CN101157939A - Method for producing 1,3-methyl glycol by resting cell conversion glycerol - Google Patents
Method for producing 1,3-methyl glycol by resting cell conversion glycerol Download PDFInfo
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- CN101157939A CN101157939A CNA2007100464203A CN200710046420A CN101157939A CN 101157939 A CN101157939 A CN 101157939A CN A2007100464203 A CNA2007100464203 A CN A2007100464203A CN 200710046420 A CN200710046420 A CN 200710046420A CN 101157939 A CN101157939 A CN 101157939A
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Abstract
The invention discloses a method for producing 1, 3-propylene glycol through microorganism cell transformation of glycerine. The microorganism cells are resting cells. The innovation of the invention is that the resting cells is added into a converting system, thus being not necessary to worry about the influence of substrate, product and poisonous by-product to the growth of cells in the converting process; the processes of cell growth and glycerine converting can be optimized independently to achieve the optimum respectively, also Ingredients of converting liquid can be simplified to facilitate abstraction and purification of products; the conversion of resting cells can be regulated manually and thalline cell density in the converting liquid can be controlled so as to raise concentration of 1, 3-propylene glycol.
Description
Technical field
The invention belongs to technical field of bioengineering, specifically, is to produce 1 about a kind of microorganism cells glycerine converting, the method for ammediol.
Background technology
1, and ammediol (1,3-propanediol; being called for short 1,3-PD) being a kind of important chemical material, itself is good solvent, antifreezing agent, protective material and lubricant; and can participate in the number of chemical building-up reactions, as organic synthesis raw material and medicine intermediate and be subjected to using widely.Its most potential application is and terephthalic acid synthetic polyester (PTT).Studies show that in recent years, PTT is than having more excellent characteristic with ethylene glycol and terephthalic acid synthetic polyester (PET), recovery of elasticity as the nylon sample, in panchromatic scope, need not to add speciality chemical and can present good continuous printing and dyeing characteristic, the tint permanence of uvioresistant, ozone and oxynitrides, anti-internal stress, low water absorption, low static and good biological degradation, reusable edible etc.
At present, produce 1, the method for 3-PD mainly comprises oxirane carbonyl method, hydrolysis of acrolein method and microbe transformation method.Wherein oxirane carbonyl method, hydrolysis of acrolein are owned by France in chemical synthesis, and its raw material is mainly from oil.The microbial transformation rule is to be raw material with renewable resourcess such as glycerine, glucose, have traditional petroleum industry incomparable superiority, meet the needs of 21 century Sustainable development.
So far, also not finding has natural bacterial strain can directly utilize glucose fermentation to produce 1, ammediol, have only the minority bacterium can directly utilize glycerine to produce 1,3-PD, these bacterium comprise Cray Bai Shi pneumobacillus (Klebsiella pneumoniae), the short lactobacillus (Lactobacilli brevis) of Fu Shi lemon bacterium (Citrobacter freundii) and lactobacillus, Lactobacillus buchneri (Lactobacillibuchneri), the clostridium butyricum of fusobacterium (Clostridia butyricum) and Pasteur's fixed nitrogen clostridium (Clostridia pastruianu).Wherein Cray Bai Shi pneumobacillus, clostridium butyricum, Fu Shi lemon bacterium have higherly 1, and 3-PD transformation efficiency and production intensity are the focuses of current research.
Microorganism utilizes glycerine to synthesize 1, and ammediol is a disproportionation pathways metabolism, comprises an oxidation branch road and a reduction branch road.Oxidative pathway mainly contains following a few step composition: (1) glycerine forms otan (DHA) under the effect of glycerol dehydrogenase (GDH); (2) otan generates di(2-ethylhexyl)phosphate pyruvic alcohol (DHAP) under the effect of dihydroxyacetone kinase; (3) the di(2-ethylhexyl)phosphate pyruvic alcohol is transformed into glyceraldehyde 3-phosphate, and glyceraldehyde 3-phosphate generates phosphoenolpyruvic acid; (4) the phosphoenolpyruvic acid metabolism generates pyruvic acid, and pyruvic acid further is metabolized to ethanol, lactic acid, acetate, butyric acid, end products such as succsinic acid again.The reduction approach mainly contains two step enzyme reactions and forms: (1) glycerol dehydratase (GDHt) is an intermediate product 3-hydroxy propanal (3-HPA) with transformation of glycerol; (2) the 3-hydroxy propanal is 1, and the effect of 3-PD oxydo-reductase (PDOR) generates purpose product 1,3-PD down.
