Production and Purification of Therapeutic Enzymes

  • M. Ângela TaipaEmail author
  • Pedro Fernandes
  • Carla C. C. R. de Carvalho
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1148)


The use of therapeutic enzymes embraces currently a vast array of applications, abridging from diggestive disorders to cancer therapy, cardiovascular and lysosomal storage diseases. Enzyme drugs bind and act on their targets with great affinity and specificity, converting substrates to desired products in a reduced time frame with minimal side reactions. These characteristics have resulted in the development of a multitude of enzyme biopharmaceuticals for a wide range of human disorders.

The advances in genetic engineering and DNA recombination techniques facilitated the production of therapeutical human-like enzymes, using different cells as host organisms. The selection of hosts generally privileges those that secrete the enzyme into the culture medium, as this eases the purification process, and those that are able to express complex glycoproteins, with glycosylation patterns and other post-translational modifications close to human proteins. Moreover, engineering approaches such as pegylation, encapsulation in micro- and nanocarriers, and mutation of amino acid residues of the native enzyme molecule to yield variants with improved therapeutic activity, half-life and/or stability, have been also addressed. Engineered enzyme products have been designed to display enhanced delivery to target sites and reduced adverse side-effects (e.g., immunogenicity) upon continuous drug administration.

Irrespectively of the production method, the final formulation of therapeutic enzymes must display high purity and specificity, and they are often marketed as lyophilized pure preparations with biocompatible buffering salts and diluents to prepare the reconstituted aqueous solution before treatment.


Therapeutic enzyme Human therapy Recombinant Engineering Production Purification 





n-acetylgalactosamine 4-sulfatase


Bovine spongiform encephalomyelitis


Cholesteryl ester storage disease


Chinese hamster ovary


Cocaine esterase


Computational protein design


Directed evolution




European Medicines Agency


Food and Drug Administration


Human butyrylcholinesterase


Half-maximal inhibitory concentration


Ion metal affinity chromatography


Turnover number


Michaelis–Menten constant






Polyethylene glycol




Plant-made pharmaceuticals


Severe combined immunodeficiency


Site-directed mutagenesis


Half-inactivation temperature


Tissue plasminogen activator


Urokinase-type plasminogen activator



CCCR de Carvalho acknowledges Fundação para a Ciência e a Tecnologia, I.P. (FCT), Portugal, for financial support under program “FCT Investigator 2013” (IF/01203/2013/CP1163/CT0002).


