CN101781398B - Enzyme method for continuously producing poly(Epsilon-caprolactone) - Google Patents

Enzyme method for continuously producing poly(Epsilon-caprolactone) Download PDF

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CN101781398B
CN101781398B CN 200910045661 CN200910045661A CN101781398B CN 101781398 B CN101781398 B CN 101781398B CN 200910045661 CN200910045661 CN 200910045661 CN 200910045661 A CN200910045661 A CN 200910045661A CN 101781398 B CN101781398 B CN 101781398B
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caprolactone
epsilon
lypase
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packed bed
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CN101781398A (en
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魏东芝
林金萍
张敏洁
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East China University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • C12M25/18—Fixed or packed bed

Abstract

The invention provides an enzyme method for continuously producing poly(Epsilon-caprolactone), comprising the steps of resolving the Epsilon-caprolactone in proper organic solvent to prepare the solution of Epsilon-caprolactone; enabling the solution of Epsilon-caprolactone flow over a lipase packed bed to do polymerization reaction so as to obtain the poly(Epsilon-caprolactone). The invention further provides a lipase packed bed reactor for continuously producing the poly(Epsilon-caprolactone), which is applied to the lipase packed bed for catalyzing polymerization reaction, a mixing tank for mixing the Epsilon-caprolactone and the organic solvent, and a peristaltic pump for sending the Epsilon-caprolactone to the lipase packed bed. The method provided by the invention realizes continuous production and is characterized by mild reacting condition, low power consumption, excellent product quality and low production cost.