Because continuing of oil price is soaring, and biological process prepares 1, ammediol further received compatriots' concern in recent years.But from domestic present present Research, utilize microbial fermentation glycerine to produce 1, ammediol also exists some problems:
(1) price of refining glycerine is higher on the market, and transformation of glycerol produces 1, and 3-PD and chemical method are than not having advantage on the cost.And if heavy addition glucose, cheap carbon sources such as sucrose because Pasteur effect, thalline can't utilize glycerine can not produce 1,3-PD.
(2), make product be difficult to improve to the transformation efficiency of glycerine because the growth needs of cell consumes substrate glycerine.
(3) substrate glycerine, product 1,3-PD, by product lactic acid, acetate, butyric acid, and the growth that poisonous intermediate metabolites such as 3-hydroxy propanal all can pair cell has a negative impact make that cell concentration is difficult to improve in the fermented liquid, thereby production concentration is generally not high yet.
(4) because the inoculum complicated component causes product separation and purification difficulty, the solution of these problems also needs the proposition and the improvement of new method for transformation.
Summary of the invention
The objective of the invention is to produce 1 at microbial fermentation glycerine, the problem that ammediol exists provides a kind of new microorganism cells glycerine converting to produce 1, the method for ammediol.
The invention provides a kind of microorganism cells glycerine converting and produce 1, the method for ammediol, described microorganism cells is a resting cell.
Wherein, described microorganism is selected from: Cray Bai Shi pneumobacillus (Klebsiella pneumoniae), Fu Shi lemon bacterium (Citrobacter freundii), clostridium butyricum (Clostridiabutyricum), Pasteur's fixed nitrogen clostridium (Clostridia pastruianu), and their genetic engineering bacterium.
Resting cell of the present invention is on-fixed cell or immobilized cell, and the preparation process of described immobilized cell is: mix in the agar-agar soln with resting cell adding fusing, promptly make immobilized cell after solidifying.
Microorganism cells glycerine converting of the present invention produces 1, and the preparation process of ammediol is:
In the conversion fluid that contains glycerine 5-120g/L, add resting cell 5-100g/L, 20-42 ℃ transforms 4-60h.
According to the present invention, in the process of described glycerine converting, bubbling air or nitrogen.
Resting cell of the present invention prepares by the following method:
Microbial strains is inoculated in substratum, and low-temperature centrifugation is collected bacterial sediment behind the cultivation certain hour, with the phosphoric acid buffer washing, promptly obtains resting cell.
According to the present invention, contain glycerine and/or non-glycerine carbon source in the described substratum.
According to the present invention, described non-glycerine carbon source comprises dextrose plus saccharose.
The present invention produces 1 with conversion of resting cells glycerine, and ammediol produces 1 with using the grown cell ferment glycerin, and ammediol is compared has following advantage:
What (1) add in the transformation system is resting cell, so conversion process needn't be worried substrate, product and the influence that the toxic byproduct cell growth is arranged;
(2) the present invention can utilize glucose, sucrose etc. to make auxiliary carbon source and cultivate thalline, thereby improves the cell concentration in the nutrient solution and reduce production costs;
(3) usually the condition of cell growth and glycerine converting is not in full accord, and these two processes are isolating among the present invention, thus cell is grown and the process of glycerine converting can independent optimization in the hope of reaching optimum condition separately;
(4) because cell growth process can produce a large amount of intermediate metabolitess, thereby and the growth of cell separates the composition that can simplify conversion fluid and is beneficial to the product separation purifying with transformation of glycerol;
(5) glycerol fermentation produces 1, during 3-PD since glycerine be not its suitableeest carbon source, and multiple product by product all has inhibition to thalli growth, so thalli growth is slow, and bacteria concentration is lower, so 1,3-PD output also is difficult to improve, and conversion of resting cells can be regulated and control in the conversion fluid somatic cells concentration artificially to improve product 1,3-PD concentration, among the present invention 1,3-PD concentration can improve 1.9 times with grown cell ferment glycerin output maximum.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
Among the present invention, " resting cell " is meant the microorganism cells after being cultured to certain hour on the growth medium, collects and wash with damping fluid with appropriate means, removed the microorganism cells of nutritive ingredient.
