Strem Chemicals, Inc. www.strem.

com

Catalog # 06-3118 Palatase® 20000 L

Note: Sold in collaboration with Novozymes A/S for research purposes only.

Palatase® 20000 L is a 1,3 specific lipase originating from Rhizmucor miehei. It is effective catalyst for the hydrolysis of small fatty
acids on the carboxylic part.

Declared activity 20000 LU-MM/g. Lipase that hydrolyzes ester bonds in glycerides. Color can vary from batch to batch. Color
intensity is not an indication of enzyme activity. Packaging must be kept intact, dry and away from sunlight. Please follow the
recommendations and use the product before the best before date to avoid the need for a higher dosage.

Other Lipase products offered by Strem: Endoprotease products offered by Strem:

06-3123 Novozym® 435 06-3110 Alcalase® 2.4 L FG

06-3155 Lipozyme® TL IM 06-3112 Alcalase® 2.5 L
06-3100 Novocor® AD L 06-3137 Savinase® 12 T
06-3120 Lipozyme® RM 06-3150 Savinase® 16 L
06-3105 Lipozyme® CALB L 06-3115 Esperase® 8.0 L
06-3140 Lipozyme® TL 100 L 06-3160 Neutrase® 0.8 L
06-3125 Resinase® HT 96-0224 Novozymes Endoprotease Screening
06-3135 Novozym® 51032 Kit (contains 6 endoprotease enzymes)
96-0220 Novozymes Lipase Screening Kit
(contains 9 lipase enzymes)

Novozymes Lipase Products

Storage

Kits should be optimally stored at 0-10C/32-50F. If stored above 25C/77F the samples
should be used within 3 months.

Introduction

Lipases (EC Number 3.1.1.3) are one of the most commonly used classes of enzymes in
biocatalysis. They have been used on a variety of substrates and show very broad substrate
specificity. 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.
 Figure 1: Regioselective hydrolysis of a triacylglycerol.

Description and optimum usage conditions

Strem
Enzyme pH Temp Substrate
Catalog Product Name Activity* Formulation
No. optimum optimum specificity
Number
1. 06-3123 Novozym® 435 10000 PLU/g Immobilized pH 5-9 30-60C Esters and
alcohols
2. 06-3155 Lipozyme® TL IM 250 IUN/g Immobilized pH 6-8 50-75C Esters
3. 06-3120 Lipozyme® RM 275 IUN/g Immobilized pH 7-10 30-50C Esters
4. 06-3105 Lipozyme® CALB L 5000 LU/g Liquid pH 5-9 30-60C Esters and
alcohols
5. 06-3118 Palatase® 20000 L 20000 LU/g Liquid pH 7-10 30-50C Esters
6. 06-3140 Lipozyme® TL 100 L 100 KLU/g Liquid pH 7-10 20-50C Esters and
diesters
7. 06-3100 NovoCor® AD L 6000 LU/g Liquid pH 5-9 30-60C Sterically
hindered esters
8. 06-3125 Resinase® HT 50 KLU/g Liquid pH 5-8 up to 90C Esters
9. 06-3135 Novozym® 51032 15 KLU/g Liquid pH 7-10 35-70C Esters

* K = Kilo, LU = Lipase unit, PLU = Propyl Laurate Unit, IUN = Interesterification Unit.
1LU is the amount of enzyme activity which liberates 1 µmol of tritratable butyric acid from the substrate glycerol tributyrate
per minute under defined standard conditions. 1LU is equal to 1IUN. 1 PLU is the amount of enzyme activity which generates 1
µmol of propyl laurate per minute under defined standard conditions.
Kinetic Resolution

Example 1. Kinetic resolution by transesterification of racemic alcohol1

 Racemic alcohol (1-2 mmol) is solubilized in organic solvent (10 mL Toluene (dry) or
other solvent)*
 Acyl donor vinyl acetate (1:3 or 1:5 molar ratio compared to racemic alcohol) is
added.
 Immobilized Lipase Enzyme1-3 (50% wt/wt with regards to substrate) is added and
the reaction is conducted under stirring.
 Typical reaction temperature is 25-50oC and typical reaction time is 36-72 hours,
depending on substrate.
 The reaction product is recovered by removing the immobilized enzyme by
filtration.

* Alternative solvents: methyl tert butyl ether (MTBE), n-hexane, iso-octane.

