COLD ACTIVE ENZYMES

IN FOOD PROCESSING
 MEMBERS OF GROUP

1. Nguyen Ngoc Hoai An – BTBTIU14009

2. Nguyen Nam Tran – BTBTIU14243
3. Cao Thi Tuyen Nhung – BTBTIU14170
 CONTENTS

1. Introduction 5.1.1 Cold Active Proteases from Fish

2. Cold Adapted Organisms as a Source
of Cold Active Enzymes
3. Basis of Cold Activity
3.1 Kinetic and Thermodynamic
Adaptations
3.2 Flexibility Versus Stability
3.3 Active Site of Cold Active
Enzymes
3.4 Structural Adaptations
4. Directed Evolution as a Tool for
Creation of Cold Active Enzymes
5. Cold Active Enzymes of Interest for
Food Processing
5.1 Proteases
5.1.2 Proteases Made Cold Active by
Mutagenesis
5.2 Glycosidases
5.2.1 Cold Active B-galactosidases
5.2.2 Cold Active Pectinases
5.2.3 Cold Active α-amylase
5.2.4 Cold Active Xylanase
5.2.5 Cold Active Endoglucanases
5.2.6 Cold Active Lysozyme
5.3 Lipase
5.4 Catalase
5.5 Other Potential Applications
6. Aspects on Production of Cold Active
Enzymes
7. References
 1. INTRODUCTION

lower energy
 The interest in “cold requirements
activity” of enzymes is
relatively recent, and has inactivation of
been promoted as a result the enzyme after
completion of the minimization of
of intense research activity process by a mild side reactions
on cold adapted organisms increase in
temperature
isolated from low
temperature environments.
Benefits
 The applications of
enzymes for low
temperature processing prevention of the
may provide various destruction of
other substances
reduced risk of
microbial
benefits. associated with spoilage
raw material

protection of
substrates or
products from
degradation
 2. COLD ADAPTED ORGANISMS AS A
SOURCE OF COLD ACTIVE ENZYMES

 Most microorganisms that inhabit permanently

cold environments are either psychrotolerant
(also called as psychrotrophic) or psychrophilic.
Psychrotolerant organisms grow well at
temperatures close to the freezing point of
water, but have optimal growth rates above
20°C while psychrophiles grow fastest at
temperature of 15°C or lower but are unable to
grow above 20°C
 Besides cold habitats, other interesting sources
of cold active enzymes include many fish,
squids, and other aquatic creatures, which are
adapted to environments that are permanently
cold.
 For example, Greenland cod remains
biologically active at about 0°C throughout the
year living in sub-Arctic latitudes
 3. BASIS OF COLD ACTIVITY

 The study of proteins and enzymes from cold adapted species

that have helped in providing insights into their molecular and
functional adaptations to low temperature.
 This has been facilitated by the availability of recombinant
enzymes for analysis by x-ray and biophysical techniques, and
comparison with mesophilic and thermophilic homologues.
 3.1 KINETIC AND THERMODYNAMIC
ADAPTATIONS

 In order for the cold active enzymes to maintain reaction rates

at low temperature that are comparable to mesophilic enzymes
at mesophilic temperatures, the loss of kinetic energy at low
temperatures needs to be overcome.
 optimization of catalytic efficiency (kcat /Km) is the main
physiological adaptation for the enzymes to be active at low
temperatures.
 The contribution of enthalpic and entropic energies to G may
further reflect adaptations to the low temperature environment
in which these enzymes function (∆G = H - T∆S).
 3.2 FLEXIBILITY VERSUS STABILITY

 Higher structural flexibility has been assumed to be a

prerequisite for catalytic activity of the enzymes from cold
adapted organisms at low temperatures.
 Higher flexibility in cold enzymes would overcome this
restriction, but would lead to poor thermostability.
 The low stability has been demonstrated by a significant
decrease in optimum temperature of activity and a higher
susceptibility to unfolding at moderate temperatures.
 The balance between flexibility and stability represents one of
the most crucial points in the adaptation of a protein to
environmental temperature.
3.3 ACTIVE SITE OF COLD ACTIVE
ENZYMES
 The structural changes necessary for cold adaptation often involve
changes in the overall conformational properties and dynamics of the
molecule.
 The greater catalytic efficiency of cold active enzymes has been explained
by better accessibility of the active site to the substrate, which may be
achieved by one or more differences like small deletions in loops bordering
the active site or replacement of bulky side chains for smaller groups at the
entrance.
 This may lead to the accommodation of substrates at lower energy costs,
and hence reduce the activation energy required for the formation of the
enzyme–substrate complex.
 Easier release of the product may also be facilitated from a larger active
site. Cold adapted enzymes have further been shown to exhibit improved
electrostatic potentials near the active site region when compared with
their mesophilic or thermophilic counterparts, which would facilitate
interaction with oppositely charged substrates.
3.4 STRUCTURAL ADAPTATIONS

