1. aggregates with other entities, and as attached

1.      Enzyme Immobilization

Enzymes are biological catalysts that promote the
transformation of chemical species in living systems. Their role in biological
processes, in health and disease, has been extensively investigated. Enzymes
have the ability to catalyze reactions under very mild conditions with a very high
degree of substrate specificity, thus decreasing the formation of by-products. (Ellis
Horwood, 1991)

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Enzymes can catalyze reactions in different states:
as individual molecules in solution, in aggregates with other entities, and as
attached to surfaces. The attached or “immobilized” state is of particular
interest due to its technical applications. Immobilized enzymes are currently
the subject of considerable interest because of their advantages over soluble
enzymes.

Enzyme
immobilization may be defined as a process of confining the enzyme molecules to
a solid support over which a substrate is passed and converted to products The immobilized enzymes refers to “enzymes physically
confined or localized in a certain defined region of space with retention of
their catalytic activities, and which can be used repeatedly and continuously.”

The industrial use of enzymes is often limited by
their high cost and rapid inactivation. To improve their economic feasibility
in industrial processes, enzymes are generally immobilized onto a matrix.
Immobilization facilitates re-use of the enzymes, and may affect the
selectivity and stability of the enzyme. Immobilization research has mainly
focused upon means to enhance the transfer of enzymes onto the support, and
upon means to ensure that the transferred enzymes remain active. (Mazeaud et al,
2006)

There are wide applications of
immobilized enzymes in industrial production, biomedical application, food
industry, textile and detergent industries 
research, biodiesel production, waste water management. Aside from the
application in industrial processes, the immobilization techniques are the
basis for making a number of biotechnology products with application in
diagnostics, bioaffinity chromatography, and biosensors. Therapeutic
applications are also foreseen, such as the use of enzymes in extra-corporeal
shunts.

Enzyme immobilization

Advantages

Disadvantages

·        
Catalyst reuse

·        
Loss or reduction in activity

·        
Easier reactor operation

·        
Diffusional limitation

·        
Easier product separation

·        
Additional cost

·        
Wider choice of reactor

·        
Problems with cofactor and regeneration

·        
High stability

 

 

2.      Need of enzyme immobilization:-

1.      Protection
from degradation and deactivation.

2.      Re-use
of enzymes for many reaction cycles, lowering the total production cost of
enzyme mediated reactions.

3.      Ability
to stop the reaction rapidly by removing the enzyme from the reaction solution.

4.      Enhanced
stability.

5.      Easy
separation of the enzyme from the product.

6.      Product
is not contaminated with the enzyme.

 

3.     
Methods
of enzyme immobilization  

The enzymes can be
attached to the support by interactions ranging from reversible physical
adsorption and ionic linkages to stable covalent bonds. One way of classifying
the various approaches to immobilizing enzymes is in two broad categories:
Physical and chemical methods

3.1.  
Physical
methods:-

Enzyme
attachment onto different matrices via
physical forces involving van der Waals forces, hydrophobic
interactions and hydrogen bonding. The process is reversible in nature by
controlling physicochemical parameters

3.1.1.     
Entrapment-

Entrapment
involves cross-linking of the enzyme to a polymer (polyacrylamide, alginate
etc.) 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. However, this method can
only be used in a limited number of enzymes.

 

3.1.2.      Adsorption-

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. The matrices used are either organic or
inorganic in nature, viz. ceramic, alumina, activated carbon, kaolinite,
bentonite, porous glass, chitosan, dextran, gelatin, cellulose, starch.

This
method of enzyme immobilization is (e.g. Asparginase on CM-cellulose) without
any conformational change in enzyme. However, it involves intensive
optimization of pH, temperature, nature of the solvent, ionic strength,
concentration of enzyme and adsorbent as they play important  a role in enzyme desorption following slight
changes in its micro-environment.

