XII. Imobilisasi Enzim (Enzyme Immobilization)

1. m.k. SATUAN PROSES XII. Imobilisasi Enzim(Enzyme Immobilization) Departemen Teknologi Industri Pertanian FATETA - IPB 2011

2. ENZYME • The use of enzymes in industrial applications has been limited by several factors : • - high cost of the enzymes • - instability • - availability in small amounts. • - soluble in aqueous media and it is difficult and expensive to recover them from reactor effluents at the end of the catalytic process. • The technological developments in the field of immobilized biocatalysts can offer the possibility of a wider and more economical exploitation of biocatalysts in industry, waste treatment, medicine, and in the development of bioprocess monitoring devices like the biosensor.

3. IMMOBILIZATION Definition: „Immobilization means that the biocatalysts are limited in moving due to chemically or physically treatment“ • transformation of enzyme to insoluble form or inclusion to definite space • method for reuse and stabilisationof enzyme • one-step reactions - domain of immobilized enzymes The attractions of immobilized enzymes from an analytical standpoint are primarily their reuseability,and hence cost saving, and the greater efficiency and control of their catalytic activity (e.g., potentially longer half-lives, predictable decay rates and more efficient multi-step reactions).

4. Immobilized enzyme • An immobilized enzyme is enzyme an that is attached to an inert, insoluble material such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride). • This can provide increased resistance to changes in conditions such as pH or temperature. • It also allows enzymes to be held in place throughout the reaction, following which they are easily separated from the products and may be used again - a far more efficient process and so is widely used in industry for reactions. • An alternative to enzyme immobilization is whole cell immobilization http://en.wikipedia.org/wiki/Immobilized_enzyme

5. ADVANTAGES OF IMMOBILIZED ENZYME • - Development of continuous processes allowing more economic organization of the operations, automation, decrease of labour, and investment/capacity ratio. • Availability of the product in greater purity. Purity of the product is very crucial in food processing and pharmaceutical industry since contamination could cause serious toxicological, sensory, or immunological problems. • - Greater control over enzymatic reaction as well as high volumetric productivity with lower residence time, which are of great significance in the food industry, specially in the treatment of perishable commodities as well as in other applications involving labile substrates, intermediates or products

6. Commercial use Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include : Convenience: Minuscule amounts of protein dissolve in the reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solventt and reaction products. Economical: The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst. Stability: Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme.

7. ENZYME IMMOBILIZATION METHOD “Carrier –binding” “Cross-linking” “Entrapment” Ikatan Ionik Ikatan Kovalen Jenis Kisi Jenis Mikrokapsul Adsorpsi Fisik

8. Aspects of the immobilization procedure : 1. The properties of the free enzyme. 2. The type of support used. 3. The methods of support activation and enzyme attachment. 0 1995 IUPAC, Pure and Applied Chemistry

9. 1. Properties of the Free Enzyme • Source of the enzyme • Purity (and method of purification) • Catalytic activity and details of other constituents - - etc. • The above information permits direct comparison of enzymes from different sources.

10. 2. Enzyme Support • The support material can have a critical effect on the stability of the enzyme and the efficiency of enzyme immobilization. • The most important requirements for a support material are that it must be insoluble in water, have a high capacity to bind enzyme, be chemically inert and be mechanically stable. • The enzyme binding capacity is determined by the available surface area, both internal (pore size) and external (bead size or tube diameter), the ease with which the support can be activated and the resultant density of enzyme binding sites. • The surface charge and hydrophilicity must be considered.

11. Parameters of Enzyme Immobilization • - Effective, easy, cheap, acceptable (non-toxic in food and medical applications) • - Rate and yield dependent on the parameters involved • (e.g., type of carrier, concentrations, pH, temperature, • method, reaction time) • Empiricaloptimization • - External protein surface properties (e.g., hydrophobicity, ionic groups, functional groups for covalent binding) • - Protein surface engineering • - Introduction of functional groups increases binding interactions, stability (e.g., nanoparticles, protecting molecules) and activity (e.g., cofactors)

12. Method Immobilization Enzyme 1. Adsorption on glass, alginate beads or matrix : Enzyme is attached to the outside of an inert material. In general, this method is the slowest among those listed here. As adsorption is not a chemical reaction, the active site of the immobilized enzyme may be blocked by the matrix or bead, greatly reducing the activity of the enzyme.

