CN103014079B - A kind of method applying to carry out Lipase absobed - Google Patents
A kind of method applying to carry out Lipase absobed Download PDFInfo
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- CN103014079B CN103014079B CN201210581083.9A CN201210581083A CN103014079B CN 103014079 B CN103014079 B CN 103014079B CN 201210581083 A CN201210581083 A CN 201210581083A CN 103014079 B CN103014079 B CN 103014079B
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Abstract
The invention discloses a kind of method applying to carry out Lipase absobed, Thermobifida fusca at is added in the acid reaction system with alcohol, at is for deriving from Thermobifida fusca, NCBI numbering is respectively AAZ54920 and AAZ54921, described reaction system is: reaction dissolvent is organic solvent, amount of water 0.03 0.08%, enzyme concentration is 10 80U/mL.The production technology of the present invention, has the advantages such as flow process is simple, conversion ratio is high, applied widely.
Description
Technical field
The present invention relates to a kind of method applying at synthetic ester, belong to technical field of enzyme engineering.
Background technology
Ester is to be reacted dehydration with alcohol and the compounds that generates by acid, is widely used in food, makes wine, drinks
Each field such as material, medicine, health product, daily-use chemical industry.The production of Ester at present uses chemical method substantially,
At high temperature it is catalyzed acid and the esterification of alcohol by organic catalyst (such as concentrated sulphuric acid) and obtains.Although producing into
This is the most relatively low, but during some unmanageable side reactions make product quality the highest, the strong acid corruption to equipment
Erosion also can increase maintenance cost, it is often more important that, the environmental pollution that this method causes does not meets current sustainable development
The main flow of exhibition.And enzymatic clarification Ester has that reaction condition is gentle, energy consumption is low, environmental pollution is little and
And the advantage of superior product quality, therefore develop the enzyme preparation important realistic meaning of application in Lipase absobed and warp
Ji is worth.
At is a kind of multi-functional esterase, both can catalyzing hydrolysis insoluble polymer cutin and various polyester
Ester bond, it is also possible to act on the triglyceride etc. of other long-chain, short-chain aliphatic ester, emulsifying.Except hydrolysis
Outside reaction, it is transesterification with alcohol with the esterification of alcohol, some soaps that at can also be catalyzed acid.The most
Having been reported that the at deriving from fungus F.solani application in short-chain ester synthesizes, wherein ethyl hexanoate closes
The conversion ratio become is the highest, is 97%, but enzyme concentration is the highest, and for 240U/mL, and the Lipase absobed of middle long-chain turns
Rate is the lowest.
This research department identifies two kinds of at Tfu_0882 deriving from Thermobifida fusca in previous work
And Tfu_0883.The invention provides this at application process in Lipase absobed.
Summary of the invention
The invention provides Thermobifida fusca at application process in Lipase absobed.
For solving the problems referred to above, technical solution of the present invention is:
Using and derive from the at of Thermobififda fusca and carry out Lipase absobed, concentration of substrate is 0.1-1M, acid and alcohol mole
Ratio is 1:1-2, amount of water 0.03-0.08%, and enzyme concentration is 10-80U/mL, 40-60 DEG C, 100-200rpm, response time 4-15
H, depending on the response time foundation of the ester of different chain length reaches the time required during maximum conversion.Conversion ratio with the acid that consumes with just
The ratio of the acid content begun is counted.
Above-mentioned enzymatic reaction condition has relation with the character of enzyme, it is possible to adjust corresponding technological parameter according to enzyme addition.
Described enzymatic reaction is carried out the most in organic solvent, and the selection of organic solvent is this area routine techniques means, more
The most preferentially select isobutyltrimethylmethane. or normal hexane.
The production method of described at is under certain condition of culture, with produce at recombination bacillus coli (Chen S,
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) fermentation certain time, warp
Cross 12000rpm, centrifugal 10min, remove thalline, supernatant is crude enzyme liquid, then through vacuum lyophilization, it is thus achieved that at
Enzyme powder.