In following examples, 1, the mensuration of 3-PD concentration adopts gas-chromatography (GC) analytical procedure, and chromatographic operational condition is as follows:
Chromatogram model: Agilent 6820
Chromatographic column: DB-WAX (30m * 0.53mm * 1.0 μ m)
Chromatographic working station: Sepu 3000 (Hangzhou Pu Hui scientific instrument company limited)
Carrier gas: nitrogen
Flow velocity: 1ml/min
Sample size: 0.5 μ l
Splitting ratio: 10: 1
Detector: hydrogen flame detector (FID)
Column temperature: 100 ℃ kept 1 minute, then with 20 ℃/min gradient increased temperature to 220 ℃
Injector temperature: 250 ℃
Detector temperature: 280 ℃
With 1, the 4-butyleneglycol is interior mark, uses inner mark method ration.
In following examples, the construction process of genetic engineering bacterium is: utilize round pcr to amplify the coding 1 of 1.14kb from Cray Bai Shi pneumobacillus (Klebsiella pneumoniae), the gene dhaT of ammediol oxydo-reductase, be connected on the expression vector pKM13, obtain recombinant plasmid pKM13-dhaT, with recombinant plasmid transformed Cray Bai Shi pneumobacillus (Klebsiella pneumoniae) ATCC25955, the Cray Bai Shi pneumobacillus ATCC25955/pKM13-dhaT that obtains recombinating is the genetic engineering bacterium that uses among the embodiment.
Concrete building process is:
(1) clone of gene dhaT:
Design two primers according to Cray Bai Shi pneumobacillus dhaT gene order, its sequence is as follows:
Primer 1:5 '-TGC
TCTAGAATGAGCTATCGTATGTTTGAT-3 '
Primer 2: 5 '-CCC
AAGCTTTCAGAATGCCTGGCGGAAAAT-3 '
With Cray Bai Shi pneumobacillus genomic dna is template, the pcr amplification goal gene
PCR reaction system (50 μ l): 10x GC Buffer 5 μ l, 2.5mmol/1 DNTPs 4 μ l, template DNA 1 μ l, Taq DNAPolymerasel μ l, each 0.1nM of primer 1 and primer 2, distilled water supply 50 μ l.
Reaction conditions: 95 ℃ of sex change 5min; 95 ℃ of 50s, 55 ℃ of 90s, 72 ℃ of 5min, 35 circulations, 72 ℃, 10min.
Obtaining the electrophoretic analysis of PCR product confirms, behind PCR product purification test kit (vast Tyke) purifying, the EcoRI enzyme is cut, reclaim the wherein fragment of 1.14kb, connection carrier pKM13 (Ping Zheng, Kirsten Wereath, et al., Process Biochemistry 2006 41:2160-2169), obtains recombinant plasmid pKM13-dhaT.
(2) expression of gene:
Recombinant plasmid pKM13-dhaT electricity is transformed into Cray Bai Shi pneumobacillus ATCC25955, and coating contains the kantlex flat board, the picking positive transformant, and Cray Bai Shi pneumobacillus ATCC25955/pKM13-dhaT obtains recombinating.
Embodiment 1, seed culture
Seed culture based formulas (g/L): peptone 10, yeast powder 5, NaCl 10, transfer pH to 7.0 with 2mol/L KOH, 115 ℃ of sterilization 20min.
Get the 250ml triangular flask, seed culture medium liquid amount 50ml presses shown in the table 1, respectively inserts the bacterial classification of a ring slant preservation with transfering loop, and culture temperature 30-37 ℃, bubbling air or nitrogen carry out aerobic or anaerobism and cultivates 10-15h, shaking speed 200r/min.
Described bacterial classification is: Cray Bai Shi pneumobacillus (Klebsiella pneumoniae) ATCC25955, Fu Shi lemon bacterium (Citrobacter freundii) DSM30040, clostridium butyricum (Clostridiabutyricum) DSM5431, Pasteur's fixed nitrogen clostridium (Clostridia pastruianu) DSM525, genetic engineering bacterium Cray Bai Shi pneumobacillus (Klebsiella pneumoniae) ATCC25955/pKM13-dhaT of structure.
Table 1, seed culture condition
| # | Insert bacterial classification | Culture temperature (℃) | Training method | Incubation time (h) | Shaking speed (rpm) |
| 1 | Cray Bai Shi pneumobacillus | 37 | Aerobic | 10 | 200 |
| 2 | Fu Shi lemon bacterium | 30 | Anaerobism | 12 | 200 |
| 3 | Clostridium butyricum | 28 | Anaerobism | 15 | 200 |
| 4 | Pasteur's fixed nitrogen clostridium | 30 | Anaerobism | 12 | 200 |
| 5 | Genetic engineering bacterium | 37 | Aerobic | 12 | 200 |
Embodiment 2, yeast culture
Yeast culture based formulas (g/L): KCl0.75, NaH
2PO
42H
2O1.5, (NH
4)
2SO
42.35, Na
2SO
40.28, MgSO
47H
2O 0.26, citric acid 0.62, and corn steep liquor 4.0, glycerine 2-80, glucose/sucrose 0-15, each 0.3ml of trace element is with the KOH accent pH to 7.0 of 2mol/L.
