CN102061278A - Methylovorus sp. MP688 and application thereof - Google Patents
Methylovorus sp. MP688 and application thereof Download PDFInfo
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Abstract
The invention discloses a new strain MP688 of Methylovorus sp., of which the preservation number is CGMCC No.4096, the preservation date is August 20th, 2010, and the preservation unit is China General Microbiological Culture Collection Center. The strain MP688 is separated from soil by using a GDH (glutamate dehydrogenase) recombinase method of PQQ (pyrroloquinoline quinone). By using the Methylovorus sp. strain disclosed by the invention, the yield of PQQ can reach 125 mg/L in a conventional culture medium under conventional culture conditions; and the Methylovorus sp. strain can be used for industrial production of PQQ, and has important meaning for promoting the industrialization of PQQ.
Description
Technical field
The present invention relates to a kind of microorganism strains and application thereof, specifically a kind of food methyl bacteria strain MP688 and application thereof.
Background technology
Pyrroloquinoline quinone (Pyrroloquinoline quinone is a kind of coenzyme and biological essential nutrients of finding more recently PQQ), have the certain micro-organisms of stimulation and animal and plant growth,
The control liver injury,
Promote nerve growth factor to generate, regulate different physiological roles such as interior free yl level and adjusting immunity, may be used for the control of diseases such as Alzheimer's disease, cardiovascular disorder and diabetes.By the new PQQ high-yield strains of seed selection, determine the synthetic relevant gene of itself and PQQ, for further improving PQQ output, reduce the PQQ production cost by the metabolic engineering means, advance the PQQ practical application significant.
Chemosynthesis PQQ step is many, and productive rate is low, and the removal of isomer and byproduct needs the multistep purifying, use multiple poisonous chemical reagent thereby seriously polluted (JACS, Vol 103, pp5599-2600,1981).New PQQ chemical synthesis process needs through the reaction of 11 steps, 5 step purifying just can obtain PQQ (the Davis Paul J of purity about 97%, Karliner Joel S, Mousa Shaker A, et al.Pyrroloquinoline quinone drugs and methods ofuse thereof:US, No.US2008/0051428A1.2008.2.28).
It is generally acknowledged that the biology synthetic method more has the industrialization meaning.Up to now, the known microorganism that can synthesize PQQ is Gram-negative bacteria, comprise paracoccus (Paracoccus), Protaminobacter (Protaminobacter), Rhodopseudomonas (Pseudomonas), achromobacter (Achromobacter), the flat born of the same parents Pseudomonas of alternate (Alteromonas), Bacillaceae (Ancylobacter), Desmobacteria (Hyphomicrobium), Methanomonas (Methanomonas), methyl bacillus (Methylobacillus), methyl Zymomonas mobilis (Methylomonas), have a liking for methyl bacterium (Methylophilus), methyl bacillus (Methylobacterium), Microcyclus (Microcyclus), the moving Pseudomonas (Mycoplana) of branch, Protomonas (Protomonas), thiobacillus (Thiobacillus) and the flat born of the same parents bacterium (Xanthobacter) that grows sturdily.At present, also there is the method that adopts strain fermentation to produce PQQ, the original strain PQQ output of using is at 0.07～7mg/L, fermentation time is (US Pat4994382 and 5344768) about 2-5 days, therefore yield poorly, the cost height is difficult to realize the industrial-scale production of PQQ, thereby needs high yield and economic PQQ production bacterial strain more.
Summary of the invention
The purpose of this invention is to provide a kind of new bacterial strain of food methyl bacterium and the application in the production pyrroloquinoline quinone thereof of production pyrroloquinoline quinone.
Bacterial strain MP688 of the present invention separates from soil by the GDH enzyme process and obtains, the new bacterial strain of food methyl bacterium (Methylovorus sp.), be preserved in (address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on August 20th, 2010, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.4096.
The present invention also provides the screening method of this bacterial strain, comprises the steps:
Clone's intestinal bacteria Hexose phosphate dehydrogenases (GDH) gene, and carry out prokaryotic expression, the GDH albumen behind the acquisition purifying.
