# CHAPTER II # 8

CHAPTER II

Review of LiteRatuRe

# CHAPTER II # 9

2.1. Enzymes

Enzymes are proteins and are nature's own biocatalysts. Enzymes have many

advantages over their chemical counterparts in that they are more specific, and generally

possess high catalytic properties. Almost all processes in a biological cell need enzymes

to occur at significant rates. More than 4000 enzymes catalyzing a wide array of reactions

are known to exist. Since the 1950s, Enzyme technology has really taken off. It is the

basis of the new industry called biotechnology. There are great benefits in using enzymes

as catalysts to make products. They can be some 10,000 times more efficient than

ordinary inorganic catalysts used in industry. One enzyme molecule can catalyze 10

million reactions in a single second!

The enzymes are classified into six major categories based on the nature of the

chemical reaction they catalyze:

1. Oxidoreductases catalyses oxidation or reduction of their substrates

2. Transferases catalyses group transfer

3. Hydrolases catalyses bond breakage with the addition of water

4. Lyases remove groups from their substrates

5. Isomerases catalyses intramolecular rearrangements

6. Ligases catalyses the joining of two molecules at the expense of chemical

energy

More than 75% of industrial enzymes are hydrolases. And amongst them, amylase

account for about 30% of the world’s enzyme production (Maarel et al., 2002). Amylases

# CHAPTER II # 10

have even found new applications but their use in detergents is the major market. Figure

2.1 illustrates the diverse use of amylase in various sectors.

Figure 2.1. Use of amylase enzyme in diverse Business areas

2.2. Amylase

Amylases are starch degrading enzymes. They are widely distributed in microbial,

plant and animal kingdoms. However, enzymes from fungal and bacterial sources have

dominated applications in industrial sectors (Pandey et al., 2000). They degrade starch

# CHAPTER II # 11

and related polymers to yield products characteristic of individual amylolytic enzymes.

Initially the term amylase was used originally to designate enzymes capable of

hydrolyzing α-1,4-glucosidic bonds of amylose, amylopectin, glycogen and their

degradation products (Bernfeld, 1955; Fisher and Stein, 1960; Myrback and Neumuller,

1950). They act by hydrolyzing bonds between adjacent glucose units, yielding products

characteristic of the particular enzyme involved. Amylases have emerged as one of the

leading biocatalysts with proven potential to find usage in a wide array of industrial

applications, such as additives in processed food industries, additives in detergents,

waste-water treatment, biopulping, bioremediations and in molecular biology. The

spectrum of amylase application has widened in many other fields, such as clinical,

medical, analytical chemistry, as well as their wide spread application in starch

sacccharification, in the textile, food, fermentation, paper, brewing and distilling

industries (Pandey et al., 2000). Figure 2.2 illustrates the disparity of sale of amylase in

industries in year 2000 and 2010.

2.2.1 History of Amylase

In 1831, Erhard Friedrich Leuchs (1800-1837) described the diastatic action of

salivary ptyalin (amylase) on starch. The modern history of enzymes began in 1833 when

French chemists described the isolation of an amylase complex from germinating barley

and named it diastase. In 1862, Danielewski separated pancreatic amylase from trypsin.

# CHAPTER II # 12

2.2.2 Classes of amylases

1. α-Amylase

Most of the α-amylases (endo-1,4-α-D-glucan glucohydrolase, EC 3.2.1.1) are

metalloenzymes, which require calcium ions (Ca2+) for their activity, structural integrity

and stability. They belong to family 13 (GH-13) of the glycoside hydrolase group of

enzymes (Bordbar et al., 2005). The specificity of the bond attacked by α-amylases

depends on the sources of the enzymes. Currently, two major classes of α-amylases are

(a)

# CHAPTER II # 13

(b)

Figure 2.2. Distribution of sales of amylase by various industries (a) in 2000 and (b) in

2010

commercially produced through microbial fermentation. Based on the points of attack in

the glucose polymer chain, they can be classified into two categories, liquefying and

saccharifying. Bacterial α-amylase randomly attacks only the α-1,4 bonds yielding

maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from

amylopectin, it belongs to the liquefying category. The hydrolysis reaction catalyzed by

this class of enzymes is usually carried out only to the extent that, for example, the starch

is rendered soluble enough to allow easy removal from starch-sized fabrics in the textile

industry. The paper industry also uses liquefying amylases on the starch used in paper

coating where breakage into the smallest glucose subunits is actually undesirable. On the

other hand, the fungal α-amylase belongs to the saccharifying category and attacks the

second linkage from the non reducing terminals (i.e. C4 end) of the straight segment,

# CHAPTER II # 14

resulting in a disaccharide called maltose. The bond breakage is thus more extensive in

saccharifying enzymes than in liquefying enzymes (Marrel et al., 2002).

Microbes are the major source of alpha-amylase. Microbial α-amylase has been shown to

produced by several fungi and moulds like Rhizopus, Aspergillus, etc. Bacterial amylase

might be produced by Bacillus, Pseudomonas, Saccharophilia, Clostridium and several

other species. But on industrial scale the strains of Bacillus species seem to be preferred

(Bordbar et al., 2005).

2. β-Amylase

Another form of amylase is β-amylase (1,4-α-D-glucan maltohydrolase, EC

3.2.1.2) is also synthesized by bacteria, fungi, and plants. Working from the non-reducing

end, β-amylase catalyzes the hydrolysis of the second α-1,4 glycosidic bond, cleaving off

two glucose units (maltose) at a time. During the ripening of fruit, β-amylase breaks

starch into maltose, resulting in the sweet flavor of ripe fruit.

Both α-amylase and β-amylase are present in seeds; β-amylase is present in an inactive

form prior to germination, whereas α-amylase and proteases appear once germination has

begun. Cereal grain amylase is key to the production of malt. Many microbes also

produce amylase to degrade extracellular starches. Animal tissues do not contain β-

amylase, although it may be present in microorganisms contained within the digestive

tract (Hill and Needham, 1970).

# CHAPTER II # 15

3. γ-Amylase

In addition to cleaving the last α-1,4 glycosidic linkages at the non-reducing end

of amylose and amylopectin, yielding glucose, γ-amylase also cleave α-1,6 glycosidic

linkage. Unlike the other forms of amylase, γ-amylase (Glucan 1,4-α-glucosidase, EC

3.2.1.3) is most efficient in acidic environments and has an optimum pH of 3.

2.2.3. Alpha Amylase Family

The α-amylase family, i.e. the clan GH-H of glycoside hydrolyses, is the largest

family of glycoside hydrolases, transferases and isomerases comprising nearly 30

different enzyme specificities (Henrissat, 1991). A large variety of enzymes are able to

act on starch. These enzymes can be divided basically into four groups: endoamylases,

exoamylases, debranching enzymes and transferases (Marrel et al., 2002);

1. endoamylases: cleave internal α-1,4 bonds resulting in α-anomeric products.

2. exoamylases: cleave α-1,4 or α-1,6 bonds of the external glucose residues resulting in

α- or β-anomeric products.

3. debranching enzymes: hydrolyze α-1,6 bonds exclusively leaving long linear

polysaccharides, and

4. transferases: cleave α-1,4 glycosidic bond of the donor molecule and transfer part of

the donor to a glycosidic acceptor forming a new glycosidic bond.

Glycoside hydrolases are able to metabolize a large variety of saccharides. They

have been divided into classes based on their mode of reaction and families based on

their well-defined amino acid sequence similarities. Most of the starch converting

enzymes belongs to GH-13 family. GH-13 family can be further classified based on a

# CHAPTER II # 16

larger unit called clan, which is the three dimensional structure of catalytic module. A

clan may consist of two or more families with the same three-dimensional structure of

catalytic domain but with limited sequence similarities, indicating that protein structure is

better preserved by evolution than amino acid sequence. Among the fourteen clans (A–N)

defined for glycosidases and transglycosidases, α-amylase family (GH-13) belongs to the

eighth clan, clan GH-H (MacGregor, 2005).

The concept of this group of enzymes as the α-amylase was proposed in 1992

(Takata, 1992). According to the definition, the members of this family must satisfy the

following requirements:

(i) They must act on α-glucosidic linkages and hydrolyze them to produce α-

anomeric monosaccharides and oligosaccharides or form α-glucosidic

linkages by transglycosylations,

(ii) Have four highly conserved regions in their primary structures consisting of

catalytic and important substrate-binding sites,

(iii) Have Asp, Glu, and Asp residues as catalytic sites corresponding to the Asp 206,

Glu 230 and Asp 297 of Taka amylase A, and

(iv) Possess a (β/α)8 or TIM barrel catalytic domain.

