0108 produce the L-L-Ala 100 cubic metres of fermentation cylinder for fermentation.
1, nutrient media components.
1.1 the composition of slant medium: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, agar 1.5%, sodium hydroxide is transferred pH to 7.0,121 ℃ of steam sterilizing 20min.
1.2 the composition of seed culture medium: peptone 0.8%, yeast extract paste 0.5%, glycerine 0.5%, potassium primary phosphate 0.2%, potassium hydrogenphosphate 1.2%, anhydrous magnesium sulfate 0.02%, sodium hydroxide is transferred pH to 7.0,121 ℃ of steam sterilizing 20min.
1.3 the composition of fermention medium: glucose 12%, potassium primary phosphate 0.2%, potassium hydrogenphosphate 1.2%, anhydrous magnesium sulfate 0.02%, sodium hydroxide is transferred pH to 7.0,115 ℃ of steam sterilizing 20min.
2, operation steps.
2.1 amplification cultivation seed liquor: with mutant strain
Lds.0108 culture transferring is cultivated 24h down at 37 ℃ to slant medium respectively.Bacterial strain on the inclined-plane is processed bacteria suspension with sterilized water.Inoculum size by 0.5% inserts in the shake-flask seed substratum, and 1000mL Erlenmeyer flask liquid amount 300mL, cultivates 18h by 37 ℃ on the shaking table of rotating speed 120rpm.
2.2 anaerobically fermenting is produced the L-L-Ala: cultured shake-flask seed liquid by in 0.5% the inoculum size access 500L seeding tank, is cultivated 37 ℃, rotating speed 200rpm, tank pressure 0.05Mpa, anaerobism is cultivated 18h.Cultured seed liquid culture transferring is continued amplification cultivation to planting to the 5000L seeding tank, cultivate 37 ℃, rotating speed 100rpm, tank pressure 0.05Mpa, anaerobism is cultivated 18h.The seed liquor of above-mentioned amplification cultivation, culture transferring to useful volume are 100 cubic metres fermentor tank, 40 ℃ of leavening temperatures, and rotating speed 56rpm, tank pressure 0.05Mpa, fermenting process pH value descends, and stream adds liquefied ammonia, keeps fermented liquid pH value 6.5 to 7.5.Whole fermentation process does not need bubbling air, keeps anaerobic state.Fermentation 52h, fermentating liquid PH value no longer reduces, and the liquefied ammonia spending rate is zero, surveys remaining sugar concentration less than 0.05%, stops fermentation, and L-L-Ala concentration is 10.68% in the performance liquid detection fermented liquid.
2.3 fermented liquid purifying treatment: above-mentioned fermented liquid is removed macromolecular substance such as somatic cells through aperture 50 nano ceramics membrane ultrafiltration, and pure water dialysis washing and filtering mother liquor is fully collected in the ultrafiltration clear liquid L-L-Ala composition wherein.The ultrafiltration clear liquid filters through 1000 molecular weight nf membrane, sloughs pigment, further removes larger molecular weight impurity, phosphoric acid salt, divalent-metal ion etc.The washing of fully dialysing of nanofiltration mother liquor, about altogether 124.5 cubes of nanofiltration liquid.
2.4 make finished product: utilize the triple effect vacuum-evaporator to above-mentioned nanofiltration liquid distillation condensing crystal, liquid concentrator is cooled to sufficient crystallising below 20 ℃, 12.2 cubic metres of suction filtration centrifuge mother liquors.Dried crystals gets L-L-Ala 7476kg, reaches 99.5% through the check product content, and specific rotatory power 14.5, quality meet GB 25543-2010.
