CN101701199B - Enterobacter aerogen and application thereof - Google Patents

Abstract

The invention relates to an enterobacter aerogen with the collection number of CGMCC No.3163. A bacterial colony is cultured on a VRBDA agar culture medium for 24 hours at the temperature of 30 DEG C and is a pink round bacterial colony; the bacterial colony is non-transparent, straight, peritrichous and Gram-negative and is in facultative anaerobe; in addition, the bacterial colony generates acid and gas when fermenting glucose; the VP test proves that the bacterial colony is gelatine hydrolysis positive and is rod-like under a common microscope. When being applied, the bacterial colony is inoculated into a test tube filled with culture medium A to stand at the temperature of 37 DEG C to be cultured for 24 hours; after being repeatedly activated five times, bacteria solution is inoculated into a culture medium B to stand at the temperature of 37 DEG C to be cultured for four days; the bacteria solution is decentralized at 1000 rpm for 10 minutes; supernate is extracted to obtain high concentration liquid; the culture medium A is enterobacteria enrichment broth added with 0.1% of lysine, and the culture medium B is enterobacteria enrichment broth added with 0.005% of phosphopyridoxal and 0.1% of lysine.

Description

One Enterobacter aerogen and application thereof

Technical field

The present invention relates to a strain mikrobe and an application thereof, belong to biological technical field.

Background technology

The chemical name of cadaverine is 1, and the 5-pentamethylene diamine can be used for organic synthesis, biological chemical reagent and medicine intermediate, and at present, the demand of China's cadaverine and verivate thereof is big, but does not domesticly still have enterprise and can produce specification product, and is very high from external import price.

Traditional cadaverine is to utilize Methionin to carry out chemosynthesis, and synthetic ratio is low, and by product is many, becomes height.

(Methionin is under the effect of the lysine decarboxylase of microorganisms to produce cadaverine through microbial metabolism; Decarboxylation produces cadaverine), obtain high density cadaverine nutrient solution, separate cadaverine then; Can reduce the production cost and the energy consumption of cadaverine, also become the major subjects of research.

Summary of the invention

The objective of the invention is to: to the problem that present cadaverine production exists, a kind of mikrobe is provided and produces cadaverine, obtain high density cadaverine nutrient solution through this microbial metabolism.

The objective of the invention is to realize like this: an Enterobacter aerogen (Enterobacter aerogenes) xlcad002, it is characterized in that: preserving number is CGMCC No.3163, and bacterium colony is on the MRS nutrient agar; Cultivate 24h, oyster white bacterium colony, 1.0-1.5 * 0.3-0.6 μ m for 30 ℃; Quarter butt, Gram-negative, glucose fermentation produces acid; This bacterial strain is shaft-like under simple microscope, and the Genbank number of landing of this bacterial strain 16S rDNA is GQ890355.

In the present invention, the VRBDA nutrient agar is: yeast extract 3g, peptone 7g, cholate 1.5g, sodium-chlor 5g, lactose 10g, toluylene red 0.03g, Viola crystallina 0.002g, agar 15g, zero(ppm) water 1000ml, pH value 7.3-7.5.

A kind of preserving number is the application of the bacterial strain of CGMCC No.3163, it is characterized in that: be inoculated in the test tube that the A substratum is housed from the inclined-plane picking colony, 37 ℃ leave standstill cultivation 24 hours, and the inoculum size in the test tube is 10 ⁵Cfu/ml.In same medium repeatedly after the activation five times; Bacterium liquid after the last activation is inoculated in the B substratum 37 ℃ of static cultivations 4 days, and inoculum size is 10 ⁶Cfu/ml; Bacterium liquid 10000 after cultivating was left the heart 10 minutes, get supernatant, obtain high density liquid, cadaverine content is 3279.24 μ g/ml;

Described A substratum increases bacterial context soup substratum for the enterobacteria that adds 0.1% Methionin; Described B liquid nutrient medium increases bacterial context soup substratum for adding 0.005% Vitazechs with the enterobacteria that adds 0.1% Methionin.

