CN102161972B - Screening of High-Glycine-Producing Strain and Its Application in Transformation of Nitrile Compounds - Google Patents

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

Screening of High-Glycine-Producing Strain and Its Application in Transformation of Nitrile Compounds

technical field

A screening method for a high-yield glycine strain and its application. The invention relates to a method for producing glycine by using microorganisms to transform glycyl nitrile, which belongs to the field of biotechnology. the

Background technique

Glycine, also known as aminoacetic acid, is a white odorless crystal, non-toxic, soluble in water, insoluble in organic solvents. It is the simplest structure among the 20 common amino acids, and it is a neutral aliphatic amino acid, which is a non-essential amino acid for the human body. Glycine is also used as the main raw material for the synthesis of glyphosate, the most widely used pesticide in the world, and its demand is very large; in addition, it can also be used as an intermediate in the preparation of food flavoring agents, flavor enhancers, high blood pressure, and liver damage. It is widely used in the fields of food, medicine and chemical industry. the

At present, the production of glycine all adopts chemical methods, such as chloroacetic acid ammonolysis method, Strecker method and so on. These methods have the characteristics of long reaction time, need for high temperature and high pressure, low product yield, need to add a large amount of acid or alkali for product recovery and purification, complicated process, and unfriendly to the environment. For example, although the ammonolysis method with chloroacetic acid is simple in process and does not require high equipment, it is difficult to separate products such as ammonium chloride, resulting in a low yield of glycine, and refining it increases the production cost. In addition, hexamethamine as a reaction catalyst cannot be recovered. , a serious waste of resources; the patent US5258550 of MITSUI TOATSU Chemical Company of Japan discloses that hydroxyacetonitrile is used as a raw material, and in the presence of water, carbon dioxide and ammonia are first pre-reacted at 80-120°C for 0.5-1h, and then the main reaction is carried out at 150-200°C. A new method for the chemical synthesis of glycine by reaction, but this method requires high temperature and high pressure, high energy consumption, high requirements for equipment, and serious environmental pollution. the

With the rapid development of biotechnology, the preparation of organic acids or amino acids by biotransformation is attracting attention. Therefore, it is very necessary to develop an efficient, green and environmentally friendly process for the production of glycine through microbial transformation or microbial catalysis, which complies with the current urgent situation. The biotransformation method mainly uses catalytic carriers such as microbial free cells or purified enzymes to convert the substrate glycyl nitrile into glycine. At present, many foreign patents have been applied for biotransformation of glycine, but there is no relevant research report on the production of glycine by biotransformation in China. Abroad, the patent US5238827 of Japanese NITTO chemical company discloses the method for the conversion of glycine into glycine by adopting bacterial strains such as Rhodococcus, Arthrobacter, Lactobacillus, and Enterobacter as catalytic enzymes, and the patent WO0148234 of ASAHI Chemical Company discloses respectively A method for preparing glycine by hydrolyzing glycyl nitrile with microorganisms such as Acinetobacter, Corynebacterium, Alcaligenes, Mycobacterium, Rhodopseudomonas and Candida and Saccharomyces, and a method for recovering and purifying glycine, such as, EP1243657, US20030040085, CN1433479, CN1840522. These have laid the foundation for further research on biotransformation to produce glycine. Although many strains used to produce glycine have been reported abroad, there is no report on the production of glycine by molds, especially Fusarium oxysporum. the

Contents of the invention

The inventors of the present invention isolated and screened a strain of Fusarium oxysporum H3 producing glycine from soil samples, and carried out fermentation research on this strain. The bacterial strain provided by the present invention is obtained from a domestic chemical plant to obtain a strain of mold by isolation and screening of soil samples. According to the physiological morphological characteristics, and 18S, ITS and 28S rDNA gene sequence determination, the bacterial strain is classified and named as Fusarium oxysporum (Fusarium oxysporum) , No. H3, was deposited in the General Microbiology Center of China Microbiological Culture Collection Management Committee on May 11, 2010, and the preservation number is CGMCC No.3829. the

The physiological form and molecular identification characteristics of bacterial strain Fusarium oxysporum (Fusarium oxysporum H3, CGMCC No.3829) provided by the invention are as follows:

