CN102161972A - Screening of high-yield glycine strains and application thereof in nitrile compound conversion - Google Patents
Screening of high-yield glycine strains and application thereof in nitrile compound conversion Download PDFInfo
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- CN102161972A CN102161972A CN2010105671502A CN201010567150A CN102161972A CN 102161972 A CN102161972 A CN 102161972A CN 2010105671502 A CN2010105671502 A CN 2010105671502A CN 201010567150 A CN201010567150 A CN 201010567150A CN 102161972 A CN102161972 A CN 102161972A
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- glycine
- fusarium oxysporum
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- 238000012216 screening Methods 0.000 title claims abstract description 25
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 20
- -1 nitrile compound Chemical class 0.000 title claims abstract description 12
- 241000223221 Fusarium oxysporum Species 0.000 claims abstract description 53
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- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to the biological technical field, and in particular relates to screening of high-yield glycine moulds and an application thereof in nitrile compound conversion. Glycine is widely applied to the fields such as food, chemical industry, medicine and the like; and the glycine is produced by utilizing a bioconversion method instead of a synthesis method, which becomes a development trend at home and abroad. The invention provides a screening method of high-yield glycine strains. By utilizing the method, a high-yield mould strain H3 for the glycine is screened for the first time, and the mould strain is identified as Fusarium oxysporum by combining morphological, physiological and biochemical characters and sequencing of genes, namely 18S, ITS and 28S rDNA; and the strain is conserved at China General Microbiological Culture Collection Center (conservation number: CGMCC 3829). The Fusarium oxysporum H3 screened by the method has the characteristics of fast growth speed, high conversion efficiency, short production cycle, mild reaction conditions, high yield, low production cost, environmental friendliness and the like, thus being applicable to industrial production of the glycine; and the after-treatment process of the product is convenient and fast without a large amount of acid or alkali.
Description
Technical field
The screening method of glycine superior strain and its are used, and the present invention relates to utilize the microbial transformation glycinonitrile to produce the method for glycine, belong to biological technical field.
Background technology
Glycine is called Padil again, and white tasteless crystal is nontoxic, water-soluble, is insoluble to organic solvent.Being that structure is the simplest a kind of in common 20 seed amino acids, is the neutral aliphatic amino acid, is the human body non-essential amino acid.Glycine conduct simultaneously is the agricultural chemicals of consumption maximum-glyphosate synthetic main raw material in the world, and its demand is very big; Also can be used as the intermediate of medication preparation such as seasonings, sweetener and hypertension in the food, liver injury in addition, use very extensive in fields such as food, medicine and chemical industry.
Chemical method is all adopted in the production of glycine at present, as the Mono Chloro Acetic Acid ammonolysis process, and Strecker method etc.There is long reaction time in these methods, needs High Temperature High Pressure, product yield is lower, product reclaim purifying need add a large amount of acid or alkali, technology complicated, to characteristics such as environment are unfriendly.Though as adopt Mono Chloro Acetic Acid ammonolysis process technology simple, not high to equipment requirements, products such as ammonium chloride are difficult to be separated, and causes the glycine productive rate lower, refining then improved production cost, the urotropine as catalysts can't reclaim serious waste of resources in addition; It is raw material that the patent US5258550 of Japan MITSUI TOATSU chemical company discloses with the hydroxyacetonitrile, in the presence of water with carbonic acid gas, ammonia at first in 80-120 ℃ of pre-reaction 0.5-1h, then 150-200 ℃ of glycine chemosynthesis novel method of carrying out main reaction, but this method needs High Temperature High Pressure, energy consumption is big, higher to equipment requirements, environmental pollution is more serious.