  1. Abdel-Mageed HM, Fahmy AS, Shaker DS, Mohamed SA (2018) Development of novel delivery system for nanoencapsulation of catalase: formulation, characterization, and in vivo evaluation using oxidative skin injury model. Artif Cells Nanomed Biotechnol 46:1–10. CrossRefGoogle Scholar
  2. Abderrezak K (2018) Therapeutic enzymes used for the treatment of non-deficiency diseases. In: Shashi Lata B, Pankaj Kumar C (eds) Research advancements in pharmaceutical, nutritional, and industrial enzymology. IGI Global, Hershey, pp 46–70. CrossRefGoogle Scholar
  3. Abdoli-Nasab M, Javaran MJ, Cusido RM, Palazon J (2016) Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants. Plant Physiol Biochem 108:139–144. PubMedCrossRefGoogle Scholar
  4. Adrio JL, Demain AL (2014) Microbial enzymes: tools for biotechnological processes. Biomol Ther 4(1):117–139. CrossRefGoogle Scholar
  5. Aggarwal S (2007) What’s fueling the biotech engine? Nat Biotechnol 25:1097–1104. PubMedCrossRefGoogle Scholar
  6. Agrawal V, Bal M (2012) Strategies for rapid production of therapeutic proteins in mammalian cells. Bioprocess Int 10(4):32–48Google Scholar
  7. Allied_Market_Research (2018) Specialty enzymes market is expected to reach $947.5 million, globally, by 2020. Accessed 17 Aug 2018
  8. Andrady C, Sharma SK, Chester KA (2011) Antibody–enzyme fusion proteins for cancer therapy. Immunotherapy 3(2):193–211. PubMedCrossRefGoogle Scholar
  9. Arnau J, Lauritzen C, Petersen GE, Pedersen J (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48(1):1–13. PubMedCrossRefGoogle Scholar
  10. Baldo BA (2015) Enzymes approved for human therapy: indications, mechanisms and adverse effects. BioDrugs 29(1):31–55. PubMedCrossRefGoogle Scholar
  11. Bonnerjea J (2004) Purification of therapeutic proteins. In: Cutler P (ed) Protein purification protocols. Humana Press, Totowa, pp 455–462. CrossRefGoogle Scholar
  12. Brady RO (2006) Enzyme replacement for lysosomal diseases. Annu Rev Med 57(1):283–296. PubMedCrossRefGoogle Scholar
  13. Brandazza A, Lee E, Ferrera M, Tillman U, Sarmientos P, Westphal H (1991) Use of the urokinase-type plasminogen activator gene as a general tool to monitor expression in transgenic animals: study of the tissue-specificity of the murine whey acidic protein (WAP) expression signals. J Biotechnol 20(2):201–212. PubMedCrossRefGoogle Scholar
  14. Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13(4):329–332. PubMedCrossRefGoogle Scholar
  15. Cheng F, Kardashliev T, Pitzler C, Shehzad A, Lue H, Bernhagen J, Zhu L, Schwaneberg U (2015a) A competitive flow cytometry screening system for directed evolution of therapeutic enzyme. ACS Synth Biol 4(7):768–775. PubMedCrossRefGoogle Scholar
  16. Cheng F, Zhu L, Lue H, Bernhagen J, Schwaneberg U (2015b) Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas. Appl Microbiol Biotechnol 99(3):1237PubMedCrossRefGoogle Scholar
  17. Chon JH, Zarbis-Papastoitsis G (2011) New Biotechnol 28(5):458–463. PubMedCrossRefGoogle Scholar
  18. Daniell H, Singh ND, Mason H, Streatfield SJ (2009) Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci 14(12):669–679. PubMedPubMedCentralCrossRefGoogle Scholar
  19. Dean SN, Turner KB, Medintz IL, Walper SA (2017) Targeting and delivery of therapeutic enzymes. Ther Deliv 8(7):577–595. PubMedCrossRefGoogle Scholar
  20. Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27(3):297–306. PubMedCrossRefGoogle Scholar
  21. Derst C, Henseling J, Röhm KH (2000) Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248. Protein Sci 9(10):2009–2017PubMedPubMedCentralCrossRefGoogle Scholar
