CN101691554B

CN101691554B - Cellulosimicrobium cellulans capable of producing acrylic acid

Info

Publication number: CN101691554B
Application number: CN2009101141805A
Authority: CN
Inventors: 黄日波; 陈云来; 左文朴; 韦宇拓; 杜丽琴; 杨登峰
Original assignee: Guangxi Academy of Sciences
Current assignee: Guangxi Academy of Sciences
Priority date: 2009-06-30
Filing date: 2009-06-30
Publication date: 2011-05-25
Anticipated expiration: 2029-06-30
Also published as: CN101691554A

Abstract

The invention discloses a CZ-2 strain which is separated and screened from the soil of a drainage ditch of a diary farm and can produce acrylic acid, the CZ-2 strain is collected in China Center for Type Culture Collection with the collection number of CCTCC No. M209039, young bacteria of the strain are shaped like slender irregular rods, and old bacteria are shaped like spheres; the CZ-2 strain is gram-positive, not acid-resistant and facultative anaerobic and does not produce spores; and the CZ-2 strain is a raised yellow bacterial colony on peptone-yeast extract agar, the strain belongs tocellulosimicrobium cellulans through analysis and identification of morphological, physiological and biochemical characteristics, as well as a rDNA sequence, the yield of the laboratory-prepared acrylic acid can achieve 5mmol/L, and the application prospect is broad.

Description

A kind of energy produces acrylic acid fibrosis fiber bacterium

Technical field

The invention belongs to technical field of bioengineering, particularly obtain to produce acrylic acid fibrosis fiber bacterium (Cellulosimicrobium cellulans) from the screening of nature points of contamination, separation, purifying.

Background technology

Vinylformic acid is a kind of important hardware and software platform compound, is unsaturated organic acid important in the industrial production, can be used for producing numerous polymeric derivatives, and be important Organic Chemicals.Main application is to produce esters of acrylic acid, polyacrylic acid and salt thereof, is widely used in tackiness agent, coating, chemical fibre, leather, papermaking, textile industry and photosensitive resin plate etc.Over nearly 10 years, China's vinylformic acid industrial development is very fast, but still can not satisfy the market requirement that increases rapidly.Also there is very big breach in the domestic vinylformic acid degree of self-sufficiency, and this breach is still constantly enlarging.The production method of vinylformic acid and ester class thereof mainly contains cyanoethanol method, Lei Pu (REPPE) method (oxo synthesis), ketenes method, acrylonitrile hydrolysis method and oxidation of propylene, and preceding 4 kinds of technologies progressively are eliminated because of technology and economic cause.Oxidation of propylene adopts propylene to be oxidised with air to vinylformic acid under catalyst action, has occupied dominant position in the production method of vinylformic acid and ester.

Yet, be that raw material production vinylformic acid obviously is not our long-term solution with the Nonrenewable resources oil along with the exhaustion day by day of the mad petroleum resources that go up of price.Therefore, numerous in the occurring in nature microorganism, it is feasible seeking therein and can producing acrylic acid microorganism.At present, all found this metabolic reaction approach in Clostridium propionicum and Megaphaera elsdenii two strain bacterium, it utilizes lactic acid to be substrate, sloughs a water molecules in born of the same parents under the effect of lactoyl CoA dehydratase, obtain acrylate, and small amount of accumulation vinylformic acid is arranged.Thereby utilize microbial method to produce the requirement that vinylformic acid adapts to social sustainable development, and might substitute and utilize petroleum chemicals to produce acrylic acid at present, help the reasonable utilization of the energy.But it is lower directly to accumulate acrylic acid concentration by lactic fermentation, finds few 1.3mmol/L of surpassing at present.Simultaneously, C.propionicum, the anaerobic condition that the growth metabolism of M.elsdenii is all strict, the key enzyme in its vinylformic acid pathways metabolism is to O ₂Also very responsive, it has brought huge resistance in order to produce in acrylic acid process in transformation to give people thus.

Thereby the microorganism that can produce acrylic acid bacterial strain that searching can be new just seems very meaningful.