13. Adsorption - Binding onto silica, clay or ion-exchange materials by weak interactions (e.g., ionic, electrostatic, hydrophobic) - Dependent on process conditions (e.g., pH, temperature, ionic strength, hydrophobicity) - Simple and cost-effective, reversible (stabilized by cross linking), but may cause enzyme unfolding Lar. Pati Karbon aktif Enzim Imobil Amilase imobil Aduk 10 0C, 1 jam Saring -amilase Gula

14. I n o r g a n i c Ca r ri e r s • 􀂉 High pressure stability • 􀂉 May undergo abrasion in stirred vessels • 􀂉 SiO2 based carriers functionalized by introduction of • amino groups (e.g., treating with aminopropyl • triethoxysilane) • 􀂉 Porous glass (Corning, Waters, Schuller) • 􀂃 Silica (Grace, Solvay, Degussa) • 􀂉 Mineralmaterials (clays) • 􀂃 Celite - adsorption and stabilisation of enzyme in organic media • 􀂃 Bentonite - excellent adsorption capacity (up to 1.5 g • protein per g bentonit) used for enzyme isolation by • dsorption/desorption • Crosslinking with glutaraldehyde prevents desorption

15. O r g a n i c C a r ri e r s f r o m Na t u r a l S o u r c e s 􀂉 Favorable compatibility with proteins 􀂉 High range of polysacharidesand derivatives used for immobilization 􀂃 Wide network structure 􀂃 Hydrophilic properties - weak interactions with proteins 􀂉 Cellulose derivatives 􀂃 DEAE-cellulose (diethylaminoethyl-cellulose) 􀂃 CM-cellulose (carboxymethyl-cellulose) 􀂉 Dextran 􀂃 widely used for enzyme immobilization 􀂃 activated by cyanogen bromide 􀂃 mechanical stability limited 􀂉 Other polysacharides 􀂃 agarose, starch, pectine and chitosan 􀂉 Proteins (gelatine)

16. O r g a n i c S y n t h e t i c C a r ri e r s 􀂉 High chemical and mechanical stability 􀂉 Wide range of carriers with good capacity and simple manipulation 􀂉 (ion-exchange) resins 􀂃 copolymerization with functional groups (e.g., nitration, sulfonation, carboxylation, epoxydation) Example : 􀂃 polystyrene 􀂃 polyvinylacetate 􀂃 acrylic polymers http://loschmidt.chemi.muni.cz/peg/lecture/biocat_lecture02.pdf

18. 2. Covalent Binding 􀂃 Better stabilization of enzyme on carrier 􀂃 Introduction of functional group (e.g., amino, epoxy, thiol, cyanide) 􀂃 Principle : 1. activation 2. derivatization 3. binding of enzyme

19. 3. Crosslinked: - The enzyme is covalent bonded to a matrix through a chemical reaction. - This method is by far the most effective method - As the chemical reaction ensures that the binding site does not cover the enzyme's active site, the activity of the enzyme is only affected by immobility.

20. Crosslinked Use reagent which usually has 2 identical functional groups  reacted with amino acid residue of the enzyme Glutaraldehyde Diisocyanate

21. 4. Entrapment • The enzyme is trapped in insoluble beads or microspheres, such as calcium alginate beads. However, this insoluble substances hinders the arrival of the substrate, and the exit of products. •  lattice type (alginat, k-caragenan, • Poliacrylamide ) •  microcapsule type • Nilon • Poliurea • Etil selulosa • Polistiren • Kolodion • Nitroselulosa • Butil asetat selulosa 1 – 300 m Permanently polymer Membran Nopermanently

22. Immobilization by Entrapment Poliacrylamide Gel

23. I n c l u s i o n i n t o Polymeric Network 􀂉 One of the most convenient method for whole cell immobilization 􀂉 Problems with enzyme diameter and leak out of the particle 􀂃 Combination with cross linking http://loschmidt.chemi.muni.cz/peg/lecture/biocat_lecture02.pdf

24. http://www.ias.ac.in/currsci/jul10/articles15.htm Techniques and supports for immobilization A large number of techniques and supports are now available for the immobilization of enzymes or cells on a variety of natural and synthetic supports. The choice of the support as well as the technique depends on the nature of the enzyme, nature of the substrate and its ultimate application. Therefore, it will not be possible to suggest any universal means of immobilization. It can only be said that the search must continue for matrices which provide facile, secure immobilization with good interaction with substrates, and which conform in shape, size, density and so on to the use for which they are intended.

25. http://www.ias.ac.in/currsci/jul10/articles15.htm Techniques and supports for immobilization Care has to be taken to select the support materials as well as the reagents used for immobilization, which have GRAS status, particularly when their ultimate applications are in the food processing and pharmaceutical industries. Macromolecular, colloidal, viscous, sticky, dense or particulate food constituents or waste streams also limit the choice of reactor and support geometries.