  22. Doran PM (2013) Chapter 11: Unit operations. In: Doran PM (ed) Bioprocess engineering principles, 2nd edn. Academic, London, pp 445–595. CrossRefGoogle Scholar
  23. Dozier KJ, Distefano DM (2015) Site-specific PEGylation of therapeutic proteins. Int J Mol Sci 16(10):25831–25864. PubMedPubMedCentralCrossRefGoogle Scholar
  24. Fang L, Chow KM, Hou S, Xue L, Chen X, Rodgers DW, Zheng F, Zhan C-G (2014) Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification. ACS Chem Biol 9(8):1764–1772. PubMedPubMedCentralCrossRefGoogle Scholar
  25. Farhadi SA, Bracho-Sanchez E, Freeman SL, Keselowsky BG, Hudalla GA (2018) Enzymes as immunotherapeutics. Bioconjug Chem 29(3):649–656. PubMedPubMedCentralCrossRefGoogle Scholar
  26. Fazel R, Zarei N, Ghaemi N, Namvaran MM, Enayati S, Mirabzadeh Ardakani E, Azizi M, Khalaj V (2014) Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris. Springerplus 3(1):395. PubMedPubMedCentralCrossRefGoogle Scholar
  27. Ferreira RG, Azzoni AR, Freitas S (2018) Techno-economic analysis of the industrial production of a low-cost enzyme using E. coli: the case of recombinant β-glucosidase. Biotechnol Biofuels 11(1):81. PubMedPubMedCentralCrossRefGoogle Scholar
  28. Fong BA, Wu W-Y, Wood DW (2010) The potential role of self-cleaving purification tags in commercial-scale processes. Trends Biotechnol 28(5):272–279. PubMedCrossRefGoogle Scholar
  29. Fuhrmann G, Leroux J-C (2014) Improving the stability and activity of oral therapeutic enzymes-recent advances and perspectives. Pharm Res 31(5):1099–1105. PubMedCrossRefGoogle Scholar
  30. Funaro MG, Nemani KV, Chen Z, Bhujwalla ZM, Griswold KE, Gimi B (2016) Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase. J Microencapsul 33(1):64–70. PubMedCrossRefGoogle Scholar
  31. Giffard M, Ferté N, Ragot F, El Hajji M, Castro B, Bonneté F (2011) Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography. PLoS One 6(5):e19013. PubMedPubMedCentralCrossRefGoogle Scholar
  32. Gomes AMV, Carmo TS, Carvalho LS, Bahia FM, Parachin NS (2018) Comparison of yeasts as hosts for recombinant protein production. Microorganisms 6(2):38. CrossRefGoogle Scholar
  33. Gonzalez NJ, Isaacs LL (1999) Evaluation of pancreatic proteolytic enzyme treatment of adenocarcinoma of the pancreas, with nutrition and detoxification support. Nutr Cancer 33(2):117–124. PubMedCrossRefGoogle Scholar
  34. Goojani HG, Javaran MJ, Nasiri J, Goojani EG, Alizadeh H (2013) Expression and large-scale production of human tissue plasminogen activator (t-PA) in transgenic tobacco plants using different signal peptides. Appl Biochem Biotechnol 169(6):1940–1951. PubMedCrossRefGoogle Scholar
  35. Goward CR, Stevens GB, Tattersall R, Atkinson T (1992) Rapid large-scale preparation of recombinant Erwinia chrysanthemi L-asparaginase. Bioseparation 2(6):335–341PubMedGoogle Scholar
  36. Grabowski GA, Barton NW, Pastores G et al (1995) Enzyme therapy in type 1 gaucher disease: comparative efficacy of mannose-terminated glucocerebrosidase from natural and recombinant sources. Ann Intern Med 122(1):33–39. PubMedCrossRefGoogle Scholar
  37. Gupta SK, Shukla P (2017) Sophisticated cloning, fermentation, and purification technologies for an enhanced therapeutic protein production: a review. Front Pharmacol 8:419. PubMedPubMedCentralCrossRefGoogle Scholar
  38. Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. Biomed Res Int 2013:329121. PubMedPubMedCentralCrossRefGoogle Scholar
  39. Hanke AT, Ottens M (2014) Purifying biopharmaceuticals: knowledge-based chromatographic process development. Trends Biotechnol 32(4):210–220. PubMedCrossRefGoogle Scholar
  40. Headon DR, Walsh G (1994) The industrial production of enzymes. Biotechnol Adv 12(4):635–646. PubMedCrossRefGoogle Scholar