The strain was inoculated on the potato dextrose agar medium plate with a hole punch inoculation method, with a hole diameter of 0.5 cm. After 4 days of cultivation at 30°C, the colony diameter was 4.9 cm, and the average growth diameter was 12.2 mm/d; after 7 days of cultivation, the colony edge was white , purple in the middle, can produce purple-red pigment; pink after growing on rice medium; aerial hyphae grow well, loose cotton-like, aerial hyphae branches, transparent, diameter 1.6-4.4μm , there are sticky spores on the aerial hyphae, the sticky spores are colorless, small conidia are born in the aerial hyphae, and they grow in a false head shape. The small conidia are nearly ovate, without septa, smooth, large Conidia are sickle-shaped, elliptic and curved. the

The 18S, ITS and 28S rDNA gene sequences of Fusarium oxysporum H3 (CGMCC No.3829), which are general primers for fungi, were used for PCR amplification to obtain fragments of 1768bp, 558bp and 925bp, respectively. By using the BLAST software on the NCBI website to compare the similarity with the existing sequences in the database, the 18S rDNA sequence of the strain is compared with that of the genus Fusarium (AB110910, GQ166777), the genus Cordyceps (AB067700) and the genus Gibberella (AB237662, AB250414) have a high degree of homology; According to the 28S rDNA gene sequence analysis, it has a high similarity with Fusarium (EF590327). Therefore, according to the analysis of the 18S and 28S rDNA gene sequences, the strain belonged to the genus Fusarium. According to the ITS sequence analysis of the strain, it has 100% homology with Fusarium oxysporum (DQ002550). Combined with morphological characteristics and physiological and biochemical characteristics, it was identified as Fusarium oxysporum H3 (CGMCC No.3829). the

The present invention also provides a screening method for glycine-producing bacteria, which is characterized in that:

Collect soil samples, and pick out the strains with a color change circle on the plate according to the color reaction between glycine and bromothymol blue; for primary screening, transfer the purified strains to nutrient-rich liquid medium, and pass paper chromatography The concentration of glycine in the supernatant was determined by the method; for re-screening, the seeds were cultured at first, and the inoculum was inserted into the liquid medium with 1% inoculum. After the cultivation, the concentration of glycine in the supernatant after the transformation was detected by high performance liquid chromatography. the

The soil samples are derived from the soil around the chemical factory building for the production of nitrile compounds, and the soil samples at 5-15 cm are collected. the

The plate screening method is as follows:

Take 1g of soil sample, put it into an Erlenmeyer flask (20mL/250mL) containing physiological saline, shake it vigorously for 10min, and let it stand still; take 1mL of soil suspension, inject it on a sterile plate, pour 25mL into the plate after sterilization and Solid screening medium cooled to 40-50°C; after the medium is completely cooled and solidified, place the plate upside down in a constant temperature incubator and culture at 30°C for 3 days; pick a single colony that produces a blue color change circle, and transfer to The fresh medium was streaked and separated, and transferred for 3-5 generations. the

The composition of the solid screening medium is: glucose 5.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, chloride Sodium 1.0g/L, glycyl nitrile 1.0g/L, bromothymol blue 0.2g/L, agar 20.0g/L, pH7.0. the

The primary screening method is as follows:

The composition of the medium is: glucose 5.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium chloride 1.0g/L , Glycyl nitrile 1.0g/L, pH7.0; culture method: 250mL Erlenmeyer bottle with 25mL volume, shaker speed 120rpm, culture at 30°C for 4 days; fermentation broth treatment method: centrifuge the fermentation broth at 12000rpm for 10min, take out the bacteria The fermentation supernatant was obtained for the determination of glycine content. the

Adopt paper chromatography method to measure glycine concentration in the supernatant in the described primary screening process, paper chromatography method is as follows:

Spot the converted fermentation broth on chromatographic filter paper (sample volume: 0.2-1.0 μL), place it in a chromatographic cylinder, and spread upward. When the chromatographic liquid goes up to about 1.0cm from the top of the filter paper, take it out and dry it at 115°C, spray the color developer evenly, put it in the oven at 115°C for 5-10min to develop the color, after the spots appear, cut off the glycine The color spots were placed in a stoppered glass test tube, eluted with 2-5mL of copper sulfate ethanol solution for 10-30min, and then the absorbance was measured on a UV spectrophotometer. the

The re-screening method is as follows:

Medium composition: seed medium, beef extract 10.0g/L, peptone 10.0g/L, yeast extract 10.0g/L, glucose 10.0g/L, sodium chloride 5.0g/L, pH 7.0;

Screening medium, glucose 5.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium chloride 1.0g/L, Glycyl nitrile 1.0g/L, pH7.0;

