CN103014079A - Method for synthesizing ester by using cutinase - Google Patents
Method for synthesizing ester by using cutinase Download PDFInfo
- Publication number
- CN103014079A CN103014079A CN2012105810839A CN201210581083A CN103014079A CN 103014079 A CN103014079 A CN 103014079A CN 2012105810839 A CN2012105810839 A CN 2012105810839A CN 201210581083 A CN201210581083 A CN 201210581083A CN 103014079 A CN103014079 A CN 103014079A
- Authority
- CN
- China
- Prior art keywords
- cutinase
- reaction
- enzyme
- acid
- ester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for synthesizing ester by using cutinase. The Thermobifida fusca cutinase is added in a reaction system of acid and alcohol, wherein the cutinase is from Thernobifida fusca and the NCBI (national center of biotechnology information) number is AAZ54920 and AAZ54921; and the reaction system is as follows: the reaction solvent is an organic solvent, the additive amount of water is 0.03-0.08%, and the addition amount of enzyme is 10-80U/mL. The method disclosed by the invention is simple in technology and has the advantages of simple process, high conversion rate, wide application scope, and the like.
Description
Technical field
The present invention relates to the method for the synthetic ester of a kind of using cutinase, belong to technical field of enzyme engineering.
Background technology
Ester is a compounds that is generated with alcohol reaction dehydration by acid, is widely used in each field such as food, wine brewing, beverage, medicine, healthcare products, daily-use chemical industry.Chemical method is adopted in the at present production of Ester substantially, is got by the esterification of at high temperature catalysis of organic catalyst (such as the vitriol oil) acid and alcohol.Although production cost is lower, some unmanageable side reactions are so that product quality is not high in the process, and strong acid also can increase maintenance cost to the corrosion of equipment, the more important thing is, the environmental pollution that this method causes does not meet the main flow of current Sustainable development.And enzyme process synthesizing ester material has the reaction conditions gentleness, energy consumption is low, environmental pollution is little and the advantage of superior product quality, therefore develops application important realistic meaning and the economic worth of zymin in ester is synthetic.
At is a kind of multi-functional esterase, but both ester bonds of the insoluble polymer cutin of catalytic hydrolysis and various polyester also can act on the triglyceride level of other long-chain, short-chain aliphatic ester, emulsification etc.Except hydrolysis reaction, at can also catalysis the transesterification of esterification, some soaps and alcohol of acid and alcohol.The application of the currently reported at that derives from fungi F.solani in short-chain ester is synthetic, wherein the synthetic transformation efficiency of ethyl hexanoate is the highest, be 97%, but enzyme concentration is very high, be 240U/mL, and the ester Synthesis conversion of middle long-chain is very low.
This research department has identified two kinds of at Tfu_0882 and the Tfu_0883 that derives from Thermobifida fusca in previous work.The invention provides the application method of this in ester is synthetic.
Summary of the invention
The invention provides the application method of Thermobifida fusca in ester is synthetic.
For addressing the above problem, technical solution of the present invention is:
It is synthetic that the at that employing derives from Thermobififda fusca is carried out ester, concentration of substrate is 0.1-1M, acid is 1:1-2 with the mol ratio of alcohol, amount of water 0.03-0.08%, enzyme concentration is 10-80U/mL, 40-60 ℃, 100-200rpm, reaction times 4-15h, required time and deciding when reaching peak rate of conversion in reaction times of the ester of different chain length.Transformation efficiency comes in the ratio of the acid that consumes and initial acid content.
The character of above-mentioned enzymatic reaction condition and enzyme has relation, also can be according to the corresponding processing parameter of enzyme add-on adjustment.
Described enzymatic reaction is preferentially carried out in organic solvent, and selecting of organic solvent be this area routine techniques means, further preferentially selects octane-iso or normal hexane.
The production method of described at is under certain culture condition, recombination bacillus coli (Chen S with the production at, Tong X, Woodard RW, Du GC, Wu J, Chen J, Identification and Characterization of Bacterial Cutinase, The Journal of Biological Chemistry, 2008,283(28) 25854-25862) the fermentation certain hour, through 12000rpm, centrifugal 10min removes thalline, and supernatant is crude enzyme liquid, pass through again vacuum lyophilization, obtain the enzyme powder of at.