26. Conclusion Enzyme immobilization is one of the most promising approaches for exploiting enzyme-based processes in biotransformation, diagnostics, pharmaceutical and food industries. Several hundred of enzymes have been immobilized in a variety of forms including penicillin G acylase, lipases, proteases, invertase, etc. and are being currently used as catalysts in various large scale processes.

27. Perubahan Sifat Enzim Terimobilisasi • Aktivitas • V1 tidak deaktivasi enzim akibat imobilisasi • V2  kemungkinan untuk mengimobilisasi enzim lebih banyak/sedikit per unit volume katalis • Penyebab penurunan aktivitas : • Konfigurasi  menghalangi substrat • Grup reaktif pada sisi aktif ikut terikat pada matriks • Terbentuk konfigurasi tidak aktif • Kondisi reaksi  denaturasi V1(aktivitas relatif)  Perbandingan aktivitas enzim imobil vs enzim larut dalam jumlah sama V2 (aktivitas spesifik absolut)  Kecepatan reaksi per unit berat atau unit volume seluruh katalis

28. b a c Aktivitas Relatif (%) → pH 7 4 • pH optimum enzim imobil • Penyebab perubahan pH :  distribusi yang tidak seragam dari ion H+, ion OH- dan substrat bermuatan • Carrier bermuatan negatif  pH optimum bersifat basa • CMC • Maleac anhydride/etilen • Asam galakturonat • Asam poliaspartat • Carrier bermuatan positif  pH lebih asam • DEAE-selulosa • Polimer polyornithyl a : enzim chymotripsin larut b : kopolimer chym – anhydride ethylene (-) c : chym – polyornithyl (+)

29. Stabilitas • Stabilitas operasi = t1/2 (half-life) = waktu dimana terjadi kehilangan 50 % dari aktivitas enzim semula k = konstanta kerusakan enzim t = waktu operasi E0 = aktivitas enzim mula-mula E = aktivitas enzim pada wktu t • Stabilitas operasi ditentukan oleh : • Jenis enzim • Cara imobilisasi • Jenis reaktor

30. Stabilitas operasi ditentukan oleh : • Jenis enzim • Cara imobilisasi • Jenis bioreaktor *) gelas berpori dilapisi ZrO2, 40 – 80 mesh,  = 550 Å

31. Kinetika Enzim Imobil Nilai Km (konstanta Michaelis-Menten)

32. BioreaktorEnzimImobil • CSTR : • Pengontrolan lbh mudah • Cocok untuk kasus inhibisi (penghambatan) substrat • Menghindari kontak enzim oleh substrat dan produk yang terlalu lama • Reaktor Curah (Batch) : • Sederhana • Viskositas tinggi & • aktivitas enzim rendah

33. Fixed-bed PFR (Unggun Diam/Terkemas) : • Sinambung  paling sering digunakan • Aliran substrat dpt dari atas, bawah atau • daur-ulang • Fluidized-bed (Unggun Terfluidisasi) : • Untuk viskositas tinggi & terbentuk gas • Laju fluidisasi perlu diatur agar enzim imobil tak rusak

34. Recycle Packed Column Reactor : - allow the reactor to operate at high fluid velocities. - a substrate that cannot be completely processed on a single pass

35. Immobilization of Microorganism Cells 􀂉 First example in 1823, Acetobacteradsorbed to wood chips (acetic production) 􀂉 Multienzymesystems (e.g., alcohol production) 􀂉 Applicable if enzyme(s) difficult to isolate or show low stability/activity outside cell (e.g., nitrilehydratase in acrylamide production) 􀂉 Continous processing with (re)synthesis of enzyme in immobilized living cells 􀂉 Mostly resting cells - limited in growth by controlling C-, N- or P- sources 􀂉 Industrial applications of immobilized viable cells: 1. Beer maturation with yeast cells 2. Anchorage-dependent mammalian cell (production of vaccines) 3. Environmental technologies using mixed cultures http://loschmidt.chemi.muni.cz/peg/lecture/biocat_lecture02.pdf

36. Immobilization of Microorganism Cells Advantages 􀂃 no enzyme isolation and purification 􀂃 multienzyme complex reactions 􀂃 cofactor regeneration in native system 􀂃 synthrophic mixed cultures Limitations 􀂃 insufficient stability, low resistance 􀂃 mass transfer limitation 􀂃 side reactions, degradation of product 􀂃 byproducts from lysis of cell or toxic metabolites 􀂃 low productivity