 There is no consensus for a common paradigm of structural

changes that confer cold activity.
 Structural models of cold active enzymes have been quite useful
in revealing the specific strategies used in each enzyme,
consisting of a set of structural adjustments to adapt to cold.
 These involve a decrease in proline residues in loops, arginine
content, salt bridges, ion pairs, H-bonds and aromatic
interactions, ion binding constants and hydrophobic
interactions, changes in α-helix dipole interactions, increased
clustering of glycine residues, and insertions.
 4. DIRECTED EVOLUTION AS A TOOL FOR
CREATION OF COLD ACTIVE ENZYMES

 Laboratory evolution has contributed significantly toward modifying

enzyme properties and functions.
 This evolution involves introduction of random mutations in a gene
and screening the resulting library for the acquisition and
improvement of a specific property.
 Cold adapted forms have been created from mesophilic enzymes by
artificial evolution.
 Ex: A mesophilic subtilisin-like protease was converted into a cold
active enzyme; only 4 amino acid substitutions were needed to obtain
a mutant enzyme with a catalytic efficiency 9.6 times greater than that
of its parent at 10°C. Even the catalytic activity of thermostable
enzymes from hyperthermophilic organisms has been improved at low
temperature by DNA shuffling and directed evolution.
 5. COLD ACTIVE ENZYMES OF INTEREST
FOR FOOD PROCESSING

 The use of cold active enzymes is not yet widespread in food

processing or other applications. The exceptions are the
digestive enzymes isolated from fish.
 The breakthrough for cold active enzymes in food processing
relies on the applications involving heat sensitive foods and
where residual enzyme activity is not desired due to potential
changes in the product.
 Proteases and glycosidases are the main groups of enzymes
with diverse applications, and several cold adapted candidates
have been studied, some of which are described here.
5.1 PROTEASES

 Various proteases are widely used for manufacturing protein

hydrolysates and extracts with improved functional properties
from cheap vegetable, meat, and fish byproducts.
 The fish industry uses proteases as gentle processing aids in a
number of operations to yield products of high quality, and
these proteases have in many cases replaced the alternative
mechanical, chemical, or manual treatment procedures.
 For example

Caviar produced using proteases has

the advantages of better recovery,
less damage to the eggs, cleanliness
of products with no residues, less
required labor, and good hygienic
conditions compared to caviar
produced mechanically. The
enzymes used are the digestive
enzymes isolated from fish, and the
processing is invariably done at low
temperatures.
 In the dairy industry, rennets are used for coagulation during
cheese production, while other proteases are used for
accelerating cheese ripening, for modifying functional properties
of some dairy products, and for modifying milk proteins to
reduce the allergenic properties of cow milk products.
 Milk oxidation is an undesirable phenomenon in the dairy
industry, and treatment of milk with trypsin improves its stability
to oxidative rancidity, presumably through improved copper
binding by the enzyme modified protein
FOOD PROCESSING OPERATIONS
SUITABLE FOR COLD ACTIVE
PROTEASES
 5.1.1 COLD ACTIVE PROTEASES FROM
FISH

The digestive proteolytic enzymes can be

recovered as byproducts from fish processing
wastes. The cold active proteolytic enzymes
like pepsin, trypsin, chymotrypsin, and
elastase, have been isolated from the Arctic fish
stomach of cold water species. These
enzymes invariably have a relatively lower
optimum temperature of activity, higher
catalytic efficiency, and much lower stability
than their mammalian counterparts. capelin

Atlantic cod (Gadus morhua) Polar cod (Boreogadus saida) Greenland cod (Gadus ogac)
 Trypsins from fish and other species of marine origin are
generally unstable at low pH values and stable under alkaline
conditions, hence differing from mammalian proteases.
 Cod pepsins have been used as rennet substitutes in cheese
production, in skin removal from fish, and as a digestive aid in
fish feed.
 Pepsins from Atlantic cod (Gadus morhua) and orange roughly
(Hoplostecthus atlanticus) have been used for caviar production.

orange roughly
 5.1.2 PROTEASES MADE COLD ACTIVE BY
MUTAGENESIS

 Mucor rennin, often used as a substitute for calf chymosin,

possesses a high thermostability that causes survival of the
enzyme activity after the curd is cooked, and thus may cause
bad flavor in cheese during long maturating periods.
 5.2 GLYCOSIDASES