  41. Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422. PubMedCrossRefGoogle Scholar
  42. Hidalgo D, Abdoli-Nasab M, Jalali-Javaran M, Bru-Martínez R, Cusidó RM, Corchete P, Palazon J (2017) Biotechnological production of recombinant tissue plasminogen activator protein (reteplase) from transplastomic tobacco cell cultures. Plant Physiol Biochem 118:130–137. PubMedCrossRefGoogle Scholar
  43. Ho SV (1990) Strategies for large-scale protein purification. In: Protein purification. vol 427. ACS symposium series, vol 427. American Chemical Society, pp 14–34. Google Scholar
  44. Hohmann H, van Dijl JM, Krishnappa L, Prágai Z (2016) Host organisms: Bacillus subtilis. In: Wittmann C, Liao JC (eds) Industrial biotechnology, vol 1. Wiley, Weinheim. CrossRefGoogle Scholar
  45. Huang C Jr, Lin H, Yang X (2012) Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J Ind Microbiol Biotechnol 39(3):383–399. CrossRefGoogle Scholar
  46. Huang Y-M, Hu W, Rustandi E, Chang K, Yusuf-Makagiansar H, Ryll T (2010) Maximizing productivity of CHO cell-based fed-batch culture using chemically defined media conditions and typical manufacturing equipment. Biotechnol Prog 26(5):1400–1410. PubMedCrossRefGoogle Scholar
  47. Jaliani HZ, Farajnia S, Mohammadi SA, Barzegar A, Talebi S (2013) Engineering and kinetic stabilization of the therapeutic enzyme Anabeana variabilis phenylalanine ammonia lyase. Appl Biochem Biotechnol 171(7):1805–1818. PubMedCrossRefGoogle Scholar
  48. Jayapal KP, Wlaschin KF, Hu WS, Yap MGS (2007) Recombinant protein therapeutics from CHO cells – 20 years and counting. Chem Eng Prog 103:40–47Google Scholar
  49. Jeyaharan D, Aston P, Garcia-Perez A, Schouten J, Davis P, Dixon AM (2016) Soluble expression, purification and functional characterisation of carboxypeptidase G2 and its individual domains. Protein Expr Purif 127:44–52. PubMedCrossRefGoogle Scholar
  50. Kakkis ED, Matynia A, Jonas AJ, Neufeld EF (1994) Overexpression of the human lysosomal enzyme α-L-iduronidase in Chinese hamster ovary cells. Protein Expr Purif 5(3):225–232. PubMedCrossRefGoogle Scholar
  51. Kaur R, Sekhon BS (2012) Enzymes as drugs: an overview. J Pharm Educ Res 3(2):29–41Google Scholar
  52. Khushoo A, Pal Y, Singh BN, Mukherjee KJ (2004) Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. Protein Expr Purif 38(1):29–36. PubMedCrossRefGoogle Scholar
  53. Kotzia GA, Labrou NE (2009) Engineering thermal stability of L-asparaginase by in vitro directed evolution. FEBS J 276(6):1750–1761. PubMedCrossRefGoogle Scholar
  54. Kumar SS, Abdulhameed S (2017) Therapeutic enzymes. In: Sugathan S, Pradeep NS, Abdulhameed S (eds) Bioresources and bioprocess in biotechnology: volume 2 : Exploring potential biomolecules. Springer, Singapore, pp 45–73. CrossRefGoogle Scholar
  55. Kunamneni A, Ogaugwu C, Goli D (2018) Chapter 15: Enzymes as therapeutic agents. In: Nunes C, Kumar V (eds) Enzymes in human and animal. Nutrition Academic Press, Oxford, pp 301–312. CrossRefGoogle Scholar
  56. Lalonde M-E, Durocher Y (2017) Therapeutic glycoprotein production in mammalian cells. J Biotechnol 251:128–140. PubMedCrossRefGoogle Scholar
  57. Langer ES (2017) Trends and growth in single-use system (SUS) adoption. Am Pharm Rev 343797Google Scholar
  58. Laukens B, Visscher CD, Callewaert N (2015) Engineering yeast for producing human glycoproteins: where are we now? Future Microbiol 10(1):21–34. PubMedCrossRefGoogle Scholar
  59. Laurent JM, Young JH, Kachroo AH, Marcotte EM (2016) Efforts to make and apply humanized yeast. Brief Funct Genomics 15(2):155–163. PubMedCrossRefGoogle Scholar