Along with the fast development of biotechnology, the employing biotransformation method prepares organic acid or amino acid just receives much concern.Therefore, the exploitation efficient, environmental protection technology of producing glycine by microbial transformation or microorganism catalysis seems very necessary, has complied with current urgent situation.Biotransformation method mainly is to utilize catalytic carriers such as microorganism free cell or purifying enzyme that the substrate glycinonitrile is converted into glycine.At present, prepare glycine about bio-transformation abroad and applied for more patent, but the domestic conversion method of lifeless matter is still produced the correlative study report of glycine.Abroad, the patent US5238827 of Japan NITTO chemical company discloses the employing rhodococcus, Arthrobacter, butter bacillus, it is the method for glycine that the bacterial isolates of Pseudomonas such as enterobacteria transforms glycinonitrile as katalaze enzyme, the patent WO0148234 of ASAHI chemical company discloses the employing acinetobacter calcoaceticus respectively, the rod bacillus, Alcaligenes, mycobacterium, the microbial hydrolytic glycinonitrile of bacterium such as rhodopseudomonas and candiyeast and Saccharomycodes prepares the method for glycine and the method that reclaims the purifying glycine, as, EP1243657, US20030040085, CN1433479, CN1840522.These all are that further research bio-transformation production glycine is laid a good foundation.Though, reported the more bacterial strain that is used to produce glycine abroad, yet do not seen mould that especially fusarium oxysporum Fusarium oxysporum produces the relevant report of glycine.
Summary of the invention
The present inventor's separation screening from soil sample obtains the fusarium oxysporum H3 that glycine is produced in a strain, and this strain bacterium has been carried out fermentation research.Bacterial strain provided by the invention is to obtain the soil sample separation screening from domestic certain chemical plant to obtain a strain mould, according to the physiology and appearance feature, and 18S, ITS and 28S rDNA gene sequencing, this strain classification called after fusarium oxysporum (Fusarium oxysporum), numbering H3, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 11st, 2010, deposit number is CGMCC No.3829.
The physiology and appearance and the Molecular Identification feature of bacterial strain fusarium oxysporum provided by the invention (Fusarium oxysporum H3, CGMCC No.3829) are as follows:
Bacterial strain adopts the inoculation of punch tool inoculation method on the potato dextrose agar flat board, aperture 0.5cm, and colony diameter is 4.9cm behind 30 ℃ of cultivation 4d, average growth diameter is 12.2mm/d; After cultivating 7d, colony edge is white in color, and the centre is red-purple, can produce the red-purple pigment; Growth back pinkiness on rice medium; The aerial hyphae well-grown is loose flocculence, aerial hyphae branch, transparent, diameter 1.6-4.4 μ m has pionnote on aerial hyphae, pionnote is colourless, microconidium is born in the aerial hyphae, false head and is given birth to, and microconidium is closely avette, do not have every, smooth, macroconidium sickleshaped, oval crooked.
Utilize 18S, ITS and the 28S rDNA gene order of fungi universal primer fusarium oxysporum (Fusarium oxysporum H3, CGMCC No.3829) to carry out pcr amplification, obtain the fragment of 1768bp, 558bp and 925bp size respectively.Carry out the similarity comparative analysis by the existing sequence of using in BLAST software and the database in the NCBI website, draw the 18S rDNA sequence and the fusarium Fusarium (AB110910 of this bacterial strain, GQ166777), (AB237662 AB250414) has homology highly for Cordyceps Cordyceps (AB067700) and Gibberella Gibberella; Show that according to 28S rDNA gene sequencing it and fusarium Fusarium (EF590327) have higher similarity.Therefore, draw this bacterial strain according to 18S and 28S rDNA gene sequencing and belong to fusarium Fusarium.ITS sequential analysis by this bacterial strain draws, and it and fusarium oxysporum Fusarium oxysporum (DQ002550) have 100% homology.Combining form feature and physio-biochemical characteristics are accredited as fusarium oxysporum (Fusarium oxysporum H3, CGMCC No.3829) with it.
The present invention also provides a kind of glycine to produce the screening method of bacterium, and its feature is as follows:
Gather soil sample,, on flat board, choose bacterial strain with variable color circle according to the color reaction of glycine and Bromothymol blue; Primary dcreening operation, the bacterial strain that purifying is good is forwarded in the nutritious liquid nutrient medium, measures glycine concentration in the supernatant liquor by Paper Chromatography; Multiple sieve at first carries out seed culture, and the inoculum size with 1% inserts liquid nutrient medium, and cultivation finishes the back and adopts high performance liquid chromatography to detect the concentration of glycine in the supernatant liquor of conversion back.
