CN105255960B - A kind of method that precursor adding synthetic prepares pyrroloquinoline quinone - Google Patents
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Abstract
The invention discloses a kind of utilize somatic cells crush supernatant and add pyrroloquinoline quinone synthesis precursor synthetic polypeptide, thus the method synthesizing pyrroloquinoline quinone in vitro, pyrroloquinoline quinone producing strains is accessed in fermentation medium according to the ratio of 5%, at 28 DEG C, 3d is cultivated in concussion, it is thus achieved that bacteria suspension；Bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, is suspended by thalline with buffer, then crushes by ultrasonic method, through high speed centrifugation, takes supernatant；The precursor of synthetic is joined in supernatant, under 35 50 DEG C of constant temperatures, reacts 12 26h.The method condition is simple, polyvinyl chloride, it is simple to large-scale industrial production, and the industrialization to promoting pyrroloquinoline quinone is significant.
Description
Technical field
The present invention relates to biological chemical field, be specifically related to before one utilizes somatic cells to crush supernatant and add PQQ
Body thing artificial synthetic polypeptide, thus the method synthesizing pyrroloquinoline quinone in vitro.
Background technology
Pyrroloquinoline quinone (Pyrroloquinoline quinine, PQQ) is a kind of water solublity quinones, is Fructus Vitis viniferae
The coenzyme of glucocorticoid dehydrogenase and ethanol dehydrogenase is it is considered to be new vitamin B group (Nature 2003；422:832).PQQ molecule
Amount is 330, is to find in microorganism the earliest, and research subsequently shows that it exists in animal and plant body, structural formula such as following formula institute
Show
It has proved that PQQ has important physiological function, such as maintain skin health, stimulate nerve growth factor, enhancing
Immunologic function, promote that cell growth, antioxidation, removing free radical, enhancing antibacterial are to the toleration of extreme environmental conditions and ginseng
With cellular signal transduction etc..At field of medicaments, PQQ has preventing and treating hepatic injury, protection nervous tissue, stimulation energy generation, strengthening
The physiological functions such as immunity.Therefore, it can for treating parkinsonism, senile dementia, heart disease, liver cirrhosis etc..Poultry
In aquaculture, PQQ as a kind of novel biostearin material, can promote body growth, improve breeding, antioxidation, anti-stress and
Strengthen immunity, in feedstuff, such as add appropriate PQQ can improve laying rate of laying hen and egg quality to a certain extent, and
The oxidation resistance of laying hen can be significantly improved.Due to physicochemical property and various physiological function of its uniqueness, can be used for food, doctor
The fields such as medicine, agricultural, industry, have development prospect widely.
PQQ produces based on chemical synthesis process in early days, but synthesis step is many, and productivity is low, going of isomer and side-product
Except needing more purification, and need to pollute with multiple toxic reagent environment (JACS, 1981；103:5599-2600).Therefore, one
As think synthetic method more industrialization meaning biology.Up to now, PQQ is principally found in gram-negative bacteria, such as first
Base shaft Pseudomonas (Methylobacterium), Bacterium gluconicum belong to (Gluconobacter), Rhodopseudomonas
(Pseudomonas) etc..Different strain PQQ synthetic quantity is different, and some strain produces trace PQQ and supplies physiological metabolism demand,
Also some bacterium but can produce excess PQQ, and is secreted into outside born of the same parents.At present, the method using fermentable is had to produce the report of PQQ
Road, but PQQ yield is the lowest, only 0.07-7mg/L.Accordingly, it would be desirable to develop new technology, produce PQQ in a large number, thus meet
Production requirement.
External synthetic technology is a kind of novel biosynthesis technology, have substrate convert that controlling is good, seriality the most also
And toleration is strong.Compared with traditional zymotic technology, can effectively overcome the target product speed limit effect to synthetic reaction, thus greatly
The big yield improving product.At present, this technology has been used in the production of some chemical products.Such as, Waleed Ahmad
Khattak etc. report the technology utilizing cell-free system technology to produce ethanol.
Summary of the invention
It is an object of the invention to provide a kind of method that precursor adding synthetic prepares pyrroloquinoline quinone, the method
Condition is simple, polyvinyl chloride, it is simple to large-scale industrial production.
For solve above-mentioned technical problem, the present invention by the following technical solutions:
A kind of method that precursor adding synthetic prepares pyrroloquinoline quinone, comprises the steps:
(1) accessing in fermentation medium by pyrroloquinoline quinone producing strains according to the ratio of 5%, at 28 DEG C, concussion is cultivated
3d, it is thus achieved that bacteria suspension；
(2) bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, is suspended by thalline with buffer, then crushes, through height by ultrasonic method
Speed is centrifugal, takes supernatant；
(3) precursor of synthetic is joined in supernatant, under 35-50 DEG C of constant temperature, react 12-26h.
