immobilized enzymes in industry alessandra basso, phd
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Immobilized enzymes in Industry
Alessandra Basso, PhDBusiness Development Manager Purolite Life SciencesPRAXISforum5 February 2020
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Purolite
• 1300 employees
• Manufacture of ion exchange resin, adsorbents and from 2012 Life Sciences products
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Wide portfolio of resins for biotech applications.
Purolite Life Sciences product portfolio
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Advantages of enzyme immobilization
Advantages Disadvantages
Easy separation of biocatalyst Loss of absolute activity due to the immobilization process
Reduced costs of down stream processing Lower activity of immobilized biocatalyst compared with non-immobilized enzymes used in processes with membrane filtration
Multiple use of biocatalyst Additional costs for carriers and the immobilization procedure
Better stability, especially towards organic solvents and higher temperatures
Lower reaction rates
Use of fixed bed or batch reactors
Simplified flow sheets
Most applications benefit from immobilized enzymesOverall process capital and operating cost of immobilized
enzyme vs. native enzyme determines best solution
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Parameters affecting immobilised enzymes
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Industrial resins for enzyme immobilization
Custom Particle size
Custom Porosity
Custom Functionality
New – IDA functionality for HIS tag immobilisation
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Industrial applications
Food – Pharma - ChemicalsApplication in water free media (organic solvent)
Application in biphasic system
Application in water mediaFluidized bed Packed bed Batch reactor
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Cosmetic industry - Surfactants
• Garcia, T. et al.; Biocat biotransf., 2009, 14, 67-85.
• O. Thum, L. Hilterhaus, A. Liese, 2008, Patent DE 2008P00002
• O. Thum, L. Hilterhaus, A. Liese, 2008, Patent DE 2008P00003
• R. Nieguth, M. Eckstein, L. O. Wiemann, O. Thum, M. B. Ansorge-Schumacher, Adv. Synth. Catal. 2011, 353, 2522 – 2528
Why immobilized enzymes?- green, no heavy chemicals- White product- No side reaction (have to smell good)
“Bubble reactor” used on pilot scale
Adsortion
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Cosmetic industry - surfactants
Myristyl miristateCetyl palmitate
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Pharma - Odanacatib
M. Truppo, G. Hughes, Org. Process Res. Dev., 2011, 15, 1033–1035.
Odanacatib: treatment of osteoporosis in post-menopausal women.
Sept 2016 - Merck announced that it is discontinuing the clinical development of its osteoporosis pipeline drug, odanacatib as its overall benefit-risk profile does not support filing or further development.
Adsortion
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Pharma - Immobilized Penicillin G Amidase
• The most used enzyme in industry
• About 100 tons immobilized enzymes used every year
• Main product: amoxicillin (Augmentin)
• Enzyme class: EC 3.5.1.11
• Size: 80KDa
• Challenges: Processes run for 500 - 1000 cycles
Covalent
• M. A. Wegman, M. H. A. Janssen, F. van Rantwijk, R. A. Sheldon*, Adv. Synth. Catal. 2001, 343, 559 – 576.
• A.I. Kallenberg, F. van Rantwijk, R. A. Sheldon, Adv. Synth. Catal. 2005, 347, 905– 926.
• Patent WO 2006069984 A2 – DSM.
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Fermentation
Penicillin G
Enzymatic hydrolysis5
(minimum 1000 cycles)
6-APA
Enzymatic synthesis
6(500 cycles)
FermentationEnzyme EcPGA
Immobilized EcPGA
beta-lactams derivatives of Penicillin G (i.e. amoxicillin or
ampicillin)
FermentationEnzyme SynPGAImmobilized
SynPGA
Isolation
Enzyme immobilization
Enzyme immobilization Isolation
beta-lactams derivatives of Penicillin G (i.e. amoxicillin or
ampicillin)
Re-crystallysation
Extraction
Pharma – Penicillin manufacture
Covalent
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Pharma – Sitagliptin (Merck antidiabetes)
Immobilized Transaminase
E.C. 2.6.1
Patent WO 2012/177527 A1 MerckAdsorption
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Pharma - Sofosbuvir
Sofosbuvir is used for the treatment of hepatitis C• First patent belongs to Gilead for USA and EU, but
does not cover emerging countries.• Hc-Pharma covers the other countries (Egypt, India)
• Patent US 2014/0017198 A1, Gilead Sciences• Patent WO 2016016327 A1, HC-Pharma Ag
Sofosbuvir
Covalent
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Pharma/Medical devices - enteral nutrition
Nutritional food
Oral ingestion
Imm
ob
ilized
en
zyme
on
a resin
Modified nutritional food
Immobilized enzyme for hydrolysis of triglycerides (fats) in patients with pancreatic diseasesfat malabsorption in patients with cystic fibrosis and exocrine pancreatic insufficiency
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Removal of Urea from dialysate
Immobilized urease
Immobilized urease in columns
Spent dialysate
Regenerated dialysate(urea removed)
CH4N2O
CO2 + NH3
Removal of uremic toxins from the spent dialysate and regeneration to fresh dialysate
Kidney Dialysis
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Chemicals - Acryl monomers
BASF Patent US2010/0273223A1
Manufactured by BASF
• Traditional chemical process employs temperatures of 80-100°C and inhibitors/acid catalysis
• Lipases do not catalyse efficiently the acrylation reaction• BASF developed a mutein of CalB that catalyse acrylation at moderate T (40°C)
Adsortion
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Chemicals – Chiral amines
Manufactured by BASF
ChiroprosChemical compounds: chiral transamidation for the synthesis of (S)-phenylethylaminehttp://www.intermediates.basf.com/chemicals/chiral-intermediates/amines
BASF Patent WO 1997010201 A1
Adsortion
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Chemicals - Pesticides
Manufactured by BASF
• Racemic Dimethenamid was developed by Sandoz, but sold to BASF in 1996• BASF implemented the single enantiomer Dimethenamid-P named Outlook®• Made in Geismar BASF site (USA)
BASF Patent DE 19837745 A1
Adsortion
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Food - Vitamin C ester
Ascorbyl palmitate is a fat-soluble form of vitamin C ("vitamin C ester“).Use:- source of vitamin C- antioxidant food additive (E number E304).