To gather and leave standstill after soil sample adds suitable sterilized water suspendible, getting supernatant adds to enrichment medium (every liter contains ammonium sulfate 3g, potassium primary phosphate 1.4g, Sodium phosphate dibasic 3g, sal epsom 0.2g, ironic citrate 30mg, calcium chloride 30mg, Manganous chloride tetrahydrate 5mg, zinc sulfate 5mg, copper sulfate 0.5mg, the 1mL vitamin solution, 8mL methyl alcohol, pH transfers to 7.0.Vitamin solution: vitamin H 2mg, 400mg calcium pantothenate, 400mgVB1,400mgVB6,200mg para-amino benzoic acid, 2mg folic acid, 2g inositol, 400mg nicotinic acid, 200mg riboflavin), 30 ℃ of anaerobism are cultivated 5d, culture centrifuging and taking supernatant.
The supernatant of getting culture filters out the high culture of PQQ output by the quick primary dcreening operation of GDH reorganization enzyme process.On the isolation medium flat board, separate the single bacterium colony in the primary dcreening operation culture, inoculate to the bottle that shakes that contains enrichment medium, 30 ℃ of shaking culture 5d, again by PQQ content in the GDH enzymatic assays Accurate Analysis culture, the high bacterial strain of screening PQQ output.Repeat to screen two-wheeled again, obtained the superior strain of a strain PQQ, called after MP688.Through shake-flask culture analysis of experiments repeatedly, this bacterium PQQ output can reach 125mg/L.
The MP688 bacterial strain is rule on the MPQ flat board 30 ℃ and is cultivated 30h, observes colonial morphology, is the moistening circular bacterium colony of little neat in edge, and bacterium colony swells and combines closely in solid medium and with substratum.Picking list bacterium colony carries out gramstaining to be observed, and thalline takes on a red color, and is little bacillus.
The MP688 bacterial strain uses Electronic Speculum to amplify 24000 * doubly observations, and thalline is a rod-short, the top amphitrichous.
16S rDNA conserved sequence according to known several food methyl bacterium designs and synthesizes primer, is template with MP688 bacterium genomic dna, by the part 16S rDNA sequence of this bacterium of PCR reaction amplification.Connect the T carrier and send order-checking, the sequence that obtains is carried out BLAST relatively in GenBank, and itself and multiple bacterium all have certain homology, chooses with its homology the higher person and sets up phylogenetic tree according to 16S rDNA sequence, and its homology with food methyl bacterium is the highest.Comprehensive morphological feature and 16S rDNA Sequence Identification MP688 are the new bacterial strain of food methyl bacterium (Methylovorus sp.).
The present invention also provides a kind of microbial inoculum that comprises this bacterial strain.
The present invention also provides the method for this bacterial strain production pyrroloquinoline quinone of a kind of usefulness, and described method comprises the steps: the fermentation with MP688, and fermented liquid is wash-out behind weakly-basic anion displacement chromatography column chromatography, and the wash-out composition is through C
18Oppositely after the chromatography column absorption, use methanol-eluted fractions, elutriant concentrates and makes its spontaneous nucleation.
Food methyl bacteria strain provided by the invention is under conventional substratum and culture condition, PQQ output can reach 125mg/L, and strain growth is good, stable yield, be expected to further improve output through strain improvement and culture optimization, can be used for industrialization PQQ production, significant to the industrialization that promotes PQQ.
Description of drawings
Fig. 1 is pedotheque GDH active part primary dcreening operation result.
Fig. 2 is the PQQ standard working curve.
Fig. 3 is the PCR product electrophoresis of GDH.Wherein 1 is λ-EcoT14 I digest Maker; 2 is GDH PCR product.
Fig. 4 is that the SDS-PAGE of GDH expression product analyzes.Wherein 1 is proteic Marker; 1 is the BL21-pET28a-gdh total protein; 2 is the BL21-pET28a-gdh supernatant liquor; 3 is the total protein of BL21; 4 is the BL21-pET28a total protein.
The proteic SDS-PAGE of GDH analyzes behind Fig. 5 purifying.
Fig. 6 is for the MP688 Electronic Speculum detects (24000 *).
Fig. 7 is the pcr amplification of MP68816S rDNA gene.Wherein 1 is DL 2000Marker; 2 is the PCR product.
Fig. 8 is the systematic evolution tree based on 16S rDNA sequence.
Fig. 9 is a PQQ product column chromatography collection of illustrative plates.
Figure 10 is the needle crystal (100 *) of PQQ product.
Figure 11 is the HPLC analytical results of product.Wherein A is the PQQ standard substance; B is the PQQ sample.