Described in described step (1), pyrroloquinoline quinone producing strains is Methylobacterium extorquens,
Klebsiella Pneumonia, Gluconobacter oxidans, Pseudomonas aeruginosa,
Bradyrhizobium sp. or Streptomyces rochei.
Buffer in described step (2) is phosphate buffer or citric acid-sodium citrate buffer, and the pH of buffer is
4.5-7.5。
In described step (3), the precursor of synthetic is one section of polypeptide, its sequence signature such as SEQ ID No.1, SEQ
Shown in ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6.
In described step (3), the precursor concentration of added synthetic is 1-30mmol/L.
Beneficial effects of the present invention: the present invention utilizes somatic cells to crush supernatant and to add PQQ precursor artificial
Synthesis polypeptide, thus synthesizes pyrroloquinoline quinone (PQQ) in vitro, conversion ratio up to 70-80%, PQQ yield up to 600mg/L,
And utilizing said method, condition is simple, polyvinyl chloride, it is simple to large-scale industrial production, and the industrialization to promoting PQQ has
Significance.
Accompanying drawing explanation
Fig. 1 be PQQ standard substance m/z under SMR pattern be the fragments characteristic quasi-molecular ions chromatogram of 285；
Fig. 2 be product m/z under SMR pattern be the fragments characteristic quasi-molecular ions chromatogram of 285；
Fig. 3 is the ultraviolet spectra at target product peak, and wherein, A is standard substance, and B is the sample that embodiment 1 prepares；
Fig. 4 is the mass spectrum at target product peak, and wherein, A is standard substance, and B is the sample that embodiment 1 prepares.
Detailed description of the invention
Embodiment 1
The method of the present embodiment " precursor adding synthetic prepares pyrroloquinoline quinone ", step is as follows:
(1) Gluconobacter oxvdans fermentation: according to the ratio of 5%, strain is accessed fermentation medium, and (every liter contains 40g
Sorbitol, 20g yeast extract, 5g (NH4)2SO4、2g KH2PO4、5g MgSO4·H2O), in, at 28 DEG C, 3d is cultivated in concussion.
According to 10% inoculation during fermentor cultivation, culture medium concentration is normal 2 times.Strain is seeded to enrichment medium, shakes at 28 DEG C
Swing cultivation 5d.
(2) biosynthesis Establishing: bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, will with the phosphate buffer that pH is 6.5
Thalline suspends, and then crushes by ultrasonic method, through high speed centrifugation, takes supernatant；
(3) biosynthesis pyrroloquinoline quinone: add the polypeptide SEQ ID of the synthetic of 30mmol/L in supernatant
No.1, reacts 12 hours in the temperature constant magnetic stirring reactor of 35 DEG C.Product analysis
The LC-MS detection of 4.1 products
Use the target product in LC-MS/MS method detection reaction system.Wherein, analysis condition controls as follows:
A) liquid-phase chromatographic analysis condition
Chromatographic column: ODS post
Flowing phase: acetonitrile-water (containing 0.1-0.2% formic acid)
Gradient elution: 0-10min, in flowing mutually, acetonitrile volumetric concentration is 30%-90%
Flow velocity: 100-200 μ L/min
Column temperature: 20-26 DEG C
B) mass spectral analysis condition
Ion source: ESI takes off solution temperature
Scan mode: anion dry gas stream speed: 5-7L/min
Sheath temperature: 350-410 DEG C of capillary voltage: 2500-3200v
Sheath gas velocity: 500-600L/h collision energy: 10-35V
Taper hole voltage: 2.5-3.5V mass scan range: 100-1700m/z, every 0.2s gathers 1 collection of illustrative plates.
By reactant liquor carries out LC-MS/MS method analysis, result shows, containing PQQ in product.By marking with PQQ
Quasi-quality modal data contrasts, and result shows, sample is completely the same with it.
Embodiment 2
The method that the precursor adding synthetic of the present embodiment prepares pyrroloquinoline quinone, step is as follows:
(1) demethylation bacillus (Methylobacterium extorquens AM1) fermentation is turned round: by strain according to 5%
Ratio accesses fermentation medium, and (every liter contains 3g (NH4)2HPO4 2g、K2HPO4、1g NaCl、10ug FeSO4·7H2O、0.2g
MgSO4·H2O、MnSO4·4H2O、Thiamine10ug、Riboflavin 20ug、Calcium pantothenate 20ug、
Pyridoxine·HCl20ug、Biotin 1ug、p-aminobenzoic acid 10ug、Nicotinic acid 20ug)
In, at 28 DEG C, 3d is cultivated in concussion.According to 10% inoculation during fermentor cultivation, culture medium concentration is normal 2 times.Strain is inoculated
To enrichment medium, at 28 DEG C, 5d is cultivated in concussion.