It is approved for use as a food additive in EU, USA, Canada, Australia, and New Zealand.The Chinese company Purun use enzymatic process instead of chemical process.
Challenge: expand the applications in food additives
H. Burham et al., J. Mol. Catal. B: Enzymatic, 2009, 58 153–157
Adsortion
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Food – Cocoa butter analogues
Vegetable oil: Derived by transesterification of palm oil to obtain semisolid oils that melt at 37C and simulate the cocoa butter (“melt in you mouth, not in your hands”)
So cocoa butter fat melts in the mouth to give cooling sensation, smooth ‘mouth appeal’ effect for e.g. chocolate.
2016
2015
AdsortionF. Hasan, A. Ali Shah, A. Hameed, Enz. Microb. Technol., 2006, 39, 235–251.
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Food - Cocoa butter analoguesLipase is selective for positions 1 and 3
• Production of palm oil -> 6.5 million t/y
• Butter fat -> 5.5 million t/y
• Cocoa butter fat is a high value product because TAGs are high in stearates, which
give a melting point of 37 ◦C. Palm oil TAGs are high in palmitate and they give a
melting point of 23°C so is oil at room temperature (low value product)
• Conversion of palm oil into cocoabutter fat substitute can be achieved by
interesterification
Patent WO 2007/080197 A2 Novozymes
Adsortion
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Food - Omega 3 from fish oil
In order to separate DHA(C22) & EPA (C20) from other fatty acids in fish oils, the tri-glyceride is first converted to ethyl esters and then the separation made using short path distillation
Adsortion
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Food - Omega 3 from fish oil
Manufactured with immobilized lipases
Adsortion
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Food – Sweetener (Allulose)
Patent WO 2014049373 A1 – Tate & Lyle
Manufactured by Tate & Lyle
Allulose is a "zero-calorie" sweetener Used in food and beverage manufacturers that currently use dextrose, fructose or HFCS in their productsAllowed in USA (not EU)
Ionic
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Food - Lactose free milk
Around 90% of humans show some kind of lactose intolerance (unable to
produce lactose)
• Yeast lactase has been immobilised by incorporation into cellulose triacetate
fibres (Snamprogetti S.p.A. in Italy)
• Used in a batchwise STR process at 5°C (Kluyveromyces lactis, pH optimum
6.4 -6.8, 90 U g-1)
• Now industry uses non-immobilized galactosidase
Ionic
P. S. Panesar, S. Kumari, R. Panesar, SAGE-Hindawi Access to Research Enzyme Research, 2010, 1 -16.
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Food - Tagatose
Ionic
Tagatose is a sweetener with 92%
sweetness of sucrose
but only 38% the calories of sugar and
much lower glycaemic index.
Tagatose can be obtained from lactose
(16% of the sweetening power of sucrose).
Lactose is a natural sugar contained in milk
in 2-8% in weight.
ECR1504
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Sweeteners on the market
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Food - Molasses
Manufactured by Manufactured by all major sugar companies (ADM, T&L, Cargill)
• Amylolytic enzymes (a-amylase and glucoamylase) convert starch to sugar by hydrolysis of a-1,4-glycosidic bonds in starch
• Glucoamylase is widely used for liquefaction and saccharification of starchy substances in the food, confectionary and beverage industries
• Production of sweetener• Reduction of dough viscosity to improve the texture and appearance of bread• The starting substrate is starch (mais)•
Ionic
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Food - high-fructose corn syrup production
Manufactured by all major sugar companies (ADM, T&L, Cargill)• HFCS (High Fructose Corn Syrup), an equilibrium mixture of glucose and fructose (1:1) is 1.3
times sweeter than sucrose and 1.7 times sweeter than glucose• The sweetening capacity of glucose is 70 to 75% that of sucrose, whereas fructose is twice as
sweet as sucrose• HFCS is manufactured from a totally nonsweet sub-stance, namely, starch• The price of HFCS is 10 to 20% lower than that of sucrose on the basis of its sweetening
power• HFCS is preferred by the food industry since it does not pose The problem of crystallization
as sucrose• The starting substrate is starch (mais)
Industry used unit: 1 GIU is 1 micromoleglucose converted to fructose in one minuteat pH 7, temperature 60°C, 300 g/l glucoseconcentration.
Ionic
L. Zittan, P. B. Poulsen, S. H. Hemmingsen, Die Starke, 1975, 27, 236 – 241.
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Food – Fructose & Glucose
Manufactured by all major sugar companies (ADM, T&L, Cargill)
• Inverted sugar can be produced by acid hydrolysis (IEX cation resins)
• Enzymatic inversion avoids the high-colour, high salt-ash, relatively low
conversion and batch variability problems of acid hydrolysis
• The starting substrate is sugar beet or cane
Covalent
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Thank you!
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