3.1.3.      Microencapsulation-

Enzymes
are immobilized by enclosing them within spherical semi-permeable polymer
membranes with controlled porosity (1–100 ?m).. Permanent membranes are
made of cellulose nitrate and polystyrene while non-permanent membranes are
made of liquid surfactant. Enzymes immobilized by encapsulation have extremely
large surface areas due to which they have higher catalytic efficiency.

Ligninolytic
enzymes like lignin peroxidase, manganese peroxidase and laccase and be
encapsulated into polyacrylamide/pectin, polyacrylamide/

Carboxymethylcellulose,
polyacrylamide/gelatin with size of particles ranging between 0.1 and 1000 lm.(Gassara-Chatti,
2013)

3.2.  
Chemical
methods:-

This
involves attachment of enzymes onto different matrices using covalent or

ionic
bonds and the process is irreversible

3.2.1.      Covalent
binding – The enzyme is attached to the matrix by means of
covalent bonds (diazotation, amino bond, Schiff’s base formation, amidation reactions,
thiol-disulfide, peptide bond and alkylation reactions). Enzyme molecules are
attached either directly to the reactive groups (e.g., hydroxyl, amide, amino,
carboxyl groups) present on the matrix or by a spacer arm, which is
artificially attached to the matrix through various chemical reactions (e.g.,
diazotization, schiff base, imine bond formation).

This method of immobilization involves
non-essential amino acids (other than active site groups) leading to minimal
conformational changes. It helps to promote the higher resistance of
immobilized enzymes towards extreme physical and chemical conditions.

3.2.2.     
Cross
linking – 

This involves formation of a number of
covalent bonds between enzyme and the matrix using bi- or multi-functional
reagents (e.g., glutardialdehyde, glutaraldehyde, glyoxal, diisocyanates,
hexamethylene diisocyanate, toluene diisocyanate). 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.

3.2.3.     
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. Sometimes, in addition to ionic
interactions, enzyme molecules are also physically adsorbed to the matrix.

Enzyme binding via ionic interactions during
immobilization depends on the pH of the solution, the concentration of the
enzyme and temperature. Commonly used matrices are: polysaccharide derivatives
(e.g., diethylaminoethylcellulose, dextran, carboxymethylcellulose, chitosan),
synthetic polymers (e.g., polystyrene derivatives, polyethylene vinylalcohol)
and inorganic materials (e.g. Amberlite, alumina, silicates, bentonite,
sepiolite, silica gel).

3.2.4.     
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.

This method is not only useful for enzyme
immobilization but also for several proteins including antibodies, cytokines,
streptavidin etc. Enzymes immobilized by this method have found various
applications in biotechnology, diagnostics and medicine.

Table of advantages and
disadvantages

 

     

Sr.
No

Method

Advantages

Disadvantages

1

Entrapment
          

·        
Simplicity
·        
No change in intrinsic enzyme
properties
·        
Involves no chemical modification
·        
Minimal enzyme requirement
·        
Matrices are available in various
shapes

·        
Enzyme leakage
·        
Only small sized
substrate/products can be used
·        
Presence of diffusional
constraints

2

Adsorption

·        
Simple and economical
·        
Limited loss of activity
·        
Can be Recycled, Regenerated
& Reused.
 

·        
Relatively low surface area for
binding.
·        
Exposure of enzyme to microbial
attack.
·        
Yield is often low due to
inactivation and desorption.

3

Microencapsulation

·        
No chemical modification.
·        
Relatively stable forms.
·        
Easy handling and re-usage.

·        
The enzyme may leak from the pores

4

Covalent

Bonding

·        
No leakage of the enzymes occurs.
·        
Stable at high ionic strength

·        
Drastic changes in conformation
·        
Change in catalytic properties of
enzyme

5

Crosslinking

·        
Simplicity
·        
Higher stability
·        
Very little desorption of enzyme

·        
Cause significant changes in the
active site
·        
Not cost effective

6

Ion
Binding

·        
Minimal changes in enzyme
conformation

·        
Detachment of enzyme at
suboptimal conditions.