Figure 12 is the absorption spectrum of PQQ product.Wherein A is the PQQ standard substance; B is the PQQ sample.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1PQQ produces the bacterium screening
To extensively gather about 3000 parts of soil samples from all parts of the country, main source comprises in Beijing and respectively distinguishes areas such as suburb, Tonghua, Jilin, Hengshui, Hebei, Zhejiang Hangzhou.1.5mL centrifuge tube with sterilization is gathered soil sample, every pipeclay sample adding lmL sterilized water is gathered and is left standstill after soil sample adds suitable sterilized water suspendible, getting 100 μ l supernatants adds to the 5ml enrichment medium (every liter contains ammonium sulfate 3g, potassium primary phosphate 1.4g, Sodium phosphate dibasic 3g, sal epsom 0.2g, ironic citrate 30mg, calcium chloride 30mg, Manganous chloride tetrahydrate 5mg, zinc sulfate 5mg, copper sulfate 0.5mg, the 1mL vitamin solution, 8mL methyl alcohol, pH transfers to 7.0.Vitamin solution: vitamin H 2mg, 400mg calcium pantothenate, 400mgVB1,400mgVB6,200mg para-amino benzoic acid, 2mg folic acid, 2g inositol, 400mg nicotinic acid, 200mg riboflavin), 30 ℃ of anaerobism are cultivated 5d, culture centrifuging and taking supernatant, with the quick primary dcreening operation of GDH reorganization enzyme process of PQQ, method is as follows:
Getting 20 μ L GDH recombinase solution adds in 96 orifice plates, add 180 μ L detection reaction liquid (pH6.8 phosphate buffer soln 50mM again, PMS 1mmol/L, sal epsom 2mM, glucose 10mM, DCIP100uM, PQQ standard solution 5 μ M or supernatant 50 μ L) begin reaction, can estimate the contents level of PQQ in the sample fast according to the fading time of reaction solution.
Fig. 1 has shown the partial results of primary dcreening operation.Enrichment medium flat board (enrichment medium+1.6% agar powder) is gone up the single bacterium colony that separates in the primary dcreening operation culture, inoculates to the bottle that shakes that contains enrichment medium, and 30 ℃ of shaking culture 5d are by PQQ content in the GDH enzymatic assays Accurate Analysis culture.Method is as follows:
Get 1mL GDH recombinase solution and add in the cuvette, add 2mL detection reaction liquid (the same) again, by the content of PQQ in the rate of change quantitative analysis sample of measuring 600nm place solution absorbance.With the positive contrast of standard substance, be blank during detection with the reaction solution that does not add PQQ.Under corresponding conditions, gradient dilution PQQ standard substance are made PQQ typical curve (Fig. 2).According to the rate of change of working sample solution absorbance, reference standard curve, the content of quantitative analysis sample P QQ.Through number wheel primary dcreening operation and multiple the screening from the PQQ superior strain.The result separates part soil sample surplus 3000 and obtains a plant height and produce bacterium, our called after MP688, and through shake-flask culture analysis of experiments repeatedly, this bacterium PQQ output can reach 125mg/L.
Wherein, the GDH recombinase prepares as follows:
According to gdh gene order design primer P1, the P2 of E.coli, sequence is introduced Nde I and Bam H I restriction enzyme site respectively at 5 ' end and 3 ' end shown in SEQ IDNo.1 and 2.With E.coli DH5 α genome is template, gets the big or small purpose fragment (Fig. 3) of 2674bp that is by pcr amplification.The PCR condition is: 94 ℃ of 4min, and 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 3min extend 5min in 72 ℃ of continuation after 30 circulations, identify the PCR product with 1.0% agarose gel electrophoresis.Product is identified correct through order-checking, through reclaiming and cutting with Nde I and Bam H I enzyme, is connected with the pET28a expression vector, and connection product transformed competence colibacillus cell DH5 α selects positive colony extraction plasmid and carries out enzyme and cut evaluation, again sequencing analysis.Sequencing result shows that the sequence that obtains of amplification is in full accord with the gdh gene order of bibliographical information, shows that the pET28a-GDH plasmid construction successfully.
