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CHAPTER 2

REVIEW OF LITERATURE

2.1 Microbial Communities

The entire life on earth is dependent on microbial life, present or past and the foundation of the

ecosystem relies on the microbial activity. Microorganisms were the first life forms on earth

about more than 3, 85 billion years ago and have been evolving since then (Mojzsis et al., 1996).

During this long history, all of the basic biochemical mechanisms of life evolved, and all life

forms have developed from these microbial ancestors (Whitman et al., 1998). Microbial diversity

is a unique asset and micro-organisms are ubiquitous in nature (Alain et al., 2002; Novikova et

al; 2006). They are capable of thriving in almost every unusual habitats, like extremes of

temperature, radiation, pressure, salt, and acidity (Figure 2. 1). These micro- organisms are

known as extremophiles and receive attributes like; temperature (psychrophiles, thermophiles),

pH (neutrophiles, acidophiles, alkaliphiles), salt (halophiles), extremely dry conditions

(xerophiles), rocks (endoliths), high sugar contents (osmophiles) and higher atmospheric

pressure (piezophiles) and can potentially serve in a verity of industrial applications (Horikoshi,

2008). As a result of adaptation to extreme environments, extremophiles have evolved unique

properties, which can provide significant commercial opportunities. Based upon their habitats,

and their unique characteristics they are a potent source of metabolites (enzymes, antibiotics,

heat and cold shock proteins, antifreeze proteins) as demonstrated by Burg, 2003.

Figure 2. 1: Habitats of microbes (A) Desert environment (B) Termite gut (C)

Freshwater lake (D) Animal gut (E) Marine environment (F) Glacial ice

(Image courtesy: Whitman et al., 1998).

A B C

D F E

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In the world consisting of massive mountains, the Himalayas are the most complex. This

mountain system covering a massive 7. 5 lakh km2 (0. 75 million km

2) running over 3, 000 km in

length and 300 km wide and >8000m high. This range comprises of 3 sub-ranges, where the

northernmost sub range is the highest and is called the Great Himalayas (Mukesh et al., 2011).

The northern Himalayas, 21°57' – 37°5' N latitudes and 72°40' – 97°25' E longitude harbors

various cold and hot water springs with largely unexplored potential (Reysenbach et al., 2000).

However, the microbial community present in the hot water springs represent extreme niches

whose biotechnological potential has largely remained unexplored and unexploited (Skirnisdottir

et al., 2000). A study of microbial diversity in hot spring samples revealed that although all the

hot springs lying in the same geographical area had temperatures of the common range (between

85oC and 95

oC) and a similar pH value (7. 8-8. 9) but they displayed absolutely different

properties in lieu with their microbial diversity (Blank et al., 2002). This study suggested that

along with the common complexity of microbial diversity in other environments it is also

affected by the geo-biochemical variations. The use of enzymes (particularly hydrolases)

obtained from microorganisms isolated from the hot springs as catalysts is well established and

documented (Eicher, 2001; Irwin et al., 2004). As many industrial reactions are carried out at

extreme conditions there is an increasing demand for enzymes that can withstand these

conditions (Nivedita et al., 2013). Most of the enzymes have been isolated from mesophilic

microbes and despite their advantages they are not the preferred ones due to their relative

instability at extreme temperatures, pH and ionic strength. However, on the other hand,

thermophiles are important sources of thermostable enzymes, which show significant stability at

high temperatures. Each group of the thermophiles has distinguished features that can be

exploited to harvest enzymes with extensive applications (Haki et al., 2003; Sellek et al., 1999).

Habitat aside, microbes are the biggest resource of enzymes on the planet and this is rationale

enough for studying their prospects for industrial applications as high-throughput enzyme

sources as most of their applications have remained unrealized.

Therefore, to gain an insight of this “latent” microbial flora there is a need of a different

molecular technique. One such new molecular tool which has emerged as a powerful centerpiece

among the methods designed to explore the genetics and physiology of organisms which cannot

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be cultured is “metagenomics. ” It is the large-scale study of the DNA of naturally existing

microbial communities rather than laboratory-cultivable organisms.