  60. Leplatois P, Le Douarin B, Loison G (1992) High-level production of a peroxisomal enzyme: Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae. Gene 122(1):139–145. PubMedCrossRefGoogle Scholar
  61. Li M (2018) Enzyme replacement therapy: a review and its role in treating lysosomal storage diseases. Pediatr Ann 47(5):e191–e197. PubMedCrossRefGoogle Scholar
  62. Li J, Chen Z, Hou L, Fan H, Weng S, Ce X, Ren J, Li B, Chen W (2006) High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli. Protein Expr Purif 49(1):55–59. PubMedCrossRefGoogle Scholar
  63. Li W, Xu S, Zhang B, Zhu Y, Hua Y, Kong X, Sun L, Hong J (2017) Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis. PLoS One 12(5):e0177877. PubMedPubMedCentralCrossRefGoogle Scholar
  64. Lim J, Sinclair A, Shevitz J, Carter JB (2011) An economic comparison of three cell culture techniques: fed-batch, concentrated fed-batch, and concentrated perfusion. BioPharm Int 24(2):54–60Google Scholar
  65. Löffler P, Schmitz S, Hupfeld E, Sterner R, Merkl R (2017) Rosetta:MSF: a modular framework for multi-state computational protein design. PLoS Comput Biol 13(6):e1005600. PubMedPubMedCentralCrossRefGoogle Scholar
  66. Lutz S, Williams E, Muthu P (2017) Engineering therapeutic enzymes. In: Alcalde M (ed) Directed enzyme evolution: advances and applications. Springer, Cham, pp 17–67. CrossRefGoogle Scholar
  67. Mane P, Tale V (2015) Overview of microbial therapeutic enzymes. Int J Curr Microbiol Appl Sci 4(4):17–26Google Scholar
  68. Market Research Engine (2018) Specialty enzymes market to touch US$ 5.5 Billion by 2022. Herald Keeper. Accessed 17 Aug 2018
  69. Merlin M, Gecchele E, Capaldi S, Pezzotti M, Avesani L (2014) Comparative evaluation of recombinant protein production in different biofactories: the green perspective. Biomed Res Int 2014:136419–136419. PubMedPubMedCentralCrossRefGoogle Scholar
  70. Mierau I, Leij P, van Swam I, Blommestein B, Floris E, Mond J, Smid EJ (2005) Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin. Microb Cell Factories 4(1):15. CrossRefGoogle Scholar
  71. Mishra P, Nayak B, Dey RK (2016) PEGylation in anti-cancer therapy: an overview. Asian J Pharm Sci 11(3):337–348. CrossRefGoogle Scholar
  72. Mitragotri S, Burke PA, Langer R (2014) Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat Rev Drug Discov 13(9):655–672. PubMedPubMedCentralCrossRefGoogle Scholar
  73. Muro S (2010) New biotechnological and nanomedicine strategies for treatment of lysosomal storage disorders. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2(2):189–204. PubMedPubMedCentralCrossRefGoogle Scholar
  74. Nazari-Robati M, Khajeh K, Aminian M, Mollania N, Golestani A (2013) Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: an insight from Ramachandran plot. BBA-Proteins Proteomics 1834(2):479–486. PubMedCrossRefGoogle Scholar
  75. NDHHS NDoHaHS (2012) Therapeutic class overview pancreatic enzymes.
  76. Nijland R, Kuipers O (2008) Optimization of protein secretion by Bacillus subtilis. Recent Pat Biotechnol 2(2):79–87. PubMedCrossRefGoogle Scholar
  77. Noppen B, Fonteyn L, Aerts F, De Vriese A, De Maeyer M, Le Floch F, Barbeaux P, Zwaal R, Vanhove M (2014) Autolytic degradation of ocriplasmin: a complex mechanism unraveled by mutational analysis. Protein Eng Des Sel 27(7):215–223. PubMedCrossRefGoogle Scholar
  78. Oh D-B (2015) Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases. BMB Rep 48(8):438–444. PubMedPubMedCentralCrossRefGoogle Scholar
  79. Oleck J, Kassam S, Goldman JD (2016) Diabetes Spectr 29(3):180–184. PubMedPubMedCentralCrossRefGoogle Scholar
