Immobilized

lipases for
biocatalysis
for smarter chemical synthesis
Biocatalysis
Biocatalysis involves the implementation of natural catalysts, such as enzymes, in place of chemical catalysts in synthetic
processes. Compared to chemical catalysts, enzymes offer:

• higher reaction rates

• milder reaction conditions
• high reaction specificity with no side products

This change can enable new, more sustainable routes for the production of intermediates and active pharmaceutical ingredients
(APIs). However, please note Novozymes products do not comply with manufacturing according to pharmaceutical standards and
Novozymes products must not be used as active pharmaceutical ingredients (APIs) or excipients.

Biocatalysis has become an increasingly important tool for medicinal, process and polymer chemists, allowing the development
of efficient and highly attractive synthetic processes on an industrial scale. Use of enzymes in catalysis is a well-established
technology within the chemical industry. An advantage of enzymes in organic synthesis is their remarkable selective properties,
which provide commercial benefits including:
• high selectivity in production of single stereoisomers
• fewer side reactions
• less reprocessing and purification steps
• easier product separation
• less pollution

The combination of all of these advantages leads to a reduction in costs.

Enzyme catalysts work by lowering the activation energy (Ea‡) for a reaction, thus dramatically increasing the rate of the
reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction
rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not
consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from
most other catalysts in that they are highly specific for their substrates.

C E
A

A. Various microorganisms can D

be used to produce natural catalysts
such as enzymes
B. The enzymes are separated from the
microorganisms and subsequently
partially purified and formulated
C. An enzyme attracts specific
substrates to its active site B

D. It catalyzes the chemical reaction by

which products are formed
E. It then allows the products
to separate from the enzyme surface
Novozym® 435
Novozym® 435 is a CALB lipase immobilised on an hydrophobic carrier (acrylic resin). CALB is a non-specific lipase
originating from Candida antarctica B.

Lipase CALB is stable over a relatively broad pH range, especially in the alkaline pH range. The enzyme exhibits a very
high degree of substrate specificity, with respect to both regio- and enantioselectivity. Lipase CALB has been used
extensively in the resolution of racemic alcohols, amines, and acids, and in the preparation of optically active compounds
from meso substrates. The resulting optically pure compounds are highly difficult to obtain by alternative routes and can
be of great synthetic value. Likewise, CALB has been used intensively as a regio-selective catalyst in selective acylation
of different carbohydrates.

Advantages of Novozym® 435

• mild and selective on multi-functional substrates

• active for both bulk liquid substances and in the presence of organic co-solvents
• performs well under anhydrous conditions and with moisture sensitive substrates
• functions across a wide temperature range (20-110°C)
• suitable for both stirred batch tank and continuous fixed bed reactors
• can be recycled 5-10 times without activity loss, depending on reaction conditions
• used in large-scale industrial production

Key applications of Novozym® 435

• dynamic kinetic resolution of alcohols coupled with ruthenium catalyst
• dynamic kinetic resolution of amine coupled with metal racemizing agent
Dynamic kinetic resolution of alcohols by transesterfication of racemic alcohol1,2:

Dynamic kinetic resolution of amines3:

Lipozyme® TL IM
Lipozyme® TL IM is a 1,3 specific lipase originating from Thermomyces lanuginosus and immobilized on a non-compressible
silica gel carrier. Lipozyme® TL IM is a highly effective catalyst for interesterification and can rearrange fatty acids preferentially,
but not uniquely in the 1- and 3- positions of the triglycerides4,5. Lipozyme® TL IM exhibits a high degree of substrate selectivity
allowing bulky side chains/large groups on the alcohol and acid part of the molecule.

Novozym® 40086
Novozym® 40086 is a 1,3 specific lipase originating from Rhizomucor mehei which is immobilized on a resin carrier.
Novozym® 40086 is a highly effective catalyst for stereoselective transesterification and ester hydrolysis.