2.2.4 Characteristics of Catalytic Domain of α-Amylases

Four conserved sequence regions covering the strands β3, β4, β5 and β7 of the

catalytic (β/α)8-barreled domain were identified and used for defining the α-amylase

family. The two dimensional structure of α-amylase prototype consists of three domains,

namely A, B and C. Domain A is the N-terminal TIM barrel structure, domain B consists

# CHAPTER II # 17

of a long loop that protrudes between β-strand 3 and α-helix 3 and C domain with a β

sheet structure linked to A domain. The (β/α)8 barrel consists of eight alternate β-strands

and α-helices. The β strands are placed parallel to one another as if on a cylinder and α-

helices lie outside the cylinder. This TIM barrel contains four highly conserved regions

closely related to the active site present in all α-amylases (Kuriki et al., 2005). They are:

(i) First region: C-terminal end of β-strand 3 and histidine residue which interacts with

the glucose residue of the substrate,

(ii) Second region: β-strand 4 with Asp residue, which acts as the nucleophile during

catalysis,

(iii) Third region: β-strand 5 with glutamic acid residue acting as proton donor/acceptor,

and

(iv) Fourth region: β-strand 7 with a histidine residue and an Asp residue that may form

hydrogen bonds with glucose residue of the substrate.

While the four original conserved sequence regions contain the catalytic and substrate

binding residues of the individual members of the family, three additional conserved

sequences were later identified and they are shown to contain amino acid residues

connected to a given enzyme specificity. Two of these three regions roughly cover the

strands of α2 and α8 of the catalytic (β/α)8 barrel and one is located near the C-terminus of

domain B (Janecek, 2002).

# CHAPTER II # 18

2.3. COLD ACTIVE ENZYMES

2.3.1. Definition of psychrophile/psychrotolerant

The definition by Ingraham and Stokes (1959), that psychrophilic bacteria should

show visible growth at 0°C after 14 days of incubation, has been renewed by the seminal

paper of Morita (1975). Further publications and reviews (Baross and Morita, 1978;

Deming, 2002; Friedmann, 1994; Herbert, 1986; Inniss and Ingraham, 1978; Russell,

1990) have set the frame for defining psychrophilic organisms and their environments.

The classical definition of Morita (1975) is frequently used in the literature. This

definition proposes that psychrophilic microorganisms have optimum growth

temperatures of <15°C and upper cardinal temperatures of ~20°C. He further

distinguished psychrophiles from psychrotrophs (psychrotolerants) on the basis of their

cardinal temperatures, in that the latter have a minimum growth temperature, which is at

or just above zero, and optimum and maximum growth temperatures above 20°C.

Bacteria Minimum temp.

(Tmin)

Optimum temp.

(Topt)

Maximum temp.

(Tmax)

Psychrophilic <0°C <15°C <20°C

Psychrotolerant <0°C >15°C >20°C

The definition of psychrophilic microorganisms is ambiguous for three main reasons.

First, the temperature limits have been arbitrarily selected and do not correspond to any

clear separation of biological processes or environmental conditions. Second, Morita’s

# CHAPTER II # 19

definition does not apply to most eukaryotes. Finally, and most important,

microorganisms behave as thermodynamic units: increasing the culture temperature

increases reaction rates and the growth rate. However, they also behave as biological

units: at a given temperature, key metabolic steps are heat labile and impair the

functioning of some pathways. It has been shown, for instance, that although the growth

rate of some cold-adapted bacteria increases with a temperature shift from 5°C to 25°C,

the physiological state is strongly altered, as judged by the decrease in viable counts,

exoenzyme production, protein synthesis and membrane permeability (Orange, 1994;

Feller et al., 1994b). This emphasizes that the use of growth rates to define the optimum

growth temperature is inappropriate.

2.3.2. Habitats of psychrophilic/psychrotolerant bacteria

Around 85% of earth is occupied by cold ecosystems including the ocean depths,

polar and alpine regions. Out of which ~70% is covered by oceans that have a constant

temperature of 4-5°C below a depth of 1000 m, irrespective of the latitude. Remaining

15% included Polar Regions to which the glacier and alpine regions must also be added.

Extremophiles successfully colonized on these eternally cold environments which we can

call as psychrophiles (which literally means cold-loving). Psychrophilic microorganisms,

including bacteria, yeasts, fungi and microalgae, can be found in soils, in water (fresh and

saline, immobile and flowing) and associated with plants and cold-blooded animals.

Numbers of pathogenic psychrotolerant pseudomonads are also reported in plant

kingdom causing variety of diseases, including soft rots and wilts. These pseudomonads

# CHAPTER II # 20

produce ice-nucleation proteins, as part of their pathogenic mechanism which has

biotechnological potential.

2.3.3. Cold active amylases

Alpha-amylases are ubiquitous enzymes produced by plants, animals and

microbes, where they play a dominant role in carbohydrate metabolism. Cold-adapted

amylolytic microorganisms produce amylases, which function effectively at low

temperatures with high rates of catalysis in comparison to the amylases from mesophiles

or thermopiles, which shows little or no activity at low temperature. These amylases have

evolved a range of structural features that confer a high level of flexibility, particularly

around the active site are translated into low activation enthalpy, low-substrate affinity,

and high specific activity at low temperatures. Moreover, the maximum level of activity

of these amylases is shifted towards lower temperatures with a concomitant decrease in

thermal stability. The knowledge of cold-active amylolytic enzymes is increasing at a

rapid and exciting rate.

2.3.4. Sources of cold-active amylases

Enzyme adapted to cold can be produced by prokaryotic as well as by eukaryotic

organisms. Up to now, most of them originate from bacteria and fish living in polar

regions and especially in Antarctic sea water which represent a permanently cold (0±2°C)

and constant temperature habitat (Feller et al., 1996).

Psychrophilic (cold-loving) or psychrotolerant (cold-adapted) microorganisms

producing cold-active amylases are found inhabiting the low temperature environments

of the Earth, including polar regions, high mountains, glaciers, ocean deeps, shallow

# CHAPTER II # 21

subterranean systems (i.e. caves), the upper atmosphere, refrigerated appliances and the

surfaces of plants and animals living in cold environments, where temperature never

exceed 5°C. The potentials of psychrophiles and psychrophilic enzymes including

amylase have been reviewed by Cavicchioli et al. (2002); Deming (2002); Margesin et al.

(2002); Feller and Gerday (2003) and Georlette et al. (2004). Many psychrophiles live in

biotopes having more than one stress factors, such as low temperature and high pressure

in deep seas (piezo-psychrophiles), or low temperature and high salt concentration in

salty seas (halo-psychrophiles) also produces amylase (Cavicchioli et al., 2002; Deming,

2002; Margesin et al., 2002; Feller and Gerday, 2003; Georlette et al., 2004).

A diverse range of psychrophilic microorganisms, belonging to bacteria, archaea,

yeast and fungi have been isolated from these cold environments (Cavicchioli et al.,

2002; Deming, 2002; Margesin et al., 2002; Feller and Gerday, 2003; Georlette et al.,

2004). In spite of the wide distribution of amylases, microbial sources, namely fungal and

bacterial amylases, are used for the industrial production due to advantages such as cost

effectiveness, consistency, less time and space required for production and ease of

process modification and optimization (Burhan et al., 2003). Among bacteria, Bacillus sp.

is widely used for α-amylase production to meet industrial needs. B. subtilis, B.

stearothermophilus, B. licheniformis and B. amyloliquefaciens are known to be good

producers of α-amylase and these have been widely used for commercial production of

the enzyme for various applications. Similarly, fungi belonging to the genus Aspergillus

have been widely used for the production of amylases for centuries (Pandey et al., 2000).

# CHAPTER II # 22

In addition, some recent examples of cold-active amylase producing bacteria are shown

in the Table 2.1.

Table 2.1: Cold-active amylase producing microbes

Microorganisms Source Reference

Bacillus cereus GA6

Microbacterium foliorum GA2

Wangia sp. C52

P. arctica GS230

β-proteobacteria

Gangotri glacier, India

Gangotri glacier, India

Southern Okinawa

Seawater from Gaogong island,

China

Roopkund Glacier, Himalayan

range, India.