Described soil sample derives from the chemical plant factory building surrounding soil of producing nitrile compounds, gathers the soil sample at 5-15cm place.
Described plate screening method is as follows:
Get the 1g soil sample, put into the Erlenmeyer flask (20mL/250mL) that contains physiological saline, concuss 10min leaves standstill; Get 1mL soil suspension, inject on the aseptic flat board, pour into 25mL in the flat board through sterilization and be cooled to 40-50 ℃ solid screening culture medium; After treating the complete cooled and solidified of substratum, place the constant temperature culture carton upside down to cultivate flat board, cultivate 3d for 30 ℃; Picking produce the blue variable colour circle single bacterium colony, be forwarded to fresh substratum line and separate, switching 3-5 generation.
Consisting of of described solid screening culture medium: glucose 5.0g/L, potassium primary phosphate 1.0g/L, sal epsom 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium-chlor 1.0g/L, glycinonitrile 1.0g/L, Bromothymol blue 0.2g/L, agar 20.0g/L, pH7.0.
Described prescreening method is as follows:
Substratum consists of: glucose 5.0g/L, potassium primary phosphate 1.0g/L, sal epsom 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium-chlor 1.0g/L, glycinonitrile 1.0g/L, pH7.0; Cultural method is: 250mL triangular flask liquid amount 25mL, and shaking speed 120rpm cultivates 4d for 30 ℃; The fermentation liquor treatment method is: with the centrifugal 10min of fermented liquid 12000rpm, the taking-up thalline obtains fermented supernatant fluid and is used for the glycine Determination on content.
The method that adopts ply of paper to analyse in the described primary dcreening operation process is measured glycine concentration in the supernatant liquor, and the ply of paper analysis method is as follows:
Fermented liquid point sample after transforming on chromatography filter paper (point sample amount 0.2-1.0 μ L), is positioned in the chromatography cylinder ascending development.When treating that chromatographic solution goes upward to apart from the 1.0cm left and right sides, filter paper top, after taking-up places 115 ℃ of oven dry, evenly spray developer, be put in the 5-10min that develops the color in 115 ℃ of baking ovens once more, after spot shows, cut glycine colour developing spot in the glass test tube of band plug, use the ethanolic soln wash-out 10-30min of the copper sulfate of 2-5mL, on ultraviolet spectrophotometer, measure its light absorption value then.
Described multiple screen method is as follows:
Substratum is formed: seed culture medium, and extractum carnis 10.0g/L, peptone 10.0g/L, yeast extract paste 10.0g/L, glucose 10.0g/L, sodium-chlor 5.0g/L, pH 7.0;
Screening culture medium, glucose 5.0g/L, potassium primary phosphate 1.0g/L, sal epsom 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium-chlor 1.0g/L, glycinonitrile 1.0g/L, pH7.0;
Cultural method: seed culture method, 250mL triangular flask liquid amount 25mL, shaking speed 120rpm cultivates 3d for 30 ℃; Liquid fermentation and culture, the inoculum size with 1% inserts fermention medium with seed,
Fermentation culture method, 250mL triangular flask liquid amount 25mL, shaking speed 120rpm cultivates 4d for 30 ℃; After cultivating end, get the centrifugal 10min collection of this nutrient solution 12000rpm thalline and be used for the bio-transformation glycinonitrile, the glycine concentration that generates in the supernatant liquor after the employing high performance liquid chromatography detects and transforms.The conversion fluid treatment process is: with the centrifugal 15min of conversion fluid 14000rpm, the taking-up thalline obtains fermented supernatant fluid and is used for the glycine Determination on content.
