ENZYMES FOR BIOCATALYSIS for smarter chemical synthesis Graphical representation of alcalase molecule Rethink Tomorrow Proteases
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). 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. A. Various microorganisms can be used to produce natural catalysts such as enzymes B. The enzymes are purified from the microorganism for industrial use C. An enzyme attracts specific substrates to its active site 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 aldehyde 1 : 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 amides 2 Formation of low molecular weight peptides 3 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 esters 4 : Kinetic resolution of strained amides 2 :
Stability of proteases The graphs below represent alcalase ph and temperature stability. Please refer to the product portfolio table overleaf for details of other products. Effect of ph on Alcalase activity 120 100 80 Relative activity (%) 60 40 20 0-20 3 4 5 6 7 8 9 10 11 12 ph Effect of temperature on Alcalase activity 120 100 Relative activity (%) 80 60 40 20 0 10 20 30 40 50 60 70 80 90 Temperature ( C) Effect of ph on Alcalase stability 120 100 80 Relative activity (%) 60 40 20 0-20 0 1 2 3 4 5 6 7 8 9 10 11 12 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 intermediate 5. 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 proteases 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 to reduced impurities in the final products High stereo-, regio-, and chemo-selectivity Less residual solvent carry over from reduced solvent use Reduction of waste products produced and solvent usage Higher energy savings
Available from Novozymes Product Name EC No. Substrate specificity Format Optimal Conditions Unit Activity Application Alcalase 2.4 L FG 3.4.21.62 liquid 30-65 C, ph 7-9 2.4 AU-A/g Stereoselective hydrolysis of amino esters and selective esters; suitable for hydrolysis of proteins; used in transesterification and transpeptidation. Alcalase 2.5 L, DX 3.4.21.62 liquid 30-65 C, ph 7-10 2.5 AU-A/g Stereoselective hydrolysis of amino esters and selective esters; suitable for hydrolysis of proteins; used in transesterification and transpeptidation. Savinase 12 T, W 3.4.21.62 granulate 30-70 C, ph 8-10 12 KNPU-S/g Stereoselective hydrolysis of amino esters and selective esters; suitable for hydrolysis of proteins, hydrolysis of strained amides Savinase 16 L, EX 3.4.21.62 granulate 30-70 C, ph 8-10 16 KNPU-S/g Stereoselective hydrolysis of amino esters and selective esters; suitable for hydrolysis of proteins, hydrolysis of strained amides Esperase 8.0 L 3.4.21.62 liquid ph 8-12.5 8 KNPU-E/g Hydrolysis of internal peptide bonds; characterized by excellent performance at elevated temperature and ph. rtrypsin 3.4.21.4 Serine protease granulate ph 7.8-8.0 800 USP/mg Neutrase 0.8 3.4.22 Metalloprotease liquid 40-50 C, ph 7 0.8 AU/g Hydrolysis of amide and ester bonds of lysine and arginine at carboxyl terminal Kinetic resolution of amino esters * 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.
Novozymes is the world leader in bioinnovation. Together with customers across a broad array of industries we create tomorrow s industrial biosolutions, improving our customers business and the use of our planet s resources. Read more at www.novozymes.com. References 1. D. A. Schichl, S. Enthaler, W. Holla, T. Riermeier, U. Kragl and M. Beller Eur. J. Org. Chem. 2008, 3506 3512 2. Mahmoud Mahmoudian, Andrew Lowdon, Martin Jones, Micheal Dawson, Christopher Wallis Tetrahedron Asymmetry 10 (1999) 1201-1206 3. Hou, R.-Z, Yang, Y. Li, G., Huang, Y.B., Wang, H., Zhang, N., Liu, Y. J., Li, X. and Zhang, X.Z., Biotechnol.Appl.Biochem., 2006, 44, 73 4. Riermeier, T., Dingerdissen, U., Gross, P., Holla, W., Beller, M., and Schichl, D. (2001) DE19955283 5. T. Nagayasu, M. Miyanaga, T. Tanaka, T. Sakiyama and K. Nakanishi, Biotechnology and Bioengineering, 1994, 43, 1118-1123 Novozymes Biopharma US Inc. (reg no. 01142208) 77 Perry Chapel Church Road Franklinton, NC 27525, USA Tel. +1 919 494 3000 Novozymes Biopharma DK A/S Krogshoejvej 36 2880 Bagsvaerd, Denmark Tel: +45 4446 2896 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, non-infringement of intellectual property rights, or regulatory/legal compliance, unless otherwise agreed in writing. Novozymes A/S No. 2014-12715-01