Example 2. Kinetic resolution of racemic alcohol by hydrolysis of acetoxy derivative

 Racemic acetoxy ester (1-2 mmol) is solubilized/dispersed in potassium phosphate

buffer (0.1 M, pH 7.5, 10 mL). For liquid substrates, emulsion or suspension will be
formed. For solid substrates, solution is prepared by adding 10% v/v of organic
solvent*
 Lipase Enzyme 1-9 is added to the substrate solution (50% wt/wt for solid enzyme or
10-20% v/v with regards to buffer for liquid enzyme)
 Reaction mixture pH is maintained at 7.5 by adjusting with 1N NaOH.
 Typical reaction temperature is 25-40◦C and typical reaction time is 24-48 hours.
 The reaction product is recovered by extraction or filtration.
*Solvent examples: IPA, acetone, tert-butanol, THF or acetonitrile
Example 3. Dynamic kinetic resolution of racemic alcohols2, 3, 4

 Ruthenium catalyst* (0.05 eq with regards to substrate), Immobilized lipase 1-3

(50% w/w with regards to substrate) and Na2CO3 (1.0 eq. with regards to substrate)
are dissolved in dry organic solvent (5 mL Toluene**) under inert atmosphere (N2)
in a closed vessel.
 Dry toluene is added and resulting mixture is stirred.
 A THF solution of t-BuOK (0.05 eq with regards to substrate) is added to reaction
mixture.
 After 30 min. of stirring, racemic alcohol (1-2 mmol) dissolved in toluene (5 mL) is
added at 25-30°C and stirring continued in 10 min.
 Vinyl acetate (3.0 eq with regards to substrate) is charged to reaction mixture at 25-
30°C.
 Reaction temperature is 50 -55°C and typical reaction time is 36 hours.
 Reaction product is recovered by filtration through filter aid and the filtrate is
concentrated to obtain crude product.
* Ruthenium catalyst options: Chlorodicarbonyl (1,2,3,4,5-pentaphenylcyclopentadienyl) ruthenium
(η5C5Ph5)Ru(CO)2Cl or Shvo catalyst - 1-Hydroxytetraphenylcyclopentadienyl-(tetraphenyl-2,4-cyclopentadien-1-
one)-μ-hydrotetracarbonyldiruthenium(II)
** Solvents have to be dried before using (moisture content should be < 0.01%)

Example 4. Kinetic Resolution by transesterification of Racemic Amine5, 6

 Acyl donor* ethyl acetate (5 mL) is charged to vessel under inert atmosphere (N 2)
 Racemic amine (1-2 mmol) and Immobilized lipase 1-3 (50% wt/wt with regards to
substrate) is added to ethyl acetate at 25-50oC under moderate stirring and inert
atmosphere.
 Typical reaction temperature is 25-40◦C and typical reaction time is 36-72 hours,
depending on substrate.
 Reaction product is recovered by filtration, whereby immobilized enzyme is
removed.
*Acyl donor solvent: α-methylbenzyl acetate, methylmethoxy acetate, ethyl acetate and methyl tert butyl ether.
Example 5. Kinetic resolution by hydrolysis of racemic carboxylic ester

 Racemic ester (1-2 mmol), organic solvent (5 mL MTBE or Toluene) and potassium
phosphate buffer (0.1 M, pH 7.0, 5 mL) is homogenized by stirring. [Two layers will
form once stirring is stopped; stir until substrate is soluble in organic phase. In case
of immobilized enzymes, solid suspension is observed.]
 Lipase Enzyme (50% wt/wt with regards to substrate for solid enzyme 1-3 or 10-20%
v/v with regards to solvent mixture for liquid enzyme 4-9) is added under stirring.
 Reaction mixture is maintained at pH 7.0 by adjusting with 1N NaOH.
 Typical reaction temperature is 20-35oC and typical reaction time is 24-48 hours,
depending on substrate.
 Reaction product is recovered by extraction or filtration.

Example 6. Desymmetrisation of diesters7, 8

 Racemic diester (1-2 mmol) and potassium phosphate buffer (0.1 M, pH 7.0, 10 mL)
is homogenized by stirring.
o For liquid substrates emulsion or suspension will be formed.
o For solid substrates a solution is prepared by adding additional solvents such.
o Biphasic reactions can be carried out by making solution in MTBE or toluene.
o Solvent free reactions can be carried out in a solid suspension.
 Lipase Enzyme (50% wt/wt for solid enzyme 1-3 or 10-20% v/v with regards to buffer
for liquid enzyme 4-9) is added and stirring continued.
 Reaction mixture is maintained at pH 7.5 by adjusting with 1N NaOH.
 Typical reaction temperature is 25-40oC and typical reaction time is 24-48 hours,
depending on substrate.
 Reaction product is recovered by extraction or filtration.
*Solvent: acetone, tetrahydrofuran (THF) or acetonitrile.
Analytical Method Principles

In-process reaction monitoring:

Depending on substrate and product, different methods can be used for in process reaction
monitoring.