Glycine adopts high performance liquid chromatography to detect in the described process, chromatographic condition is: Agilent 1100 type high performance liquid chromatographs, adopt the pre-column derivatization mode, derivative reagent is o-phthalaldehyde(OPA) (OPA) and chloroformic acid fluorenes methyl esters (FMOC-Cl), chromatographic column is Hypersil AA-ODS C18 post (250 * 4.6mm, 5 μ m), 40 ℃ of column temperatures, flow velocity is 1.0mL/min, ultraviolet detection wavelength 338nm, mobile phase A is sodium acetate-acetic acid-triethylamine solution mutually, and B is sodium acetate-acetic acid-acetonitrile-methanol solution mutually, and type of elution adopts gradient elution.
The invention provides a kind of glycine and produce the application method of bacterial strain, its feature is as follows:
Fusarium oxysporum (Fusarium oxysporum H3) is inserted nutritious substratum, obtain the fermented liquid of high reactivity high-biomass; To ferment
Liquid is handled, and obtains thalline,, utilizes thalline that glycinonitrile is carried out bio-transformation under certain condition.
Described nutritious substratum consists of: glycerine 10-20g/L, yeast extract paste 1.0-10g/L, peptone 1.0-10g/L, sal epsom 0.1-0.5g/L, potassium primary phosphate 1.0-5.0g/L, sodium-chlor 0.5-1.0g/L, ferrous sulfate 0.01-0.1g/L, hexanolactam 0.5-1.0g/L, pH 5.0-8.0.
The treatment process of described fermented liquid is: the centrifugal 5-20min of 6000-15000rpm, to collect the thalline that obtains through cultivation, with the phosphoric acid buffer (pH 5.0-9.0) of 0.01-0.5M, wash thalline 2-4 time, and thalline be suspended in this damping fluid again.
The preparation method of described glycine substrate is: the glycinonitrile solution of preparation 5-100g/L, regulate pH to 5.0-9.0 with sodium hydroxide or the hydrochloric acid soln of 0.5-5.0M.
Described bio-transformation condition is: with bacteria suspension and substrate solution mixing, control reaction pH remains on 5.0-9.0, and temperature of reaction remains on 20-60 ℃, rotating speed 50-250rpm, reaction times 6-48h.
Advantage of the present invention and beneficial effect:
To propose first with the glycinonitrile in glycine is produced the screening process of bacterial strain be only nitrogen source, with the glycinonitrile-Bromothymol blue solid screening culture medium of Bromothymol blue as indicator, set up that rapid screening can transform that glycinonitrile is produced the strain culturing system of glycine and to the detection method of purpose bacterial strain; Obtain bacterial strain---fusarium oxysporum (the Fusarium oxysporum H3 that glycine is produced in a strain through primary dcreening operation, multiple sieve, CGMCCNo.3829), it is short that this bacterial strain has growth cycle, the transformation efficiency height, characteristics such as the transformation period is short, greatly reduce the production cost of glycine, be particularly suitable for the scale operation of glycine; This technology also has the reaction conditions gentleness, productive rate height, characteristics such as production cost is low, and is environmentally friendly, and last handling process need not to add a large amount of acid or alkali, product separation and use convenient and swift.
Description of drawings:
Fig. 1 is biomass and an enzyme change procedure alive in the bacterial strain Fusarium oxysporum H3 fermenting process provided by the invention.
Embodiment
The isolation identification of embodiment 1 fusarium oxysporum (Fusarium oxysporum H3) and the preservation of bacterial strain
(1) the enrichment screening process of fusarium oxysporum (Fusarium oxysporum H3).
Gather nitrile compounds factory soil sample on every side, remove veneer of soil with scuppit earlier during sampling, the soil sample at collection 5-15cm place.Get the 1g soil sample, put into the Erlenmeyer flask (20mL/250mL) that contains physiological saline, concuss 10min leaves standstill.Get 1mL soil suspension, inject on the aseptic flat board, pour into 25mL in the flat board through sterilization and be cooled to 40-50 ℃ solid screening culture medium.After treating the complete cooled and solidified of substratum, place the constant temperature culture carton upside down to cultivate flat board, cultivate 3d for 30 ℃.Consisting of of solid screening culture medium: glucose 5.0g/L, potassium primary phosphate 1.0g/L, sal epsom 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium-chlor 1.0g/L, glycinonitrile 1.0g/L, Bromothymol blue 0.2g/L, agar 20.0g/L, pH 7.0, the single bacterium colony that produces the blue variable colour circle are that glycine produces bacterium.Picking produce the blue variable colour circle single bacterium colony, be forwarded to fresh substratum line and separate, switching 3-5 generation.