(2) biosynthesis Establishing: bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, will with the phosphate buffer that pH is 4.5
Thalline suspends, and then crushes by ultrasonic method, through high speed centrifugation, takes supernatant；
(3) biosynthesis pyrroloquinoline quinone: add the SEQ ID of 20mmol/L synthetic in above-mentioned supernatant
No.2, reacts 16 hours in the temperature constant magnetic stirring reactor of 37 DEG C.
Product analysis method is with embodiment 1.
By reactant liquor carries out LC-MS/MS method analysis, result shows, containing PQQ in product.By marking with PQQ
Quasi-quality modal data contrasts, and result shows, sample is completely the same with it.
Embodiment 3
The method that the precursor adding synthetic of the present embodiment prepares pyrroloquinoline quinone, step is as follows:
(1) Cray primary formula pneumobacillus (Klebsiella pneumoniae CICC 10011) fermentation: by strain according to
The ratio of 5% accesses fermentation medium, and (every liter contains glucose 100g, Semen Maydis powder 6g, KCl 0.4g, CaCl210mg、0.1g
MgSO4In), at 28 DEG C, 3d is cultivated in concussion.According to 10% inoculation during fermentor cultivation, culture medium concentration is normal 2 times.By bacterium
Planting and be seeded to enrichment medium, at 28 DEG C, 5d is cultivated in concussion.
(2) biosynthesis Establishing: bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, with citric acid-Fructus Citri Limoniae that pH is 7.5
Thalline is suspended by acid sodium buffer, then crushes by ultrasonic method, through high speed centrifugation, takes supernatant；
(3) biosynthesis pyrroloquinoline quinone: add the SEQ ID of 15mmol/L synthetic in above-mentioned supernatant
No.3, reacts 18 hours in the temperature constant magnetic stirring reactor of 42 DEG C.
Product analysis method is with embodiment 1.
By contrasting with PQQ standard quality modal data, result shows, sample is completely the same with it.
Embodiment 4
The method that the precursor adding synthetic of the present embodiment prepares pyrroloquinoline quinone, step is as follows:
(1) sieve formula streptomycete (Streptomyces rochei CGMCC 4.6554) fermentation: by strain according to 5% ratio
Example access fermentation medium (every liter containing starch 2g, cotton seed meal 0.5g, glucose 0.5g, Semen Maydis pulp 0.5g, yeast powder 0.5g,
Calcium carbonate 0.2g) in, at 28 DEG C, 3d is cultivated in concussion.According to 10% inoculation during fermentor cultivation, culture medium concentration is normal 2
Times.Strain is seeded to enrichment medium, and at 28 DEG C, 5d is cultivated in concussion.
(2) biosynthesis Establishing: bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, with the phosphate buffer that pH is 6 by bacterium
Body suspends, and then crushes by ultrasonic method, through high speed centrifugation, takes supernatant；
(3) biosynthesis pyrroloquinoline quinone: add the SEQ ID of 10mmol/L synthetic in above-mentioned supernatant
No.4, reacts 20 hours in the temperature constant magnetic stirring reactor of 45 DEG C.
Product analysis method is with embodiment 1.
By contrasting with PQQ standard quality modal data, result shows, sample is completely the same with it.
Embodiment 5
The method that the precursor adding synthetic of the present embodiment prepares pyrroloquinoline quinone, step is as follows:
(1) pseudomonas aeruginosa (Pseudomonas aeruginosa PAO1) fermentation: by strain according to 5% ratio
Example accesses fermentation medium, and (every liter contains Na2HPO4 12g、KH2PO4 5g、NH4Cl 1g、MgSO4·7H2O 0.5g、CaCl2
In 0.005g), at 28 DEG C, 3d is cultivated in concussion.According to 10% inoculation during fermentor cultivation, culture medium concentration is normal 2 times.Will
Strain is seeded to enrichment medium, and at 28 DEG C, 5d is cultivated in concussion.
(2) biosynthesis Establishing: bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, with citric acid-citric acid that pH is 7
Thalline is suspended by sodium buffer, then crushes by ultrasonic method, through high speed centrifugation, takes supernatant；
(3) biosynthesis pyrroloquinoline quinone: add the SEQ ID No.5 of 5mmol/L synthetic in above-mentioned supernatant,
The temperature constant magnetic stirring reactor of 24 DEG C reacts 26 hours.