Summary of the invention

The objective of the invention is from physical environment, to separate a strain new can produce acrylic acid bacterial strain, for acrylic acid production preparation provides a kind of new available microorganism, utilizing the Nonrenewable resources oil with alleviation is the acrylic acid limitation of raw material production.

The present invention is achieved in that

The applicant separates from the waterways soil of the Guangxi University diary farm that is arranged in Nanning City, Guangxi Zhuang Autonomous Region and obtains a strain and can produce acrylic acid CZ-2 bacterial strain; Identify through morphologic observation, Physiology and biochemistry and 16S rDNA, the CZ-2 bacterial strain belongs to fibrosis fiber bacterium (Cellulosimicrobium cellulans) on taxonomy, the applicant is preserved in Chinese typical culture collection center in the Wuhan University of Wuhan City, Hubei Province (being called for short CCTCC) on March 16th, 2009 with this bacterial strain, and its deposit number is: CCTCC NO:M 209039.

CZ-2 bacterial strain children thalline in age is elongated irregular shaft-like, and old and feeble thalline is spherical; Gram-positive is not given birth to spore, and is not antiacid; Amphimicrobian; It on peptone-yeast extract paste agar the yellow bacterium colony of projection.

The CZ-2 bacterial strain is a facultative anaerobe, can survive in temperature-20 ℃～50 ℃, pH value are 4.5～9.0 environment, and the alcohol tolerance level reaches the 6-8% of alcohol volume ratio, and suitable growth temperature is 20 ℃～40 ℃, and the appropriate pH value is 6.0～8.0.

CZ-2 bacterial strain 16S rDNA sequence has the nucleotide sequence shown in the sequence table 1, with fibrosis fiber bacterium homology more than 97%.

Outstanding substantive distinguishing features and the obvious improvement that the present invention has is:

1, successfully separated a kind of energy from occurring in nature and produced acrylic acid new bacterial strain, widened and utilized microorganism to prepare acrylic acid bacterial classification selection kind, helped advancing having now and utilize non-renewable petrochemical industry resource to prepare acrylic acid technological change.

2, the present invention utilizes the new bacterial strain of CZ-2 is produced acrylic acid technology having carried out useful exploration and test of laboratory means success, and the vinylformic acid yield is apparently higher than the yield of producing of existing other bacterial strain, and the technical progress effect is remarkable, has a extensive future.

Description of drawings

Fig. 1 is that the 16S rDNA sequential system of CZ-2 is grown tree.

Fig. 2 is a vinylformic acid standard specimen gas chromatogram.The appearance time of vinylformic acid standard specimen is 6.009 minutes.

Fig. 3 is the gas chromatogram of CZ-2 cell extract.The appearance time of the 3rd chromatographic peak is 6.005 minutes, and with the appearance time basically identical of vinylformic acid standard specimen in the accompanying drawing 2, this shows in the CZ-2 cell extract may exist vinylformic acid, judge that this peak is that vinylformic acid need further be proved by the makings chromatogram.

Fig. 4 is a vinylformic acid standard specimen makings color atlas.The appearance time of vinylformic acid standard specimen is 3.480 minutes.

Fig. 5 is a vinylformic acid standard specimen mass spectroscopy feature spectrogram.X-coordinate is mass-to-charge ratio (m/z) among the figure, and ordinate zou is a strength of signal.

Fig. 6 goes out peak figure for the makings chromatogram of CZ-2 cell extract.The appearance time of the 3rd chromatographic peak is 3.458 minutes, with the appearance time basically identical of vinylformic acid standard specimen in the accompanying drawing 4.

Fig. 7 is the mass spectroscopy feature spectrogram at the 3rd peak in the accompanying drawing 6.X-coordinate is mass-to-charge ratio (m/z) among the figure, and ordinate zou is a strength of signal.By can seeing among the figure that the material property peak among Fig. 7 is consistent with the characteristic peak of standard specimen among Fig. 5, this shows and includes vinylformic acid in the CZ-2 cell extract really.

