-costul minim- conditie esentiala pentru mentinerea pe piata a unui produs

Enzime disponibile comercial

- industria detergentilor, industria alimentara, industria textila

- biotransformari- hidrolaze, izomeraze, oxidoreductaze;

-celule intregi- cost redus

- reactii secundare

- viteza de reactie redusa

-enzime purificate- reproductibilitate avansata

- costisitoare

- necesita cofactori enzimatici

Surse de enzime: -organe animalelor

- tesuturi ale plantelor

- microbiene(90%)

Enzyme production and purification

Enzyme production and purification

Surse de biocatalizatori

Enzyme EC number Source Intra/extra Scale of production Industrial use -cellularc

Animal enzymes Catalase 1.11.1.6 Liver I - Food Chymotrypsin 3.4.21.1 Pancreas E - Leather Lipase 3.1.1.3 Pancreas E - Food Rennet 3.4.23.4 Abomasum E + Cheese Trypsin 3.4.21.4 Pancreas E - Leather Plant enzymes Actinidin 3.4.22.14 Kiwi fruit E - Food a-Amylase 3.2.1.1 Malted barley E +++ Brewing b-Amylase 3.2.1.2 Malted barley E +++ Brewing Bromelain 3.4.22.4 Pineapple latex E - Brewing b-Glucanase 3.2.1.6 Malted barley E ++ Brewing Ficin 3.4.22.3 Fig latex E - Food Lipoxygenase 1.13.11.12 Soybeans I - Food Papain 3.4.22.2 Pawpaw latex E ++ Meat Bacterial enzymes a-Amylase 3.2.1.1 Bacillus E +++ Starch b-Amylase 3.2.1.2 Bacillus E + Starch Asparaginase 3.5.1.1 Escherichia coli I - Health Glucose isomerase 5.3.1.5 Bacillus I ++ Fructose syrup Penicillin amidase 3.5.1.11 Bacillus I - Pharmaceutical Protease 3.4.21.14 Bacillus E +++ Detergent Pullulanase 3.2.1.41 Klebsiella E - Starch Fungal enzymes a-Amylase 3.2.1.1 Aspergillus E ++ Baking Aminoacylase 3.5.1.14 Aspergillus I - Pharmaceutical Glucoamylase 3.2.1.3 Aspergillus E +++ Starch Catalase 1.11.1.6 Aspergillus I - Food Cellulase 3.2.1.4 Trichoderma E - Waste Dextranase 3.2.1.11 Penicillium E - Food Glucose oxidase 1.1.3.4 Aspergillus I - Food Lactase 3.2.1.23 Aspergillus E - Dairy Lipase 3.1.1.3 Rhizopus E - Food Rennet 3.4.23.6 Mucor miehei E ++ Cheese Pectinase 3.2.1.15 Aspergillus E ++ Drinks Pectin lyase 4.2.2.10 Aspergillus E - Drinks Protease 3.4.23.6 Aspergillus E + Baking Raffinase 3.2.1.22 Mortierella I - Food Yeast enzymes Invertase 3.2.1.26 Saccharomyces I/E - Confectionery Lactase 3.2.1.23 Kluyveromyces I/E - Dairy Lipase 3.1.1.3 Candida E - Food Raffinase 3.2.1.22 Saccharomyces I - Food

Microbial enzymes and their application

Utilizarea practic a enzimelor

Scheme for the production of enzymes. F: fermentation; S: solidliquid separation; E: cell extraction; C: concentration; Pi: operations of purification; D: drying; Fi: formulation; - - - - -:extracellular enzyme; : intracellular enzyme; cell tissue or

fluid - - - - -

Enzyme Production

Obtinerea enzimelor poate fi impartite in patru etape:

Producerea enzimei: reprezinta etapa de crestere si producere a celulelor

Extractia enzimei: reprezinta extractia enzimei din mediu de crestere a

enzimei si presupune separari solid-lichid, etape de extractie si/sau

concentrare;(izolarea, concentrare si stabilizarea produsului)

Purificarea enzimei: reprezinta o serie de operatii dupa extractie, cu scopul de

a inlatura contaminantii nedoriti(proteine, acizi nucleici).