Claims (5)
1. a mutagenesis lactic acid produces the method that bacterium produces the L-L-Ala, it is characterized in that, produces the bacterium lactobacillus delbruockii subspecies bulgaricus with lactic acid
Lactobacillus delbrueckii subsp. bulgaricusBe the bacterial classification that sets out; Mutant strain → amplification cultivation seed liquor → anaerobically fermenting production L-L-Ala → fermented liquid purifying treatment of the bacterium but activation is set out → low energy ion injection method mutagenic treatment cell → retroactive inhibition assay method screening production L-L-Ala → make finished product comprises following concrete steps:
A, the activation bacterium that sets out: will set out the bacterium lactobacillus delbruockii subspecies bulgaricus on slant medium 42 ℃ be cultured to logarithmic phase, process bacteria suspension with 0.85% saline water, be diluted to the 1mL bacteria suspension and contain the bacterium number and be about 10
7, behind the assorted bacterium of microscopy bacterial classification stalwartness and nothing, draw the 0.1mL bacteria suspension and evenly coat on the aseptic blank petridish the drying one-tenth mycoderm of aseptic wind;
B, low energy ion inject mutagenesis mutagenic treatment cell: microscopy is acellular overlapping after, under the vacuum condition somatic cells is carried out ion implantation processing, injecting ion is the N ion, dosage is 1.5 * 10
14-5 * 10
16Ions/cm
2, energy is 15-20keV, pulse is injected, and injects 5s at every turn, pitch time 15-40s; The mycoderm that will pass through after the ion implantation processing carries out wash-out with the 1mL SPSS, gets the 0.1mL elutriant then and coats on the solid plate, places 37 ℃ of incubators to cultivate;
But the mutant strain of L-L-Ala is produced in C retroactive inhibition assay method screening: single bacterium colony of selecting after ion implantation is numbered, and is inoculated in the fermention medium of reference numeral, and fermented liquid pH value is at 6.5-7.5,37 ℃ of anaerobically fermenting 48h; Filtering fermentation liquor is removed thalline, and filtrating adds 1.5% agar through 121 ℃ of steam sterilizing 20min, behind 115 ℃ of steam sterilizing 20min, adds the 0.01-0.05% suppressor factor, and is dull and stereotyped, and the coating indicator, places 35-40 ℃ of incubator to cultivate; The mutant strain of choosing the dull and stereotyped pairing numbering of indicator growth carries out continuous passage to be cultivated, and through stability test, finally obtains two strains and is numbered
Lds.0108 with
Lds.1109 L-L-Ala superior strain;
D, amplification cultivation seed liquor:
Lds.0108 with
Lds.1109 pass through slant culture and seed tank culture, 37 ℃ of culture temperature, slant culture time 24h, seed tank culture time 18h respectively;
E, anaerobically fermenting are produced the L-L-Ala: the seed liquor after will increasing is inoculated in the fermention medium, and the substrate glucose concn is 6-12%, and leavening temperature is 35-40 ℃; The medium pH value is 6.5-7.5; Keep anaerobic environment, treat that remaining sugar concentration is less than 0.1% fermentation completion, fermentation time 30-52h;
F, fermented liquid purifying treatment: adopt ceramic super-filtering film or organic tubular ultra-filtration membrane to filter earlier, further filter through nf membrane then;
G, make finished product: nanofiltration liquid is through the vacuum concentration evaporation, and the L-L-Ala is separated out in crystallization, through centrifugal, and dry finished product.
2. a kind of mutagenesis lactic acid according to claim 1 produces the method that bacterium produces the L-L-Ala, it is characterized in that, but described retroactive inhibition assay method is a suppressor factor with L-amino-ethyl sulfonic acid, with proteus vulgaris
Proteus vulgarisBe indicator.
3. a kind of mutagenesis lactic acid according to claim 1 produces the method that bacterium produces the L-L-Ala, it is characterized in that, stream adds ammoniacal liquor or liquefied ammonia in the described anaerobic fermentation process.
4. a kind of mutagenesis lactic acid according to claim 1 produces the method that bacterium produces the L-L-Ala, it is characterized in that, described ceramic super-filtering film or organic tubular ultra-filtration membrane adopt below the 20-50 nano aperture.
5. a kind of mutagenesis lactic acid according to claim 1 produces the method that bacterium produces the L-L-Ala, it is characterized in that, described nf membrane adopts below the 600-1000 molecular weight aperture.
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| CN107164419A (en) * | 2017-06-15 | 2017-09-15 | 安徽旭辰生物科技有限公司 | A kind of method of activated strains fermenting and producing L alanine |
| CN108642041A (en) * | 2018-06-20 | 2018-10-12 | 秦皇岛华恒生物工程有限公司 | A method of improving recombination bacillus coli fermenting production l-Alanine ability |
| CN110804602A (en) * | 2019-11-18 | 2020-02-18 | 江南大学 | L-aspartic acid β -decarboxylase mutant and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103214498A (en) * | 2013-04-20 | 2013-07-24 | 河北美邦工程科技有限公司 | Penicillin fermentation broth treating technology |
| CN103214498B (en) * | 2013-04-20 | 2015-03-11 | 河北美邦工程科技有限公司 | Penicillin fermentation broth treating technology |
| CN103602609A (en) * | 2013-09-05 | 2014-02-26 | 淮北新旗氨基酸有限公司 | High-yield strain for producing L-alanine by fermentation and preparation method thereof |
| CN107164419A (en) * | 2017-06-15 | 2017-09-15 | 安徽旭辰生物科技有限公司 | A kind of method of activated strains fermenting and producing L alanine |
| CN108642041A (en) * | 2018-06-20 | 2018-10-12 | 秦皇岛华恒生物工程有限公司 | A method of improving recombination bacillus coli fermenting production l-Alanine ability |
| CN108642041B (en) * | 2018-06-20 | 2022-04-19 | 秦皇岛华恒生物工程有限公司 | Method for improving L-alanine fermentation production capacity of recombinant escherichia coli |
| CN110804602A (en) * | 2019-11-18 | 2020-02-18 | 江南大学 | L-aspartic acid β -decarboxylase mutant and application thereof |
| CN110804602B (en) * | 2019-11-18 | 2021-01-29 | 江南大学 | L-aspartic acid beta-decarboxylase mutant and application thereof |
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