Cultivation method: seed cultivation method, 250mL Erlenmeyer bottle liquid volume 25mL, shaking table speed 120rpm, 30°C cultivation for 3 days; liquid fermentation cultivation, insert the seeds into the fermentation medium with 1% inoculation amount,

Fermentation culture method, 250mL Erlenmeyer bottle liquid volume 25mL, shaker speed 120rpm, 30°C culture for 4 days; after the culture, take the culture solution and centrifuge at 12000rpm for 10min to collect bacteria for bioconversion of glycyl nitrile, and use high performance liquid chromatography to detect Concentration of glycine produced in the supernatant after transformation. The transformation liquid treatment method is as follows: the transformation liquid is centrifuged at 14000 rpm for 15 minutes, and the bacterial cells are taken out to obtain the fermentation supernatant for the determination of glycine content. the

Glycine adopts high performance liquid chromatography to detect in the described process, and chromatographic condition is: Agilent 1100 type high performance liquid chromatography, adopts pre-column derivatization mode, derivatization reagent is o-phthalaldehyde (OPA) and fluorenylmethyl chloroformate ( FMOC-Cl), the chromatographic column is Hypersil AA-ODS C18 column (250×4.6mm, 5μm), the column temperature is 40°C, the flow rate is 1.0mL/min, the ultraviolet detection wavelength is 338nm, and the mobile phase A is sodium acetate-acetic acid- Triethylamine solution, phase B is sodium acetate-acetic acid-acetonitrile-methanol solution, and the elution method adopts gradient elution. the

The present invention provides a kind of application method of glycine production bacterial strain, its feature is as follows:

Insert Fusarium oxysporum H3 into a nutrient-rich medium to obtain a fermented liquid with high activity and high biomass;

The liquid is processed to obtain bacteria, and under certain conditions, the bacteria are used to biotransform glycyl nitrile. the

The nutrient-rich medium consists of: glycerol 10-20g/L, yeast extract 1.0-10g/L, peptone 1.0-10g/L, magnesium sulfate 0.1-0.5g/L, potassium dihydrogen phosphate 1.0-5.0g/L L, sodium chloride 0.5-1.0g/L, ferrous sulfate 0.01-0.1g/L, caprolactam 0.5-1.0g/L, pH 5.0-8.0. the

The treatment method of described fermented liquid is: 6000-15000rpm centrifugal 5-20min, to collect the thallus obtained through culture, with the phosphate buffer (pH 5.0-9.0) of 0.01-0.5M, wash thalline 2-4 times, The cells were resuspended in the buffer. the

The preparation method of the glycine substrate is as follows: preparing 5-100 g/L glycine nitrile solution, and adjusting the pH to 5.0-9.0 with 0.5-5.0 M sodium hydroxide or hydrochloric acid solution. the

The biotransformation conditions are as follows: uniformly mix the bacterial suspension and the substrate solution, control the reaction pH at 5.0-9.0, keep the reaction temperature at 20-60° C., rotate at 50-250 rpm, and react for 6-48 hours. the

Advantages and beneficial effects of the present invention:

In the screening process of glycine production strains, the glycylnitrile-bromothymol blue solid screening medium was proposed for the first time, using glycylnitrile as the only nitrogen source and bromothymol blue as the indicator, and established a rapid screening method for the conversion of glycine The strain culture system for producing glycine and the detection method for the target strain; a glycine-producing strain—Fusarium oxysporum H3 (CGMCCNo.3829) was obtained after primary screening and rescreening. This strain has a short growth cycle, The characteristics of high conversion efficiency and short conversion cycle greatly reduce the production cost of glycine, and are especially suitable for large-scale production of glycine; this process also has the characteristics of mild reaction conditions, high yield, low production cost, and environmental friendliness. There is no need to add a large amount of acid or alkali during the treatment process, and the product separation and application are convenient and quick. the

Description of drawings:

Fig. 1 is the change process of biomass and enzyme activity in bacterial strain Fusarium oxysporum H3 fermentation process provided by the present invention. the

Detailed ways

Example 1 Isolation and Identification of Fusarium oxysporum H3 and Preservation of the Strain