Product analysis method is with case study on implementation 1
By contrasting with PQQ standard quality modal data, result shows, sample is completely the same with it.
Embodiment 6
The method that the precursor adding synthetic of the present embodiment prepares pyrroloquinoline quinone, step is as follows:
(1) chronic root nodule bacteria (Bradyrhizobium sp.BTAi1) fermentation: strain is accessed according to the ratio of 5% and sends out
(every liter contains sucrose 40g, peptone 6g, yeast extract 34g, KH to ferment culture medium2PO4 0.4g,K2HPO40.4g,(NH4)2SO4
6g,MgSO4In 75mg), at 28 DEG C, 3d is cultivated in concussion.According to 10% inoculation during fermentor cultivation, culture medium concentration is normal
2 times.Strain is seeded to enrichment medium, and at 28 DEG C, 5d is cultivated in concussion.
(2) biosynthesis Establishing: bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, will with the phosphate buffer that pH is 6.2
Thalline suspends, and then crushes by ultrasonic method, through high speed centrifugation, takes supernatant；
(3) biosynthesis pyrroloquinoline quinone: add the SEQ ID No.6 of 1mmol/L synthetic in above-mentioned supernatant,
The temperature constant magnetic stirring reactor of 50 DEG C reacts 24 hours.
Product analysis method is with case study on implementation 1
By contrasting with PQQ standard quality modal data, result shows, sample is completely the same with it.
The ultimate principle of the present invention and principal character and advantages of the present invention have more than been shown and described.The technology of the industry
Personnel, it should be appreciated that the present invention is not restricted to the described embodiments, simply illustrating this described in above-described embodiment and description
The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and these become
Change and improvement both falls within scope of the claimed invention.Claimed scope by appending claims and
Equivalent defines.
Claims (3)
1. the method that the precursor adding synthetic prepares pyrroloquinoline quinone, it is characterised in that comprise the steps:
(1) accessing in fermentation medium by pyrroloquinoline quinone producing strains according to the ratio of 5%, at 28 DEG C, 3d is cultivated in concussion, it is thus achieved that
Bacteria suspension；
(2) bacteria suspension is through high speed centrifugation, it is thus achieved that thalline, is suspended by thalline with buffer, then crushes by ultrasonic method, through at a high speed from
The heart, takes supernatant；
(3) precursor of synthetic is joined in supernatant, under 35-50 DEG C of constant temperature, react 12-26h；Described step
Suddenly in (3), the precursor concentration of added synthetic is 1-30mmol/L；
In described step (3), the precursor of synthetic is one section of polypeptide, its sequence signature such as SEQ ID No.1, SEQ ID
Shown in No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6.
The method that the precursor of interpolation synthetic the most according to claim 1 prepares pyrroloquinoline quinone, it is characterised in that:
Described in described step (1), pyrroloquinoline quinone producing strains isMethylobacterium extorquens, Klebsiella Pneumonia, Gluconobacter oxidans, Pseudomonas aeruginosa, Bradyrhizobium sp. or Streptomyces rochei。
The method that the precursor of interpolation synthetic the most according to claim 1 prepares pyrroloquinoline quinone, it is characterised in that:
Buffer in described step (2) is phosphate buffer or citric acid-sodium citrate buffer, and the pH of buffer is 4.5-7.5.
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| PCT/CN2016/104219 WO2017076273A1 (en) | 2015-11-06 | 2016-11-01 | Method for preparing pyrroloquinoline quinine by adding artificially synthesized precursor |
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| CN102816780A (en) * | 2012-09-07 | 2012-12-12 | 天津大学 | Combination gene of gene cluster ABCDEN by synthesizing sorbose dehydrogenase genes, sorbosone dehydrogenase genes and pyrroloquinoline quinone |
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Non-Patent Citations (9)
| Title |
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| WP_002905689;无;《GenBank》;20150322;序列 * |
| WP_003106462;无;《GenBank》;20150322;序列 * |
| WP_010502998;无;<GenBank>;20150322;序列 * |
| WP_011113089;无;《GenBank》;20130515;序列 * |
| WP_012029362;无;《GenBank》;20141225;序列 * |
| WP_012317526;无;《GenBank》;20150911;序列 * |
| 吡咯喹啉醌研究进展;杨璐;《生物技术通讯》;20091130;第877-878页 * |
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| 新辅酶吡咯喹啉醌研究进展;周怡雯;《中国生化药物杂志》;20080820;第279-280页 * |
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