The good bacterial strain of picking 25 strain purifying is forwarded in the nutritious liquid nutrient medium to be cultivated.Consisting of of liquid screening substratum: glucose 5.0g/L, potassium primary phosphate 1.0g/L, sal epsom 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium-chlor 1.0g/L, glycinonitrile 1.0g/L, pH 7.0, liquid amount 25mL/250mL, shaking speed 120rpm cultivates 4d for 30 ℃.Cultivate and finish the centrifugal removal thalline in back, adopt the glycine concentration in the Paper Chromatography mensuration supernatant liquor.
Carry out multiple sieve again, at first carry out seed culture, inoculum size with 1% inserts liquid nutrient medium, 3 bottles of every strains, after cultivating end, get the centrifugal 10min collection of this nutrient solution 12000rpm thalline and be used for the bio-transformation glycinonitrile, adopt high performance liquid chromatography to detect and transform the glycine concentration that generates in the supernatant liquor of back.Obtain the highest strain bacterial strain of output, called after fusarium oxysporum (Fusarium oxysporum H3, CGMCC No.3829).
Seed culture medium: extractum carnis 10.0g/L, peptone 10.0g/L, yeast extract paste 10.0g/L, glucose 10.0g/L, sodium-chlor 5.0g/L, pH 7.0.Liquid amount 25mL/250mL, culture condition are 30 ℃, and 120rpm cultivates 3d.
(2) form of fusarium oxysporum (Fusarium oxysporum H3)
Fusarium oxysporum (Fusarium oxysporum H3) bacterial strain adopts the inoculation of punch tool inoculation method on the potato dextrose agar flat board, aperture 0.5cm, and colony diameter is 4.9cm behind 30 ℃ of cultivation 4d, average growth diameter is 12.2mm/d; After cultivating 7d, colony edge is white in color, and the centre is red-purple, can produce the red-purple pigment; Growth back pinkiness on rice medium; The aerial hyphae well-grown is loose flocculence, aerial hyphae branch, transparent, diameter 1.6-4.4 μ m has pionnote on aerial hyphae, pionnote is colourless, microconidium is born in the aerial hyphae, false head and is given birth to, and microconidium is closely avette, do not have every, smooth, macroconidium sickleshaped, oval crooked.
(3) Molecular Identification of fusarium oxysporum (Fusarium oxysporum H3)
Be collected in the mycelium of growing on the potato glucose solid medium flat board, extract total DNA with fungal gene group DNA extraction test kit.Amplimer is respectively the universal primer of 18S, the universal primer of ITS sequence and the universal primer of 28S rDNA sequence amplification.
50 μ L reaction systems are adopted in the PCR reaction,
10×buffer(Mg
2+) 5.0μL
dNTPs(2.5mM) 4.0μL
Forward primer (20 μ M) 2.0 μ L
Reverse primer (20 μ M) 2.0 μ L
ExTaq archaeal dna polymerase (5U/ μ L) 1.0 μ L
Template DNA 2.0 μ L
ddH
2O 34μL
Pcr amplification condition: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 60s, 55 ℃ of annealing 60s, 72 ℃ are extended 90s, totally 30 circulations, last 72 ℃ are extended 10min eventually.The purifying of pcr amplification product is undertaken by a small amount of glue recovery PCR product purification test kit explanation that worker biotech company is given birth in Shanghai, and order-checking is finished by the big gene of Shanghai China.
18S, the ITS and the 28S rDNA gene order that obtain after checking order are carried out the BLAST comparison and are determined its kind in Genbank.
