Enzyme immobilization involves attaching enzymes to an inert, insoluble material which provides benefits like increased pH and temperature resistance. It allows the enzyme to be reused through multiple reactions by easily separating it from reaction products. There are physical and chemical methods for immobilization like entrapment within a polymer lattice or covalent attachment to a matrix. Immobilized enzymes have properties like higher stability and altered selectivity compared to free enzymes. Creative Enzymes Inc. offers custom enzyme immobilization services at any scale with high enzyme loading and variable particle sizes.

1. Enzyme Immobilization
Creative Enzymes Inc.

2. Introduction of Enzyme
Immobilization
An immobilized enzyme is an enzyme attached to an
inert, insoluble material—such as calcium alginate.
This can provide increased resistance to changes in
conditions such as pH or temperature. It also lets
enzymes 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 enzyme
catalysed reactions.
Enzyme Immobilization

3. History and Commercialization of
Immobilized Enzymes
Figure 2. Enzyme-based biosensors: Significance in various sectors as
well as their market in various countries across the world.
Figure 1. Publication of articles (research and review) on
enzyme immobilization (PubMed)

4. Methods of Enzyme Immobilization
Figure 3. Overview on the techniques being used for enzyme immobilization.

5. Physical Method
1 2 3
This involves cross-linking of the
enzyme to a polymer in every direction,
covering almost every side chain
present on the surface of the enzyme
by physical entrapment within the
polymer lattice. It allows permeation of
appropriately sized substrate and
release of product molecules, which
ensures continuous transformation.
Entrapment
Enzyme is attached to the support
material by non-covalent linkages
including ionic or hydrophobic
interactions, hydrogen bonding, and
van der Waals forces without any pre-
activation of support. Here, the
enzyme is directly added to the
surface (active adsorbent) without the
removal of any non-adsorbed enzyme
during washing.
Adsorption
Enzymes are immobilized by enclosing
them within spherical semi-permeable
polymer membranes with controlled
porosity (1–100 μm). Semi-permeable
membranes can either be permanent
or nonpermanent membranes based
on the constituents. These membranes
are also used in the encapsulation of
dyes, drugs, and other chemicals.
Microencapsulation

6. Chemical Method
Covalent
Attachment
The enzyme is attached to the
matrix by means of covalent
bonds. Enzyme molecules are
attached either directly to the
reactive groups present on the
matrix or by a spacer arm,
which is artificially attached to
the matrix through various
chemical reactions. Matrices
commonly used are either
natural or synthetic.
Cross-Linking
This involves formation of a
number of covalent bonds
between enzyme and the
matrix using bi- or multi-
functional reagents.
Generally, amino groups of
lysine, sulfhydryl groups of
cysteine, phenolic OH groups
of tyrosine, or imidazol group
of histidine are used for
enzyme binding under mild
conditions.
Ionic binding
This is based on ionic
interactions between enzyme
molecules with a charged
matrix. Here, higher the
surface charge density on the
matrix, the greater would be
the amount of enzyme being
bound to the matrix. Enzyme
binding via ionic interactions
during immobilization depends
on the pH of the solution, the
concentration of the enzyme
and temperature.
Conjugation by
affinity ligands
Attachment of the enzyme to
the matrix using specific
ligands; viz, his-tag on
enzyme to a metal-containing
matrix, lectin-containing
domain to carbohydrate
moieties present on the matrix
or sometimes substrate-
mimicking chemical
compounds are also used as
ligands.

7. Properties of Immobilized Enzymes
Figure 4. Effects on enzymes being immobilized on various matrices by either method of
immobilization.

8. Enzyme Modification by
Immobilization Service
Creative Enzymes Inc.
 Quick and rational design
 Custom immobilization of enzyme of interest
 Available at any scale with short lead times
 Variable particle size of immobilized enzyme:
0.05-1mm
 High loading rate: > 45%, up to 85%

9. Enzyme Modification by
Immobilization Service

10. Contact Us
Address: 45-1 Ramsey Road, Shirley, NY 11967, USA
Email: info@creative-enzymes.com
Tel: 1-631-562-8517 1-516-512-3133
Fax: 1-631-938-8127
www.creative-enzymes.com

11. THANKS
Creative Enzymes Inc.