 Thin Layer Chromatography (TLC) is a simple method for monitoring reaction progress
and completion.
 To quantitatively estimate product formation and consumption of reactant, HPLC or GC
can be used for monitoring.
 Chiral HPLC is recommended to estimate chiral purity or consumption of isomers of
racemic mixture.
 Final chiral purity can be obtained by analyzing product isolated by using an appropriate
chiral column.
Key parameters for Enantiomeric excess (ee) and Enantioselectivity (E) can be calculated from
the areas in chiral HPLC:

% ee = ((R-S)/(R+S)) × 100 where R and S stand for the individual optical isomer in the
mixture (and R +S = 1)
Where R = area for R isomer and S = area for S isomer

Screening Procedure

Listed below is recommended equipment for conducting the screens, however, pH-stat system
gives more consistent results.

Simple equipment Advanced equipment

Reaction vessel (25 mL round bottom or Thermostated reaction vessel (25 mL)
Erlenmeyer flask or test tubes)
pH-meter or pH-paper (range 5 - 9) Autotitrator/pH-stat system (pH-meter,
automatic burette/addition funnel)
Burette or calibrated addition funnel Recording device (e.g., x/y-plotter)
Propeller mixer or magnetic needle Propeller mixer
Buffer Preparation

0.1M Potassium Phosphate Buffer at 25oC 0.1M Sodium Phosphate Buffer at 25oC
pH Volume of 1M Volume of 1M pH Volume of 1M Volume of 1M
K2HPO4 (mL) KH2PO4 (mL) Na2HPO4(mL) NaH2PO4 (mL)
5.8 8.5 91.5 5.8 7.9 92.1
6.0 13.2 86.8 6.0 12.0 88.0
6.2 19.2 80.8 6.2 17.8 82.2
6.4 27.8 72.2 6.4 25.5 74.5
6.6 38.1 61.9 6.6 35.2 64.8
6.8 49.7 50.3 6.8 46.3 53.7
7.0 61.5 38.5 7.0 57.7 42.3
7.2 71.7 28.3 7.2 68.4 31.6
7.4 80.2 19.8 7.4 77.4 22.6
7.6 86.6 13.4 7.6 84.5 15.5
7.8 90.8 9.2 7.8 89.6 10.4
8.0 94.0 6.0 8.0 93.2 6.8
Dilute combined 1M stock solutions to 1 L with distilled Dilute combined 1M stock solutions to 1 L with distilled
H2O. H2O.

References

1. H. V. Ferreira, L. C. Rocha, R.P. Severino and André L. M. Porto Molecules 2012, 17, 8955-
8967
2. A.Traff, R. Lihammar, and J.E. Backvall J. Org. Chem. 2011, 76, 3917–3921
3. Mahn-Joo Kim, Yangsoo Ahn and Jaiwook Park Current Opinion in Biotechnology 2002,
13:578–587
4. Kiwon Han, Cheolwoo Kim, Jaiwook Park and Mahn-Joo Kim J. Org. Chem. 2010, 75, 3105–
3108
5. Javier Gonzalez-Sabın, Vicente Gotor and Francisca Rebolledo Tetrahedron: Asymmetry 16
(2005) 3070–3076
6. Mahn-Joo Kim, Won-Hee Kim, Kiwon Han, Yoon Kyung Choi, and Jaiwook Park Org. Lett., 9,
No. 6, 2007
7. M. J. Homann, R. Vail, B. Morgan, V. Sabesan, C. Levy, D. R. Dodds, A. Zaks, Adv. Synth.
Catal. 2001, 343, 744-749
8. A. Goswami & T.P.Kissick Org. Proc. Res. Dev. 2009, 13, 483
The products and services described in this document are the responsibility of Novozymes Biopharma DK A/S, Krogshoejvej 36, 2880 Bagsvaerd,
Denmark (company registration no. 29603537) - a wholly owned subsidiary of Novozymes A/S. The information in this document is based on
data we believe to be reliable. They are offered in good faith, but without warranty, as conditions and methods of use of the products are
beyond our control. Furthermore, laws, regulations, and/or third-party rights may prevent the recipient from using the information herein in a
given manner. Thus, the information contained herein is provided “AS IS” and Novozymes makes no representation or warranty whatsoever
with regard to said information, hereunder the accuracy, fitness for a particular purpose, noninfringement of intellectual property rights, or
regulatory/legal compliance, unless otherwise agreed in writing.