Proteases 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 Proteases Proteases (EC 3.4.21.62) are enzymes which conduct proteolysis by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein. Proteases belong to the class of enzymes known as hydrolases catalyzing the reaction of hydrolysis of various bonds with the participation of a water molecule.
Proteases can be used in organic synthesis to resolve a pair of enantiomeric forms in racemic mixtures through kinetic resolution where one enantiomer in the mixture is more rapidly transformed than the other.
Protease catalysts can resolve enantiomers through a variety of reactions such as:
• hydrolysis of esters or amides of carboxylic acid • esterification or transesterification reactions • amide/peptide bond formation
Proteolysis of a peptide bond:
Key applications of proteases Kinetic resolution of amino acids by hydrolysis of racemic amino esters which can be converted into dynamic kinetic resolution by addition of catalytic aldehyde1:
Kinetic resolution of carboxylic acid by hydrolysis of carboxylic ester:
Kinetic resolution of amino acid by hydrolysis of racemic amino ester:
Other potential applications for proteases include:
• hydrolysis of selective amides2 • formation of low molecular weight peptides3 • transesterifications
Serine proteases Serine proteases contain a serine group in their active site which is essential for substrate binding and cleavage. Serine proteases are characterized by their broad substrate specificity and their activity extends beyond purely peptidase to include esterase and amidase activities. The common reaction mechanism is in the form of a catalytic center containing serine as a nucleophile, aspartate as an electrophile and histidine as a base. The reaction mechanism involves the formation of covalently linked enzyme substrate intermediate through acylation resulting in loss of the corresponding amino acid or peptide fragment. Nucleophilic attack on the intermediate by water results in deacylation thereby completing hydrolysis of the peptide. Subtilisin A Subtilisin A (E.C. 3.4.21.62) is an alkaline non-specific serine protease from Bacillus subtilis that initiates the nucleophilic attack on the peptide bond through a serine residue at the active site; it catalyzes the hydrolysis of proteins and peptide amides.
Alcalase® Alcalase® acts as an esterase, enabling it to catalyze stereoselective hydrolysis of some esters. Alcalase also efficiently hydrolyzes amino esters which include heterocyclic amino esters.
Savinase® Savinase® catalyzes stereoselective hydrolysis of some esters as well as strained amides under alkaline conditions.
Esperase® Esperase® is an endo-peptidase with a broad specificity which performs well in alkaline conditions and at elevated temperatures as compared to other microbial serine proteases.
Kinetic resolution of carboxylic acids by hydrolysis of carboxylic esters4:
Kinetic resolution of strained amides2:
Stability of proteases The graphs below represent alcalase pH and temperature stability.
Effect of pH on alcalase activity Effect of temperature on alcalase activity Effect of pH on alcalase stability
100 100 100
80 80 80
60 60 60
40 40 40
20 20 20 Relative activity (%) Relative Relative activity (%) Relative Relative activity (%) Relative
0 0 0 4 5 6 7 8 9 10 10 11 12 10 20 30 40 50 60 70 80 90 100 3 4 5 6 7 8 9 10 11 12
pH Temperature (°C) pH Neutrase® Neutrase® (E.C.3.4.24) is a neutral, zinc metallo endo-protease from Bacillus amyloliquefaciens that randomly hydrolyses internal peptide bonds and also facilitates enzymatic synthesis of oligopeptides by the reverse proteolysis reaction with zinc metal as co-catalyst. Neutrase® belongs to the same protease family as thermolysin, a zinc dependent metallo endo-protease.
Thermolysin in an immobilized form has been used successfully in industrial processes for synthesis of an Aspartame intermediate5. The reaction takes place in organic solvent and involves kinetic resolution of an amine methyl ester with high enatioselectivity and high regioselectivity in the amide bond formation of the a-carbonyl in Aspartic acid preferred over the b-carbonyl. rTrypsin® rTrypsin® (EC 3.4.21.4) is an endopeptidase that preferentially hydrolyses ester bonds whose carboxyl groups are contributed by lysine (Lys) or arginine (Arg) except when either is followed by proline. The enzymatic mechanism of action is similar to other serine proteases. The aspartate residue located in the catalytic pocket of rTrypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is thus responsible for the specificity of the enzyme.
Benefits of enzymes as biocatalysts in organic transformations
Cost savings Improved productivity Improved quality of API/intermediate Environmental friendliness
• reduction in raw material input • shortened synthesis routes • fewer or no by-products, leading • reduction of waste products • avoids use of costly chiral • more batches resulting in increased to reduced impurities in the final produced and solvent usage resolving agents or costly metal capacity products • higher energy savings based catalysts • avoids laborious protection and • high stereo-, regio-, and chemo- • lower equipment, labor and de-protection selectivity energy costs • higher yields • less residual solvent carry over from reduced solvent use
Available from Novozymes
Product EC Optimum usage Type Form Activity Applications name number conditions
Alcalase 3.4.21.62 Serine endo-peptidase Liquid 30–65°C, 2.4 AU-A/g Stereoselective hydrolysis of amino esters 2.4 L FG (mainly subtilisin A) pH 7–9 and selective esters
Alcalase 3.4.21.62 Serine endo-peptidase Liquid 30–65°C, 2.5 AU-A/g Stereoselective hydrolysis of amino esters 2.4 L, DX (mainly subtilisin A) pH 7–9 and selective esters
Savinase 3.4.21.62 Serine endo-peptidase Granulate 30–70°C, 12 KN-PU- Stereoselective hydrolysis of amino esters 12 T (mainly subtilisin A) pH 8–10 S/g and selective esters
Savinase 3.4.21.62 Serine endo-peptidase Liquid 30–70°C, 16 KN-PU- Stereoselective hydrolysis of amino esters 16 L (mainly subtilisin A) pH 8–10 S/g and selective esters
Esperase 3.4.21.62 Serine endo-peptidase Liquid 30–70°C, 8 KNPU-E/g Serine endoprotease that hydrolyzes internal 8.0 L (mainly subtilisin A) pH 8–10 peptide bonds
Neutrase 3.4.24.28 Metalloprotease Liquid 40–50°C, pH 7 0.8 AU-N/g Metallo endoprotease that hydrolyz-es internal 0.8 L peptide bonds
rTrypsin 3.4.21.4 Serin protease Granulate pH 7.8–8.0 800 USP/mg Hydrolysis of amid end ester bonds of lysine and arginine at carboxyl terminal
* K = Kilo, AU = Anson Unit, NPU = Novo Protease Unit, 1 AU = 1NPU, ASNU = Asparaginace Unit, USP = Trypsin activity unit using USP Crystallised Trypsin Reference Standard. The activity is determined relative to a protease A standard. The result is given in the same units as the standard. 1 ASNU is the amount of enzyme that produces 1 µmol Ammonia per minute under the standard reaction conditions. 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.
About Novozymes +45 44460044(DK) Novozymes A/S Novozymes is the world leader in biological solutions. Together with customers, partners and the +1 9194943331(US) Krogshoejvej 36 global community, we improve industrial performance while preserving the planet’s resources and +91 2025604401(IN) DK – 2880 Bagsvaerd helping to build better lives. As the world’s largest provider of enzyme and microbial technologies, +86 13817879003 (CN) Denmark our bioinnovation enables higher agricultural yields, low-temperature washing, energy-efficient [email protected] production, renewable fuel and many other benefits that we rely on today and in the future. We call it Rethink Tomorrow. June 2016 - No. 2014-12715-04 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 novozymes.com are used as process aids, it is the responsibility of the API or excipient manufacturer to assure that the enzymes are suitable for the 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.