The measuring method of described at activity is: take p-nitrophenyl butyric ester (pNPB) as substrate, adopt spectrophotometry to measure under 20 ° of C.Reaction system is 1mL, comprises 960 μ L damping fluids (20mM Tris – HCl, 10mM NaCl, 50mM sodium taurodeoxycholate, pH8.0), 20 μ L enzyme liquid, 20 μ L pNPB (1mM, preserve in the acetonitrile), at the 405nm place, the generating rate of record p-nitrophenol.Enzyme is lived and defined: under 20 ° of C, the enzyme amount that per minute catalysis p-nitrophenyl butyric ester generates 1 μ M p-nitrophenol is an enzyme activity unit.
Described organic reagent and substrate acid, alcohol all need to dewater with 4A type molecular sieve, prevent the impact on water-content in the reaction system.
The detection method of described acid is vapor detection: instrument is GC-2010, GAS CHROMATOGRAPH SHIMADZU, and chromatographic column is that RTX-WAX(30m * 0.32mm), detector is FID, carrier gas is N
2, flow velocity 3mL/min, the column temperature condition is 50 ℃ of (3min)-10 ℃ min
-1-220 ℃ (1min).
Embodiment
Embodiment 1
The present embodiment carries out catalyzed reaction with the at Tfu_0883 in Thermobifida fusca source.
It is synthetic for ester afterwards that organic reagent and substrate acid, alcohol adopt 4A type molecular sieve to dewater.Reaction conditions: 0.1M acid, 0.2M are pure, and 0.03%, 50 ℃ of amount of water, 150rpm, enzyme 30U/ml, reaction solvent octane-iso, concrete outcome see Table 1, table 2.
Table 1
Table 2
Embodiment 2
The present embodiment carries out catalyzed reaction with the at Tfu_0882 in Thermobifida fusca source.
Reaction conditions: 0.15M acid, 0.3M are pure, and 0.05%, 50 ℃ of amount of water, 150rpm, enzyme 40U/ml, reaction solvent octane-iso, concrete outcome see Table 3, table 4.
Table 3
Table 4
Embodiment 3
The present embodiment carries out catalyzed reaction with the at Tfu_0883 in Thermobifda fusca source.
Reaction conditions: 0.5M is sour, 0.5M is pure, 0.08%, 50 ℃ of amount of water, 150rpm, enzyme 60U/ml, reaction solvent octane-iso.Reaction 5h, 15h, the propyl butyrate transformation efficiency is respectively 92.8% and 91.5%.
Claims (9)
1. a using cutinase carries out the synthetic method of ester, it is characterized in that, adds Thermobifida fusca in the reaction system of acid and alcohol.
2. method according to claim 1 is characterized in that, described at is for deriving from Thermobifida fusca, and the NCBI numbering is respectively AAZ54920 and AAZ54921.
3. method according to claim 2 is characterized in that, the carbon chain lengths of described acid is C1-C18.
4. method according to claim 2 is characterized in that, the carbon chain lengths of described alcohol is C1-C10.
5. method according to claim 2 is characterized in that, described acid is 0.5-1 with the mol ratio of alcohol.
6. method according to claim 2 is characterized in that, described reaction system is: reaction solvent is organic solvent, and amount of water 0.03-0.08%, enzyme concentration are 10-80U/mL.
7. method according to claim 6 is characterized in that, described organic solvent is octane-iso or normal hexane.
8. method according to claim 2 is characterized in that, reaction system is octane-iso, and amount of water 0.03-0.08%, enzyme concentration are 10-80U/mL.