Description

A kind of enzyme process continuous production gathers the method for (6-caprolactone)
Technical field
The invention belongs to biological chemical field, specifically, relate to the method that a kind of enzyme process continuous production gathers (6-caprolactone).
Background technology
Gather (6-caprolactone) and have application very widely, the present container that uses the product that gathers (6-caprolactone) to comprise toiletry bottle, flexible packages and trees plantation and transplant etc.; Simultaneously, gather (6-caprolactone) and multiple superpolymer and have good consistency, can prepare the blend of multiple excellent property, for example utilize it with the blend manufacturing environment-friendly type shopping bag of starch etc.; In addition, have non-toxicity, histocompatibility and numerous medicines are had high osmosis owing to gather (6-caprolactone), it is remarkable day by day that the function of its biological medicine also becomes.
At present, gather synthetic main chemical method and the catalyzed by biological enzyme of (6-caprolactone) the catalysis ring-opening polymerization is realized under body or solution condition, specific as follows:
Figure G2009100456615D00011
Wherein:
Chemical method: acid or the metal catalyst used in the chemical method can remain in the product, not only can influence its degradation rate, reduce product performance, more because its potential toxicity has limited the application in biomedical materials field.
Biological enzyme: biological enzyme, especially wherein lipase-catalyzed is because advantage such as reaction conditions is gentle, catalytic efficiency (is high, anacidity or metal catalyst are residual obtains the concern of more and more researchers.Investigators report first can be 6-caprolactone by the monomer of lipase-catalyzed ring-opening polymerization; So far studied the reaction of multiple lypase divided ring polymerization 6-caprolactone; For example: porcine pancreatic lipase (porcine pancreatic lipase; PPL), (Candida antartica lipase B, Novozyme 435 for antarctic candidia lipase TM), Rhizomucor miehei (RM) and Pseudomonas fluorescens lypase (Pseudomonas fluorescens lipase; PFL); Confirmed that organic solvent, concentration of substrate and temperature have very big influence to reaction, set up the reaction model of lipase-catalyzed polyreaction simultaneously.But existing research has all adopted airtight reaction vessel to criticize formula reaction, will filter between every batch reaction, evaporate, the operation of dissolving, reprecipitation, just can obtain product; And in batch formula reaction; The concussion that generally can adopt or the mode of stirring make mixing of materials evenly and reduce the resistance to mass transfer between substrate and the enzyme; But the shearing force that these modes produced can cause the fragmentation of the inactivation or the immobilized enzyme particle of enzyme; And causing the operational stability of enzyme not high, repeating utilization factor reduces.
Criticize in the reaction process of formula catalytic reaction polymerization 6-caprolactone at lypase, operating procedure complicacy and repeating utilization factor are low, have influenced its application in large-scale industrialization is produced.
Summary of the invention
The objective of the invention is to, provide a kind of enzyme process continuous production to gather the method for (6-caprolactone), produce the operating procedure that gathers (6-caprolactone), improve transformation efficiency to simplify.
A further object of the invention is to provide a kind of lypase packed bed reactor that continuous production gathers (6-caprolactone) that is used for.
The method that enzyme process continuous production provided by the invention gathers (6-caprolactone) may further comprise the steps: 6-caprolactone is dissolved in the suitable organic solvent, is mixed with the solution of 6-caprolactone; The solution even flow that makes said 6-caprolactone, obtains to gather (6-caprolactone) to carry out polyreaction through the lypase packed bed.
According to a preferred embodiment of the present invention, the lypase of use comprises porcine pancreatic lipase (PPL), Pseudomonas fluorescens lypase (PFL) and antarctic candidia lipase (Novozyme 435 TM).
According to a preferred embodiment of the present invention; Method provided by the invention also comprises the step that the reaction solution that gathers (6-caprolactone) that obtains is carried out separation and purification; Comprise that the reaction solution to gathering (6-caprolactone) is rotated evaporation process; Collecting precipitation also is dissolved in methylene dichloride, re-uses methanol extraction, obtains highly purified gathering (6-caprolactone).
Lypase packed bed reactor provided by the invention comprises the lypase packed bed that is used for catalyzed reaction, be used for 6-caprolactone and organic solvent carries out the blended mixing kettle and mixed 6-caprolactone solution sent into the peristaltic pump of lypase packed bed.
According to the present invention, the said lypase packed bed that provides comprises the core layer of degreasing cotton layer, intermediary lypase layer and the below of top, and wherein, said lypase layer is used for catalyzed reaction.
Working method provided by the invention adopt lypase as catalyzer avoided acid or heavy metal catalyst with flowing of reactant outflow reactor and remaining in the product; Packed bed reactor makes the operational stability of lypase improve greatly simultaneously; And realized the production of serialization, so this method has the reaction conditions gentleness, energy consumption is low; Product property is good, the advantage that production cost is low.
Description of drawings
Fig. 1 is the structural representation of lypase packed bed reactor provided by the invention.
Fig. 2 is the structural representation of lypase packed bed provided by the invention.
Fig. 3 is the end product that obtains among the embodiment 1 1H NMR figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.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 following embodiment of the present invention, the structure of the lypase packed bed reactor of use is as shown in Figure 1, comprises packed bed 1, mixing kettle 2, the chuck 4 around parcel packed bed 1, peristaltic pump 6 and the well heater 5 that is connected with chuck 4; Wherein, stirring rake 3 is arranged in the mixing kettle 2, be used to mix to obtain the solution of 6-caprolactone; And the solution of the 6-caprolactone that mixes got in the packed beds 1 through peristaltic pump 6 react; Wherein, the structure of packed bed 1 is as shown in Figure 2, comprises 3 layers; Be respectively degreasing cotton layer 7, lypase layer 8 and core layer 9 from top to bottom; Wherein, lypase layer 8 is used for catalyzed reaction, and lypase wherein can be selected from porcine pancreatic lipase (PPL), Pseudomonas fluorescens lypase (PFL) and antarctic candidia lipase, and (Novozyme 435 TM) waiting lypase, above-mentioned lypase is packed in the reaction column through the mode of natural subsidence, and the internal diameter of reaction column is 0.5-2cm, and height is 4-60cm.