Kuddus et al. (2012)

Roohi et al. (2011)

Liu et al. (2011b)

Lu et al. (2010)

Suman et al. (2010)

Nocardiopsis aegyptia

Marine sediment of Abu Qir Bay,

Alexandria, Egypt

Abou-Elela et al.

(2009)

Streptomyces 4 Alga Soil and vegetation (East Antarctica) Cotarlet et al. (2009)

Micrococcus antarcticus Antarctica Fan et al. (2009)

Bacterial strains Sediment samples from Midtre

Lovenbreen Arctic glacier

Reddy et al. (2009)

Culturable bacteria Sediment and a soil from

Kongsfjorden and Ny-Alesund,

Svalbard, Arctic.

Srinivas et al.

(2009)

Nocardiopsis sp. 7326 Deep sea sediment of Prydz Bay,

Antarctic.

Zhang and Zeng

(2008)

# CHAPTER II # 23

Lactobacillus plantarum

MTCC 1407

Central Tuber Crop Research

Institute, Bhubaneswar, India

Smita et al. (2008)

Amy I and Amy II Eisenia foetida

Earthworm, Osaka, Japan.

Ueda et al. (2008)

Arthrobacter

psychrolactophilus

ATCC 700733

Pennsylvania soil Michael et al.

(2005)

Aspergillus oryzae Culture from University of

Agriculture, Abeokuta, Nigeria

Akpan and Adelaja,

(2004)

Gamma-Proteobacteria Permanently cold fjords of

Spitzbergen, Arctic Ocean.

Tatiana et al. (2004)

Aspergillus ochraceus Soil Nahas and Mirela

(2002)

Alteromonas sp. Antarctic sea water Chessa et al. (1999)

2.4. STRUCRURAL MODIFICATIONS FOR COLD ADAPTATION

Temperature is one of the most important environmental factors for life, as it

influences most biochemical reactions. A reduction in temperature slows down most

physiological processes, changes protein-protein interactions, reduces membrane fluidity

and provokes an increased viscosity of water. Moreover, Enzymes are also subject to cold

denaturation, leading to the loss of enzyme activity at low temperatures (Yancey and

Somero, 1978; Somero, 1981). Psychrophilic microorganisms producing cold-active

amylases are structurally modified by an increasing flexibility of the polypeptide chain

# CHAPTER II # 24

enabling an easier accommodation of substrates at low temperature. They must modify

their lipid composition to maintain membrane fluidity at environmental temperatures.

This can be done in many ways;

a) Unlike cold-adapted proteins, which improve their structural mobility, the thermal

adaptation of membrane lipids does not involve the synthesis of fatty acyls that have

increased degrees of freedom, but rather the introduction of steric constraints that reduce

the packing of acyl chains in the membrane. These steric constraints destabilize the

membrane and reduce the lipid viscosity (Margesin et al., 2002; Russell and Hamamoto,

1998; Russel, 1997).

b) Presence of a cis-unsaturated double bond in the chain that induces a 30° bend. Such

bending creates a cavity in the lipid layer and perturbs the packing density. Trans-

unsaturated double bonds are also observed, but are less efficient as they only produce a

modest kink of the acyl chain.

c) The occurrence of branched lipids mainly methyl-branched fatty-acyl chains also

perturbs the compactness of neighbouring chains owing to the steric hindrance that is

caused by the side-chain group. The position of this branching along the chain also

modulates the gel-phase transition temperature.

d) Finally, shorter fatty-acyl chains reduce the contacts between adjacent chains and

increase fluidity (Jagannadham, 2000; Fong et al., 2001).

Besides the variations in membrane structure and its lipid composition, another

important feature in these cold-adapted microorganisms is the presence of cold-

acclimation proteins (CAPs). These are a set of ~20 proteins which is permanently

synthesized during steady-state growth at low temperatures, but not at milder

# CHAPTER II # 25

temperatures (Hebraud and Potier, 2000; Hebraud et al., 1994; Berger et al., 1996).

Interestingly, some of the few CAPs that have been identified in cold-adapted bacteria

actually acting as cold-shock proteins in mesophiles, such as the RNA chaperone CspA

(Berger et al., 1997). It has been proposed that these CAPs are essential for the

maintenance of both growth and the cell cycle at low temperatures (Hebraud and Potier,

2000) but their function is still poorly understood.

Cold-shock proteins (Csp) are an additional type of adaptation for psychrophilic

organisms in cold regions. These proteins act mainly on the regulation of cellular protein

synthesis, particularly at the level of transcription and the initiation of translation; and

they also act as chaperone by preventing the formation of mRNA secondary structures.

Advantage of these Csp’s is that the synthesis of housekeeping gene products is not

inhibited by cold-shock which is normally occurring in their mesophilic and thermophilic

homologues (Cavicchioli et al., 2000; Berger et al., 1996; Mayr et al., 1996).

Antifreeze proteins, AFPs (are peptides and glycopeptides of various sizes) are

more frequently occurring in fishes, insects, plants, fungi and some microorganisms

which decrease the freezing point of cellular water by binding to ice crystals and prevent

the destruction of cell membranes and the disruption of osmotic balance. Besides

contributing to freeze resistance and freeze tolerance, AFPs also helped to increase

species diversity in some of the harshest and most inhospitable environments (Barrett,

2001; Jia and Davies, 2002). Although antifreeze proteins have been reported in several

eukaryotes, there is no supporting evidence for the occurrence of such glycopeptides in

psychrophilic prokaryotes.

# CHAPTER II # 26

To define the properties of a cold-active enzyme, the effect of temperature on the

activity of psychrophilic and mesophilic enzymes is illustrated in Figure 2.3.

Figure 2.3. Temperature dependence of enzyme activity.

[Psychrophilic enzymes (left curve; blue) are up to tenfold more active at low and

moderate temperatures (up to 20–30°C) than their mesophilic homologues (right curve;

red). Such high activity compensates the cold-induced inhibition of reaction rates.

However, the activity of cold-adapted enzymes is also heat labile, as judged by the

downshift of the apparent optimal temperature of activity (Feller et al., 1992)].

# CHAPTER II # 27

2.4.1. Structural adaptations at the active site

To date, five crystal structures of bacterial psychrophilic enzymes have been

solved by X-ray crystallography. These include the bacterial α-amylase (Aghajari et al.,

1998), malate dehydrogenase (Kim et al., 1999), citrate synthase (Russell, 1998), triose-

phosphate isomerase (Alveraz et al., 1998) and the fish trypsin (Smalas et al., 1994).

Unfortunately, only these limited data of X-ray structures have been compared with

mesophilic or thermophilic homologues and analyzed in detail with regards to the

structural parameters related to cold activity. Nevertheless, this limited set of data has

already provided valuable insights into the molecular basis of cold adaptation (Russell,

2000; Schroder et al., 2000; Leiros et al., 1999; Gianese et al., 2002).

As far as the active site of these psychrophilic enzymes is concerned, all reactive

side chains as well as most side chains pointing towards the catalytic cavity are strictly

conserved. This means that the overall catalytic mechanism and reaction pathway are not

modified in cold-active enzymes. This was demonstrated by the X-ray structure solved at

high resolution of both the cold-active α-amylase and of its closest structural homologue

from pig (Qian et al., 1994; Aghajari et al., 2002).

Two other types of molecular adaptation have been shown by X-ray structures.

Frequently, the catalytic cavity seems to be larger and more accessible to ligands in

psychrophilic enzymes than in mesophilic enzymes (Russell, 1998; Aghajari, 2003). This

is achieved by the deletion of residues in loops bordering the active site, by the distinct

conformation of these loops or by the replacement of bulky side chains with smaller

groups at the entrance to the active site. This improved accessibility is thought not only to

# CHAPTER II # 28

be responsible for the accommodation of the substrate at low energy cost, but also to

facilitate the release and exit of the reaction products. In some enzymes, the electrostatic

potential around the active-site region is also improved, so as to attract the oppositely

charged ligand and channel the substrate towards the catalytic cavity (Russell, 1998;

Aghajari, 2003; Kim et al., 1999; Smalas et al., 1994; Brandsdal et al., 2001). As a result

of the low temperature of unfolding, which prevents aggregation, psychrophilic enzymes

frequently show a high degree of unfolding reversibility that allows analysis of their

stability in the context of equilibrium thermodynamics (Feller et al., 1999). Such an

analysis is illustrated in Figure 2.4 by the stability curves of psychrophilic, mesophilic

and thermophilic proteins.