In the present invention: described enterobacteria enrichment broth substratum: peptone 10g, glucose 5g, potassium hydrogenphosphate 2g, SODIUM PHOSPHATE, MONOBASIC 8g, zero(ppm) water 1000mL, pH value 7.2～7.4 is at 115 ℃ of sterilization 15min.

The invention has the advantages that: because enteroaerogen (Enterobacter aerogenes) xlcad002 product cadaverine ability is strong; In the nutrient solution that aforesaid method makes; Cadaverine content can reach 3279.24 μ g/ml, produces cadaverine with this method and will greatly reduce production costs and reduce pollution.

Description of drawings

Fig. 1 is the photo of enteroaerogen under simple microscope;

Fig. 2 is the dna segment electrophorogram that detects the enteroaerogen amplification;

Fig. 3 is the high-efficient liquid phase chromatogram of biogenic amine standard specimen;

Fig. 4 is that performance liquid detects enteroaerogen nutrient solution color atlas.

The canonical plotting that Fig. 5 cadaverine detects.

Embodiment

Embodiment 1

The screening of bacterial strain and preservation

Substratum

The enterobacteria enrichment broth substratum: peptone 10g, glucose 5g, potassium hydrogenphosphate 2g, SODIUM PHOSPHATE, MONOBASIC 8g, zero(ppm) water 1000mL, pH value 7.2～7.4 is at 115 ℃ of sterilization 15min;

Lower floor's substratum: peptone 5g, yeast extract paste 5g, Carnis Bovis seu Bubali cream 5g, sodium-chlor 2.5g, glucose 0.5g, tween 1g, sal epsom 0.4g, manganous sulfate 0.03g, potassium hydrogenphosphate 2g, Triammonium citrate 2g, lime carbonate 0.1g, ferrous sulfate 0.04g, vitamins B ₁0.01g, Vitazechs 0.05g, agar 18 grams.Rotten propylhomoserin, each 5 gram, 1000ml zero(ppm) water.PH 5.0 (115 ℃ autoclaving 15 minutes);

Upper strata substratum: purpurum bromocresolis 0.06g, agar 20g, 1000ml zero(ppm) water, pH5.0 (121 ℃ of sterilization 10min);

VRBDA nutrient agar: yeast extract 3g, peptone 7g, cholate 1.5g, sodium-chlor 5g, lactose 10g, toluylene red 0.03g, Viola crystallina 0.002g, agar 15g, zero(ppm) water 1000ml, pH value 7.3-7.5.

Under aseptic condition, get 20 gram traditional Chinese sausages; Shred under the aseptic condition to be placed on and 180ml is housed in the triangular flask of sterile saline, room temperature 230 is changeed shaking tables and was shaken 10 minutes, gets 1ml and is inoculated in the enterobacteria enrichment broth substratum; Cultivated 24 hours for 37 ℃; Get the 1ml nutrient solution with 9ml saline water stepwise dilution to 10-100cfu/ml concentration, coat and produce cadaverine and detect on lower floor's substratum, cultivated 48 hours for 37 ℃.Slow impouring skim upper strata substratum, outcome record finishes in 10 minutes, and the bacterium colony that shows purple is positive, and displaing yellow is negative, and wherein, positive in producing the bacterial strain of putrescine, negative is the bacterial strain that can not produce putrescine.

The picking positive strain, was cultivated 24 hours for 37 ℃ with enterobacteria hyperplasia broth culture more than three times at purifying on the VRBDA solid medium, and centrifugal back obtains enteroaerogen (Enterobacter aerogenes) xlcad002 bacterial strain.

The bacterium colony of this bacterial strain is cultivated 24h for 30 ℃ on the VRBDA nutrient agar, pink circular bacterium colony is opaque, straight-bar, and peritrichous, Gram-negative, amphimicrobian, glucose fermentation produce sour aerogenesis, VP test, gelatin liquefaction positive.