7

Conjugation
by
Affinity
ligand

·        
Minimal changes in enzyme
conformation
·        
High stability and catalytic
efficiency

·        
Not cost effective

 

4.      Selection of matrix:-

During enzyme immobilization, enzymes are either
attached to the surface of matrices or entrapped inside them by physical or
chemical methods. The ideal matrix should be inert, physically strong and
stable, cost effective, regenerable and should reduce product inhibition.

Matrices can be classified as

      
I.           
Inorganic matrix: – They show high
pressure stability and may undergo abrasion.

e.g. Commercially SiO2 available
materials- Porous glass, Silica.

Mineral materials- Celite
,Centonite

   
II.           
Organic :- There are two types

a)      Organic
natural carriers:- They are favourable
and compatible with proteins.

e.g. Cellulose derivatives-  DEAE-cellulose , CM-cellulose.

Dextran, Polysacharides, Agarose,
Starch, Pectine, Chitosan

b)      Organic Synthetic Carriers:-

e.g. Polystyrene, Polyvinyl
acetate, Acrylic polymers

 

The
physical characteristics of the matrices (such as mean particle diameter, swelling
behavior, mechanical strength, compression behavior) will be of major
importance for the performance of the immobilized systems and determine the
type of reactor used under technical conditions. The choice of
the most suited

method
and the chemical nature of support material clearly depends on the application
that enzyme is devoted to.

 

5.     
Applications
of immobilized enzyme:-

5.1.   Production
of fine and bulk chemicals:

Immobilised enzymes are widely used
in industrial production of antibiotics, beverages, amino acids etc. In
production of cephalosporin, cephalosporin C acylase (CPCA) and cephalosporin C
deacetylase (CAH) were immobilized epoxy carrier (LH-EP) by covalent binding
for conversion of cephalosporin C (CPC) to deacetyl-7-aminocephalosporanic acid
(D-7-ACA) in single pot. They found that A D-7-ACA yield of 78.39% was achieved
in 30 min in a single reactor under the optimized enzyme amount. The specific
productivity of D-7-ACA reached 10.85 g g?1 h?1 L?1,
which increased approximately 4-fold compared to that obtained from the two-pot
enzymatic process. ( Xiaoqiang Ma et al, 2015)

A new and efficient process was
developed involving lipase-catalyzed transacylation to resolve ethyl
8-chloro-6- hydroxy octanoate (ECHO) to produce an important chiral precursor
for the synthesis of (R)-?-lipoic acid. Lipase immobilized on acrylic resin was
used and achieved in 94% Enantiomeric excess (ee), 35% isolated yield and 38 g
L?1 d?1 space-time yield of (ECHO). (Zhou et al, 2014)

5.2.   Biomedical
applications:

Immobilized enzymes are widely used
in the diagnosis and treatment of many diseases. Immobilized enzymes can be
used to overcome inborn metabolic disorders by the supply of immobilized
enzymes. Cholesterol biosensors have been developed which immobilize
cholesterol oxidase onto a gold multi walled carbon nanotube electrode covered
with a cross linked matrix of chitosan room temperature ionic liquid.

A flux based glucose sensor was
demonstrated using single walled carbon nanotubes (SWCNT) non-covalently
functionalized by glucose oxidase enzyme The addition of glucose resulted in
the modulation of the fluorescent emission of the glucose oxidase-SWCNT,
allowing for real-time monitoring of glucose concentrations. (Oliveira et al,
2015)

5.3.   Food
industry: Enzymes like pectinases, cellulases, glucose isomerase, lactase   immobilized on suitable carriers are
successfully used in the production of jams, jellies, fruit juice, cheese.

Glucose isomerase immobilized on
onsilica/chitosan hybrid microspheres showed relative enzyme activity to be
above 90% with a wide pH range of 5.8–8.0, temperature range of 40–80?C,
storage range of 3 months and gives higher conversion of glucose to fructose.
(Zhao et al, 2016)

Enzymes like ?-glucosidase,
?-arabinosidase, and ?-rhamnosidase from a commercial Aspergillus niger
preparation, were immobilized onto acrylic beads to enhance flavor of wine. (González-Pombo
et al, 2014).