Figure 2.4. Gibbs free energy of denaturation of a psychrophilic α-amylase from

Antarctic Pseudoalteromonas haloplanktis (blue line), of mesophilic proteins (yellow

lines) and of one thermophilic protein (red line). ΔG is given in specific units (cal mol–1

of residues). By increasing order of melting temperatures (Tm) from left to right: the

# CHAPTER II # 29

psychrophilic Pseudoalteromonas haloplanktis α-amylase, the mesophilic proteins T4

lysozyme, barnase, Rnase T1, Rnase A, spectrin SH3, barstar, phosphocarrier HPr,

chymotrypsin inhibitor ACI2, Protein G IgG binding domain, ovomucoid third domain,

thioredoxin, ubiquitin and the thermophilic protein Sso7d (Feller et al., 1999).

2.4.2. Structural alterations create low stability

All the structural factors that stabilize a protein molecule can be attenuated in

both strength and number in psychrophilic enzymes (Smalas et al., 2000; Russell, 2000).

The number of proline and arginine residues (which restrict backbone rotations and can

form multiple hydrogen bonds and salt bridges, respectively) is reduced, whereas clusters

of glycine residues (which essentially have no side chain) provide localized chain

mobility. All weak interactions (ion pairs, aromatic interactions, hydrogen bonds and

helix dipoles) are less abundant, and non-polar core clusters have a weaker

hydrophobicity, making the protein interior less compact. Frequently, stabilizing

cofactors bind weakly, and loose or relaxed protein extremities seem to favor unzipping.

The protein surface is generally characterized by the disappearance of several solvent

exposed ion pairs, the exposure of a higher proportion of non-polar groups to the

surrounding medium (an entropy-driven destabilizing factor) and an excess of negative

charges that favor interactions with the solvent. These factors are thought to improve the

resilience or the breathing of the external shell. In multimeric enzymes, the cohesion

between monomers is also reduced by decreasing the number and strength of interactions

that are involved in association (Bell, 2002). However, each protein family adopts its own

strategy to decrease stability by using one or a combination of these structural alterations

# CHAPTER II # 30

(Leiros et al., 1999; Schroder-Leiros et al., 2000; Gianese et al., 2001; Gianese et al.,

2002).

2.4.3. Kinetic optimization of cold-active enzymes

Improving the turnover number kcat (catalytic constant; kcat is the maximal

enzyme reaction rate at a given temperature, which is expressed as the number of

substrate molecules that are transformed by one molecule of enzyme per unit of time) is

the main physiological adaptation, because it offsets the inhibitory effect of low

temperatures on reaction rates and therefore provides adequate raw metabolic activity to

the growing organism. However, both kcat and Km is the substrate concentration that is

required to produce 50% of the maximal activity. The specificity constant kcat/Km is

generally a better indication of catalytic efficiency than kcat alone. In principle, cold-

adapted enzymes can optimize the kcat/Km ratio by increasing kcat, decreasing Km or by

changes in both kcat and Km (Hoyoux et al., 2004).

2.4.4. Structure of Antarctic psychrophile Alteromonas haloplanctis α-amylase

The cold-active α-amylase from the Antarctic psychrophile, Alteromonas

haloplanctis has been studied extensively (Feller et al., 1992). The enzyme has a

molecular mass of 49 kDa with few salt bridges, aromatic interactions, small hydrophobic

cluster, few arginine residues and weak stabilization of helix dipoles. It is the first cold-

active α-amylase, which has been successfully crystallized and the 3-D structure resolved

at 1.85 Å (Aghajari et al., 1996). Also, this α-amylase was successfully expressed in

mesophilic host E. coli preserving genuine properties of a psychrophilic enzyme (Feller

et al., 1998).

# CHAPTER II # 31

The overall fold of A. haloplanctis α-amylase is very similar to those reported for

mesophilic α-amylases (Aghajari et al., 1998). Three characteristic domains as well as

ion-binding sites are found: domain A (residues 1-86 and 147-356); the central N-

terminal domain with a (β/α)8-barrel fold; a minor domain B (residues 87-146, an

insertion between α3 and β3) that protrudes from domain A and comprises a loop

structure, short β-strands and a short α-helix; and the C-terminal domain C consisting of

eight β-strands that form a Greek-key motif (the number of β-strands in other α-amylases

varies from five in barley to ten in human salivary). The largest variations in primary

structures between these enzymes from different species have been found in domain C

(Jespersen et al., 1993; MacGregor, 1988) and domain B (Janecek et al., 1997), but it

should also be mentioned that, throughout the α-amylase family, only eight residues are

invariant in the (β/α)8 barrel (Svendsen, 1994). These include seven residues at the active

site and a structurally important glycine. As in the mammalian α-amylases, binding sites

for calcium and chloride ions have been located in the structure of A. haloplanctis α-

amylase (Figure 2.5 a,b) (Aghajari et al., 1998).

# CHAPTER II # 32

Figure 2.5 (a) Figure 2.5 (b)

Figure 2.5(a). The three dimensional structure of amylase from Aspergillus sp.

(Matsuura et al., 1984)

Figure 2.5(b). Overall structure of psychrophilic AHA with residues (Gln 58 and Ala 99)

that replace the cysteines involved in the disulfide bridge between domains A and B in

MAA highlighted (in the upper right part of the figure). Domain A is colored in cyan,

domain B in pink and domain C in blue. The active site with the three catalytic amino

acids is shown, as are the calcium ion (yellow sphere) and chloride ion (green sphere)

(Aghajari et al., 1998). Several laboratories are now applying the techniques of gene

cloning and over expression (Table 2.2), followed by enzyme purification and

crystallization. Moreover draft genome sequences have been produced from two cold-

adapted Archaea: Methanogenium frigidum and Methanococcoides burtonii (Saunders et

al., 2003).

# CHAPTER II # 33

Table 2.2. Enzymes from psychrophilic bacteria, which have been cloned and

sequenced

Enzyme Bacterium Reference

Uracil-DNA glycosylase Psychrophilic strain

BMTU3346

Jaeger et al. (2000)

β-galactosidase Carnobacterium piscicola BA

Coombs and

Brenchley (1999)

Subtilisin (proteinase) Bacillus sp. TA39 Davail et al. (1994)

Lactate dehydrogenase Bacillus psychrosacchardyticus Vckovski et al.

(1990)

Esterase Pseudomonas sp. LS107d2 Mckay et al. (1992)

Isocitrate dehydrogenase Vibrio sp. ABE-1 Ishii et al. (1993)

β-Galactosidase Arthrobacter sp. B7 Gutshall et al.

(1995)

3-Isopropylmalate

dehydrogenase

Vibrio sp. 15 Wallon et al. (1997)

Alcohol dehydrogenase Moraxella sp. TAE 123 Tsigos et al. (1998)

Citrate synthase Arthrobacter sp. DS2-3R Gerike et al. (1997)

Malate dehydrogenase Aquaspirillium articum Kim et al. (1999)

Elongation factor 2 Methanococcus burtonii Thomas and

Cavicchioli (1998)

α-Amylase Alteromonas haloplanctis Feller et al. (1992)

# CHAPTER II # 34

Lipase* Moraxella sp. TA144

Psychrobacler immobilis

Pseudomonas sp. B11-1

Feller et al. (1991)

Arpigny et al. (1993)

Choo et al. (1998)

Triosephosphate

isomerase

Moraxella sp. TA137 Rentier et al. (1993)

β -Lactamase Psychrobacter immobilis Feller et al. (1996)

* Three isozymes were cloned

2.5. OPTIMIZATION PRODUCTION OF COLD-ACTIVE ALPHA-

AMYLASE ENZYME

Cold-active amylases are mostly extracellular and are highly influenced by

nutritional and physicochemical factors such as temperature, agitation, pH, nitrogen

source, carbon source, inducers, inorganic sources and dissolved oxygen. To meet the

demand of industries, low-cost medium is required for the production of α-amylase. Both

solid state fermentation (SSF) and submerged fermentation (SmF) could be used for the

production of α-amylases, although traditionally these have been obtained from

submerged cultures because of ease of handling and greater control of environmental

factors such as temperature and pH. SSF has been used for long to convert moist

agricultural polymeric substrates such as wheat, rice, soy, cassava, etc. (Table 2.3) into

fermented food products including industrial enzymes (Pandey et al., 1995). Solid state

fermentation is generally defined as the growth of microorganisms on moist solid

substrates with negligible free water (Selvakumar et al., 1998). The solid substrate may

# CHAPTER II # 35

provide only support or both support and nutrition. SSF constitutes an interesting

alternative since the metabolites so produced are concentrated and purification

procedures are less costly (Pandey et al., 1995; Benjamin and Pandey, 1997; Pandey,

1991). SSF is preferred to SmF because of simple technique, low capital investment,

lower levels of catabolite repression and end-product inhibition, low waste water output,

better product recovery, and high quality production (Zadrazil and Puniya, 1995). Among

the different substrates used for SSF, wheat bran has been reported to produce promising

results (Duenas et al., 1995; Muniswaran et al., 1994).