Resolution of ibuprofen by transesterfication6:

Novozym® 40086
Lipases
Lipases (EC Number 3.1.1.3) are one of the most commonly used classes of enzymes in biocatalysis. Lipases catalyze the
hydrolysis of triacylglycerols to diacylglycerol, monoacylglycerol, glycerol and free fatty acids. The reaction reverses under
anhydrous conditions and the enzyme is able to synthesize new molecules by esterification, alcoholysis and transesterification.
All reactions can be performed with high regio- and enantioselectivity under mild reaction conditions. Lipases have been used
on a variety of substrates and show very broad substrate specificity due to the ubiquity in nature and the heterogeneity of
lipases from different sources.

Regioselective hydrolysis of a triacylglycerol:

Benefits of enzymes as biocatalysts in organic transformations

Cost savings Improved productivity Improved quality of API/intermediate Environmental friendliness

• reduction in raw material input
• avoids use of costly chiral
resolving agents or costly metal
based catalysts
• lower equipment, labor and
energy costs
• shortened synthesis routes
• more batches resulting in increased
capacity
• avoids laborious protection and
de-protection
• higher yields
• fewer or no by-products, leading • reduction of waste products
to reduced impurities in the final produced and solvent usage
products • higher energy savings
• high stereo-, regio-, and chemo-
selectivity
• less residual solvent carry over from
reduced solvent use

Key advantages of immobilized lipases

Immobilization of lipase enzymes on a solid carrier has shown to increase enzyme stability in non-aqueous solvents.
Furthermore, immobilization onto a solid carrier facilitates recovery of products, recycling and reuse of catalyst and thereby
decreases the cost of enzyme use in chemical processes. Advantages of immobilized lipases are:

• better performance in non-aqueous solvents compared to native enzyme formulations

• efficient recovery and separation of reaction product
• economically viable in reuse/recycling of enzymes
• minimising or eliminating protein contamination of product
• enhanced storage and operational stability from denaturation by heat, organic solvents or autolysis
• higher catalyst productivity (kg product/kg enzyme) determining enzyme cost per kg of product
• convenient and safer handling of the enzyme

Novozymes offers a product portifolio of three immobilized lipase enzymes

Product name Activity* pH optimum Temp optimum Substrate specificity

Novozym® 435 10000 PLU/g pH 5-9 30-60°C Esters and alcohols

Lipozyme ® TL IM 250 IUN/g pH 6-8 50-75°C Esters

Novozym 40086
®
275 IUN/g pH 7-10 30-50°C Esters

* LU = Lipase unit, PLU = Propyl Laurate Unit, IUN = Interesterification Unit. 1 PLU is the amount of enzyme activity which
generates 1 μmol of Propyl laurate per minute under defined standard conditions. 1PLU is equal to 1IUN.
References
1. Pietruszka, J., Simon, R.C., Kruska, F., and Braun, M. (2010) Euro.J.Org.Chem., 6217-6224
2. Schonherr, H., Mollitor, J., and Schneider, C.(2010) Eur.J.Org.Chem., 20, 3908-3918
3. Gedey, S., Lijebald, A., Lazar, L., Fulop, F., and Kanerva, L.T.,(2002), Can.J, Chem., 80, 565-570
4. Ref. Carlos A. Martinez, Shanghui Hu, Yves Dumond, Junhua Tao, Patrick Kelleher, Liam Tully Organic
Process Research & Development 2008, 12, 392-398
5. Hari Krishna, S., Persson, M., and Bornscheuer, U.T. (2002) Tetrahedron Asymmetry, 13, 2693-269
6. Ozdermirhan, D., Sezer, S., and Sonmez, Y.(2008) Tetrahedron Asymmetry ,19, 2717-2720.

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June 2016 - No. 2014-12576-04
Novozymes does not promote or support the use of enzymes as Active Pharmaceutical Ingredients (APIs) or excipients.
Novozymes enzymes are industrial bulk products and are not produced according to pharmaceutical standards. If enzymes are
used as process aids, it is the responsibility of the API or excipient manufacturer to assure that the enzymes are suitable for the novozymes.com
intended use, including but not limited to evaluation of purity and absence of carry over in the final product. Laws, regulations,
and/or third party rights may prevent customers from importing, using, processing, and/or reselling the products described
herein in a given manner.
Without separate, written agreement between the customer and Novozymes to such effect, this document does not constitute
a representation or warranty of any kind and is subject to change without further notice.