This bacterial strain is according to Gen bank comparison result; Called after enteroaerogen (Enterobacter aerogenes) xlcad002; On July 6th, 2009 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and the address is: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Postcode 100101, culture presevation number is CGMCC No.3163.The Genbank number of landing of this bacterial strain 16S rDNA is GQ890355.

This bacterial strain adds aseptic glycerine, preserves at-80 ℃ of refrigerators.

Embodiment 2

The gene test of enteroaerogen (Enterobacter aerogenes) xlcad002

Detect the primer of lysine decarboxylase gene

CAD1-f：5‘-TTYGAYWCNGCNTGGGTNCCNTAYAC-3’；

CAD1-r：5‘-CCRTGDATRTCNGTYTCRAANCCNGG-3’；

Detection method:

25 μ l reaction systems comprise: GoTaq Green Master Mix 12.5 μ l, every kind of each 1.0 μ l of primer, DNA masterplate 2 μ l, de-ionized distilled water 8.5 μ l.

The pcr amplification program: 94 ℃ of preparatory sex change 5min, 30 circulations comprise: 95 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 2min, and final 72 ℃ are extended 7min, are cooled to 4 ℃.

With the Auele Specific Primer CAD1-f and the CAD1-r that detect lysine decarboxylase, can successfully amplify the gene fragment of 1098bp, in Fig. 2, the gene fragment that swimming lane 1 increases out for this research, the negative contrast of swimming lane n.Thus it is clear that, in enteroaerogen, have the lysine decarboxylase gene.

Embodiment 3

High density cadaverine inoculum preparation method

Enterobacteria enrichment broth substratum (with embodiment 1);

The preparation method

Be inoculated in the test tube that the A substratum is housed from the inclined-plane picking colony, 37 ℃ leave standstill cultivation 24 hours, and the inoculum size in the test tube is 10 ⁵Cfu/ml.In same medium repeatedly after the activation five times; Bacterium liquid after the last activation is inoculated in the B substratum 37 ℃ of static cultivations 4 days, and inoculum size is 10 ⁶Cfu/ml; Bacterium liquid 10000 after cultivating is left the heart 10 minutes, gets supernatant, obtain high density cadaverine liquid.

Described A substratum increases bacterial context soup substratum for the enterobacteria that adds 0.1% Methionin; Described B liquid nutrient medium increases bacterial context soup substratum for the enterobacteria that adds 0.005% Vitazechs and 0.1% Methionin.

Embodiment 4

Putrescine detection method of content (performance liquid detection)

1, the preparation of biogenic amine standardized solution

Accurately take by weighing each 50mg of tryptamines, phenylethylamine, tyrasamine, cadaverine, putrescine, spermidine and spermine, with the perchloric acid (HClO of 0.4mol/L ₄) be settled to 50mL, it is subsequent use to process the 1mg/ml storing solution.Get above standard substance storing solution respectively, use 0.4mol/L HClO ₄Be mixed with final concentration and be respectively the standardized solution of 5.0,10,20,30,40,50 μ g/ml, lucifuge, 4 ℃ of refrigerators are preserved.

2, the derivatize of sample solution

The sample 1mL that gets embodiment 3 is in the 5ml volumetric flask; The 2N NaOH that adds 200 μ L makes it to be alkalescence; The saturated sodium bicarbonate solution that adds 300 μ L then cushions; Add dansyl chloride (dansyl chloride) solution (concentration is 10mg/mL, and solvent is an acetone) of 2ml again, be positioned over reaction treatment 40min in 40 ℃ of water-bath dark then.Reaction finishes the ammoniacal liquor stopped reaction that the back adds 100 μ L, removes residual dansyl chloride solution.At last with the acetonitrile constant volume to 5mL.Behind organic membrane filtration with 0.22 μ m after the derivation process, obtain the verivate of sample solution.