  80. Pan Y, Gao D, Yang W, Cho H, Yang G, Tai H-H, Zhan C-G (2005) Computational redesign of human butyrylcholinesterase for anticocaine medication. Proc Natl Acad Sci U S A 102(46):16656–16661. PubMedPubMedCentralCrossRefGoogle Scholar
  81. Patil PN (2012) Discoveries in pharmacological sciences. Discoveries in pharmacological sciences. World Scientific Publishing Co., Singapore. CrossRefGoogle Scholar
  82. Patti T, Bembi B, Cristin P, Mazzarol F, Secco E, Pappalardo C, Musetti R, Martinuzzi M, Versolatto S, Cariati R, Dardis A, Marchetti S (2012) Endosperm-specific expression of human acid beta-glucosidase in a waxy rice. Rice 5(1):34. PubMedPubMedCentralCrossRefGoogle Scholar
  83. Persistence Market Research (2018) Therapeutic enzymes market: global industry trend analysis 2012 to 2017 and forecast 2017–2025. Accessed 14 Sept 2018
  84. Petersen C (2015) History of health. Manuf Today :90–92Google Scholar
  85. Phue J-N, Shiloach J (2004) Transcription levels of key metabolic genes are the cause for different glucose utilization pathways in E. coli B (BL21) and E. coli K (JM109). J Biotechnol 109(1):21–30. PubMedCrossRefGoogle Scholar
  86. Qi Y, Chilkoti A (2015) Protein-polymer conjugation – moving beyond PEGylation. Curr Opin Chem Biol 28:181–193. PubMedPubMedCentralCrossRefGoogle Scholar
  87. Qiu J, Swartz JR, Georgiou G (1998) Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl Environ Microbiol 64(12):4891–4896PubMedPubMedCentralGoogle Scholar
  88. Ramirez-Paz J, Saxena M, Delinois LJ, Joaquín-Ovalle FM, Lin S, Chen Z, Rojas-Nieves VA, Griebenow K (2018) Thiol-maleimide poly(ethylene glycol) crosslinking of L-asparaginase subunits at recombinant cysteine residues introduced by mutagenesis. PLoS One 13(7):e0197643. PubMedPubMedCentralCrossRefGoogle Scholar
  89. Richette P, Bardin T (2006) Successful treatment with rasburicase of a tophaceous gout in a patient allergic to allopurinol. Nat Clin Pract Rheumatol 2(6):338–342.; quiz 343. PubMedCrossRefGoogle Scholar
  90. Rodríguez-Martínez JA, Rivera-Rivera I, Solá RJ, Griebenow K (2009) Enzymatic activity and thermal stability of PEG-α-chymotrypsin conjugates. Biotechnol Lett 31(6):883–887. PubMedPubMedCentralCrossRefGoogle Scholar
  91. Roque ACA, Lowe CR, Taipa MÂ (2004) Antibodies and genetically engineered related molecules: production and purification. Biotechnol Prog 20(3):639–654. PubMedCrossRefGoogle Scholar
  92. Roque ACA, Silva CSO, Taipa MÂ (2007) Affinity-based methodologies and ligands for antibody purification: advances and perspectives. J Chromatogr A 1160(1):44–55. PubMedCrossRefGoogle Scholar
  93. Roxas M (2008) The role of enzyme supplementation in digestive disorders. Altern Med Rev 13(4):307–314PubMedGoogle Scholar
  94. Ryan BJ, Henehan GT (2013) Overview of approaches to preventing and avoiding proteolysis during expression and purification of proteins. Curr Protoc Protein Sci 71(1):5.25.21–25.25.27. CrossRefGoogle Scholar
  95. Sabalza M, Christou P, Capell T (2014) Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 36(12):2367–2379. PubMedCrossRefGoogle Scholar
  96. Samish I (2017) Achievements and challenges in computational protein design. In: Samish I (ed) Computational protein design. Springer, New York, pp 21–94. CrossRefGoogle Scholar
  97. Shahaboddin ME, Khajeh K, Maleki M, Golestani A (2017) Improvement of activity and stability of Chondroitinase ABC I by introducing an aromatic cluster at the surface of protein. Enzym Microb Technol 105:38–44. CrossRefGoogle Scholar
  98. Shan L, Bethune M, Khosla C, Gass J (2005) Therapeutic enzyme formulations and uses thereof WO/2005/107786Google Scholar