5.4.   Production
of bio-diesel:-

The enzymes like cellulases and
lipases can be used for biodiesel production. Lipase-catalyzed
transesterification of oil feed stocks has been considered as one of the most
promising techniques for producing biodiesel. Biodiesel from Soybean Oil was
produced by using lipase immobilised on magnetic composite poly
(styrene-methacrylic acid) microsphere It was found that the oil conversion

of 86% was attained at a reaction
temperature of 35 °C for 24 h.(
Xie and Wang, 2014)

Biodiesel was obtained from Nannochloropsis gaditana microalgae
using lipases immobilized on calcined mesoporous material and a fatty acid
ethyl ester (FAEE) content of 71 and 74.1 wt % were achieved with free and immobilized
Cal B lipase. (Bautista
et al, 2015)

5.5.   Waste
water management: 

Major
concerns about micropollutants in the environment are related to potentially
hazardous, undesirable biological activities such as, e.g., endocrine
disruption. To eliminate
critical contaminants from water, recent trends favor environmentally friendly technologies.
The use of enzymes is environmentally benign, efficient, and more selective
compared to chemical catalysts

Laccase
was immobilized on biocatalytic membrane PVDF with redox mediators Syr and
ABTS. It helps in removal of phenolic compounds like bisphenol A. There was
first order relationship in bisphenol A and laccase concentration.( Jahangiri et al, 2014)

Synthetic
dyes are extensively used in a number of industries, such as textile dyeing.
Due to their low biodegradability, they cause serious environmental pollution.
Laccase was entrapped into calcium alginate beads and applied to the
decolorization of different synthetic dyes. While
by free enzyme, this anthraquinone dye was decolorized to 74.6% and increased
up to 90.3% using immobilized laccase after 90 min of treatment.( Daassi et al, 2014)

5.6.  
Textile industry: Immobilized enzymes
can be used for scouring, bio-polishing and desizing of fabrics. Eudragit S-100
has been covalently bound to the cellulase enzyme to form immobilized cellulase
enzyme and then the effect of the treatment on ramie fabric properties is
studied. The tensile strength of ramie yarns treated with the immobilized
cellulase enzyme (141cN) is found remarkably higher than that treated with
native cellulase enzyme (118cN) and immobilized enzymes show comparatively
lower weight loss.(Ni et al, 2016)

Enzymes like cellulase and laccase
can be used in the denim washing to produce the desired aged look in the
indigo-dyed denim garments. In this study, cellulase and laccase were
co-immobilized onto the reversibly soluble polymers (Eudragit S-100 or Eudragit
L-100). Eudragit-cellulaselaccase could retain more than 35 % of the original
enzyme activities after five cycles of repeated uses, showing certain reusability.(Yu
et al 2017)

5.7.   Detergent
industry:

Lipase is immobilized for effective
dirt removal from cloths. To make enzymes
more stable with detergents, solvents used in detergent powders enzymes are
immobilized A thermo-tolerant and alkaline lipase
enzyme was purified from Lactobacillus
brevis and immobilized onto modified ?-Fe3O4 florisil nanoparticles.
Olive
oil was removed by the detergent alone and by the detergent and immobilised
lipase at ratios of 45% and 72%, respectively (Soleiman et al, 2017)

6.     
Application
of immobilized enzymes in fruit juice clarification:-

Nowadays, with increasing in
natural fruit juice consumption, the developments of modern technologies have
attracted considerable attention for the improvement in quality of fruit juice industry.

Physical chemical methods

If physical or chemical methods of
fruit juice clarification are used the processed juices are not always stable,
but rather tend to produce pronounced haze and enzymatic and nonenzymatic browning,
caused by reactive phenolic compounds that cannot be removed.