3, the derivatize of standardized solution standard specimen

The standardized solution sample 1mL that gets 5.0,10,20,30,40,50 μ g/ml respectively is in the 5ml volumetric flask; The 2N NaOH that adds 200 μ L makes it to be alkalescence; The saturated sodium bicarbonate solution that adds 300 μ L then cushions; Add dansyl chloride (dansyl chloride) solution (concentration is 10mg/mL, and solvent is an acetone) of 2ml again, be positioned over reaction treatment 40min in 40 ℃ of water-bath dark then.Reaction finishes the ammoniacal liquor stopped reaction that the back adds 100 μ L, removes residual dansyl chloride solution.At last with the acetonitrile constant volume to 5mL.Behind organic membrane filtration with 0.22 μ m after the derivation process, obtain the verivate of six groups of sample solutions.

4, detection method is:

Waters Alliance2695 Liquid Detection system, chromatographic column are AgilentZORBAXXDB-C18 (4.6 * 250mm2,5 μ m); Flow velocity is 1mLmin-1, and the ultraviolet detection wavelength is 254nm, sample size 20 μ L; 30 ℃ of column temperatures, mobile phase A are water, and Mobile phase B is an acetonitrile; Adopt gradient elution, elution program is seen table 1.

Table 1 gradient elution program

In the performance liquid figure of the biogenic amine standard specimen of Fig. 3: 1 is tryptamines, and 2 is phenylethylamine, and 3 is putrescine, and 4 is cadaverine, and 5 is histamine, and 6 is tyrasamine, and 7 is spermidine, and 8 is spermine.Sample solution performance liquid detected result of the present invention is as shown in Figure 4.Visible by Fig. 4, contain cadaverine in the sample solution.

Utilize above-mentioned detection method to detect, response value is 17403574.28 (referring to Fig. 5), and calculating the result according to typical curve is 327.924 μ g/mg, but owing in testing process, diluted 10 times to former state, so final cadaverine content is 3279.24ug/mg.

More than each embodiment be not to concrete qualification of the present invention.

Claims (3)

1. an Enterobacter aerogen (Enterobacter aerogenes), it is characterized in that: preserving number is the bacterial strain of CGMCC No.3163, bacterium colony is cultivated 24h for 30 ℃ on the VRBDA nutrient agar; The circular bacterium colony of pink, opaque, straight-bar, peritrichous; Gram-negative, amphimicrobian, glucose fermentation produce sour aerogenesis; VP test, gelatin liquefaction positive, this bacterial strain is shaft-like under simple microscope, and the Genbank number of landing of this bacterial strain 16S rDNA is GQ890355.

2. an Enterobacter aerogen according to claim 1 (Enterobacter aerogenes), it is characterized in that: the VRBDA nutrient agar is: yeast extract 3g, peptone 7g; Cholate 1.5g, sodium-chlor 5g, lactose 10g; Toluylene red 0.03g, Viola crystallina 0.002g, agar 15g; Zero(ppm) water 1000ml, pH value 7.3-7.5.

3. the preserving number Enterobacter aerogen that is CGMCC No.3163 the application that produces high density cadaverine liquid is characterized in that: be inoculated in the test tube that the A substratum is housed from the inclined-plane picking colony, 37 ℃ leave standstill and cultivated 24 hours, and the inoculum size in the test tube is 10 ⁵Cfu/ml is in same medium repeatedly after the activation five times; Bacterium liquid after the last activation is inoculated in the B substratum 37 ℃ of static cultivations 4 days, and inoculum size is 10 ⁶Cfu/ml; Bacterium liquid 10000 after cultivating was left the heart 10 minutes, get supernatant, obtain high density liquid, cadaverine content is 3279.24 μ g/ml;

Described A substratum increases bacterial context soup substratum for the enterobacteria that adds 0.1% Methionin; Described B liquid nutrient medium increases bacterial context soup substratum for adding 0.005% Vitazechs with the enterobacteria that adds 0.1% Methionin;

Described enterobacteria enrichment broth substratum: peptone 10g, glucose 5g, potassium hydrogenphosphate 2g, SODIUM PHOSPHATE, MONOBASIC 8g, zero(ppm) water 1000mL, pH value 7.2～7.4 is at 115 ℃ of sterilization 15min.

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