  99. Shanley N, Walsh G (2005) Applied enzymology, an overview. In: McGrath BM, Walsh G (eds) Directory of therapeutic enzymes. CRC Press, Boca Raton, pp 1–16Google Scholar
  100. Sharabi O, Erijman A, Shifman JM (2013) Chapter 3: Computational methods for controlling binding specificity. In: Keating AE (ed) Methods in enzymology, vol 523. Academic, Burlington, pp 41–59. CrossRefGoogle Scholar
  101. Sharifi-Sirchi GR, Jalali-Javaran M (2016) Selecting appropriate hosts for recombinant proteins production: review article. HMJ 20(3):214–222Google Scholar
  102. Sharma SK, Bagshawe KD (2017) Antibody directed enzyme prodrug therapy (ADEPT): trials and tribulations. Adv Drug Deliv Rev 118:2–7. PubMedCrossRefGoogle Scholar
  103. Singh R, Kumar M, Mittal A, Mehta PK (2016) Microbial enzymes: industrial progress in 21st century. 3 Biotech 6(2):174–174. PubMedPubMedCentralCrossRefGoogle Scholar
  104. Sizer IW (1972) Medical applications of microbial enzymes. In: Perlman D (ed) Advances in applied microbiology, vol 15. Academic, New York, pp 1–11. CrossRefGoogle Scholar
  105. Smelko JP, Wiltberger KR, Hickman EF, Morris BJ, Blackburn TJ, Ryll T (2011) Performance of high intensity fed-batch mammalian cell cultures in disposable bioreactor systems. Biotechnol Prog 27(5):1358–1364. PubMedCrossRefGoogle Scholar
  106. Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5(1):2–15. PubMedCrossRefGoogle Scholar
  107. Tobin PH, Richards DH, Callender RA, Wilson CJ (2014) Protein engineering: a new frontier for biological therapeutics. Curr Drug Metab 15(7):743–756PubMedPubMedCentralCrossRefGoogle Scholar
  108. Tripathi NK (2016) Production and purification of recombinant proteins from Escherichia coli. ChemBioEng Rev 3(3):116–133. CrossRefGoogle Scholar
  109. Tripathi N, Kannusamy S, Jana A, Rao PVL (2009) High yield production of heterologous proteins with Escherichia coli. Def Sci J 59(2):137–146. CrossRefGoogle Scholar
  110. Tundisi LL, Coêlho DF, Zanchetta B, Moriel P, Pessoa A, Tambourgi EB, Silveira E, Mazzola PG (2017) L-Asparaginase purification. Sep Purif Rev 46(1):35–43. CrossRefGoogle Scholar
  111. Varga I, Bobalova M, Michalkova E, Jakubcova M (2011) Method of polymyxin B recovery from fermentation broth. USA Patent US7951913B2, 2011-05-31Google Scholar
  112. Vellard M (2003) The enzyme as drug: application of enzymes as pharmaceuticals. Curr Opin Biotechnol 14(4):444–450. PubMedPubMedCentralCrossRefGoogle Scholar
  113. Verma N, Kumar K, Kaur G, Anand S (2007) L-asparaginase: a promising chemotherapeutic agent. Crit Rev Biotechnol 27(1):45–62. PubMedCrossRefGoogle Scholar
  114. Vidya J, Ushasree MV, Pandey A (2014) Effect of surface charge alteration on stability of L-asparaginase II from Escherichia sp. Enzym Microb Technol 56:15–19. CrossRefGoogle Scholar
  115. Vidya J, Sajitha S, Ushasree MV, Binod P, Pandey A (2017) 12 – Therapeutic enzymes: l-Asparaginases. In: Pandey A, Negi S, Soccol CR (eds) Current developments in biotechnology and bioengineering. Elsevier, Amsterdam, pp 249–265. CrossRefGoogle Scholar
  116. Wang A, Lewus R, Rathore AS (2006) Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth. Biotechnol Bioeng 94(1):91–104. PubMedCrossRefGoogle Scholar
  117. Waugh DS (2011) An overview of enzymatic reagents for the removal of affinity tags. Protein Expr Purif 80(2):283–293. PubMedPubMedCentralCrossRefGoogle Scholar
  118. Wells E, Robinson AS (2017) Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement. Biotechnol J 12(1):1600105. CrossRefGoogle Scholar
  119. Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. BBA-Mol Cell Res 1694(1):299–310. CrossRefGoogle Scholar
  120. Wijma HJ, Janssen DB (2013) Computational design gains momentum in enzyme catalysis engineering. FEBS J 280(13):2948–2960. PubMedCrossRefGoogle Scholar