2. 2 Metagenomics

The advent of microbiological techniques, including various culture techniques has been of

prime importance to the development of industrially viable processes and products. The ability to

cultivate microbes and their manipulation to obtain metabolites and by-products is essentially the

backbone of fermentation industry. Scientists across the R&D community have isolated an

infinitesimal list of microbes from soil, water and many other natural resources accessible to

man. But that is, not by far, the end of the story. Every milligram of soil is home to a wide

plethora of microorganisms, and this fact was proven right by “the great plate anomaly” (also

referred to as microbial unculturability) (Staley et al., 1985) in a semi-quantitative manner as

well as 16S r-RNA world theory in a quantitative aspect. Theoretically, only 1% of the soil

microbiome can be cultivated using conventional culture techniques, but there is always more to

where this vast reserve is harbored (Table 2. 1).

Table 2.1: Cultivability of microbes from different habitats (Table courtesy: Amann et al.,

1995)

Thus, a new approach has been developed in the last decade to access the total genetic resources

or the metagenome of a microbial community without the need to culture. This approach has

been termed as metagenomics (Handelsman et al., 1998). Metagenomics is a term that describes

an area of biotechnological research and a collection of techniques that allow cultivation-

independent exploration of a microbial consortium/community in any ecosystem (Sleator et al.,

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2008). It is the access to, and study of collective genomes of environmental microorganisms

(Handelsman et al., 1998; Riesenfeld et al., 2004a). The research in metagenomics has been

extensively carried out in the last 10 to 15 years enabling researchers to have an unimaginable

access to the “uncultivated majority” (Rappe et al., 2003) of our microbial counterparts in our

habitats and closer to home, on and in our own bodies.

Metagenomic analysis involves the basic steps like (1) the selection of an environmental niche

(2) the extraction of DNA directly from an environmental sample (3) manipulation of the genetic

material (4) library construction and (5) the analysis of genetic material in the metagenomic

library for specific function(s) (Srivastava et al., 2013). All these steps are illustrated

schematically in (Figure 2. 2).

Figure 2. 2: Steps in metagenomic analysis (Image courtesy: Sjoling and Cowman, 2008)

2. 3 Selection of niche

The selection of the niche for the sample is an important part of metagenomics. Although any

environmental sample offers a rich source of diversity, it could be further reduced based on the

requirement and interest of the researcher. The metagenomic libraries constructed from a vast

range of metagenomes have earlier been studied to get a foresight of the potential of the

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microbiota like sediment samples (Parsley et al., 2010), marine environments (Breitbart et al.,

2004; Venter et al., 2004; Martin-Cuadrado et al., 2007; Jiang et al., 2011), soil (Lämmle et al.,

2007; Van Elsas et al., 2008; Fan et al., 2011), animal guts (Qin et al., 2012; Bao et al., 2011)

and freshwater samples (Wexler et al., 2005). Also, the glacial ice, an example of extreme

environment (Simon et al., 2009), hypersaline environments (Ferrer et al., 2005) and a thermo-

pond (Robe et al., 2003) have been exploited by approaches based on metagenomics

2.4 Metagenomics of soil and sediment

Soil and sediment offers probably the most complex and challenging microenvironment for the

resident species with respect to microbiological research. It has been estimated that there are as

many as 40 million prokaryotes harboring each gram of a forest soil (Richter and Markevitz,

1995) and over 2 billion prokaryotes in cultivated grassland soils (Paul and Clarke, 1989). These

populations are represented by a whopping 2000-18000 distinct prokaryotic species (Torsvik et

al., 2002). So the dominant portion of soil biomass is formulated by the prokaryotic population.

The microbes harbor the surface of soil and sediment aggregates, the interstitial spaces between

aggregates and inside the aggregates also (Hassink et al., 1993; Foster, 1988). Analysis of non-

culturable soil and sediment micro biodiversity showed that most native soil and sediment

bacteria belong to phyla Firmicutes, Acidobacteria, Proteobacteria, Actinobacterium and

Verrucomicrobia, meaning that numerous 16S rDNA clones were isolated from uncultivated

bacterial species (Delmont et al., 2011; Janssen, 2006). Mostly generally, the population of

microbes in a given soil and sediment type depends on many factors including the pH, mineral

content, organic content and aeration. Although most soils are rich in their organic content, the

“bioavailability” of these nutrients is detrimental to the microbial diversity existing in the soil. It

is well known that much of the available organic carbon in soil is abiotically as well as biotically

converted to humus, which is inaccessible to microbes for nutrition (Paul and Clark, 1989). The

microbial density is theoretically the highest when the soil and sediment conditions are near

neutral, as compared to acidic or alkaline (Lauber et al., 2009). But that is not the rule of the

thumb. The microbial distribution varies from phylum to phylum. The abundance of bacteria in a

given soil and sediment alkalinity can be very different from that of archaebacteria, fungi and

protista (Rousk et al., 2010). Such analyses of soil and sediment samples help the researchers to

biogeographically locate the source of soil and sediment for a particular phenotype of the

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organisms they are looking for. For example, if one is looking for an alkaline protease, one

would source the soil and sediment from an alkaline habitat, pH is not the only factor influencing

microbial growth.