Formularea enzimei: constituie o serie de operatiuni cu scopul de a da

enzimei o forma finala; include operatiuni de finisare, stabilizare si

standardizare;

Obtinerea enzimelor(proteinelor)

Obtinerea enzimelor(proteinelor)

Extractia enzimei(to isolate, concentrate, and stabilize the target product)

-Spargerea peretelui celular

-Ultrasonarea, french press

separarea solid-lichid- centrifugare- organisme unicelulare

( bacterii, drojdii)

- filtrare- organsime multicelulare

-enzime extracelulare

-concentrarea enzimelor extracelulare

-evaporare sub vid (Lambert and Meers 1983; Schaffeld et al. 1988)

-concentrarea prin inghetarea apei (Darbyshire 1981; Whitaker 1994)

- ultrafiltrarea (Ehsani et al. 1996; Euzenat et al. 1998)

-enzime intracelulare

-extractia enzimelor intracelulare

- soc osmotic

- extractia enzimelor membranare

- detergenti, solventi

-separarea solid-lichid- centrifugare- organisme unicelulare( bacterii, drojdii)

- filtrare- organsime multicelulare

Producerea enzimei - cresterea plantelor

- cresterea animalelor

- fermentatie - enzime microbiene

- enzime obtinute prin expresia genelor straine in

microorganisme

Methods of Extraction of Intracellular Enzymes

The results obtained from cell lysis depend on several factors, including sample volume, cell

concentration, time, temperature, energy input (speed of agitation, pressure, etc.), and physical

properties of the cell lysis device.

-Use procedures that are as gentle as possible because too vigorous cell or tissue

disruption may denature the target protein or lead to the release of proteolytic enzymes

and general acidification.

-Extraction should be performed quickly, at sub-ambient temperatures, in the presence

of a suitable buffer to maintain pH and ionic strength and stabilize the sample.

-Add protease inhibitors before cell disruption.

-The release of nucleic acids may cause viscosity problems (addition of DNase may

decrease viscosity). Frequently, protease inhibitors are needed to reduce protein

breakdown during extraction. Fractional precipitation may reduce the presence of proteases.

- In bacterial and yeast expression systems, the recombinant protein may often be

contained in inclusion bodies. Extraction requires solubilization of the inclusion bodies,

usually in the presence of denaturants, followed by refolding before or after purification.

Purificare enzimei

-enzime extracelulare- se vand ca si preparate crude

-enzime intracelulare

- amestec de proteine, acizi nucleici si alti constituenti ai celulei

-indepartarea acizilor nucleici:

- folosirea nucleazelor si precipitarea cu diferiti agenti(Harve and Bajpai 2000)

- folosirea detergentilor cationici (cationic detergent cetyltrimethyl

ammonium bromide, streptomycin sulfate and protamine sulfate )(Burgess 1969;Yang et al. 1987;

Cordes et al. 1990)

- bovine pancreatic nucleases- cea mai potrivita metoda

-indepartarea ionilor :

-Dializa

-Diafiltararea

-Cromatografie de excluziune sterica

-passage of solutes through a semi-permeable membrane (cellulose tube)

-dialysis membrane with certain pore size

-molecules smaller than the pore size pass through the membrane (water, salts,

protein fragments ...); protein stays in the tube

Dializa

Dialysis is based on the fact that due to their size, protein molecules are unable to pass through the

pores of a semipermeable membrane, while lower-molecular substances distribute themselves

evenly between the inner and outer spaces over time. After repeated exchanging of the external

solution, the conditions inside the dialysis tube (salt concentration, pH, etc.) will be the same as in the

surrounding solution.

-indepartarea altor proteine( cresterea puritatii-scaderea activitatii enzimei)

- precipitarea proteinelor:- prin adaugarea de saruri( salting in-out) (Coulon et al. 2004),

- cu solventi organici(Water-miscible organic solvent

precipitation) (Drapeau et al. 1972; Omar et al. 1987; Iizumi et al. 1990)

- temperaturi joase (near or below 0C)

- acetona, etanol;

-la pH izolelectric

- polimeri organici (PEG)

- cromatografia de lichide(Janson and Ryden 1998; Kastner 2000; Ahuja 2003;Cutler 2004

-precipitarea fractionata se bazeaza pe diferentele de solubilitate

- Their solubility increases as the ionic strength increases, because more

and more of the well-hydrated anorganic ions (blue circles) are bound to the

proteins surface, preventing aggregation of the molecules (salting in). At very high ionic strengths, the salt withdraws the hydrate water from the

proteins and thus leads to aggregation and precipitation of the molecules

(salting out).