The assay method of described Cutinase activity is: with p-nitrophenyl butyrate (pNPB) as substrate, uses spectrophotography to exist
Measure under 20 ° of C.Reaction system is 1mL, including 960 μ L buffer (20mM Tris HCl, 10mM NaCl, 50mM
Sodium taurodeoxycholate, pH8.0), 20 μ L enzyme liquid, 20 μ L pNPB (1mM preserves in acetonitrile), at 405nm
Place, the generating rate of record paranitrophenol.Enzyme is lived and is defined: under 20 ° of C, and catalysis p-nitrophenyl butyrate per minute generates 1 μM
The enzyme amount of paranitrophenol is an enzyme activity unit.
Described organic reagent and substrate acid, alcohol are both needed to 4A type molecular sieve except water, prevent the impact of water content in reaction system.
The detection method of described acid is vapor detection: instrument is GC-2010, GAS CHROMATOGRAPH SHIMADZU,
Chromatographic column is RTX-WAX(30m × 0.32mm), detector is FID, and carrier gas is N2, flow velocity 3mL/min, column temperature bar
Part is 50 DEG C (3min)-10 DEG C of min-1-220 DEG C (1min).
Detailed description of the invention
Embodiment 1
At Tfu_0883 that the present embodiment is originated with Thermobifida fusca carries out catalytic reaction.
Organic reagent and substrate acid, alcohol use 4A type molecular sieve except being used for Lipase absobed after water.Reaction condition: 0.1M acid,
0.2M alcohol, amount of water 0.03%, 50 DEG C, 150rpm, enzyme 30U/ml, reaction dissolvent isobutyltrimethylmethane., specifically tie
Fruit is shown in Table 1, table 2.
Table 1
Table 2
Embodiment 2
At Tfu_0882 that the present embodiment is originated with Thermobifida fusca carries out catalytic reaction.
Reaction condition: 0.15M acid, 0.3M alcohol, amount of water 0.05%, 50 DEG C, 150rpm, enzyme 40U/ml,
Reaction dissolvent isobutyltrimethylmethane., concrete outcome is shown in Table 3, table 4.
Table 3
Table 4
Embodiment 3
At Tfu_0883 that the present embodiment is originated with Thermobifda fusca carries out catalytic reaction.
Reaction condition: 0.5M acid, 0.5M alcohol, amount of water 0.08%, 50 DEG C, 150rpm, enzyme 60U/ml,
Reaction dissolvent isobutyltrimethylmethane..Reaction 5h, 15h, propyl butyrate conversion ratio is respectively 92.8% and 91.5%.
Claims (2)
1. apply the method that at carries out Lipase absobed for one kind, it is characterised in that in the reaction system of acid and alcohol, add Thermobifida
Fusca at；Described reaction system is: reaction dissolvent is organic solvent, amount of water 0.03-0.08%, and enzyme concentration is
10-80U/mL；Described at for derive from Thermobifida fusca, NCBI numbering be respectively AAZ54920 and
AAZ54921；The carbon chain lengths of described acid is C1-C18；The carbon chain lengths of described alcohol is C1-C10；Described acid and alcohol
Mol ratio is 0.5-1；Described organic solvent is isobutyltrimethylmethane. or normal hexane；Enzyme-catalyzed reaction condition is 40-60 DEG C, 100-200
rpm；Described sour and described alcohol concentration is 0.1-1M, response time 4-15h.
Method the most according to claim 1, it is characterised in that reaction system is isobutyltrimethylmethane., amount of water 0.03-0.08% adds
Enzyme amount is 10-80U/mL.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210581083.9A CN103014079B (en) | 2012-12-28 | 2012-12-28 | A kind of method applying to carry out Lipase absobed |
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| CN201210581083.9A CN103014079B (en) | 2012-12-28 | 2012-12-28 | A kind of method applying to carry out Lipase absobed |
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| CN103014079A CN103014079A (en) | 2013-04-03 |
| CN103014079B true CN103014079B (en) | 2016-08-24 |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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 |
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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 |
|---|
| Synthesis of alkyl esters by cutinase in miniemulsion and organic solvent media;Dragana PC, de Barros et al;《Biotechnology Journal》;20090531;第4卷(第5期);674–683 * |
| Thermobifida fusca角质酶基因的鉴定及功能研究;吴敬;《2008年中国微生物学会学术年会》;20081231;114-115 * |
| 角质酶的研究进展;李江华;《生物工程学报》;20091225;第25卷(第12期);1829-1837 * |
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