The explanation of preservation information

A kind of fibrosis fiber bacterium Cellulosimicrobium cellulans CZ-2 is deposited in Chinese typical culture collection center (being called for short CCTCC) at present, the address is a Wuhan City, Hubei Province Wuhan University, preservation date is on March 16th, 2009, and deposit number is CCTCC NO:M209039.

Embodiment

Embodiment 1: the separation of bacterial strain CZ-2, screening and evaluation

The first step: sampling

Gather soil sample and water sample from the waterways of Guangxi University diary farm.

Second step: primary dcreening operation

Preparation is carbon source and the liquid synthetic medium that contains several mineral materials with vinylformic acid, contaminated soil is joined in the synthetic medium, wherein can utilize the bacterium of vinylformic acid growth to breed in a large number, with its cultivation 5～10 times of going down to posterity repeatedly, the mixed bacterium of cultivating according to going down to posterity determines further to carry out the object of strains separation purifying to acrylic acid ability of utilization.Concrete steps are as follows:

The preparation acrylic acid concentration is the liquid synthetic medium of 20mmol/L, and the mineral salt composition is identical in the substratum, and each set of dispense ratio is: NH ₄Cl 1.5g/L, Na ₂HPO ₄6g/L, KH ₂PO ₄3g/L, NaCl 1.0g/L, CaCl ₂0.05mmol/L, MgSO ₄10mmol/L, pH7.5-8.0.To respectively get 1 gram from the pedotheque that different points of contamination are adopted, join respectively in 50 milliliters the primary dcreening operation substratum, at 37 ℃, cultivated 2-4 days in 200 rev/mins the shaking table, get 1 milliliter of bacteria suspension again, join in 50 milliliters the primary dcreening operation substratum cultivation of going down to posterity under the same conditions.Go down to posterity and cultivate 5～10 times, wherein can all can utilize vinylformic acid as carbon source by prolific bacterial strain.

The 3rd step: multiple sieve

Preparation vinylformic acid substratum, medium component is identical with second step, but acrylic acid concentration progressively is increased to 100mmol/L by 20mmol/L, and the agar that adds 10-15g/L therein, make it become solid medium and in watch-glass, be paved into solid plate, with the direct spread plate of mixed bacterium suspension that second step obtained after going down to posterity, culture temperature is 37 ℃, and incubation time is 3-7 days.

The 4th step: separation and purification

Obtain 18 single bacterium colonies from the multiple sieve substratum in the 3rd step, the single colonial morphology unanimity of part is arranged, each single bacterium colony of picking respectively uses the cultivation of going down to posterity respectively of the liquid nutrient medium of identical component.From 18 strain bacterial strains, identify the bacterium that obtains 3 strain different generas by morphologic observation and 16S rDNA result, but finding when utilizing gas-chromatography that the tunning of this 3 strain bacterium is measured has 1 strain bacterium energy metabolism to produce vinylformic acid, this strain number is CZ-2, after mass spectrum confirms that this bacterial strain can produce vinylformic acid really.

The 5th step: identify

Morphological specificity: bacterial strain CZ-2 children thalline in age is elongated irregular shaft-like, and old and feeble thalline is spherical; Gram-positive is not given birth to spore, and is not antiacid; Amphimicrobian; It on peptone-yeast extract paste agar the yellow bacterium colony of projection.

Physiological and biochemical property:

L-arabinose

+

Cellobiose

+

Glycerine

-

Synanthrin

+

Seminose

+

Melizitose

-

Melibiose

-

Raffinose

+

The L-rhamnosyl

-

Sorbyl alcohol

-

D-ribose

+

Catalase

+

Mierocrystalline cellulose decomposes

+

Nitrate reduction

+

The casein hydrolysis

+

Mobility

-

Urase

+

Gelatin hydrolysis

+

35 ℃ of growths

+

(annotate: "+" is positive in the table, and "-" is negative)

Sequential analysis:

Bacterial strain CZ-2 grows tree as shown in Figure 1 with relevant kind 16S rDNA sequential system.