salt precipitation

ammonium sulfate (NH4)2SO4 good solubility inexpensive readily obtained pure prevents proteolysis stabilises proteins

proteins "salted out" by hydrophobic interaction ordering of water molecules around hydrophobic amino acid residues on protein surface (for instance, Ile, Leu, Met, Phe, Tyr, Val) maintain the protein in solution freely available water molecules scare when salt is added; ordered "frozen" water was removed hydrophobic areas are exposed, and residues interact with one another protein aggregate use different salt saturation ranges for separation (0 to 20%, 20 to 40%, etc.), determine the amount of protein recovered and enzyme activity for each fractionation range and select the fraction with the best enzyme specific activity

organic solvent precipitation

acetone, ethanol

water miscible solvents reduce the water activity ordered water structure around hydrophobic areas displaced by organic solvent molecules decrease solubility of water-soluble proteins and lead to aggregation and precipitation (larger molecules aggregate sooner)

organic polymers polyethyleneglycol (PEG) PEG 6000 or PEG 20000

considerable success with low-solubility proteins disadvantage

difficulty in removing PEG

isoelectric point

proteins tend to be least soluble at their isoelectric point pI and therefore most likely to precipitate out of solution

pH at which the average charge on the population of amino acids is zero average of the pKa's that lie to either side of the neutral form of the amino acid

Examples of precipitation techniques

Ammonium sulfate precipitation

Ammonium sulfate precipitation is frequently used for initial sample concentration and

cleanup. As the concentration of the salt is increased, proteins will begin to salt out.

Cromatografia de lichide pentru separarea proteinelor

-Cromatografia de excluziune sterica

-Cromatografia de schimb ionic

-Cromatografia de interactiune hidrofoba

-Cromatografia de afinitate

CHROMATOGRAPHIC INSTRUMENTATION

Cromatografia de excluziune sterica( Gel filtararea)

-Separarea proteinelor(enzimelor) pe baza masei moleculare

-Elutia este simpla nu necesita folosirea unui gradient

Chromatogram of ZAP-70 gel filtration on a Sephadex 200 column.

Protein (~1mg) at a flow rate of 0.5ml/min (blue trace). The retention times of protein

standard markers run separately are indicated in a separate chromatogram (red trace).

Cromatografia de schimb ionic

-is a process that allows the separation of ions and polar molecules based on their charge.

-retains analyt molecules on the column based on coulombic (ionic) interactions

-the separation is based on the reversible interaction between a charged protein and an

oppositely charged chromatography medium.

- Proteins bind as they are loaded onto a column. Conditions are then

altered so that bound substances are eluted differentially. Elution is usually

performed by increasing salt concentration or changing pH. Changes are

made stepwise or with a continuous gradient.

-Most commonly, samples are eluted with salt (NaCl), using a gradient

elution

Cromatografia de interactiune hidrofoba (Reversed phase chromatography (RPC), Hydrophobic Interaction Chromatography (HIC)

- RPC separates proteins and peptides with differing hydrophobicity based on their reversible interaction with the hydrophobic surface of a chromatographic medium.

- Due to the nature of the reversed phase matrices, binding is usually

very strong.

-Binding may be modulated by the use of organic solvents and other additives

(ion pairing agents).

-Elution is usually performed by increases in organic solvent concentration,

most commonly acetonitrile

Cromatografia de afinitate

polyacrylamide gel electrophoresis (PAGE)

electrophoretic separation of proteins is most commonly performed in polyacrylamide gels separation of molecules on the polyacrylamide gel matrix by applying an electric field

polyacrylamide gels: successful separation can be accomplished by electrophoresis in various gels (semisolid suspensions

in water) rather than in a liquid solution gels are cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into

polyacrylamide chains and simultaneously cross-linking the chains into a semisolid matrix gel pore size can be varied by adjusting the concentrations of polyacrylamide and the cross-linking

reagent highly cross-linked polyacrylamide gel = pores are quite small such a gel could resolve small proteins and peptides, but large proteins would not be able to

move through it smaller proteins migrate faster than larger proteins through the gel gel's pore size and strength of the electric field influence the rate of movement

SDS-PAGE

sodium dodecyl sulfate (SDS) coats proteins with negative charges coated polypeptide chains can then separated by molecular mass (method to determine molecular weight) determine the approximate molecular weight of a polypeptide chain as well as the subunit composition of a complex protein

even chains that differ by less than 10 percent in molecular weight can be separated ! molecular weight estimation by distance comparison

compare distance 1 (migration through the gel) with distance 2 (migration of proteins with known molecular weight)

-separation procedure:

-proteins are exposed to the ionic detergent SDS before and during gel

electrophoresis

-SDS denatures proteins, causing multimeric proteins to dissociate into their

subunits all polypeptide chains are then forced into extended conformations with similar

charge / mass ratios

- SDS treatment eliminates the effect of differences in shape

-chain length = unique determinant of the migration rate of proteins

individual polypeptide chains migrate as a negatively charged SDS-protein complex

through the porous polyacrylamide gel speed of migration is proportional to the size of the

proteins

-smaller polypeptides running faster than larger polypeptides