Among the following embodiment of the present invention, adopt the molecular weight and the MWD thereof of the product of following detection method detection reaction acquisition:
Adopt Agilent 1100series liquid chromatographic system, the GPC post that is equipped with light scattering detector and waters HR3 and HR4 detects, wherein,
Analysis condition: moving phase is THF, and flow velocity is 1ml/min, 30 ℃ of column temperatures, sampling volume 20 μ L.The atomization temperature of light scattering detector is 70 ℃, and vaporization temperature is 100 ℃, nitrogen flow rate 1.5SLM.
The PS of employing standard relative molecular mass is as the relative molecular mass standard, and wherein the PS molecular mass is 580g/mol, 930g/mol, 1260g/mol; 1940g/mol, 1790g/mol, 4920g/mol, 7200g/mol; 10100g/mol, 19900g/mol, 29700g/mol.
Specimen preparation: get reaction solution 50 μ L or reaction product 50mg, be diluted to 1ml, adopt disposable filter,, get the filtrating sample introduction through 0.45 μ m organic membrane filter with THF.
Among the following embodiment of the present invention, substrate conversion efficiency is expressed as the per-cent that the 6-caprolactone that has reacted accounts for initial 6-caprolactone, obtains through computes:
C＝(n o-n x)/n o
Wherein, C is a substrate conversion efficiency; n oAmount for initial 6-caprolactone; n xAmount for final 6-caprolactone.
Embodiment 1-6, use Novozyme 435 TMLipase-catalyzed 6-caprolactone
6-caprolactone is mixed in mixing kettle 2 with organic solvent, obtain the solution of 6-caprolactone, then the solution of 6-caprolactone is got into Novozyme 435 with certain flow velocity peristaltic pump 6 of flowing through TMCarry out polyreaction in the packed column 1 of lypase, and collect the reaction solution that gathers (6-caprolactone) that obtains, concrete reaction conditions is as shown in table 1.
Table 1, Novozyme 435 TMLipase-catalyzed preparation gathers the reaction conditions of (6-caprolactone)
Embodiment Mixing temperature 6-caprolactone Organic solvent Flow velocity Temperature of reaction
1 50℃ 0.1mol 200ml toluene 0.01ml/min 50℃
2 50℃ 0.4mol 200ml toluene 0.3ml/min 50℃
3 60℃ 0.2mol The 200ml isopropyl ether 0.005ml/min 60℃
4 50℃ 0.1mol 200ml toluene 1ml/min 50℃
5 30℃ 0.1mol 200ml toluene 0.01ml/min 30℃
6 55℃ 0.05mol The 200ml hexane 0.01ml/min 55℃
Embodiment 7, use porcine pancreatic lipase catalysis 6-caprolactone
Under 30 ℃; The 10.6ml 6-caprolactone is mixed in mixing kettle 2 with the 200ml isopropyl ether; Then the solution of 6-caprolactone is got in the packed column 1 of porcine pancreatic lipases and carries out polyreaction with the flow velocity of the 0.01ml/min peristaltic pump 6 of flowing through; The internal diameter of packed column 1 is 1cm, the high 10cm of being, control reaction temperature is 60 ℃, collects the reaction solution that gathers (6-caprolactone) that obtains.
Embodiment 8, use the lipase-catalyzed 6-caprolactone of Pseudomonas fluorescens
Under 30 ℃; The 5.3ml 6-caprolactone is mixed in mixing kettle 2 with the 200ml isopropyl ether; Then the solution of 6-caprolactone is got in the packed column 1 of Pseudomonas fluorescens lypase and carries out polyreaction with the flow velocity of the 0.01ml/min peristaltic pump 6 of flowing through; The internal diameter of packed column 1 is 0.5cm, the high 16cm of being, control reaction temperature is 40 ℃, collects the reaction solution that gathers (6-caprolactone) that obtains.
Embodiment 9, product purification
Respectively the transformation efficiency of the substrate in the reaction solution that obtains among the embodiment 1-8, the weight-average molecular weight and the MWD of product are detected, detected result is as shown in table 2.
The detected result of reaction solution before table 2, the purifying
Embodiment The transformation efficiency of substrate The weight-average molecular weight of product MWD
1 99.8％ 4520 1.6
2 99.6％ 15600 2.1
3 98.3％ 21430 1.8
4 99.8％ 7500 1.9
5 95％ 2653 2.2
6 94％ 20396 1.7
7 99.8％ 26562 1.5
8 99.8％ 4662 2.2
The reaction solution that obtains among the embodiment 1-8 is handled through rotary evaporation, and collects distillate and deposition respectively, wherein, comprises solvent and a spot of small-molecule substance in the distillate of collection, can continue on for the successive reaction among the present invention.
Then, deposition is dissolved in methylene dichloride after, again through methanol extraction, collecting precipitation also detects the product behind the above-mentioned purifying, detected result is as shown in table 3.
The detected result of table 3, purifying afterreaction liquid
Embodiment The weight-average molecular weight of product MWD
1 6700 1.2
2 18230 1.5
3 23500 1.1
4 9201 1.4
5 3502 1.6
6 21940 1.1
7 31200 1.2
8 6325 1.7
For confirming end product, the contriver has carried out the end product that embodiment 1-8 obtains 1H NMR detects, the result shows that the end product of acquisition is and gather (6-caprolactone), wherein the product of embodiment 1 1H NMR detected result is as shown in Figure 3.
For detecting the continuous production time of high conversion, the contriver presses the catalytic condition of embodiment 2 and 8 at random, has carried out continual production.
The result shows, in successive reaction behind the 672h, under above-mentioned catalytic condition, the lypase packed bed reactor still can keep very high transformation efficiency, maintains more than 98%.
Though; The structure of the lypase packed bed reactor that uses in the present invention is as shown in Figure 1, but as long as the lypase packed bed reactor that uses can be used for continuous production, then also can realize the object of the invention; This is conspicuous to those skilled in the art; Therefore, for the purpose of the present invention, need not stipulate the structure of specific lypase packed bed reactor.
Working method provided by the invention adopt lypase as catalyzer avoided acid or heavy metal catalyst with flowing of reactant outflow reactor and remaining in the product; Packed bed reactor makes the operational stability of lypase improve greatly simultaneously; And realized the production of serialization, so this method has the reaction conditions gentleness, energy consumption is low; Product property is good, the advantage that production cost is low.