Table 2.3. Agro substrates used for cold-active α-amylase production

Substrate Organism Activity Reference

Wheat bran Paenibacillus

amylolyticus

275.95

(U/g/min)

Haq et al. (2012)

Wheat bran Aspergillus flavus, F2Mbb 119.1 (U/mg/ml) Sidkey et al. (2010)

Brown Lentils Bacillus sp. B4M bl 89 (U/mg/ml) Sidkey et al. (2010)

Maize meal Penicillium sp. 137 (U/g) Gouda and Elbahloul (2008)

Wheat bran:

Groundnut oil

cake (1:1)

Bacillus

amyloliquefaciens ATCC

23842

62470 (U/g) Gangadharan et al. (2006)

Wheat bran Bacillus sp. PS-7 464000 (U/g) Sodhi et al. (2005)

Rice bran Bacillus sp. PS-7 145000 (U/g) Sodhi et al. (2005)

# CHAPTER II # 36

2.5.1. Temperature

The influence of temperature on amylase production is related to the growth of

the organism. Hence, the optimum temperature depends on whether the culture is

mesophilic, thermophilic or psychrophilic. Among the fungi, most amylase production

studies have been done with mesophilic fungi within the temperature range of 25–37°C

(Francis et al., 2003; Ramachandran et al., 2004). Bacterial α-amylases are produced at a

Corn bran Bacillus sp. PS-7 97600 (U/g) Sodhi et al. (2005)

Coconut oil

cake

A. oryzae 3388 (U/g) Ramachandran et al. (2004)

Spent brewing

grain

A. oryzae NRRL 6270 6583 (U/g) Francis et al. (2003)

Rice husk B. subtilis 21 760 (U/g) Baysal et al. (2003)

Amaranthus

grains

Aspergillus flavus 1920 (U/g) Vishwanathan & Surlikar

(2001)

Maize bran B. coagulans 22956 (U/g) Babu and Satyanarayana

(1995)

Mustard oil

cake

B. coagulans 5953 (U/g) Babu and Satyanarayana

(1995)

Gram bran B. coagulans 8984 (U/g) Babu and Satyanarayana

(1995)

# CHAPTER II # 37

much wider range of temperature. B. amyloliquefaciens, B. subtilis, B. licheniformis and

B. stearothermophilus are among the most commonly used Bacillus sp. reported to

produce α-amylase at temperatures 37-60°C (Syu and Chen, 1997; Mishra et al., 2005;

Mendu et al., 2005; Mielenz, 1983). A cold-active α-amylase from Antarctic

psychrophile Alteromonas haloplanktis was reported to exhibit maximum α-amylase

production at 4°C (Feller et al., 1998).

2.5.2. pH

pH is also one of the important factors that determine the growth and morphology

of microorganisms as they are sensitive to the concentration of hydrogen ions present in

the medium. Earlier studies have revealed that fungi required slightly acidic pH and

bacteria required neutral pH for optimum growth. pH is known to affect the synthesis and

secretion of α-amylase just like its stability (Fogarty, 1983). Bacterial cultures such as B.

subtilis, B. licheniformis and B. amyloliquefaciens required an initial pH of 7.0 (Syu and

Chen, 1997; Tanyildizi et al., 2005; Haq et al., 2005). Rhodothermus marinus was

reported to yield good enzyme levels at initial pH range 7.5 to 8.0 (Gomes et al., 2003).

Hyperthermophilic archae such as Pyrococcus furiosus, P. woesei and Thermococcus

profundus yielded optimum α-amylase at pH 5.0 (Vieille and Zeikus, 2001).

Thermophilic anaerobic bacteria Clostridium thermosulfurogenes gave maximum titres of

α-amylase at pH 7.0 (Swamy and Seenayya, 1996). A list of various cold active α-

amylase producing psychrophillic and psychrotrophic bacteria and their production

parameters are presented in Table 2.4.

# CHAPTER II # 38

Table 2.4. Production parameters for cold-active α-amylase

Microbes Incubation

period (hr)

Optimum

temp.(°C)

Optimum

pH

Reference

Bacillus cereus GA6 96 20 10.0 Kuddus et al. (2012)

Microbacterium

foliorum GA2

120 20 9.0 Roohi et al. (2011)

Wangia sp. C52 NM 20 7.18 Liu et al. (2011a)

Pseudoalteromonas

arctica GS230

24 20 8.0 Lu et al. (2010)

Nocardiopsis aegyptia 48 25 5.0 Abou-Elela et al.

(2009)

Streptomyces sp. 1 20 NM Cotarlet et al. (2009)

Micrococcus

antarcticus

64 12 8.0 Fan et al. (2009)

Lactobacillus

plantarum

36 35 7.0 Smita et al. (2008)

Penicillium sp. 48 30 4.0 Gouda and Elbahloul

(2008)

Nocardiopsis sp. 7326 NM 20 8.0 Zhang and Zeng (2008)

P. amylolyticus 0.5 35 6.0 Xiaohong et al. (2007)

Arthrobacter

psychrolactophilus

NM 22 NM Michael et al. (2005)

# CHAPTER II # 39

NM: not mentioned

2.5.3. Carbon and Nitrogen sources

These are necessary for the growth and metabolism of organisms. Various carbon

sources are tried to optimize the maximum production of cold-active α-amylase for

different bacterial species. For enhanced growth and metabolism of organisms, nitrogen

also play very important role just like carbon. A large variety of carbon and nitrogen

sources for bacterial species are available which give rise to maximum production of

cold-active α- amylase (Table 2.5 and 2.6).

Aspergillus oryzae NM 4 NM Akpan & Adelaja

(2004)

Penicillum sp. FS

010441

NM 15 6.0 Zhang et al. (2002)

Aspergillus ochraceus 48 30 5 Nahas and Mirela

(2002)

Flavobacterium

balustinum A201

NM 30 NM Morita et al. (1997)

Bacillus sp. A-001 NM 35 7.5 Lealem and Gashe

(1994)

# CHAPTER II # 40

Table 2.5. Carbon sources used for maximum cold-adapted α-amylase production

Bacterial species Best C-source Reference

Bacillus cereus GA6 Glycerol Kuddus et al. (2012)

Microbacterium foliorum GA2 Lactose Roohi et al. (2011)

Pseudoalteromonas arctica GS230 Soluble starch Lu et al. (2010)

Bacillus sp. strain TSCVKK Dextrin Kiran and Chandra (2008)

Bacillus sp.1 Starch Varalakshmi et al. (2008)

Nocardiopsis aegyptia Starch Abou-Elela et al. (2009)

B. subtilis IMG22 Starch and Glycerol Sodhi et al. (2005)

Bacillus sp. PS-7 Starch and Glycerol Tanyildizi et al. (2005)

Bacillus sp. I-3 Starch and Glycerol Goyal et al. (2005)

Bacillus sp. Lactose Hamilton et al. (1999)

B. stearothermophilus Soluble starch Srivastava & Baruah (1986)

B. licheniformis Galactose, glycogen and inulin Chandra et al. (1980)

# CHAPTER II # 41

Table 2.6. Nitrogen sources used for maximum cold-active α-amylase production

Bacterial species Best N-source Reference

Bacillus cereus GA6 Ammonium acetate Kuddus et al. (2012)

Microbacterium foliorum GA2 Yeast extract Roohi et al. (2011)

Wangia sp. C52 Yeast extract Liu et al. (2011a)

Pseudoalteromonas arctica

GS230

Beef extract Lu et al. (2010)

Pseudoalteromonas

haloplanktis

Glutamic acid Wilmes et al. (2010)

Nocardiopsis aegyptia Potassium nitrate Abou-Elela et al. (2009)

Bacillus sp. strain TSCVKK Yeast extract Kiran and Chandra (2008)

Bacillus sp. I-3 Soya bean meal Sodhi et al. (2005) and

Francis et al. (2003)

B. licheniformis SPT 278 Peptone Aiyer (2004)

A. oryzae A1560 Casein hydrolysate Pederson and Nielson

(2000)

B. amylolyticus Peptone and yeast extract Dettori et al. (1992)

# CHAPTER II # 42

2.5.4. Other fermentative conditions

Surfactants in the fermentation medium are known to increase the production of

extracellular amylase enzymes by increasing cell membrane permeability. Some common

surfactants are Tween 80, polyethylene glycols, Cholic acid which is used in different

concentrations. Supplementation of salts of certain metal ions provided good growth of

microorganisms and thereby better enzyme production (as most α-amylases are known to

be metalloenzymes). Some frequently used metal ions are CaCl2, NaCl, LiSO4, MgSO4,

FeCl3, Mn2+, Zn2+, etc. In SSF system some additional fermentative conditions play very

vital role viz selection of a suitable substrate and microorganism; pre-treatment of the

substrate; particle size (inter-particle space and surface area) of the substrate; water

content; relative humidity; type and size of the inoculum; removal of metabolic heat;

period of cultivation; maintenance of uniformity in the environment of SSF system, and

the gaseous atmosphere, i.e. oxygen consumption rate and carbon dioxide evolution rate.