  121. Wildt S, Gerngross TU (2005) The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3:119–128. PubMedCrossRefGoogle Scholar
  122. Willson RC, Ladisch MR (1990) Large-scale protein purification. In: Protein purification. vol 427. ACS Symposium Series, vol 427. American Chemical Society, pp 1–13. Google Scholar
  123. Xiao H, Bao Z, Zhao H (2015) High throughput screening and selection methods for directed enzyme evolution. Ind Eng Chem Res 54(16):4011–4020. PubMedCrossRefGoogle Scholar
  124. Xue L, Ko M-C, Tong M, Yang W, Hou S, Fang L, Liu J, Zheng F, Woods JH, Tai H-H, Zhan C-G (2011) Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine. Mol Pharmacol 79(2):290–297. PubMedPubMedCentralCrossRefGoogle Scholar
  125. Yang H, Li J, Du G, Liu L (2017) Chapter 6: Microbial production and molecular engineering of industrial enzymes: challenges and strategies. In: Brahmachari G (ed) Biotechnology of microbial enzymes. Academic, Amsterdam, pp 151–165. CrossRefGoogle Scholar
  126. Yari M, Ghoshoon MB, Vakili B, Ghasemi Y (2017) Therapeutic enzymes: applications and approaches to pharmacological improvement. Curr Pharm Biotechnol 18(7):531–540. CrossRefGoogle Scholar
  127. Zhang L, Liu M, Jamil S, Han R, Xu G, Ni Y (2015) PEGylation and pharmacological characterization of a potential anti-tumor drug, an engineered arginine deiminase originated from Pseudomonas plecoglossicida. Cancer Lett 357(1):346–354. PubMedCrossRefGoogle Scholar
  128. Zhao K-W, Neufeld EF (2000) Purification and characterization of recombinant human α-N-acetylglucosaminidase secreted by Chinese hamster ovary cells. Protein Expr Purif 19(1):202–211. PubMedCrossRefGoogle Scholar
  129. Zheng F, Zhan C-G (2008) Rational design of an enzyme mutant for anti-cocaine therapeutics. J Comput Aided Mol Des 22(9):661–671. PubMedCrossRefGoogle Scholar
  130. Zhu MM, Mollet M, Hubert RS (2007) Industrial production of therapeutic proteins: cell lines, cell culture, and purification. In: Kent JA (ed) Kent and Riegel’s handbook of industrial chemistry and biotechnology. Springer, Boston, pp 1421–1448. CrossRefGoogle Scholar
  131. Zhu L, Tee KL, Roccatano D, Sonmez B, Ni Y, Sun Z-H, Schwaneberg U (2010) Directed evolution of an antitumor drug (arginine deiminase PpADI) for increased activity at physiological pH. Chembiochem 11(5):691–697. PubMedCrossRefGoogle Scholar
  132. Zhu MM, Mollet M, Hubert RS, Kyung YS, Zhang GG (2017) Industrial production of therapeutic proteins: cell lines, cell culture, and purification. In: Kent JA, Bommaraju TV, Barnicki SD (eds) Handbook of industrial chemistry and biotechnology. Springer, Cham, pp 1639–1669. CrossRefGoogle Scholar
  133. Zimran A, Brill-Almon E, Chertkoff R, Petakov M, Blanco-Favela F, Terreros Muñoz E, Solorio-Meza SE, Amato D, Duran G, Giona F, Heitner R, Rosenbaum H, Giraldo P, Mehta A, Park G, Phillips M, Elstein D, Altarescu G, Szleifer M, Hashmueli S, Aviezer D (2011) Pivotal trial with plant-cell–expressed recombinant glucocerebrosidase, taliglucerase alfa, a novel enzyme replacement therapy for Gaucher disease. Blood 118(22):5767–5773. PubMedCrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  • M. Ângela Taipa
    • 1
    Email author
  • Pedro Fernandes
    • 1
    • 2
  • Carla C. C. R. de Carvalho
    • 1
  1. 1.iBB-Institute for Biosciences and Bioengineering, Department of Bioengineering, Instituto Superior TécnicoUniversidade de LisboaLisbonPortugal
  2. 2.Faculty of EngineeringUniversidade Lusófona de Humanidades e TecnologiasLisbonPortugal

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