Wherein said trace element solution is formed (g/L): ZnCl
234.2, FeCl
36H
2O 2.7, MnCl
24H
2O 10, CuCl
22H
2O 0.85, CoCl
26H
2O 23.8, H
3BO
30.31, Na
2MoO
42H
2O0.25.
Get the 500ml triangular flask, substratum liquid amount 200ml presses condition shown in the table 2, insert the seed liquor that embodiment 1 obtains respectively, inoculum size 5-10%, culture temperature 30-37 ℃, bubbling air or nitrogen carry out aerobic or anaerobism is cultivated 10-18h, shaking speed 200r/min.
Table 2, yeast culture
| # | Substratum glycerol content (g/L) | Glucose/sucrose and content (g/L) | Inoculum size (%) | Culture temperature (℃) | Incubation time (h) | Training method |
| 1 | 5 | Glucose (15) | 3 | 37 | 10 | Anaerobism |
| 2 | 10 | - | 5 | 30 | 15 | Anaerobism |
| 3 | 20 | - | 5 | 30 | 18 | Anaerobism |
| 4 | 10 | Sucrose (10) | 10 | 30 | 15 | Anaerobism |
| 5 | 20 | - | 5 | 37 | 12 | Aerobic |
Embodiment 3, resting cell preparation
Get the nutrient solution that embodiment 2 obtains, in 15-20 ℃, the centrifugal 5min of 8000rpm, the bacterial sediment of gained washs three times with the phosphate buffer solution of pH7.00, is to transform through the microorganism cells that washs the back gained and uses resting cell.
Get sample No. 3, make the agar gel immobilized cell, specific as follows:
Take by weighing 2.0g agar in the 100ml small beaker, add water 50ml, after the heat fused, be cooled to about 50 ℃ on the fire, add the above-mentioned resting cell that makes of 12g, mix, pour in the plate, being cut into size after fully solidifying is 3 * 3 * 3mm
3Bulk, deionized water wash 3 times makes to transform and uses the immobilization resting cell.
Embodiment 4, conversion of resting cells glycerine produces 1,3-PD
As shown in table 3, in pack into the glycerine solution 200ml of 5-120g/L of 1000ml triangular flask, add resting cell or immobilization resting cell 5-100g/L that embodiment 3 obtains, the 200r/min vibration, 20-42 ℃, bubbling air or nitrogen carry out aerobic or anaerobism transforms 4-60h, the centrifugal 5min of 12000rpm, get supernatant liquor and measure 1,3-PD concentration the results are shown in Table 3.
Table 3, conversion of resting cells glycerine produce 1,3-PD
| # | Glycerol content in the conversion fluid (g/L) | Resting cell or immobilized cell and dosage (g/L) | Invert point (℃) | Transformation time (h) | Transform mode | 1,3-PD concentration (g/L) |
| 1 | 40 | Resting cell (18) | 37 | 15 | Aerobic | 28.2 |
| 2 | 120 | Resting cell (100) | 30 | 60 | Anaerobism | 44.5 |
| 3 | 20 | Immobilized cell (12) | 42 | 10 | Anaerobism | 11.8 |
| 4 | 5 | Resting cell (5) | 20 | 4 | Anaerobism | 3.2 |
| 5 | 60 | Resting cell (20) | 37 | 18 | Aerobic | 35.3 |
| Mean value | 24.6 |
Embodiment 5, grown cell batch fermentation glycerine produces 1,3-PD (in contrast)
Fermentative medium formula (g/L): KCl 0.75, NaH
2PO
42H
2O1.5, (NH
4)
2SO
42.35, Na
2SO
40.28, MgSO
47H
2O 0.26, citric acid 0.62, and corn steep liquor 4.0, glycerine 5-120g/L, each 0.3ml of trace element is with the KOH accent pH to 7.0 of 2mol/L.
Wherein said trace element solution is formed (g/L): ZnCl
234.2, FeCl
36H
2O 2.7, MnCl
24H
2O 10, CuCl
22H
2O 0.85, CoCl
26H
2O 23.8, H
3BO
30.31, Na
2MoO
42H
2O0.25.