37. E x a m p l e s : i n d u s t r i a l W h o l e C e l l I m m o b i l i z a t i o n s

38. Applications of immobilized enzymes

39. L Amino Acid Resolution • The first industrial use of an immobilized enzyme is amino acid acylasefor the resolution of racemic mixtures of chemically synthesized amino acids. • Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid, that can be easily separated, racemized and recycled. • Some of the methods of enzyme immobilization used for this purpose : • ionic binding to DEAE-sephadex • entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate . • immobilized on macroporous beads made of flexiglass-like material

40. Example : L-Methionine Production • Amino acid needed by the body but not manufactured naturally by it, L-Methionine can be acquired through proper diet and supplements. • Methionine improves the body’s ability to synthesize muscle protein. It is a source of sulfur required for the synthesis of other substances important for energy production like choline, creatine and carnitine • Comercially, methionine produce by chemical reaction which produce racemic mixture of acetylated DL Methionine  separated by immobilized aminoacylase (using DEAE-Sephadex)  deacylated of L-methionine  L-methionine

41. High Fructose Syrups Production • The most important application of immobilized enzymes in industry • The conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase.  the most of the commercial preparations use either the adsorption or the cross- linking technique. • Application of glucose isomerase technology has gained considerable importance, especially in nontropical countries that have abundant starch raw material.

42. (http://www.scribd.com/doc/22862853/Applications-of-Immobilized-Enzymes)(http://www.scribd.com/doc/22862853/Applications-of-Immobilized-Enzymes)

43. Low Lactose Milk & Whey Production Preparation of lactose-hydrolysed milk and whey, using β –galactosidase (lactase) Lactose hydrolysis also enhances the sweetness and solubility of the sugars, and can find future potentials in preparation of a variety of dairy products. Lactose-hydrolysed whey may be used as a component of whey-based beverages, leavening agents, feed stuffs, or may be fermented to produce ethanol and yeast, thus converting an inexpensive byproduct into a highly nutritious, good quality food ingredient A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of Kluyveromyces fragilis has also been developed (http://www.scribd.com/doc/22862853/Applications-of-Immobilized-Enzymes)

44. (http://www.scribd.com/doc/22862853/Applications-of-Immobilized-Enzymes)(http://www.scribd.com/doc/22862853/Applications-of-Immobilized-Enzymes)

45. L-Aspartic Acid Production • L -Aspartic acid is widely used in the food and pharmaceutical industries and is needed for the production of aspartame (low -calorific sweetener ) • The enzyme aspartase catalyses addition of ammonia to the double bond of fumaric acid. • The enzymes have been immobilized using the whole cells of Escherichia coli onto e.g. phenolformal-dehyde resin, for adsorbing aspartase (http://www.scribd.com/doc/22862853/Applications-of-Immobilized-Enzymes)

46. Enzyme Immobilization on Polymeric Resins - Amberlite™ and Duolite™ Strive to Improve Catalysis Economics Through Reuse http://www.rohmhaas.com/ionexchange/pharmaceuticals/enzymes.htm

47. Malic Acid Production - The immobilized fumarase is used for the production of malic acid (for pharmaceutical use). - These processes make use of immobilized nonviable cells of B. flavus as a source of fumarase. - An active biocatalyst for the synthesis of L-malic acid from fumaric acid was obtained based on cells immobilized in carrageenan. (http://www.scribd.com/doc/22862853/Applications-of-Immobilized-Enzymes)

48. ABSTRACT The yeast Saccharomyces cerevisiae was entrapped within polyacrylamide gel beads by employing a procedure that uses sodium dodecylsulfate as a detergent to improve the spherical configuration of the beads. The resulting preparation showed a rate of fumarate bioconversion to L-malic acid about 60 times higher than that found for the free cells. Almost all fumarate was converted in 30 min of incubation (Oliveira et al.) http://www.springerlink.com/content/n6655r1x12451534/

49. ANTIBIOTIC PRODUCTION Example : 6-aminopenicillanic acid (6-APA) Production Production of 6-aminopenicillanic acid (6-APA) by the deacylation of the side chain in either penicillin G or V, using penicillin acylase(penicillin amidase).  One of the major reasons for its success is in obtaining a purer product, thereby minimizing the purification costs. A number of immobilized systems have been patented or commercially produced for penicillin acylase which make use of a variety of techniques either using the isolated enzyme or the whole cells. (http://www.scribd.com/doc/22862853/Applications-of-Immobilized-Enzymes)

50. Lecture 14 Application of Immobilized Enzyme www.youtube.com/watch?v=_hM8I-yzOAo28 Nov 2008 - 47 menit - Diunggah oleh nptelhrdLecture Series on Enzyme Science and Engineering by Prof.Subhash Chand, Department of Biochemical ... . Lecture - 26 Applications