Another important parameter is the temperature, which essentially is the temperature of the

environment. The microbial diversity is rich near moderate to tropical temperatures and reduces

as we reach either extreme. Thus understanding the biogeographic distribution is extremely

important to understanding the phenotypes of microorganisms. Albeit, recent research shows that

knowledge of biogeographic distribution of microbiota is not sufficient to explain why the

functional capabilities of the microbes vary substantially across biomes. Ideally, the functionality

of microbes should vary proportionally with the taxonomic diversity but that is not the case.

Many closely related taxonomic groups are known to have markedly different phenotypes,

physiology and environmental tolerance, whereas, unrelated taxa might share functional

attributes (Philippot et al., 2010). Thus, although important, the bio-geographical distribution of

microbial functions is not the gold standard for allocating absolute attributes to the native

species. To address this limitation, scientists have now turned to an advanced technique called

the shotgun metagenomic sequencing. Microbial communities are complex and hence the

assembly of their sequences is till date, a farfetched reality. This is because of the high gene

density of prokaryotes. Studies have reported the microbial gene density for prokaryotes to be as

high as 1 ORF per kb of DNA and modern sequencing techniques yield us a read length of

approximately 800bp. This means that every time we sequence a metagenomic read, we end up

having at least one gene per individual sequence. Thus direct sequencing has a significant

limitation and is not the ideal tool for constituting metagenome assemblies (Goo et al., 2004).

Shotgun sequencing allows a comprehensive analysis of the microbial genotypes and

phenotypes, pertaining to an ecological niche, which the microbes employ for their survival and

sustenance (Tringe et al., 2005; Mackelprang et al., 2011).

2.5 Metagenomics of water

Soil is the most diverse ecosystem in our biosphere and harbors the dominant population of

environmental microbes. But soil is not an ecosystem that can be considered in isolation. 70% of

earth’s surface is covered with water, which makes it sizeably the largest ecosystem on the

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planet. Water can provide the most extreme of habitats prevailing on earth. Its temperature can

range from -90 oC in the Antarctic glaciers to over 100

oC in the natural hot springs of Bulgaria

(Steig et al., 2009). Water is the universal solvent and is critical for sustaining life. Water bodies

are known to harbor an enormous plethora of microbial population, which has previously been

ignored for decades. Microbiologists as well as environmentalists worldwide have shown

mushrooming interest in the microbiota of the aqueous ecosystem. In constant contact with the

soil ecosystem, water has important roles to play for balancing the biogeochemical cycles, which

is responsible for recycling of nutrients, oxidation, storage and release of organic carbon,

thereby, regulating the carbon turnover in the ecosystem (Cole et al., 2007). This makes water a

potential reservoir of various biogeochemically active principles, novel enzymes and genes

essential for survival.

It seems now that hot springs are gaining a lot of attention vis-à-vis metagenomic research

because the native microbes are thermophilic and/or hyperthermophilic, which thrive at

temperatures >55 °C and >80 °C respectively. The corollary derived from this fact is that these

organisms could very well be sources of industrially useful enzymes. The most striking example

is that of Taq polymerase, which is obtained from Thermus aquaticus and is used in the

polymerase chain reaction (PCR) (Chien et al., 1976). The hot springs offer diverse niches and

habitats including sediment, microbial mats and hot water which differ in their physicochemical

properties and hence the microbial diversity. Every hot spring differs from others in temperature

and chemical compositions. Hot springs comprise several habitats, such as thermal fluids,

microbial mats and sediments. (Olalla et al., 2013).

Cultivation independent methods have been employed in order to tap into the bioresources

available in the form of soil, sediment and water microbiota. Ideally speaking, the total DNA

isolated from a microbial community represents a culmination of the gene pool of that ecosystem

(Rondon et al., 1999). Thus, for gaining access to the metagenome, research groups across the

globe have been working on efficient isolation of total metagenomic DNA from soils of different

ecological niches. This is particularly of a challenging nature, taking into consideration the

microbial biodiversity, prokaryotic population and the complexity of the soil and sediment

matrix, which is composed of contaminating humic acids and fulvic acids. Altogether, these

factors make the isolation of complete metagenomic DNA the “Achilles” heel for metagenomics

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researchers (Daniel, 2005). So, to overcome this hurdle various methods for isolating

metagenomic DNA from different soil and sediment types have been reported.