Claims (3)

1. the method that the enzyme process continuous production gathers (6-caprolactone) is characterized in that, said method comprising the steps of:
A) 6-caprolactone is dissolved in the suitable organic solvent, is mixed with the solution of 6-caprolactone;
B) the solution even flow that makes said 6-caprolactone, obtains to gather (6-caprolactone) to carry out polyreaction through the lypase packed bed;
Wherein, comprise with the said organic solvent of said 6-caprolactone blended: toluene, isopropyl ether and hexane; The concentration of 6-caprolactone is 0.05-2mol/L in the solution of said 6-caprolactone; Said lypase comprises porcine pancreatic lipase, Pseudomonas fluorescens lypase and antarctic candidia lipase; The flow velocity of the solvent of said 6-caprolactone is 0.005-5ml/min, and the temperature of reaction of said polyreaction is 20-70 ℃.
2. the method for claim 1 is characterized in that, said method also comprises the step that the reaction solution that gathers (6-caprolactone) that obtains is carried out separation and purification.
3. method as claimed in claim 2; It is characterized in that said purification procedures comprises that the reaction solution that gathers (6-caprolactone) to obtaining is rotated evaporation process, collecting precipitation and be dissolved in methylene dichloride after; Re-use methanol extraction, obtain highly purified gathering (6-caprolactone).
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CN104152350B (en) * 2014-08-25 2016-01-13 鲁东大学 A kind of is the method for filling bed type enzyme reactor by stirred-tank fermenter system assembles
CN105969816A (en) * 2016-05-06 2016-09-28 南京工业大学 Method for preparing thiol-functional polylactone by virtue of micro-reaction device
CN106520851B (en) * 2016-11-08 2019-09-10 南京工业大学 A method of enzymatic and organic catalysis synthetic segmented copolymer are coupled using microreactor
CN109541084B (en) * 2018-11-28 2021-11-16 湖北航天化学技术研究所 Device for dynamically analyzing and detecting gas-phase product

Citations (5)

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US6486295B1 (en) * 2000-01-24 2002-11-26 Richard A. Gross Lipase-catalyzed transesterifications to regulate copolymer structure
CN1818026A (en) * 2005-11-09 2006-08-16 中国科学院广州能源研究所 Production of biological diesel oil by fixed enzyme method
CN101020740A (en) * 2007-03-27 2007-08-22 吉林大学 New-type super thermophilic esterase catalyzed prepn of poly-epsilon-caprolactone
CN101146847A (en) * 2005-03-23 2008-03-19 巴斯福股份公司 Two-step method for producing polyesterols
CN101189277A (en) * 2005-06-06 2008-05-28 巴斯福股份公司 Method for producing an aqueous polymer dispersion

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6486295B1 (en) * 2000-01-24 2002-11-26 Richard A. Gross Lipase-catalyzed transesterifications to regulate copolymer structure
CN101146847A (en) * 2005-03-23 2008-03-19 巴斯福股份公司 Two-step method for producing polyesterols
CN101189277A (en) * 2005-06-06 2008-05-28 巴斯福股份公司 Method for producing an aqueous polymer dispersion
CN1818026A (en) * 2005-11-09 2006-08-16 中国科学院广州能源研究所 Production of biological diesel oil by fixed enzyme method
CN101020740A (en) * 2007-03-27 2007-08-22 吉林大学 New-type super thermophilic esterase catalyzed prepn of poly-epsilon-caprolactone

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