Get the 1000ml triangular flask, substratum liquid amount 200ml presses shown in the table 4, inserts the seed liquor that the method by embodiment 1 obtains, inoculum size 10%, and culture temperature 20-42 ℃, bubbling air or nitrogen carry out aerobic or anaerobically fermenting, shaking speed 200r/min.
Table 4, grown cell batch fermentation glycerine produce 1,3-PD
| # | Fermented liquid glycerol content (g/L) | Inoculum size (%) | Leavening temperature (℃) | Fermentation time (h) | Transform mode | 1,3-PD concentration (g/L) |
| 1 | 40 | 3 | 37 | 22 | Aerobic | 15.1 |
| 2 | 120 | 5 | 30 | 72 | Anaerobism | 31.4 |
| 3 | 20 | 5 | 42 | 18 | Anaerobism | 8.9 |
| 4 | 5 | 10 | 20 | 12 | Anaerobism | 2.7 |
| 5 | 60 | 5 | 37 | 30 | Aerobic | 21.3 |
| Mean value | 15.9 |
By the result of table 3, table 4 as can be seen, under the situation that contains same amount glycerine, the immobilized cell that utilizes resting cell or make by resting cell produce 1, ammediol concentration is all than obtaining 1 with the grown cell fermentative production, the final concentration height of ammediol, 1.5 times of average out to, reach as high as 1.9 times, improved 1 greatly, the yield of ammediol, this be because:
What (1) add in the transformation system is resting cell, so conversion process needn't be considered substrate, product and the influence that the toxic byproduct cell growth is arranged;
(2) growth and to transform two processes be isolating among the present invention, thus the process of cell growth and glycerine converting can independent optimization in the hope of reaching optimum condition separately;
(3) conversion of resting cells can be regulated and control in the conversion fluid somatic cells concentration artificially to improve product 1,3-PD concentration;
(4) the present invention can utilize glucose, sucrose etc. to make auxiliary carbon source and cultivate thalline (wherein need add the generation that an amount of glycerine is induced glycerine metabolic enzyme system), thereby improves the cell concentration in the nutrient solution and reduce production costs.
Claims (10)
1. a microorganism cells glycerine converting produces 1, and the method for ammediol is characterized in that, described microorganism cells is a resting cell.
2. the method for claim 1, it is characterized in that, described microorganism is selected from: Cray Bai Shi pneumobacillus (Klebsiella pneumoniae), Fu Shi lemon bacterium (Citrobacter freundii), clostridium butyricum (Clostridia butyricum), Pasteur's fixed nitrogen clostridium (Clostridiapastruianu), and their genetic engineering bacterium.
3. the method for claim 1 is characterized in that, described resting cell is the on-fixed cell.
4. the method for claim 1 is characterized in that, described resting cell is an immobilized cell.
5. method as claimed in claim 4 is characterized in that, the preparation process of described immobilized cell is: mix in the agar-agar soln with resting cell adding fusing, promptly make immobilized cell after solidifying.
6. the method for claim 1 is characterized in that, described microorganism cells glycerine converting produces 1, and the process of ammediol is:
In the conversion fluid that contains glycerine 5-120g/L, add resting cell 5-100g/L, 20-42 ℃ transforms 4-60 hour.
7. method as claimed in claim 6 is characterized in that, bubbling air or nitrogen in the described preparation process.
8. the method for claim 1 is characterized in that, described resting cell prepares by the following method:
Microbial strains is inoculated in substratum, and low-temperature centrifugation is collected bacterial sediment behind the cultivation certain hour, with the damping fluid washing, promptly obtains resting cell.
9. method as claimed in claim 8 is characterized in that, contains glycerine and/or non-glycerine carbon source in the described substratum.
10. method as claimed in claim 9 is characterized in that, described non-glycerine carbon source comprises dextrose plus saccharose.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011042434A1 (en) * | 2009-10-05 | 2011-04-14 | Metabolic Explorer | 1,3-propanediol production with immobilised cells reactors |
| CN108060203A (en) * | 2018-01-03 | 2018-05-22 | 江苏大学 | A kind of method of full mixing with cells glycerine converting production 1,3- propylene glycol |
-
2007
- 2007-09-26 CN CNA2007100464203A patent/CN101157939A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011042434A1 (en) * | 2009-10-05 | 2011-04-14 | Metabolic Explorer | 1,3-propanediol production with immobilised cells reactors |
| CN108060203A (en) * | 2018-01-03 | 2018-05-22 | 江苏大学 | A kind of method of full mixing with cells glycerine converting production 1,3- propylene glycol |
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