2. 6 Metagenomic Methodology: DNA isolation, library construction and

screening

2. 6. 1 DNA isolation methods

The important point that need to be considered for metagenomic DNA isolation from

environmental soils/sediments/water samples are: 1. The isolated DNA must have the genetic

material of all the species dwelling in the ecosystem; 2. The isolated DNA must be intact, must

have high molecular weight, which can be ensured by using a gentle DNA isolation method; 3.

The metagenomic DNA should not have any interfering contaminants such as humic acids and

phenolic compounds, as such substances are known to inhibit PCR, restriction digestion and

ligation of DNA. (Furrie, 2006; Bertrand, 2005).

Basically, there are two methods to obtain metagenomic DNA. The first one is the direct

isolation of the DNA from the environmental sample, followed by the removal of humic acids i.

e to purify the DNA. The second method includes the removal of bacterial cells from the

environmental samples, followed by cell lysis and then isolation of the DNA (Lorenz and

Schleper, 2002).

Figure 2. 3 depicts the direct and indirect isolation of metagenomic DNA.

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Figure 2.3: Direct and Indirect methods of DNA isolation from environmental samples

(Image courtesy: Robe et al., 2003)

2. 6. 1. 1 Direct method of DNA isolation: Methods of microbial cell lysis

Three methods of microbial cell lysis can be undertaken viz. chemical, enzymatic and physical.

These can be employed as separate protocols or in combinations in order to increase the

efficiency of the process.

Cell lyses by chemical methods are often used in combination with enzymatic or physical

methods or separately for DNA extraction. The most common detergent used for

isolation of DNA is SDS, which causes the removal of the lipids that occur in the

microorganisms cell membrane (Roose-Amsaleg et al., 2001). SDS can be used in

varying concentrations from (0. 1% – 20%) and at different temperatures and especially

in combination with chelating chemical compounds such as ETDA- a chelating chemical

substance or Chelex® 10 -a chelating resin in a sodium form (Maarit et al., 2001).

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There are different methods of isolation of DNA by enzymatic lysis (Roose-Amsaleg et

al., 2001). Most common enzyme used is lysozyme which cleaves the polysaccharides of

the microorganisms that are found in the cell wall. One more enzyme used in this method

equally is proteinase K. Achromopeptidase and pronase (Roose-Amsaleg et al., 2001) are

the least proteolytic enzymes that are used in this method.

The physical method of lysis of cells usually involves the access to each cells, which

increases the efficiency of isolation of DNA. Freezing-thawing and freezing-boiling

cycles are the physical methods which are often applied (More et al., 1994). Applying

ultrasounds, liquid nitrogen grinding and grinding in mortar, (Robe et al., 2003) are some

of the other methods used for extracting metagenomic DNA.

2. 6. 1. 2 Indirect method of DNA isolation

The metagenomic DNA extraction by an indirect method includes: dispersion of the metagenome

collected from the environment, separation of the cells and lysis, DNA isolation and purification

of the isolated DNA (Robe et al., 2003).

2. 6. 1. 2. a Dispersion

This method involves two procedures chemical and physical. With the use of rotating rubber

pestle treatment or homogenization with blender are commonly used for releasing the cells from

the microbes (Hardeman and Sjoling, 2007; Lindahl and Bakken, 1995; Berry et al., 2003).

Mechanical methods are used in combination with chemical method. PEG, SDS, sodium cholate

and deoxycholate can be used to increase the rate of the process of dispersion (Robe et al., 2003;

Bertrand et al., 2005).

2. 6. 1. 2. b Microbial cell separation

One more method of separating bacteria from environmental samples is centrifuging the samples

at an increased g-factor value (Carac- Ciolo et al., 2005; Robe et al., 2003). This method is based

on a (1. 3 g/cm3) high density of the Nycodenz®, which has a greater value than the density of

the microbes (Lasken et al., 2005).

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The characteristics of metagenomic DNA obtained by different extraction methods is depicted in

Table 2. 2.