(1) Enrichment screening process of Fusarium oxysporum H3. the

Collect soil samples around the nitrile compound production plant. When sampling, first remove the top soil with a small shovel, and collect soil samples at 5-15cm. Take 1g of soil sample, put it into an Erlenmeyer flask (20mL/250mL) containing physiological saline, shake it vigorously for 10min, and let it stand still. Take 1mL of soil suspension, inject it on a sterile plate, and pour 25mL of sterilized and cooled to 40-50°C solid screening medium into the plate. After the culture medium was completely cooled and solidified, the plate was placed in a constant temperature incubator and cultured upside down at 30°C for 3 days. The composition of solid screening medium is: glucose 5.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium chloride 1.0 g/L, glycyl nitrile 1.0g/L, bromothymol blue 0.2g/L, agar 20.0g/L, pH 7.0, the single colony producing blue color circle is glycine producing bacteria. Pick a single colony that produces a blue color-changing circle, transfer it to a fresh medium, and separate it by streaking, and transfer it for 3-5 generations. the

Pick 25 purified strains and transfer them to nutrient-rich liquid medium for cultivation. The composition of the liquid screening medium is: glucose 5.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.02g/L, calcium chloride 0.02g/L, sodium chloride 1.0 g/L, glycyl nitrile 1.0g/L, pH 7.0, liquid volume 25mL/250mL, shaker speed 120rpm, culture at 30°C for 4 days. After the cultivation, the cells were removed by centrifugation, and the concentration of glycine in the supernatant was determined by paper chromatography. the

Carry out re-screening again, at first carry out seed culture, insert liquid culture medium with 1% inoculum size, 3 bottles of every plant, after the cultivation finishes, get this culture liquid 12000rpm centrifugation 10min and collect thalline and be used for bioconversion glycyl nitrile, adopt The concentration of glycine produced in the supernatant after conversion was detected by high performance liquid chromatography. Obtain a bacterial strain with the highest yield, named as Fusarium oxysporum (Fusarium oxysporum H3, CGMCC No.3829). the

Seed medium: beef extract 10.0g/L, peptone 10.0g/L, yeast extract 10.0g/L, glucose 10.0g/L, sodium chloride 5.0g/L, pH 7.0. The liquid volume is 25mL/250mL, and the culture conditions are 30°C and 120rpm for 3 days. the

(2) Morphology of Fusarium oxysporum H3

The Fusarium oxysporum H3 strain was inoculated on the potato dextrose agar medium plate with a hole punch inoculation method, with a hole diameter of 0.5 cm. After culturing at 30°C for 4 days, the colony diameter was 4.9 cm, and the average growth diameter was 12.2 mm/d ; after 7 days of cultivation, the colony edge is white, the middle is purple red, and can produce purple red pigment; after growing on the rice medium, it is pink; Branches, transparent, 1.6-4.4μm in diameter, there are myxospore clusters on the aerial hyphae, the myxospore clusters are colorless, small conidia are born in the aerial hyphae, false heads are attached, the small conidia are nearly Oval, without septum, smooth, large conidia sickle-shaped, elliptic curved. the

(3) Molecular identification of Fusarium oxysporum H3

The mycelium grown on the potato dextrose solid medium plate was collected, and the total DNA was extracted with a fungal genomic DNA extraction kit. The amplification primers were 18S general primers, ITS sequence general primers and 28S rDNA sequence general primers. the

The PCR reaction uses a 50 μL reaction system,

10×buffer(Mg ²⁺ ) 5.0μL

dNTPs(2.5mM) 4.0μL

Forward primer (20μM) 2.0μL

Reverse primer (20μM) 2.0μL

ExTaq DNA polymerase (5U/μL) 1.0μL

Template DNA 2.0μL

ddH ₂ O 34 μL

PCR amplification conditions: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 60 s, annealing at 55°C for 60 s, extension at 72°C for 90 s, a total of 30 cycles, and a final extension at 72°C for 10 min. Purification of PCR amplification products was carried out according to the instructions of Shanghai Sangon Biotechnology Co., Ltd. Small Volume Gel Recovery PCR Product Purification Kit, and the sequencing was completed by Shanghai Huada Genomics. the

The 18S, ITS and 28S rDNA gene sequences obtained after sequencing were compared by BLAST in Genbank to determine their species. the

Example 2 The method of producing glycine by fermentation of Fusarium oxysporum H3

This example illustrates the method of culturing Fusarium oxysporum H3 into a nutrient-rich medium to obtain highly active and high-biomass bacteria, and biotransforming glycylnitrile to generate glycine. the

The composition of the medium: glycerol 10.0g/L, yeast extract 1.0g/L, peptone 1.0g/L, magnesium sulfate 0.1g/L, potassium dihydrogen phosphate 1.0g/L, sodium chloride 0.5g/L, sulfite Iron 0.01g/L, caprolactam 0.5g/L, pH 7.5;