The method of embodiment 2 fusarium oxysporums (Fusarium oxysporum H3) fermentative production glycine
The explanation of this example inserts nutritious substratum to fusarium oxysporum (Fusarium oxysporum H3) have been carried out cultivating the thalline that obtains the high reactivity high-biomass, and the bio-transformation glycinonitrile generates the method for glycine.
The composition of substratum: glycerine 10.0g/L, yeast extract paste 1.0g/L, peptone 1.0g/L, sal epsom 0.1g/L, potassium primary phosphate 1.0g/L, sodium-chlor 0.5g/L, ferrous sulfate 0.01g/L, hexanolactam 0.5g/L, pH 7.5;
Fusarium oxysporum (Fusarium oxysporum H3) is inserted in the substratum with 1% inoculum size, 30 ℃, 120rpm supports well and cultivates 3d, promptly cultivate and finish, the centrifugal 10min of 12000rpm collects cultured thalline, with the phosphoric acid buffer of 0.5M washing thalline 2 times, and thalline is suspended in this damping fluid again makes bacteria suspension.
The glycinonitrile solution of preparation 7.8g/L is regulated pH to 5.0 with sodium hydroxide or the hydrochloric acid soln of 2.0M, with this substrate as catalyzed reaction.With this substrate solution and bacteria suspension mixing, making final biomass is 1%, and control mixed system pH is 7.2,30 ℃ of temperature, reacts 48h under the condition of rotating speed 120rpm, and glycine concentration is 6.6g/L in the reaction solution, and productive rate has reached more than 60%.
Embodiment 3 changes fermentation condition, fusarium oxysporum (Fusarium oxysporum H3) fermentative production glycine
Present embodiment adopts the method identical with embodiment 2, change reaction times.
By the centrifugal thalline of from nutrient solution, collecting, and, suspend again and make bacteria suspension with the phosphoric acid buffer of 0.5M washing 2 times, glycinonitrile solution mixing with 7.8g/L at 30 ℃, reacts 12h under the condition of 120rpm, glycine concentration is 8.5g/L in the reaction solution, and productive rate has reached 80%.
Embodiment 4 changes fermentation condition, fusarium oxysporum (Fusarium oxysporum H3) fermentative production glycine
The method of present embodiment is identical with embodiment 2, changes the method therefor parameter.
Substratum is formed: glycerine 10.0g/L, and yeast extract paste 5.0g/L, peptone 10.0g/L, sal epsom 0.1g/L, potassium primary phosphate 4.0g/L, sodium-chlor 1.0g/L, ferrous sulfate 0.02g/L, hexanolactam 1.0g/L, pH 7.5;
Fusarium oxysporum (Fusarium oxysporum H3) is inserted in the substratum with 2% inoculum size, 30 ℃, 120rpm supports well and cultivates 3d, promptly cultivate and finish, the centrifugal 10min of 12000rpm collects cultured thalline, with the phosphoric acid buffer of 0.5M washing thalline 2 times, and thalline is suspended in this damping fluid again makes bacteria suspension.
The glycinonitrile solution of preparation 23.4g/L is as the catalyzed reaction substrate.With this substrate solution and bacteria suspension mixing, and control pH is 7.2,30 ℃ of temperature, reacts 12h under the condition of rotating speed 120rpm, and the glycinonitrile in the reaction mixture almost all transforms, and the output of glycine has reached 24.6g/L.
Embodiment 5 changes fermentation condition, fusarium oxysporum (Fusarium oxysporum H3) fermentative production glycine
The method of present embodiment is identical with embodiment 2, changes the method therefor parameter.
The glycinonitrile solution of preparation 46.8g/L is as the catalyzed reaction substrate.With this substrate solution and bacteria suspension mixing, inoculum size is 1%, and control pH is 7.2,30 ℃ of temperature, reacts 12h under the condition of rotating speed 120rpm, and the substrate glycinonitrile almost all transforms, and the output of glycine has reached 39.8g/L.