To sum up, this bacterial strain CZ-2 morphology, physiological and biochemical property meet fiber Microbacterium (cellulosimicrobium) feature, and near fibrosis fiber bacterium (Cellulosimicrobium cellulans), and sequential analysis shows that bacterial strain CZ-2 and fibrosis fiber bacterium (Cellulosimicrobium cellulans) homology are more than 97%.Identify that thus bacterial strain CZ-2 belongs to fibrosis fiber bacterium (Cellulosimicrobium cellulans).

Embodiment 2 bacterial strain CZ-2 metabolism vinylformic acid gas chromatography-mass spectrums detect

1, gas chromatographic detection

Standard specimen is produced: with inoculation in containing 10ml substratum (yeast powder 1.5g/L, peptone 3g/L, NH ₄Cl 1.0g/L, lactic acid 60mmol/L, Na ₂HPO ₄6g/L, KH ₂PO ₄3g/l, NaCl 1.0g/L, CaCl ₂0.05mmol/L, MgSO ₄10mmol/L in straight type bottle pH7.0), under 37 ℃ of conditions, cultivated 48 hours.Collect the bacterial sediment of 50ml fermented liquid, precipitation is resuspended in the fermented liquid of 10ml, placed precooling on ice 5 minutes, with JY92-II type ultrasonic cell disruption instrument power broken born of the same parents 10 minutes during for 300W, utilize high speed freezing centrifuge high speed centrifugation 15 minutes under the 12000r/pm condition, collect the sample of supernatant liquor as gas chromatographic analysis.The vinylformic acid (Sigma company analytical pure product) that standard specimen is is 100mmol/L with ultrapure water-reducible concentration.

Chromatographic instrument: Agilent6890+ type high resolution gas chromatography instrument (U.S.);

Chromatographic column: capillary gas chromatographic column, model are the INNOWAX post, internal diameter Diameter 0.32mm, length 30.0m.;

Analysis condition: sample size 1ul, 200 ℃ of injector temperatures, column temperature with 10 ℃/minute by constant temperature 4min behind 90 ℃ of cascade raising temperatures to 140 ℃, detector temperature is 250 ℃, carrier gas is that purity is 99.99% high pure nitrogen;

Experimental result: standard specimen is carried out gas chromatographic analysis, shown in accompanying drawing 2,3, detect vinylformic acid from the CZ-2 cell extract, concentration is about 1.5～5mmol/L.

2, the makings chromatogram detects

Standard specimen is produced: it is identical with the preparation method to be used for the preparation method of the standard specimen of makings stratographic analysis and the sample standard specimen used with being used for gas chromatographic detection.

Makings chromatographic instrument: GC-MS/QP5050A type makings chromatographic instrument (day island proper Tianjin);

The nonpolar chromatographic column of chromatographic column: DB-1, model: 30 meters * 0.2mm * 0.25 μ m;

The mass spectrum condition: 10 minutes analysis times, standard substance are quantitative, sample introduction 1 μ L, not split stream sampling.Temperature programming, initial column temperature is 90 ℃, is warming up to 140 ℃ with the stepwise speed of 10 ℃/min, injection port and detector temperature are respectively 200 ℃ and 250 ℃, carrier gas: purity is high-purity helium of 99.99%;

Experimental result: corresponding chromatographic peak is carried out mass spectroscopy, shown in accompanying

drawing

4,5,6,7, have vinylformic acid to exist in the cell extract of CZ-2 bacterial strain.

Detect conclusion: detect through gas chromatography-mass spectrum and find that bacterial strain CZ-2 has acrylic acid ability of producing.

Sequence table

＜110〉Guangxi Academy Of Sciences

＜120〉a kind of energy produces acrylic acid fibrosis fiber bacterium

<160>1

<210>1

<211>1463

<212>DNA

＜213〉fibrosis fiber bacterium (Cellulosimicrobium cellulans)