9. method according to claim 8 is characterized in that, enzyme-catalyzed reaction condition is 40-60 ℃, 100-200rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210581083.9A CN103014079B (en) | 2012-12-28 | 2012-12-28 | A kind of method applying to carry out Lipase absobed |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210581083.9A CN103014079B (en) | 2012-12-28 | 2012-12-28 | A kind of method applying to carry out Lipase absobed |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103014079A true CN103014079A (en) | 2013-04-03 |
| CN103014079B CN103014079B (en) | 2016-08-24 |
Family
ID=47963194
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210581083.9A Active CN103014079B (en) | 2012-12-28 | 2012-12-28 | A kind of method applying to carry out Lipase absobed |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103014079B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105821087A (en) * | 2016-04-29 | 2016-08-03 | 江南大学 | Method for synthesizing short-chain flavor ester with cutinase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1946850B (en) * | 2004-03-03 | 2010-12-08 | 陶氏康宁公司 | Methods for forming structurally defined organic molecules |
-
2012
- 2012-12-28 CN CN201210581083.9A patent/CN103014079B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1946850B (en) * | 2004-03-03 | 2010-12-08 | 陶氏康宁公司 | Methods for forming structurally defined organic molecules |
Non-Patent Citations (3)
| Title |
|---|
| DRAGANA PC, DE BARROS ET AL: "Synthesis of alkyl esters by cutinase in miniemulsion and organic solvent media", 《BIOTECHNOLOGY JOURNAL》 * |
| 吴敬: "Thermobifida fusca角质酶基因的鉴定及功能研究", 《2008年中国微生物学会学术年会》 * |
| 李江华: "角质酶的研究进展", 《生物工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105821087A (en) * | 2016-04-29 | 2016-08-03 | 江南大学 | Method for synthesizing short-chain flavor ester with cutinase |
| CN105821087B (en) * | 2016-04-29 | 2019-03-15 | 江南大学 | A method of using the short chain aromatic ester of cutin enzymatic synthesis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103014079B (en) | 2016-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Khoo et al. | Algae biorefinery: Review on a broad spectrum of downstream processes and products | |
| Lima et al. | An overview on the conversion of glycerol to value‐added industrial products via chemical and biochemical routes | |
| Barth et al. | Effect of hydrolysis products on the enzymatic degradation of polyethylene terephthalate nanoparticles by a polyester hydrolase from Thermobifida fusca | |
| Manikandan et al. | Recent development patterns, utilization and prospective of biofuel production: Emerging nanotechnological intervention for environmental sustainability–A review | |
| da Costa et al. | Poly (ethylene terephthalate)(PET) degradation by Yarrowia lipolytica: Investigations on cell growth, enzyme production and monomers consumption | |
| Christopher et al. | Enzymatic biodiesel: Challenges and opportunities | |
| Badgujar et al. | Thermo-chemical energy assessment for production of energy-rich fuel additive compounds by using levulinic acid and immobilized lipase | |
| Harding et al. | A life-cycle comparison between inorganic and biological catalysis for the production of biodiesel | |
| Martins et al. | Improved production of butyl butyrate with lipase from Thermomyces lanuginosus immobilized on styrene–divinylbenzene beads | |
| Romero et al. | Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in n-hexane | |
| Narwal et al. | Production and characterization of biodiesel using nonedible castor oil by immobilized lipase from Bacillus aerius | |
| Xin et al. | Strategies for production of butanol and butyl-butyrate through lipase-catalyzed esterification | |
| Damnjanović et al. | Covalently immobilized lipase catalyzing high-yielding optimized geranyl butyrate synthesis in a batch and fluidized bed reactor | |
| Ferraz et al. | Application of home-made lipase in the production of geranyl propionate by esterification of geraniol and propionic acid in solvent-free system | |
| Wang et al. | An effective method for reducing free fatty acid content of high-acid rice bran oil by enzymatic amidation | |
| Kosamia et al. | Perspectives for scale up of biorefineries using biochemical conversion pathways: Technology status, techno-economic, and sustainable approaches | |
| Pereira et al. | Enzymatic esterification for the synthesis of butyl stearate and ethyl stearate | |
| Sjöblom et al. | Catalytic upgrading of butyric acid towards fine chemicals and biofuels | |
| Bento et al. | Single cell oil production and modification for fuel and food applications: Assessing the potential of sugarcane molasses as culture medium for filamentous fungus | |
| Diaz et al. | A pervaporation-assisted bioreactor to enhance efficiency in the synthesis of a novel biolubricant based on the enzymatic transesterification of a castor oil based biodiesel | |
| Radzi et al. | Green synthesis of butyl acetate, a pineapple flavour via lipase-catalyzed reaction | |
| Mustafa et al. | Highly selective synthesis of glyceryl monostearate via lipase catalyzed esterification of triple pressed stearic acid and glycerin | |
| Zhang et al. | Environmentally-friendly strategy for separation of 1, 3-propanediol using biocatalytic conversion | |
| D'Ambrosio et al. | Ethyl hexanoate rich stream from grape pomace: A viable route to obtain fine chemicals from agro by-products | |
| Chang et al. | Optimization of lipase-catalyzed biodiesel by isopropanolysis in a continuous packed-bed reactor using response surface methodology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |