WO2012036973A2 - Process for immobilization of candida antarctica lipase b - Google Patents

Process for immobilization of candida antarctica lipase b Download PDF

Info

Publication number
WO2012036973A2
WO2012036973A2 PCT/US2011/050906 US2011050906W WO2012036973A2 WO 2012036973 A2 WO2012036973 A2 WO 2012036973A2 US 2011050906 W US2011050906 W US 2011050906W WO 2012036973 A2 WO2012036973 A2 WO 2012036973A2
Authority
WO
WIPO (PCT)
Prior art keywords
sepabeads
enzyme
immobilization
candida antarctica
antarctica lipase
Prior art date
Application number
PCT/US2011/050906
Other languages
French (fr)
Other versions
WO2012036973A3 (en
WO2012036973A8 (en
Inventor
Matthew Truppo
Original Assignee
Beeler, Nicole M.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Beeler, Nicole M. filed Critical Beeler, Nicole M.
Publication of WO2012036973A2 publication Critical patent/WO2012036973A2/en
Publication of WO2012036973A8 publication Critical patent/WO2012036973A8/en
Publication of WO2012036973A3 publication Critical patent/WO2012036973A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction

Definitions

  • ⁇ -fluoroleucine-a-amino acids The asymmetric synthesis of ⁇ -fluoroleucine-a-amino acids is a proven technology for the production of potential pharmaceutical compounds that have a wide array of biological uses, including enzyme inhibitors, receptor antagonists and lipophilicity enhancing agents.
  • enzyme mediated dynamic kinetic resolution ring opening of azalactones has been demonstrated as an effective way of introducing stereochemistry in ⁇ -fluoroleucine ethyl ester compounds.
  • the fiuoroleucine ethyl esters are useful for the preparation of odanacatib, a cathepsin K inhibitor.
  • the instant invention describes an immobilization method for the enzyme Candida Antarctica lipase B for the production of (S)- ⁇ -fluoroleucine ethyl ester via selective enzyme mediated dynamic kinetic resolution ring opening of the azalactone.
  • the use of the enzyme can be utilized in both a batch reactor and a continuous plug flow column reactor packed with the immobilized Candida Antarctica lipase B preparation.
  • This invention solves the problem of enzyme deactivation in both batch and plug flow column reactor processes used to make ⁇ -fluoroleucine ethyl ester compounds.
  • enzyme deactivation at high temperature and in organic solvent limited the useful life of the catalyst for the production process of fiuoroleucine.
  • the deactivated enzyme and immobilization solid support needed to be disposed of, adding to the cost of the overall process.
  • this invention exhibits enhanced enzyme stability in organic solvent and high temperature compared to alternative methods to immobilize the enzyme catalyst. Also, this invention provides the ability to process more fiuoroleucine intermediate with less catalyst than alternative immobilized forms of Candida Antarctica lipase B.
  • the immobilization resin is selected from the group consisting of Sepabeads® EXE- 120, Sepabeads® EC-EP, Sepabeads® EC-HFA, Sepabeads® EXE- 1 19 and Sepabeads® EXA-252.
  • the Sepabeads® immobilization resins are available from Mitsubishi Chemical Corporation.
  • the immobilization resin is Sepabeads® EXE120.
  • Rl is halo and R i lk l
  • Candida antarctica lipase B which has been immobilized on an
  • Rl is fiuoro and R2 is ethyl.
  • the immobilization resin is selected from the group consisting of Sepabeads® EXE120, Sepabeads® EC-EP, Sepabeads® EC-HFA, Sepabeads® EXEl 19 and Sepabeads® EXA252.
  • the immobilization resin is Sepabeads® EXEl 20.
  • a procedure for the immobilization of Candida Antarctica lipase B has been developed to make the enzyme more stable under the reaction conditions.
  • the immobilization of the enzyme on immobilization resin Sepabeads® EXE 120 (CI 8 functionalized resin) provides for an enzyme with high stability under the reaction conditions that can be reused many times.
  • Alternative resins can also be used including immobilization resins Sepabeads® EC-EP, Sepabeads® EC-HFA, Sepabeads® EXEl 19 and Sepabeads® EXA252.
  • the Sepabeads® EXE 120 immobilized enzyme is created by mixing a liquid Candida antarctica lipase B prep from Novozymes with the Sepabeads® EXE 120 resin for between 1 and 40 hours. The resin is then filtered away from the liquid enzyme solution and rinsed with buffer (pH 7). The immobilized enzyme is then stored between -20°C and room temperature. In one embodiment of the invention, the process is run as a fed batch reaction. The fed batch reaction is carried out in a temperature controlled stirred tank reactor where agitation is carried out with overhead stirring via a pitched blade impeller. This agitation needs to be sufficient to suspend the immobilized enzyme resin.
  • the fed batch reaction is carried out at a temperature of about 50° to about 65 °C. In a subclass of the invention, the fed batch reaction is carried out at a temperature of about 65°C.
  • the fed batch reaction can be carried out in an organic solvent such as MTBE THF, DMF, toluene, C ⁇ CN and mixtures thereof. In a class of the invention, the fed batch reaction is carried out in MTBE.
  • the process is a continuous plug flow column reaction.
  • the immobilized enzyme is slurried in MTBE and then packed into the column under atmospheric pressure.
  • Two feed solutions are made, the first solution comprising an azalactone of formula II and a second solution comprising amine base and EtOH.
  • the amine base is triethylamine, DBU, 2,6-lutidine or DABCO.
  • the amine base is triethylamine.
  • the EXE 120 immobilized enzyme exhibits an excellent stability profile, losing only 0.15% activity per hour under these reaction conditions. Novozyme-435 for comparison loses activity at 3% per hour (20X greater deactivation).
  • alkyl shall mean a substituting univalent group derived by conceptual °removal of one hydrogen atom from a straight or branched-chain acyclic saturated hydrocarbon
  • halo shall include iodo, bromo, chloro and fluoro.
  • immobilized Candida Antarctica lipase B (“CAL-B”) enzyme
  • 50 g of immobilization resin (Mitsubishi Sepabeads® EXE- 120) was added to 150 mL
  • Novozymes liquid CAL-B The Novozymes liquid CAL-B prep is purchased directly from Novozymes. The mixture was stirred with overhead stirring for 20 hours at room temperature. The liquid was then drained from the resin and the resin was then rinsed with 3X volumes of 50 mM pH 7.5 potassium phosphate buffer. Next, the resin was dried over a filter with a nitrogen sweep for 5 hours. The immobilized enzyme can then be stored at 5°C.
  • Azalactone substrate A is dissolved in MTBE. EtOH and Et 3 N are then added to the azlacetone in MTBE solution. Immobilized enzyme from Candida antarctica lipase B is then added so that the final concentrations in the resulting solution are 80 g/L azalactone A, 86 g/L EtOH, 7.6 g/L Et 3 N, and 80 g/L immobilized enzyme.
  • the solution is heated to 50°C and mixed with agitation sufficient to suspend the immobilized enzyme.
  • the reaction is aged for 0.5 hours and an addition of azalactone A and EtOH is added (i.e. 80 g azalactone and 17.2 g of EtOH for a 1 L reaction).
  • the outlet at the bottom of the column is fed through a back pressure regulator set at 20psi to prevent the solution from boiling. After going through the back pressure regulator, the solution is fed to a quench tank containing IN ⁇ S0 4 . The quench tank is then assayed for product B.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The instant invention describes an immobilization method for the enzyme Candida Antarctica lipase B for the production of (S)- γ-fluoroleucine ethyl ester via selective enzyme mediated dynamic kinetic resolution ring opening of the azalactone. The enzyme can be utilized in either a batch reactor and a continuous plug flow column reactor packed with the immobilized Candida Antarctica lipase B preparation.

Description

TITLE OF THE INVENTION
PROCESS FOR IMMOBILIZATION OF CANDIDA ANTARCTICA LIPASE B
BACKGROUND OF THE INVENTION
The asymmetric synthesis of γ-fluoroleucine-a-amino acids is a proven technology for the production of potential pharmaceutical compounds that have a wide array of biological uses, including enzyme inhibitors, receptor antagonists and lipophilicity enhancing agents. The use of enzyme mediated dynamic kinetic resolution ring opening of azalactones has been demonstrated as an effective way of introducing stereochemistry in γ-fluoroleucine ethyl ester compounds. The fiuoroleucine ethyl esters are useful for the preparation of odanacatib, a cathepsin K inhibitor.
The instant invention describes an immobilization method for the enzyme Candida Antarctica lipase B for the production of (S)- γ-fluoroleucine ethyl ester via selective enzyme mediated dynamic kinetic resolution ring opening of the azalactone. The use of the enzyme can be utilized in both a batch reactor and a continuous plug flow column reactor packed with the immobilized Candida Antarctica lipase B preparation.
This invention solves the problem of enzyme deactivation in both batch and plug flow column reactor processes used to make γ-fluoroleucine ethyl ester compounds. In previously disclosed methods, enzyme deactivation at high temperature and in organic solvent limited the useful life of the catalyst for the production process of fiuoroleucine. The deactivated enzyme and immobilization solid support needed to be disposed of, adding to the cost of the overall process.
In addition, this invention exhibits enhanced enzyme stability in organic solvent and high temperature compared to alternative methods to immobilize the enzyme catalyst. Also, this invention provides the ability to process more fiuoroleucine intermediate with less catalyst than alternative immobilized forms of Candida Antarctica lipase B.
SUMMARY OF THE INVENTION
By this invention, there is provided a process for immobilization of Candida Antarctica lipase B using an immobilization resin.
DETAILED DESCRIPTION OF THE INVENTION
By this invention, there is provided a process for immobilization of Candida Antarctica lipase B using an immobilization resin.
In a class of the invention, the immobilization resin is selected from the group consisting of Sepabeads® EXE- 120, Sepabeads® EC-EP, Sepabeads® EC-HFA, Sepabeads® EXE- 1 19 and Sepabeads® EXA-252. The Sepabeads® immobilization resins are available from Mitsubishi Chemical Corporation. In a subclass of the invention, the immobilization resin is Sepabeads® EXE120.
By this invention, there is provided a process for preparing a compound of formula I:
Figure imgf000003_0001
comprising an enzyme mediated ring opening of an azalactone of formula II:
Figure imgf000003_0002
wherein Rl is halo and R i lk l
Figure imgf000003_0003
and the enzyme is Candida antarctica lipase B, which has been immobilized on an
immobilization resin.
In a class of the invention, Rl is fiuoro and R2 is ethyl.
In a class of the invention, the immobilization resin is selected from the group consisting of Sepabeads® EXE120, Sepabeads® EC-EP, Sepabeads® EC-HFA, Sepabeads® EXEl 19 and Sepabeads® EXA252. In a subclass of the invention, the immobilization resin is Sepabeads® EXEl 20.
A procedure for the immobilization of Candida Antarctica lipase B has been developed to make the enzyme more stable under the reaction conditions. The immobilization of the enzyme on immobilization resin Sepabeads® EXE 120 (CI 8 functionalized resin) provides for an enzyme with high stability under the reaction conditions that can be reused many times. Alternative resins can also be used including immobilization resins Sepabeads® EC-EP, Sepabeads® EC-HFA, Sepabeads® EXEl 19 and Sepabeads® EXA252.
The Sepabeads® EXE 120 immobilized enzyme is created by mixing a liquid Candida antarctica lipase B prep from Novozymes with the Sepabeads® EXE 120 resin for between 1 and 40 hours. The resin is then filtered away from the liquid enzyme solution and rinsed with buffer (pH 7). The immobilized enzyme is then stored between -20°C and room temperature. In one embodiment of the invention, the process is run as a fed batch reaction. The fed batch reaction is carried out in a temperature controlled stirred tank reactor where agitation is carried out with overhead stirring via a pitched blade impeller. This agitation needs to be sufficient to suspend the immobilized enzyme resin. In a class of the invention, the fed batch reaction is carried out at a temperature of about 50° to about 65 °C. In a subclass of the invention, the fed batch reaction is carried out at a temperature of about 65°C. The fed batch reaction can be carried out in an organic solvent such as MTBE THF, DMF, toluene, CなCN and mixtures thereof. In a class of the invention, the fed batch reaction is carried out in MTBE.
With the fed batch reaction, it is preferable to always maintain a high enzyme to substrate ratio and to feed the substrate over time, as opposed to a higher starting concentration of substrate, to minimize background ethanolysis and hydrolysis.
In another embodiment of the invention, the process is a continuous plug flow column reaction. The immobilized enzyme is slurried in MTBE and then packed into the column under atmospheric pressure. Two feed solutions are made, the first solution comprising an azalactone of formula II and a second solution comprising amine base and EtOH. In a class of the invention, the amine base is triethylamine, DBU, 2,6-lutidine or DABCO. In a subclass of the invention, the amine base is triethylamine. The EXE 120 immobilized enzyme exhibits an excellent stability profile, losing only 0.15% activity per hour under these reaction conditions. Novozyme-435 for comparison loses activity at 3% per hour (20X greater deactivation).
With the column reaction, it is preferable to keep the two feeds (i.e., the azalactone feed and the Et3N/EtOH feed) separate before entering the column to minimize background ethanolysis. Background ethanolysis can result when the azalactone comes in contact with the EtOH. An advantage of the column reaction system is the elimination of enzyme degradation or deactivation due to shear from mixing in batch systems, which decreases enzyme deactivation rate by >20 fold.
Important to both the fed batch and column reactions is running them at high (~65°C) temperatures to increase enzymatic rate relative to background rates of ethanolysis and hydrolysis. Running the reactions at high temperature boosts yield and ee.
The term "alkyl" shall mean a substituting univalent group derived by conceptual °removal of one hydrogen atom from a straight or branched-chain acyclic saturated hydrocarbon
Figure imgf000004_0001
The term "halo" shall include iodo, bromo, chloro and fluoro.
An illustration of the processes of the present invention is described by the following general scheme, using appropriate materials. The specific examples following the scheme further exemplify the processes of the present invention. The compounds illustrated in the scheme and examples are not, however, to be construed as forming the only genus that is considered as the invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are degrees Celsius unless otherwise noted.
EXAMPLE 1
SYNTHESIS OF IMMOBILIZED CANDIDA ANTARCTICA LIPASE B ENZYME
To make 50 g of immobilized Candida Antarctica lipase B ("CAL-B") enzyme, 50 g of immobilization resin (Mitsubishi Sepabeads® EXE- 120) was added to 150 mL
Novozymes liquid CAL-B, The Novozymes liquid CAL-B prep is purchased directly from Novozymes. The mixture was stirred with overhead stirring for 20 hours at room temperature. The liquid was then drained from the resin and the resin was then rinsed with 3X volumes of 50 mM pH 7.5 potassium phosphate buffer. Next, the resin was dried over a filter with a nitrogen sweep for 5 hours. The immobilized enzyme can then be stored at 5°C.
EXAMPLE 2
FED BATCH PROCESS
Figure imgf000005_0001
Azalactone substrate A is dissolved in MTBE. EtOH and Et3N are then added to the azlacetone in MTBE solution. Immobilized enzyme from Candida antarctica lipase B is then added so that the final concentrations in the resulting solution are 80 g/L azalactone A, 86 g/L EtOH, 7.6 g/L Et3N, and 80 g/L immobilized enzyme. The solution is heated to 50°C and mixed with agitation sufficient to suspend the immobilized enzyme. The reaction is aged for 0.5 hours and an addition of azalactone A and EtOH is added (i.e. 80 g azalactone and 17.2 g of EtOH for a 1 L reaction). The reaction is then aged for 1 hour and an addition of azalactone A and EtOH is added (i.e. 80 g azalactone and 17.2 g of EtOH for a 1 L reaction). The reaction is aged for 1.5 hours and an addition of azalactone A and EtOH is added (i.e. 80 g azalactone and 17.2 g of EtOH for a 1 L reaction). The reaction is aged for 3 hours and assayed for completion and the formation of product B. EXAMPLE 3
CONTINUOUS PLUG FLOW COLUMN PROCESS
Figure imgf000006_0001
50g of immobilized enzyme from Candida antarctica lipase B is slurried in MTBE and packed in a jacketed column under atmospheric pressure. 1kg of azalactone A is dissolved in MTBE so that 6.25L of a solution containing 160 g/L azalactone A in MTBE is made. 6.25L of a second solution of 172 g/L EtOH and 15.2 g/L Et3N in MTBE is made. The column jacket is set to 65°C and the two solutions are fed at equal rates and mixed just before entering the top of the column. The two solutions can be fed via pumps or pressurized holding tanks and the total volume is fed over 20 hours. The outlet at the bottom of the column is fed through a back pressure regulator set at 20psi to prevent the solution from boiling. After going through the back pressure regulator, the solution is fed to a quench tank containing INなS04. The quench tank is then assayed for product B.

Claims

WHAT IS CLAIMED IS:
1. A process for immobilization of Candida Antarctica lipase B using an immobilization resin.
2. The process of Claim 1 wherein the immobilization resin is selected from the group consisting of Sepabeads® EXE 120, Sepabeads® EC-EP, Sepabeads® EC-HFA, Sepabeads® EXE119 and Sepabeads® EXA252.
3. The process of Claim 2 wherein the immobilization resin is Sepabeads® EXE120.
4 A process for reparing a compound of formula I:
Figure imgf000007_0001
comprising an enzyme mediated ring opening of an azalactone of formula II :
Figure imgf000007_0002
wherein Rl is halo and
Figure imgf000007_0003
and the enzyme is Candida antarctica lipase B, which has been immobilized on an
immobilization resin.
5. The process of Claim 4 wherein the immobilization resin is selected from the group consisting of Sepabeads® EXE120, Sepabeads® EC-EP, Sepabeads® EC-HFA, Sepabeads® EXE 119 and Sepabeads® EXA252.
6. The process of Claim 5 wherein the immobilization resin is
Sepabeads® EXE120.
PCT/US2011/050906 2010-09-15 2011-09-09 Process for immobilization of candida antarctica lipase b WO2012036973A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38294710P 2010-09-15 2010-09-15
US61/382,947 2010-09-15

Publications (3)

Publication Number Publication Date
WO2012036973A2 true WO2012036973A2 (en) 2012-03-22
WO2012036973A8 WO2012036973A8 (en) 2012-05-03
WO2012036973A3 WO2012036973A3 (en) 2012-06-14

Family

ID=45832177

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/050906 WO2012036973A2 (en) 2010-09-15 2011-09-09 Process for immobilization of candida antarctica lipase b

Country Status (1)

Country Link
WO (1) WO2012036973A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112695020A (en) * 2020-12-18 2021-04-23 安徽丰原生物技术股份有限公司 Preparation method of chemically modified lipase, lipase and application of lipase in synthesis of L-lactide

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6605452B1 (en) * 1997-09-24 2003-08-12 Enzymotec, Ltd. Fatty acid polyol ester-coated lipase complex immobilized on insoluble matrix
US7455998B2 (en) * 2004-03-03 2008-11-25 Dow Corning Corporation Methods for forming structurally defined organic molecules
US20090133322A1 (en) * 2007-11-28 2009-05-28 Transbiodiesel Ltd. Robust multienzyme preparation for the synthesis of fatty acids alkyl esters

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6605452B1 (en) * 1997-09-24 2003-08-12 Enzymotec, Ltd. Fatty acid polyol ester-coated lipase complex immobilized on insoluble matrix
US7455998B2 (en) * 2004-03-03 2008-11-25 Dow Corning Corporation Methods for forming structurally defined organic molecules
US20090133322A1 (en) * 2007-11-28 2009-05-28 Transbiodiesel Ltd. Robust multienzyme preparation for the synthesis of fatty acids alkyl esters

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
HERNANDEZ ET AL.: 'Lipase B from Candida antarctica immobilized on octadecyl Sepabeads: A very stable biocatalyst in the presence of hydrogen peroxide.' PROCESS BIOCHEMISTRY vol. 46, no. 4, April 2011, pages 873 - 878 *
HUGHES ET AL.: 'Production of Candida antarctica lipase B gene open reading frame using automated PCR gene assembly protocol on robotic workcell and expression in an ethanologenic yeast for use as resin-bound biocatalyst in biodiesel production.' J LAB AUTOM. vol. 16, no. 1, 21 July 2010, pages 17 - 37 *
KEIDEL ET AL.: 'Equalizer technology--Equal rights for disparate beads.' PROTEOMICS vol. 10, no. 11, June 2010, pages 2089 - 2098 *
'ReliZyme and SEPABEADS EC.' RESINDON, [Online] 18 July 2011, Retrieved from the Internet: <URL:http://www.resindion.com/images/storie s/resindion/download/RESINDION_ ReliZyme_Sepabea dsEC_lug11_web.pdf> [retrieved on 2012-03-16] *
SUN ET AL.: 'Immobilization of Candida antarctica lipase B by adsorption in organic medium.' NEW BIOTECHNOL. vol. 27, no. 1, 11 December 2009, pages 53 - 58 *
TRUPPO ET AL.: 'Development of an Improved Immobilized CAL-B for the Enzymatic Resolution of a Key Intermediate to Odanacatib.' ORG. PROCESS RES. DEV. vol. 15, no. 5, 14 July 2011, pages 1033 - 1035 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112695020A (en) * 2020-12-18 2021-04-23 安徽丰原生物技术股份有限公司 Preparation method of chemically modified lipase, lipase and application of lipase in synthesis of L-lactide
CN112695020B (en) * 2020-12-18 2023-01-31 安徽丰原生物技术股份有限公司 Preparation method of chemically modified lipase, lipase and application of lipase in synthesis of L-lactide

Also Published As

Publication number Publication date
WO2012036973A3 (en) 2012-06-14
WO2012036973A8 (en) 2012-05-03

Similar Documents

Publication Publication Date Title
Wallert et al. Ionic liquids as additives in the pig liver esterase (PLE) catalysed synthesis of chiral disubstituted malonates
CN109735582B (en) Method for synthesizing cyclohexanol beta-amino alcohol derivatives on line by lipase catalysis
Petri et al. Asymmetric synthesis of a high added value chiral amine using immobilized ω-transaminases
CN104788332B (en) A kind of preparation and purification method of oleic monoethanolamide
CN102557952B (en) Method for preparing di-tert-butyl dicarbonate
CN106834372A (en) A kind of transaminase living things catalysis prepare (S) or（R）The method of 2 amino-butanamides
Ong et al. Enantioseparation of (R, S)‐ketoprofen using Candida antarctica lipase B in an enzymatic membrane reactor
Lundell et al. Room temperature ionic liquids in the kinetic resolution of adrenaline‐type aminoethanols by burkholderia cepacia lipase under normal and microwave conditions
CN105969836B (en) Method for splitting abacavir chiral intermediate vinelactone by enzymatic method
CN109706194B (en) Method for synthesizing phenethyl alcohol beta-amino alcohol derivatives on line based on mobile chemical enzymatic ammonolysis reaction
WO2012036973A2 (en) Process for immobilization of candida antarctica lipase b
CN109988787B (en) Method for synthesizing 2-phenylamino cyclohexanol on line under catalysis of lipase
CN101130803B (en) Method for enzymatically synthesizing beta-lactam antibiotic in organic solvent
CN104830940A (en) An enzymatic synthesis process of Amoxicillin
CN113005169B (en) Chiral resolution method of isobutyrate compound
JP5266875B2 (en) Process for producing optically active organic carboxylic acid from organic carboxylic acid ester
US7238503B2 (en) Process for making fluoroleucine ethyl esters
CN109762853B (en) Method for synthesizing isopropanol beta-alkamine derivative on line by lipase catalysis
Lavrov et al. Novel biocatalytic process of N-substituted acrylamide synthesis
CN108330154B (en) Application of esterase EstP00714 in catalytic resolution of racemic linalyl acetate to obtain (S) -linalool
KR101774674B1 (en) Method for producing acrylamide
CN102559833A (en) Method for preparing (S)-(+)-2,2-dimethyl cyclopropane methanoic acid by biological resolution in ionic liquid cosolvent
JPH09283A (en) Enzymatic reaction in super-critical carbon dioxide atmosphere
Ye-Wang et al. Effect of substrate concentration on the synthesis of cefaclor by penicillin acylase with in situ product removal
CN101012181A (en) Resolution method of DL-p-chlorophenylalanine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11825712

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11825712

Country of ref document: EP

Kind code of ref document: A2