The pectic substances, located
primarily in the middle lamella between cells in higher plant tissues, are
complex polysaccharides. They include the negatively charged
rhamnogalacturonans, and the neutral arabinogalactans I and II and l-arabinans.
These polysaccharides add viscosity to juices but may also form hazes and
precipitates and retard maximum recovery of juices from the fruit.

However, the problems in enrichment
raw juices clarification are caused mainly by the presence of polysaccharides
such as pectins, starch and hemicellulosic components which tend to settle
during storage and led to decrease quality of them.

The raw fruit juice obtained after
pressing is very turbid viscous and contains a significant amount of colloidal
compounds, mainly pectin that causes cloudiness; therefore, clarification of
fruit juices involves the removal of juice haze by enzyme hydrolysis with
pectolytic enzymes. Tannin content in the fruit juices that
related to the bitterness is the major obliging factor to the consumers.
Positively charged proteins and negatively charged proteins interact and leads
to formation of complexes and increases turbidity of juice.

Immobilization provides an
excellent base for enzymes exploitation by increasing their reusability,
enhancing their structural and catalytic stability in different environmental
conditions, and reducing product inhibition (Sheldon, 2007). Apart from being affordable,
immobilization generates continuous economic operations,

automation, high investment/capacity
ratio and recovery of product with greater purity (D’Souza, 1998). Several
methods are used for enzyme immobilization and various factors influence the
performance of the immobilized. The choice of the most suited method and the
chemical nature of support material clearly depends on the application that
enzyme is devoted to. Particularly, covalent binding is the most widely applied
method in industrial.

6.1.  
Role
of Role of enzymes used in fruit juice clarification

Sr. No.

Enzyme

Role of enzyme

1

Pectinase and Polygalacturanase

Pectinolytic
enzymes are a group of enzymes that hydrolyze
pectin, one of
the main polysaccharides in plant cell wall  The most widely occurring enzymes are polygalacturonase
(PG), pectin methylesterase (PME) and pectate.
Pectinase is a
collective term that refers to a group of enzymes that degrade pectic
substances in the cell wall of higher plants. Pectic substance is a
polysaccharide composed of ?-1,4-linked d-galacturonic acid. It can be
divided broadly into two groups: one is pectic acid, which is a polymer of
galacturonic acid, and the other is pectin, which is a polymer of
galacturonic acid whose carboxyl groups are methyl-esterified.

2

Xylanase

Xylanase
(endo-?-1,4-D-xylan xylanohydrolase; EC
3.2.1.8)
catalyzes the hydrolytic cleavage of glycosidic bonds in the xylan backbone
to produce a mixture of xylooligosaccharides of various chain lengths and
xylose.
Xylan,
the major renewable hemicellulosic polysaccharide
in
secondary plant cell walls, consists of a ?-1,4-linked
D-xylopyranose
backbone substituted predominantly with ?-
L-arabinofuranosyl,
acetyl and glucuronosyl residues

3

Laccase

Laccases
(EC 1.10.3.2) are copper-containing oxidase enzymes found in many plants,
fungi, and microorganisms. Laccases act on phenols and similar molecules
Laccases are widespread enzymes able to oxidize a wide range of phenolic
substrates, using only molecular oxygen as cofactor and generating water as
unique by-product

4

Tannase

Tannase
(EC, 3.1.1.20) is an inducible, intracellular/extracellular microbial enzyme
that catalyzes the hydrolysis of ester and depside linkages of tannin, which
is the sole responder of bitterness, turbidity, and undesirable cloudiness in
fruit juices and wine.

5

Papain

In
the food industry, papain (EC 3.4.22.2) is commonly used
for
meat tenderization, protein hydrolysate production, and the clarification of
juice and beer. It is a

 

 

 

7.      Recent methods of enzyme
immobilization used in fruit juice clarification:-

 

7.1.  
Immobilization
on nanoparticles:-

7.2.  
Immobilization
on magnetic microspheres

7.3.  
Immobilization
on functionalized supports

7.4.  
Immobilization
of multiple enzymes

7.5.  
Immobilization
on natural polymers

7.6.   Immobilization on inorganic matrix

x

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