 Although the starch industry accounts for a significant part of

the applications requiring heat stable glycosidases (glycoside
hydrolases), various other processes are potential targets for
cold active enzymes.
 The hydrolysis of lactose in milk and whey using -galactosidase
is a well known process to obtain better quality dairy products
and ones that are more easily digested by lactose intolerant
individuals.
 Pectinases are now an integral part of fruit juice industries,
being used to facilitate juice extraction and clarification.
 In bread making, the concentrations of starch, hemicelluloses, and
gluten are very important for the quality of baking products. Amylases,
along with xylanases and proteases, have therefore been applied in
baking processes.
 These enzymes can be used to reduce the dough fermentation time and
to improve the properties of dough and crumb, and can aid in the
retention of aromas and levels of moisture. A clear advantage of cold
active enzymes here is the ease of inactivating the enzymes by heat, so
that the enzymatic activity can be controlled better and a prolonged
action of the enzymes prevented, which otherwise can result in the
crumb becoming too soft or too sticky.
 Glycosidases with antimicrobial activity have a clear potential for
prolonging the shelf life of foods stored in cold or ambient
temperatures. For example, lysozyme has been used as a food
preservative in food products such as cheese and sausages because of
its antibacterial activity.
 5.2.1 COLD ACTIVE B-
GALACTOSIDASES
 Detailed characterization of the -galactosidase from P. haloplanktis isolated
from Antarctica has revealed significant similarities with the mesophilic
enzyme from Escherichia coli, including the multimeric form and subunit
mass, active site residues involved in catalysis, and strict requirement for
divalent metal ions.
 5.2.2 COLD ACTIVE PECTINASES

 Reports on pectinases from cold adapted organisms were until

recently restricted to psychrotolerant spoilage bacteria such as
different strains of Pseudomonas fluorescens.
 Endopolygalacturonases from psychrophilic fungi, Sclerotinia
borealis and Mucor flavus have been reported which are active
on both polygalacturonic acid and esterified pectin to different
extents
5.2.3 COLD ACTIVE -AMYLASE

 The -amylase from psychrophilic A. haloplanctis is an enzyme

that has been examined in great detail with respect to its
structure and thermodynamic properties.
 The amylase is among the most thermolabile enzymes known
 5.2.4 COLD ACTIVE XYLANASE

 The enzymes exhibit high activity at low temperature, poor

stability, and high flexibility, like the other cold adapted
biocatalysts.
 5.2.5 COLD ACTIVE
ENDOGLUCANASES
 Very few reports are available on cold active endoglucanases
(cellulases) from cold adapted organisms.
 5.2.6 COLD ACTIVE LYSOZYME

 A cold active lysozyme-like ~11kDa enzyme designated

chlamysin has been isolated from viscera of the marine bivalve
Chlamys islandica that is a marine species located 20 m or
deeper in the southern part of an arctic region having sea
temperatures rarely exceeding 4°C.
 Interestingly, the enzyme was completely stable during storage
at room temperature for a month as well as after heating to
70°C for 15 min, suggesting high structural stability.
 5.3 LIPASE

 Lipases are mainly used in the treatment of fats and oils for
which activity at high temperature is normally desirable due to
the solubility characteristics of these materials.
 Lipases are also used to some extent in cheese ripening. A lipase
from an Antarctic psychrophilic bacterium Psychrobacter
immobilis sp.
 5.4 CATALASE

 The enzyme catalase is important in the treatment of dairy and

liquid egg products for the removal of residual hydrogen
peroxide used for low temperature pasteurization.
 The enzyme’s own oxygen-generating capacity is also exploited
in its application as a leavening agent for baked goods.
5.5 OTHER POTENTIAL APPLICATIONS

 Transglutaminase is used to impart unique rheological and

functional properties, such as increase in elasticity and firmness,
texture improvement, gelation capability, and water holding
capacity, by forming cross links between protein molecules.
 A bacterial (Streptoverticillium sp.) transglutaminase is widely
used in seafood surimi (fish paste) products, meat products
(sausage and ham), dairy products, and baked goods, and has
potential in the manufacture of yogurt, cheese, and frozen dairy
products with improved quality.
 Alpha-L-rhamnosidases and laccases are applicable in the
processing of fruit juices.
 6. ASPECTS ON PRODUCTION OF COLD
ACTIVE ENZYMES

 Application of cold active enzymes places demand on their cost

effective production.
 Cloning and expression in Escherichia coli has provided
sufficient quantities of the enzymes from cold adapted
organisms to facilitate structural studies.
 Some efforts are being made to develop cloning or expression
systems in cold adapted bacteria that may circumvent the low
stability of the product encountered during heterologous
production in mesophilic hosts.
 Subsequent to their production, strategies for purification of
cold active enzymes need to be carefully evaluated because
of their sensitive nature. Use of separation techniques
allowing rapid and gentle purification are essential to
minimize losses.
 Eventually, conditions for storage of the purified enzymes
prior to their use would also need to be considered in specific
cases.
 In conclusion, it may be stated that cold active enzymes, even
though having existed as long as the existance of life in cold
environments, have been “discovered” relatively recently.
 Their potential in food processing is yet to be fully exploited.
It is very likely that evolution in the laboratory will be used to
assist the natural evolution or even gain preference in the
design of cold active enzymes with required stability for the
applications of interest.
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