Insert Fusarium oxysporum H3 into the culture medium with 1% inoculum amount, and cultivate it for 3 days at 30°C and 120rpm. After the cultivation is over, collect the cultured cells by centrifuging at 12000rpm for 10min, and wash them with 0.5M phosphoric acid. The bacteria were washed twice with the buffer, and the bacteria were resuspended in the buffer to prepare the bacteria suspension. the

Prepare 7.8g/L glycyl nitrile solution, adjust the pH to 5.0 with 2.0M sodium hydroxide or hydrochloric acid solution, and use it as the substrate of the catalytic reaction. Mix the substrate solution with the bacterial suspension so that the final bacterial volume is 1%, and control the pH of the mixed system to be 7.2, react for 48 hours at a temperature of 30°C and a rotational speed of 120rpm, and the concentration of glycine in the reaction solution is 6.6g /L, the productive rate has reached more than 60%. the

Example 3 Change the fermentation conditions, Fusarium oxysporum (Fusarium oxysporum H3) fermentation to produce glycine

This embodiment adopts the same method as in embodiment 2, changing the reaction time. the

The bacteria were collected from the culture medium by centrifugation, washed twice with 0.5M phosphate buffer, resuspended to make a bacterial suspension, mixed with 7.8g/L glycyl nitrile solution, at 30°C, 120rpm After 12 hours of reaction, the concentration of glycine in the reaction liquid was 8.5 g/L, and the yield reached 80%. the

Example 4 Change the fermentation conditions, Fusarium oxysporum (Fusarium oxysporum H3) fermentation to produce glycine

The method of this embodiment is the same as that of Embodiment 2, and the parameters of the method used are changed. the

Medium composition: glycerol 10.0g/L, yeast extract 5.0g/L, peptone 10.0g/L, magnesium sulfate 0.1g/L, potassium dihydrogen phosphate 4.0g/L, sodium chloride 1.0g/L, ferrous sulfate 0.02g/L, caprolactam 1.0g/L, pH 7.5;

Insert Fusarium oxysporum H3 into the culture medium with 2% inoculum amount, and cultivate it for 3 days at 30°C and 120rpm. After the cultivation is over, collect the cultured cells by centrifuging at 12000rpm for 10min, and wash them with 0.5M phosphoric acid. The bacteria were washed twice with the buffer, and the bacteria were resuspended in the buffer to prepare the bacteria suspension. the

Prepare 23.4g/L glycyl nitrile solution as the catalytic reaction substrate. The substrate solution was mixed with the bacterial suspension, and the pH was controlled to be 7.2. The reaction was carried out at a temperature of 30° C. and a rotation speed of 120 rpm for 12 hours. Almost all glycylnitrile in the reaction mixture was converted, and the yield of glycine reached 24.6 g/m. L. the

Example 5 Change the fermentation conditions, Fusarium oxysporum (Fusarium oxysporum H3) fermentation to produce glycine

The method of this embodiment is the same as that of Embodiment 2, and the parameters of the method used are changed. the

Prepare 46.8g/L glycyl nitrile solution as the catalytic reaction substrate. The substrate solution was mixed with the bacterial suspension, the inoculation amount was 1%, and the pH was controlled to be 7.2, and the reaction was carried out at a temperature of 30° C. and a rotation speed of 120 rpm for 12 hours. The substrate glycylnitrile was almost completely converted, and the yield of glycine reached 39.8g/L. the

Claims (3)

1. a plant height produces the bacterial strain of glycocoll; This bacterial strain is fusarium oxysporum (Fusarium oxysporum) H3; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.3829 at present, and preservation date is on May 10th, 2010.

2. the cultural method of the described fusarium oxysporum of 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.

3. utilizing the described fusarium oxysporum of claim 1 (Fusarium oxysporum) H3 to transform glycinonitrile is the method for glycocoll, 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) fusarium oxysporum (Fusarium oxysporum) H3 microorganism collection: cultural method according to claim 2 carries out fusarium oxysporum (Fusarium oxysporum) H3 yeast culture; With the centrifugal 5-20min of 6000-15000rpm; To collect the thalline that obtains through cultivating; Use 0.01-0.5M, the phosphoric acid buffer washing thalline of pH 6.0-8.0 2-4 time;

(3) transform: fusarium oxysporum (Fusarium oxysporum) H3 thalline is suspended in the above-mentioned damping fluid again; And with the substrate solution mixing, the reaction pH remain on 6.0-8.0, temperature of reaction remains on 20-60 ℃; Mixing speed is 50-250rpm, reaction times 6-48h.

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