2.6. COLD-ACTIVE AMYLASES: PURIFICATION AND

CHARACTERIRIZATION

Industrial enzymes produced in bulk generally require little downstream

processing and hence are relatively crude preparations. The commercial use of α-amylase

generally does not require purification of the enzyme, but enzyme applications in

pharmaceutical and clinical sectors require high purity amylases. The enzyme in purified

form is also a prerequisite in studies of structure-function relationships and biochemical

# CHAPTER II # 43

properties. The purification of α-amylases from microbial sources in most cases has

involved classical purification methods. These methods involve separation of the culture

from the fermentation broth, selective concentration by precipitation using ammonium

sulfate or organic solvents such as chilled acetone. The crude enzyme is then subjected to

chromatography, usually affinity, ion exchange and/or gel filtration. The need for large-

scale cost effective purification of proteins has resulted in evolution of techniques that

provide fast, efficient and economical protocols in fewer processing steps (Amritkar et

al., 2004). Purification techniques that produce homogeneous preparation of α-amylases

in a single step are given in Table 2.7.

Table 2.7. Methods of one-step purification of α-amylases

Method

Adsorbent Yield (%)

Purification fold

Reference

Affinity

adsorption

chromatography

β-cyclodextrin-

iminodiacetic acid-Cu2+

95 - Liao and Syu (2005)

Expanded bed

chromatography

Alginic acid-cellulose cell

beads

69 51 Amritkar et al. (2004)

High speed

counter current

chromatography

PEG4000-aqueous two-

phase system

73.1 - Zhi et al. (2005)

Magnetic affinity

adsorption

Magnetic alginate

microparticles

88 9 Safarikova et al.

(2003)

Substitute affinity

method

Insoluble corn starch at 4°C 78 163 Najafi and Kembhavi

(2005)

# CHAPTER II # 44

Ultrafiltration α-Cyclodextrin coupled with

Sepharose 6B (pH 7.0)

78 140 Iefuji et al. (1996)

Gel filtration

chromatography

Sephacryl-S200 HR 17 9 Marco et al. (1996)

Ion exchange

chromatography

DEAE-Cellulose DE52 (pH

5.3)

66 33 Morgan and Priest

(1981)

Ultrafiltration Cellulose 35 20 Giraud et al. (1993)

Mono Q

adsorption

chromatography

Sephadex G-25 (pH 7.5 50 6.9 Freer (1993)

Drum humidifier

chromatography

25% (NH4)2SO4, 70%

acetone

- 1.036 Robyt and Ackerman

(1971)

The pH optima of α-amylases vary from 2.0 to 12.0 (Vihinen and Mantsala, 1989).

Alpha-amylases from most bacteria and fungi have pH optima in the acidic to neutral

range (Pandey et al., 2000). Alpha-amylases from Alicyclobacillus acidocaldarius

showed an acidic pH optima of 3.0 (Schwermann et al., 1994), in contrast to the alkaline

amylase with optima of pH 9.0-10.5 as reported from an alkalophilic Bacillus sp. (Saito,

1973; Krishnan and Chandra, 1983; Lee et al., 1994; Shinke et al., 1996). Extremely

alkaliphilic α-amylase with pH optima of 11.0-12.0 has been reported from Bacillus sp.

GM8901 (Kim et al., 1995). In some cases, the pH optimum was observed to be

dependent upon temperature as in the case of Bacillus stearothermophilus DONK BS-1

(Ogasahara et al., 1970) and on calcium as in the case of B. stearothermophilus (Pfueller

and Elliott, 1969). Alpha-amylases are generally stable over a wide range of pH from 4.0

to 11.0 (Fogarty and Kelly, 1979; Vihinen and Mantsala, 1989; Hamilton et al., 1999a;

# CHAPTER II # 45

Hamilton et al., 1999b; Saito, 1973; Khoo et al., 1994), however, α-amylases with

stability in a narrow range have also been reported (Coronado et al., 2000; Krishnan and

Chandra, 1983; Robyt and Ackerman, 1971).

The optimal activity temperature of Nocardiopsis sp. 7326 amylase was 35°C,

and the enzyme was stable between pH 5.0 and 10.0 with maximal activity at pH 8.0

(Zhang and Zeng, 2008). The activity of α-amylase from Streptococcus bovis JB1 was

optimal at pH 5.0 to 6.0. The enzyme was relatively stable at temperatures below 50°C

(Freer 1993). Ueda and his coworkers (2008) purified and characterized the novel cold-

adapted α-amylase (Amy I and Amy II) which are most active at pH 5.5 and stable at pH

7.0-9.0. Both Amy I and Amy II exhibited activities at 10°C. A cold-active α-amylase

from Antarctic psychrophile Alteromonas haloplanktis was reported to exhibit maximum

α-amylase production at 4°C (Feller et al. 1998).

The optimal temperature and pH for the purified amylase from Micrococcus

antarcticus were 30°C and 6.0, respectively (Fan et al. 2009). It still showed high activity

at low temperature 10-15°C. It was sensitive to high temperature but was stable at pH

6.0-10.0 with at least 70% activity remained. Liu et al. (2011b) reported a 58 kDa cold-

active amylase from marine Wangia sp. C52 having an optimum pH and temperature of

6.0 and 30°C, also in the presence of Ca2+ and Co2+, the enzyme activity was stimulated

while Cu2+, Hg2+, Mn2+, Zn2+, Fe3+, Al3+, EDTA, EGTA and SDS reduced the activity.

Km and Vmax values of the purified enzyme for soluble starch were 2.08±0.3 mg/ml and

1.26±0.02 mg/ml/min, respectively. Nouadri et al. (2010) purified 60.5 kDa α-amylase

from Penicillium camemberti PL21 with specific activity of 154.2 units/ml/mg protein

with 38.5 folds purification. The optimum substrate concentration for soluble starch was

# CHAPTER II # 46

1% (w/v) at the optimum temperature of 30ºC. The purified α-amylase enzyme had a

maximum activity at pH 6.0 and the Km value for soluble starch was found to be 0.92

mg/ml.

Alpha-amylase from Alteromonas sp. TAC 240B collected form Antarctic sea water

showed a molecular mass of about 50 kDa and a pI of 5.2. The enzyme is stable from pH

7.5 to 9.0 and has a maximal activity at pH 7.5. This psychrophilic alpha-amylase

requires both Cl- and Ca2+ for its amylolytic activity. Br- is also quite efficient as an

allosteric effector. The comparison of the amino acid composition with those of other

alpha-amylases from various organisms shows that the cold-active alpha-amylase has the

lowest content of Arg and Pro residues (Chessa et al., 1999). Psychrophilic amylase

produced by Penicillum sp. FS010441 isolated from sea mud of the Yellow Sea and the

East China Sea. The optimum growth temperature and pH for this α-amylase enzyme is

15°C and 6.0, respectively and even at 0°C, the enzyme shows some activities (Zhang et

al., 2002). A cold-active amylase producing P. amylolyticus strain showed an optimum

temperature of 35°C and pH 6.0 when incubated for 30 hours. This amylase was not

stable when the temperature was above 40°C (Xiaohong et al., 2007). Alpha-amylases

from B. subtilis, B. amyloliquefaciens I and B. amyloliquefaciens II were strongly

inhibited by Zn2+, Ag+, Cu2+ and Fe2+ (Elif and Velittin, 2000). However, the activity of

cold-adapted Nocardiopsis sp. 7326 amylase was not affected by Zn2+, Ni2+ and Fe2+, but

was even activated by Cd2+ and Cu2+ (Zhang and Zeng, 2008).