The expression plasmid that makes up is converted into BL21 (DE3), single bacterium colony of picking positive colony is connected to the LB substratum that 5mL contains kantlex, 37 ℃ of shaking culture are spent the night, all inserting 500mL contains in the kantlex LB substratum, 37 ℃ of shaking culture to OD values are about 0.6, adding IPTG is 1.0mmol/L to final concentration, 37 ℃ of inducing culture 4h.Collect thalline in 10 ℃ of centrifugal 10min of 5000r/min.Phosphate buffer soln with 20mmol/L pH7.0 washs thalline 2 times, is suspended into the same damping fluid of 20mL, carrying out ultrasonic bacteria breaking.Broken bacterium liquid is through centrifugal, by DEAE Sepharose ion exchange column purifying target protein from supernatant liquor.Chromatography column after balance with the NaCl gradient elution of 1mol/L and collect each elution peak component.Has enzyme component alive after testing through the drainage column desalination.The SDS-PAGE check and analysis, the molecular weight of expression product and purifying protein is about 87kDa, consistent with bibliographical information (Fig. 4～5).
Embodiment 2PQQ produces the evaluation of bacterium MP688
The MP688 bacterial strain is rule on the MPQ flat board 30 ℃ and is cultivated 30h, observes colonial morphology, is the moistening circular bacterium colony of little neat in edge, and bacterium colony swells and combines closely in solid medium and with substratum.Picking list bacterium colony carries out gramstaining to be observed, and thalline takes on a red color, and is little bacillus.The MP688 bacterial strain uses Electronic Speculum to amplify 24000 * doubly observations, and thalline is a rod-short, top amphitrichous (Fig. 6).16S rDNA conserved sequence according to known several food methyl bacterium designs and synthesizes primer, is template with MP688 bacterium genomic dna, by the part 16S rDNA sequence (Fig. 7) of this bacterium of PCR reaction amplification.Its primer sequence is shown in SEQ ID No.3 and 4.Connect the T carrier and send order-checking, its sequence is shown in SEQ ID No.5.The sequence that obtains is carried out BLAST relatively in GenBank, reach 99% (Fig. 8) with the 16SrDNA sequence homology of eating methyl Pseudomonas two bacterial strains.
The extraction of embodiment 3PQQ, purifying and evaluation
1.MP688 fermentation culture
Bacterial classification is inserted fermention medium according to 5% ratio, and (every liter contains yeast powder 5g, peptone 5g, ammonium sulfate 3g, potassium primary phosphate 1.4g, Sodium phosphate dibasic 3g, sal epsom 0.2g, ironic citrate 30mg, calcium chloride 30mg, Manganous chloride tetrahydrate 5mg, zinc sulfate 5mg, copper sulfate 0.5mg, the 1mL vitamin solution, 8mL methyl alcohol, pH transfers to 7.0.Vitamin solution: vitamin H 2mg, 400mg calcium pantothenate, 400mg VB
1, 400mg VB
6, 200mg para-amino benzoic acid, 2mg folic acid, 2g inositol, 400mg nicotinic acid, 200mg riboflavin) in, 30 ℃ of shaking culture 5d.According to 10% inoculation, substratum concentration is normal 2 times during fermentor cultivation.
2.PQQ preparation
MP688 is seeded to enrichment medium, 30 ℃ of shaking culture 5d, 10 ℃ of centrifugal 10min of 8000r/min of culture, supernatant liquor adsorbs through weakly-basic anion displacement chromatography post AmberlystA21, with Tris-HCl (pH 8.0) the buffered soln balance of 20mmol/L, carry out gradient elution with 1mol/L NaCl again.The wash-out composition of collecting is again with Seppak C
18Oppositely chromatography column absorption (Fig. 9), with methanol-eluted fractions, elutriant makes its spontaneous nucleation after concentrating, and grows the needle-like crystal (Figure 10) of product as a result in the concentrated solution.
3.PQQ analysis
3.1 the HPLC of product analyzes
Adopt Waters Symmetry 300C
18The reverse chromatograms post, with water: methyl alcohol: phosphoric acid (80: 20: 0.1) is moving phase, flow velocity 1ml/min by HPLC compartment analysis PQQ, adopts gradient elution method and 254nm wavelength to detect and can obtain stratographic analysis result preferably (Figure 11).The result shows that the retention time at sample spectra peak is identical with standard substance, and interpret sample is PQQ.
3.2 the ultra-violet absorption spectrum of product
The PQQ that produces with Sigma company in contrast, the uv absorption spectra (Figure 12) that utilizes the ultraviolet-visible spectrophotometric to make the 200-400nm length scanning can to obtain the PQQ product.