Claims (9)
1. the screening of high yield glycine bacterial strain and the application in nitrile compounds transforms thereof, it is characterized in that: gather the chemical plant factory building pedotheque on every side of producing nitrile compounds, at glycinonitrile is that only nitrogen source and the solid primary dcreening operation substratum that adds Bromothymol blue screen, and obtains a plant height and produces glycine bacterial strain fusarium oxysporum H3 (Fusarium oxysporum).This bacterial strain is cultivated, and be that substrate carries out glycine and transforms with the glycinonitrile.This bacterial strain also can be converted into other nitrile compounds corresponding acid.
2. the screening of high yield glycine bacterial strain according to claim 1 is characterized in that the screening soil sample of using is the pedotheque around the chemical plant factory building of nitrile compounds.
3. the screening of high yield glycine bacterial strain according to claim 1, it is characterized in that the solid primary dcreening operation substratum that uses be with glycinonitrile as only nitrogen source and added Bromothymol blue, its glycinonitrile addition is 0.1g/L～2.0g/L, Bromothymol blue 0.05～0.5g/L.
4. the screening of high yield glycine bacterial strain according to claim 1, it is characterized in that the solid primary dcreening operation substratum that uses be with glycinonitrile as only nitrogen source and added Bromothymol blue, also comprised an amount of glucose, potassium primary phosphate, sal epsom, ferrous sulfate, calcium chloride, sodium-chlor, agar.
5. the screening of high yield glycine bacterial strain according to claim 1, it is characterized in that the described soil sample of claim 2 is made soil supension, get an amount of suspension and be poured into the described solid primary dcreening operation of claim 3 substratum, mixing is also cultivated 3-5d, picking produce the blue variable colour circle single bacterium colony, be forwarded to fresh substratum line and separate, transfer 3-5 generation, obtain the pure culture bacterial strain.
6. a plant height according to claim 1 produces the glycine bacterial strain, it is characterized in that this bacterial strain is fusarium oxysporum H3 (Fusarium oxysporum), now be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.3829. but the described high yield glycine of claim 1 bacterial strain is not limited to fusarium oxysporum H3 (Fusarium oxysporum).
7. the cultivation of fusarium oxysporum according to claim 1 (Fusarium oxysporum H3) is characterized in that:
(1) substratum: glycerine 10-20g/L, yeast extract paste 1.0-10g/L, peptone 1.0-10g/L, sal epsom 0.1-0.5g/L, potassium primary phosphate 1.0-5.0g/L, sodium-chlor 0.5-1.0g/L, ferrous sulfate 0.01-0.1g/L, hexanolactam 0.5-1.0g/L, pH 5.0-8.0;
(2) culture temperature 25-45 ℃, incubation time 2-6d.
8. according to claim 1 is that substrate carries out the method that glycine transforms with the glycinonitrile, it is characterized in that:
(1) substrate solution: the glycinonitrile solution of preparation 5-100g/L, regulate pH to 5.0-9.0 with sodium hydroxide or the hydrochloric acid soln of 0.5-5.0M;
(2) microorganism collection: substratum according to claim 7 and cultural method carry out yeast culture, with the centrifugal 5-20min of 6000-15000rpm, to collect the thalline that obtains through cultivation, use 0.01-0.5M, the phosphoric acid buffer washing thalline of pH 6.0-8.0 2-4 time.
(3) transform: thalline is suspended in the above-mentioned damping fluid again, and with the substrate solution mixing, reaction pH remains on 6.0-8.0, temperature of reaction remains on 20-60 ℃, mixing speed is 50-250rpm, reaction times 6-48h.
9. fusarium oxysporum according to claim 1 (Fusarium oxysporum H3) also can be converted into corresponding carboxylic acid with other nitrile compounds.
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| Title |
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| 《Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing》 20091210 Grace DeSantis et al Applications of Nitrile Hydratases and Nitrilases 153-181 6-9 , * |
| GRACE DESANTIS ET AL: "《Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing》", 10 December 2009 * |
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