# CHAPTER II # 47

2.7. BIOTECHNOLOGICAL ASPECTS OF COLD ACTIVE ALPHA-

AMYLASE

An emerging area of research in the field of enzymology is to develop radically

different and novel biocatalysts through various molecular approaches including

recombinant DNA technology, protein engineering, directed evolution and the

metagenomic approach. As a whole, amylase biotechnology has just reached the end of

lag phase and the beginning of the exponential phase: it demands extension in terms of

both quality and quantity. Qualitative improvements in restructuring amylase gene and its

protein can be achieved by employing already established recombinant DNA technology

and protein engineering. Quantitative enhancement needs strain improvement, especially

through site-directed mutagenesis and standardizing the nutrient medium for the

overproduction of cold-active α-amylases.

2.7.1. Gene cloning

To date, a very few number of cold-active α-amylase genes were isolated and the

related studies have been carried out. Early successes in the production of heterologous

proteins were achieved using Escherichia coli as host and various kinds of proteins were

expressed in E. coli. However, expression of eukaryotic proteins in E. coli became very

difficult due to formation of inclusion bodies, protein misfolding and safety issues. Other

expression systems were developed among yeasts, fungi, plants and animals. Cloning and

expression of the cold-active amylase gene from Alteromonas haloplanctis has been

reported (Feller et al., 1992, 1998).

# CHAPTER II # 48

2.7.2. Protein engineering

Psychrophilic organisms and their enzymes have, in recent years, increasingly

attracted the attention of the scientific community due to their peculiar properties that

render them particularly useful in investigating the possible relationship existing between

stability, flexibility and specific activity and as valuable tools for biotechnological

purposes. Although α-amylase carry significant commercial value, biotechnologically

produced or engineered cold-active α-amylases may represent the focus of industrial

interest in future. Cold-active α-amylases could generate avenues for industrial

applications, once their specific properties are improved through enzyme engineering.

Determination of three dimensional structures of more cold-active amylases would allow

the detailed analysis of protein adaptation to temperatures at molecular level. This may

include increased thermolabile nature and/or catalytic activity at low temperatures, or the

modification of pH profiles. Cold-active α-amylases from microorganisms retaining high

catalytic activity at low temperatures are successfully produced using site directed

mutagenesis and directed evolution. Alpha-amylase from the Antarctic psychrophile

Alteromonas haloplanctis is synthesized at 0±2°C by the wild strain. This heat-labile α-

amylase folds correctly when over expressed in Escherichia coli, providing the culture

temperature is sufficiently low to avoid irreversible denaturation (Feller et al., 1998). The

thermal stability of the cold-active α-amylase (AHA) secreted by the Antarctic bacterium

Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular

dichroism and differential scanning calorimetry. It was found that this heat-labile enzyme

is the largest known multidomain protein exhibiting a reversible two-state unfolding

(Feller et al., 1998).

# CHAPTER II # 49

2.8. BIOTECHNOLOGICAL POTENTIAL OF PSYCHRO-

TOLERANT ALPHA-AMYLASES

Amylases are among the most important hydrolytic enzymes for all starch based

industries and the commercialization of amylases is oldest with first use in 1984, as a

pharmaceutical aid for the treatment of digestive disorders. In the present day scenario,

amylases find application in all the industrial processes such as in food, detergents,

textiles and in pulp-paper industry, for the hydrolysis of starch, etc. In this light,

microbial amylases have completely replaced chemical hydrolysis in the starch

processing industry. They can also be of potential use in the pharmaceutical and fine

chemical industries. Today, amylases have the major world market share of enzymes

(Aehle and Misset, 1999). Several different amylase preparations are available with

various enzyme manufacturers for specific use in varied industries.

2.8.1. Detergent applications

Dirt and stains on clothes comes in many forms and includes proteins, starches

and lipids. In addition, clothes that have been starched must be freed of the starch. Using

detergents in water at high temperatures and with vigorous mixing, it is possible to

remove most types of dirt and stains but the cost of heating the water is high and lengthy

mixing or beating will shorten the life of clothing and other materials. The use of

enzymes allows lower temperatures to be employed and shorter periods of agitation are

needed, often after a preliminary period of soaking. Psychrophilic enzymes can be very

useful for domestic processes as amylases; especially cold-active alkaline α-amylases can

be used in detergents since washing clothes at low temperatures protect the colors of

# CHAPTER II # 50

fabrics and reduce energy consumption. In general, enzymatic detergents remove stains

from clothes far more effectively than non-enzymatic detergents.

2.8.2. Bread and baking industry and as an antistaling agent

The baking industry has made use of these enzymes for hundreds of years to

manufacture a wide variety of high quality products. For decades, enzymes such as malt

and microbial α-amylases have been widely used in the baking industry (Hamer et al.,

1995; Si, 1999). These enzymes were used in bread and rolls to give these products a

higher volume, better color and a softer crumb. It is the malt preparation that has led the

way and opened the opportunities for many enzymes to be used commercially in baking.

Today, many enzyme preparations such as proteases, lipases, xylanases, pullulanases,

cellulases, glucose oxidases, lipoxygenases etc. are being used in the bread industry for

varied purposes (Kulp, 1993; Prieto et al., 1995; Si, 1999; Monfort et al., 1996), but none

had been able to replace α-amylases.

Till date, α-amylases used in baking have been cereal enzymes from barley malt

and microbial enzymes from fungi and bacteria (Hebeda et al., 1990, 1991). Fungal α-

amylases have been permitted as bread additives since 1955 in the US and in 1963 in UK

after confirmation of their GRAS status (Pritchard, 1992). Presently they are used all over

the world to different extents. Supplementation of flour with exogenous fungal α-amylase

having higher activities is common in the present day modern and continuous baking

process (Pritchard, 1992). Cold-active α-amylase supplementation in flour not only

enhances the rate of fermentation but also consume less amount of energy at industrial

scale and moreover reduces the viscosity of dough, resulting in improvements in the

# CHAPTER II # 51

volume and texture of the product, also generates additional sugar in the dough, which

improves the taste, crust color and toasting qualities of the bread (Van and Hille, 1992).

One of the new applications of α-amylase in the industry has been in retarding the

staling of baked products, which reduces the shelf life of these products. Upon storage the

crumb becomes dry and firm, the crust loses its crispness and the flavor of the bread

deteriorates. All these undesirable changes in the bread are together known as staling.

The importance of retrogradation of starch fraction in bread staling has been emphasized

(Kulp and Ponte, 1991). A loss of more than US $1 billion is incurred in USA alone

every year due to the staling of bread. Conventionally various additives are used to

prevent staling and improve the texture and flavor of baked products. Additives include

chemicals, small sugars, enzymes/their combinations, milk powder, emulsifiers,

monoglycerides/diglycerides, sugar esters, lecithin, etc; granulated fat, anti-oxidant

(ascorbic acid or potassium borate), sugars/salts (Spendler and Jorgensen, 1997).

Recently emphasis has been given to the use of enzymes in dough improvement as anti-

staling agents, e.g. α-amylase (Cole, 1982), branching enzymes (Okada et al., 1984) and

debranching enzymes (Carroll et al., 1987), maltogenic amylases (Oleson, 1991), β-

amylases (Wursch and Gumy, 1994) amyloglucosidases (Vidal and Gerrity, 1979).

Pullulanases and α-amylase combination are used for efficient antistaling property

(Carroll et al., 1987). However, a slight excess of α-amylases was also used which is

undesirable as it causes stickiness in bread (Oleson, 1991). Therefore, a recent trend is to

use intermediate temperature stable (ITS) α-amylases or better cold-active α-amylase

(Kulp, 1993; Hebeda et al., 1990 and 1991; Ahuja et al., 1998). They are active after

starch gelatinization and become inactive much before the completion of the baking

# CHAPTER II # 52

process. Further, the dextrin with 4-9 degree of polymerization produced by these shows

the anti-staling properties. Although a wide variety of microbial α-amylases is known, α-

amylase with ‘ITS’ property has been reported from only a few microorganisms (Prieto et

al., 1995; Monfort et al., 1996; Kraus and Hebeda, 1993; Gigras et al., 2002).