The result shows that the charateristic avsorption band of sample and standard substance is almost completely consistent.
The preparation of embodiment 4MP688 microbial inoculum
1, actication of culture
Use enrichment medium, MP688 is inoculated on the solid enrichment medium inclined-plane, cultivate 3d for 30 ℃.
2, culture of seed liquid
The seed culture enrichment medium, the MP688 that activation is good is mixed with 10 with stroke-physiological saline solution
9The bacteria suspension of CFU/mL, the inoculum size with 5% are inoculated in the liquid enrichment medium, and 30 ℃ of shaking table concussions are cultivated, and rotating speed is 200rpm, and incubation time is 3d.
3, ferment tank
Fermentor cultivation is used fermention medium.Per minute air flow volume), tank pressure 1.2-1.8F/cm cultured seed liquid is inserted in the fermentor tank with 10% inoculum size, and 30 ℃, stirring velocity is 200rpm, and air flow is 1: 0.8-1.0 (fermentating liquid volume:
2Fermentation 5d, the bacterium amount reaches 5-10 * 10
9CFU/mL obtains the microbial inoculum of MP688 bacterial strain.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. eat methyl bacterium (Methylovorus sp.) bacterial strain MP688, its preserving number is CGMCCNo.4096.
2. the microbial inoculum that contains the described bacterial strain of claim 1.
3. the application of the described bacterial strain of claim 1 in the production pyrroloquinoline quinone.
4. utilize the method for the described bacterial strain production of claim 1 pyrroloquinoline quinone.
5. method according to claim 4 is characterized in that, comprises the steps: the fermentation with MP688, and fermented liquid is wash-out behind weakly-basic anion displacement chromatography column chromatography, and the wash-out composition is through C
18Oppositely after the chromatography column absorption, use methanol-eluted fractions, elutriant concentrates and makes its spontaneous nucleation.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103224965A (en) * | 2013-03-27 | 2013-07-31 | 中国人民解放军军事医学科学院生物工程研究所 | Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same |
| CN103361390A (en) * | 2013-03-27 | 2013-10-23 | 中国人民解放军军事医学科学院生物工程研究所 | Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method |
| CN103740780A (en) * | 2013-12-20 | 2014-04-23 | 浙江大学 | Method for synthesizing pyrroloquinoline quinone through lactobacillus fermentation |
| CN104892597A (en) * | 2015-05-14 | 2015-09-09 | 郑州轻工业学院 | Complex extraction method for separation and purification of pyrroloquinoline quinine in fermentation broth |
| CN106755169A (en) * | 2016-12-14 | 2017-05-31 | 吴银娣 | A kind of method for improving PQQ yield |
| CN113337432A (en) * | 2021-06-04 | 2021-09-03 | 华熙生物科技股份有限公司 | Methylophagus for producing pyrroloquinoline quinone and application thereof |
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| CN103224965A (en) * | 2013-03-27 | 2013-07-31 | 中国人民解放军军事医学科学院生物工程研究所 | Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same |
| CN103361390A (en) * | 2013-03-27 | 2013-10-23 | 中国人民解放军军事医学科学院生物工程研究所 | Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method |
| CN103224965B (en) * | 2013-03-27 | 2015-04-01 | 中国人民解放军军事医学科学院生物工程研究所 | Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same |
| CN103740780A (en) * | 2013-12-20 | 2014-04-23 | 浙江大学 | Method for synthesizing pyrroloquinoline quinone through lactobacillus fermentation |
| CN103740780B (en) * | 2013-12-20 | 2016-09-14 | 浙江大学 | A kind of method by lactic acid bacteria fermentation synthesis pyrroloquinoline quinone |
| CN104892597A (en) * | 2015-05-14 | 2015-09-09 | 郑州轻工业学院 | Complex extraction method for separation and purification of pyrroloquinoline quinine in fermentation broth |
| CN104892597B (en) * | 2015-05-14 | 2016-05-04 | 郑州轻工业学院 | PQQ in complexing abstraction separation and purification zymotic fluid |
| CN106755169A (en) * | 2016-12-14 | 2017-05-31 | 吴银娣 | A kind of method for improving PQQ yield |
| CN113337432A (en) * | 2021-06-04 | 2021-09-03 | 华熙生物科技股份有限公司 | Methylophagus for producing pyrroloquinoline quinone and application thereof |
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