<400>

CGGGGGGGGG?GCGCGCAAAA?CACGTTAAAG?TCGAACGATG?ATAGCCCAGC 50

TTGCTGGGCG?GTTTAGTGGC?GAACGGGTGA?GTAACACGTG?AGTAACCTGC 100

CCTTGACTTC?GGGATAACTC?CGGGAAACCG?GGGCTAATAC?CGGATATGAG 150

CCGTCCTCGC?ATGGGGGTGG?TTGGAAAGTT?TTTCGGTCAG?GGATGGGCTC 200

GCGGCCTATC?AGCTTGTTGG?TGGGGTGATG?GCCTACCAAG?GCGACGACGG 250

GTAGCCGGCC?TGAGAGGGCG?ACCGGCCACA?CTGGGACTGA?GACACGGCCC 300

AGACTCCTAC?GGGAGGCAGC?AGTGGGGAAT?ATTGCACAAT?GGGCGAAAGC 350

CTGATGCAGC?GACGCCGCGT?GAGGGATGAA?GGCCTTCGGG?TTGTAAACCT 400

CTTTCAGCAG?GGAAGAAGCG?CAAGTGACGG?TACCTGCAGA?AGAAGCGCCG 450

GCTAACTACG?TGCCAGCAGC?CGCGGTAATA?CGTAGGGCGC?AAGCGTTGTC 500

CGGAATTATT?GGGCGTAAAG?AGCTCGTAGG?CGGTTTGTCG?CGTCTGGTGT 550

GAAAACTCGA?GGCTCAACCT?CGAGCTTGCA?TCGGGTACGG?GCAGACTAGA 600

GTGCGGTAGG?GGAGACTGGA?ATTCCTGGTG?TAGCGGTGGA?ATGCGCAGAT 650

ATCAGGAGGA?ACACCGATGG?CGAAGGCAGG?TCTCTGGGCC?GCAACTGACG 700

CTGAGGAGCG?AAAGCATGGG?GAGCGAACAG?GATTAGATAC?CCTGGTAGTC 750

CATGCCGTAA?ACGTTGGGCA?CTAGGTGTGG?GGCTCATTCC?ACGAGTTCCG 800

TGCCGCAGCA?AACGCATTAA?GTGCCCCGCC?TGGGGAGTAC?GGCCGCAAGG 850

CTAAAACTCA?AAGGAATTGA?CGGGGGCCCG?CACAAGCGGC?GGAGCATGCG 900

GATTAATTCG?ATGCAACGCG?AAGAACCTTA?CCAAGGCTTG?ACATGCACGA 950

GAAGCCACCA?GAGATGGTGG?TCTCTTTGGA?CACTCGTGCA?CAGGTGGTGC 1000

ATGGTTGTCG?TCAGCTCGTG?TCGTGAGATG?TTGGGTTAAG?TCCCGCAACG 1050

AGCGCAACCC?TCGTCCCATG?TTGCCAGCGG?GTTATGCCGG?GGACTCATGG 1100

GAGACTGCCG?GGGTCAACTC?GGAGGAAGGT?GGGGATGACG?TCAAATCATC 1150

ATGCCCCTTA?TGTCTTGGGC?TTCACGCATG?CTACAATGGC?CGGTACAAAG 1200

GGCTGCGATA?CCGTAAGGTG?GAGCGAATCC?CAAAAAGCCG?GTCTCAGTTC 1250

GGATTGGGGT?CTGCAACTCG?ACCCCATGAA?GTCGGAGTCG?CTAGTAATCG 1300

CAGATCAGCA?ACGCTGCGGT?GAATACGTTC?CCGGGCCTTG?TACACACCGC 1350

CCGTCAAGTC?ACGAAAGTCG?GTAACACCCG?AAGCCCATGG?CCCAACCGTT 1400

CGCGGGGGGA?GTGGTCGATG?GTGGGACTGG?CGATTGGACT?AAGTCGAACA 1450

ACGAGCCCCC?GCC 1463

Claims

1. one kind can be produced acrylic acid bacterial strain, it is characterized in that its classification called after fibrosis fiber bacterium (Cellulosimicrobiumcellulans), bacterial strain CZ-2, from the soil of waterways, diary farm, separate and get, be deposited in Wuhan City, Hubei Province China typical culture collection center, preservation date is on March 16th, 2009, and deposit number is CCTCC NO:M209039.

2. the application of the described bacterial strain of claim 1 in producing vinylformic acid.