2.8.3. Manufacturing of maltose

Maltose is a disaccharide made up of glucose units. It is the main component of

malt-sugar syrup (Matsumoto et al., 1982). Maltose is commonly used as sweetener and

also as intravenous sugar supplement. It has a great value in food industries since it is

non-hygroscopic and does not easily crystallize. For the manufacturing of variety of

grade of maltose, starch (potato, sweet potato, corn and cassava starches are frequently

used) is hydrolyzed by the use of alpha-amylase. To reduce the time and energy

consumption and to increase the efficiency, cold-active α-amylase is better option. For

production of medical grade, the concentration of starch slurry is adjusted to be 10-20%

maltose and for food grade, 20-40% maltose is used.

2.8.4. Manufacturing of high fructose containing syrups

High fructose containing syrups 42 F (Fructose content, 42%) is prepared by

enzymatic isomerization of glucose with glucose isomerase. The starch is first converted

to glucose by α-amylase enzymatic liquefaction and saccharification.

# CHAPTER II # 53

2.8.5. Manufacturing of maltotetraose syrup

Maltotetraose syrup (G4 syrup) is produced by breaking of starch into

maltotetraose by the action of α-amylase enzyme. The sweetness of the syrup is as low as

20% of sucrose. Therefore in foods, G4 syrup can be successfully used in place of

sucrose which reduces the sweetness without altering their inherent taste and flavor. It

has high moisture retention power which maintains integrity of starch particles and

retains suitable moisture in foods. G4 syrup improves the food texture because of its high

viscosity than sucrose. It further lowers down the freezing point of water than sucrose or

high fructose syrup, so can be used to control the freezing points of frozen foods. G4

syrup imparts gloss and can be used in industry such as a paper sizer.

2.8.6. Manufacturing of high molecular weight branched dextrins

High molecular weight branched dextrins are used as extender for production of

powdery foods and a glazing agent for rice cakes. These are produced by the action of α-

amylase on corn starch. Degree of hydrolysis depends on the type of starch and the

physical properties desired. Branched dextrins can be collected as powder after

chromatography and spray drying.

2.8.7. Removal of starch sizer from textile (desizing)

Modern production processes for textiles introduce a considerable strain on the

warp during weaving. The yarn must, therefore, be prevented from breaking. For this

purpose a removable protective layer is applied to the threads. The materials that are used

for this size layer are quite different. Starch is a very attractive size, because it is cheap,

easily available in most regions of the world, and it can be removed quite easily. Good

# CHAPTER II # 54

desizing of starch sized textiles is achieved by the application of α-amylases, which

selectively remove the size and do not attack the fibers. It also randomly cleaves the

starch into dextrins that are water soluble and can be removed by washing. It also

prevents the loss of string by friction, cutting and generation of static electricity on the

string by giving softness to the surface of string due to laid down warp. The use of α-

amylases in warp sizing of textile fibres for manufacturing fibers with great strength has

been reported (Hendriksen et al., 1999).

2.8.8. Direct fermentation of starch to ethanol

For large-scale processing, the bioconversion of biomass resources, especially

starchy materials, to ethanol, is very useful because it can be used as a bio-fuel and as the

starting material for various chemicals. However, in the present scenario its cost of

production is very high because of three main reasons; ethanol production from starchy

materials via fermentation consists of two or three steps, large quantity of α-amylase is

needed and starchy materials need to be cooked at a high temperature (140° to 180°C).

Now methods are developed in which cost of production can be minimized; by

fermenting starch to ethanol in one step using co-cultures of two different strains and by

using low temperature-cooking fermentation systems (that succeeded in reducing energy

consumption by approximately 50%), but it is still necessary to add large amounts of

amylolytic enzymes to hydrolyze the starchy materials to glucose (Matsumoto et al.,

1982, 1985).

# CHAPTER II # 55

2.8.9. Treatment of starch containing waste water

Food processing wastewater offers a unique challenge to any treatment system.

Often containing multiple types of contaminants that pose serious threats to the ability of

a standard sewage treatment facility, causes pollution problem also. Biotechnological

treatment of food processing starch waste water by the action of α-amylase can produce

valuable products such as microbial biomass protein and also purifies the effluent at low

temperature (Aiyer, 2005).

2.8.10. Starch liquefaction and saccharification

The major market for α-amylases lies in the production of starch hydrolysates

such as glucose and fructose. Starch is converted into high fructose corn syrups (HFCS).

Because of their high sweetening property, these are used in huge quantities in the

beverage industry as sweeteners for soft drinks. The process requires the use of α-

amylase for starch liquefaction. The use of α-amyalse in starch liquefaction is well

established and has been extensively reviewed (Marrel et al., 2002; Pandey et al., 2000).

2.8.11. Paper and pulp industry

The use of α-amylase for the production of low viscosity, high molecular weight

starch for coating of paper is reported (Bruinenberg et al., 1996). The use of amylases in

the pulp and paper industry is in the modification of starches for coated paper. As for

textiles, sizing of paper is performed to protect the paper against mechanical damage

during processing. It also improves the quality of the finished paper. The size enhances

the stiffness and strength in paper. It also improves the erasibility and is a good coating

# CHAPTER II # 56

for the paper. Starch is also a good sizing agent for the finishing of paper. Starch is added

to the paper in the size press and paper picks up the starch by passing through two rollers

that transfer the starch slurry. A constant viscosity of the starch is required for

reproducible results at this stage. The mill also has the flexibility of varying the starch

viscosity for different paper grades. The viscosity of the natural starch is too high for

paper sizing and is adjusted by partially degrading the polymer with α-amylases in a

batch or continuous processes. The conditions depend upon the source of starch and the

type of α-amylase at different temperatures (Tolan, 1996).

2.8.12. Analysis in medicinal and clinical chemistry

With the advent of new frontiers in biotechnology, the spectrum of amylase

applications has expanded into many other fields, such as clinical, medicinal and

analytical chemistry. There are several processes in the medicinal and clinical areas that

involve the application of amylases. The application of a liquid stable reagent, based on

α-amylase for the Ciba Corning Express clinical chemistry system has been described

(Becks et al., 1995). A process for the detection of higher oligosaccharides, which

involved the application of amylase, was also developed (Giri et al., 1990). This method

was claimed to be more efficient than the silver nitrate test. Biosensors with an

electrolyte isolator semiconductor capacitor (EIS-CAP) transducer for process monitoring

were also developed (Menzal et al., 1998).

# CHAPTER II # 57

2.8.13. Other applications

In food industry cold-active α-amylase can be used for the reduction of haze

formation in juices. Psychrophilic microorganisms have also been proposed for the

bioremediation of polluted soils and waste waters during the winter in temperate

countries, when the degradative capacity of the endogenous microflora is impaired by

low temperatures. Glycosidases are often used in the baking industry, but can retain

residual activity after cooking that alters the structure of the final product during storage;

this can be avoided by the use of psychrophilic glycosidases. An important achievement

in the field has been the construction of a host-vector system that allows the over

expression of genes in psychrophilic bacteria (Tutino, 2001): expression at low

temperatures prevents the formation of inclusion bodies and protects heat-sensitive gene

products. Using enzymes having highest activity below 20°C in food processing, limits

the growth of other contaminating microorganisms, shorten the process times, and avoid

designing expensive heating steps. Cold-active α-amylases could be used in the brewing

industry to speed the mashing phase at low temperatures. The expansion of experimental

models to include plants, nematodes, some cold blooded animals such as fish and frogs,

and other microorganisms may create the need for enzymes with higher activities at

lower temperatures. In addition, reporter genes making cold-active enzymes would be

valuable additions to the arsenal of molecular tools.

# CHAPTER II # 58

2.9. Future prospects

The emerging picture of cold adaptation indicates that to catalyze biochemical

reactions at low temperatures, psychrophilic enzymes improve the flexibility of the

structural elements that are involved in the catalytic cycle, thereby resulting in an activity

that is markedly heat labile. The high flexibility of cold-active enzymes is strongly

supported by experiments using dynamic quenching of fluorescence that show increased

permeability to a small quencher molecule (D’Amico et al., 2003; Collins et al., 2003;

Georlette et al., 2003). Obviously, the next challenge will be to define this flexibility in

terms of the type, amplitude and timescale of molecular motions. In fact, the intricate

relationships between these factors and activity or stability are still poorly understood,

not only in psychrophilic enzymes but also in proteins and enzymes in general (Jaenicke,

2000; Hernandez et al., 2000; Tehei et al., 2001). As a result of their biophysical

peculiarities, psychrophilic enzymes are interesting and useful models in protein research

for protein evolution, folding and dynamic studies.