2. 6. 1. 2. c Extraction and Purification of DNA

After lysis, the metagenomic DNA isolated by both direct and indirect methods is subjected to

separation, purification followed by precipitation. Isolation of DNA can be carried out with the

deproteinisation in solvents like phenol or mixtures: PCI (25:24:1) (Zhou et al., 1996), phenol:

chloroform(50:50) (Ranjan et al., 2005) are used most commonly in the physical as well as

chemical methods of isolation of DNA. This can also be used for the removal of proteins from

the DNA. Isolated DNA is purified by precipitation at the same time with isopropanol, ethanol,

PEG or sodium acetate (Porteous and Armstrong, 1991; Roose-Amsaleg et al., 2001). poly

ethylene glycol is used instead of isopropanol because alcohol precipitates the DNA along with

humus substances found in the soil (Porteous and Armstrong, 1991). To improve the purity of

the DNA at the time of precipitation sodium acetate is added.

Marketa et al., (2008) recommended the use of calcium carbonate for removal of humic

substances. After this pretreatment step, the DNA was isolated using the CTAB lysis buffer

method (Zhou et al., 1996). Pretreatment with aluminium ammonium sulphate was used by Braid

(Braid et al., 2003) who reported reduced co-purification of PCR inhibitors when aluminium

ammonium sulphate was added during the extraction step. Dong and his co-workers ( Dong et

al., 2006) isolated the DNA from 300 mg of soil by re-suspending it in both acidic (pH 6. 6) and

alkaline (pH 8. 0) phosphate buffers containing 50, 100 and 200µL of 100 mM aluminium

sulphate followed by bead beating lysis. Miller proposed DNA extraction by bead beating

homogenization followed by SDS-Chloroform extraction and sephadex G-200 column

purification (Miller et al., 1999). DNA extraction by modified CTAB extraction buffer method

complemented with 0. 5 volumes of 50% PEG and 0. 1 vol of 1M NaCl precipitation and

purification by one step post treatment with 2% CaCl2 (Singh et al., 2014) is deemed the

successful method in removing humic acids. This method absolutely removes the humic acid

Table 2.2:Metagenomic DNA characteristics obtained by different methods of extraction

(Table courtesy: Monika and Marek, 2008).

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contamination because CaCl2 prevents the humic substances to undergo oxidation forming

quinones, which covalently bind to the DNA, thus hampering the DNA and Taq polymerase.

DNA extraction directly from unculturable microbial species present in environmental water

samples is much easier as there is less space for humic acid contamination in aquatic ecosystem.

The water has to be filtered before it is preceded for DNA extraction. There are different types of

filter papers that are used:

a) PVDF SYRINGE FILTERS:-

Good heat endurance and chemical stability.

Strong hydrophobicity.

They are individually wrapped sterile.

They are RNase and DNase free.

b) PES MEMBRANE FILTERS:-

Polyether sulfone membranes are hydrophilic.

They are designed to remove particulates.

It has low protein and drug binding capability.

It has superior thermostability and high flow rate.

c) NYLON MEMBRANE FILTER :-

It has high strength and heat resistance.

They are hydrophilic membranes which are made up of polyester web with nylon.

It is compatible with aqueous, alcoholic solvents and solutions.

2. 6. 2 Metagenomic library construction

Once the DNA is isolated, the immediate next step is construction of metagenomic DNA

libraries. Summarily, metagenomic library construction involves DNA fragmentation by

restriction digestion, followed by cloning the fragments into a suitable plasmid, cosmid or a BAC

construct (Rondon et al., 2000). The method used for the construction of metagenomic libraries

involves the same techniques as that in cloning from genomic DNA of culturable and isolated

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microorganisms. This method includes metagenomic DNA fragmentation by restriction

digestion, ligation of desired fragments into an appropriate vector followed by transformation of

a suitable host. High molecular weight DNA becomes even more credible in this case as it yields

ligatable DNA fragments of appropriate size (Wilkinson et al., 2002). The most commonly used

host strain in most recombinant DNA approaches is E. coli.

The plasmid, cosmid or BAC libraries are screened for various functional gene types viz.

proteases, lipases, cellulases, amylases, oxidoreductases, nitrilases and many more. These

screening strategies mainly involve microbiological methods like substrate plate screening and

biochemical methods involving enzyme activity assays (Table 2. 3).

Table 2.3: List of enzymes identified through metagenomic approaches (Table courtesy:

Reevander et al., 2013).

Function Habitat Library type Substrate Reference

Alkaline pectate

lyase

Soil Plasmid Pectin Wang et al., 2013

Amylase Soil Cosmid Starch Sharma et al., 2010

α Amylase Soil Fosmid Starch Vidya et al., 2011

Cellulase Soil Plasmid Carboxymethyl-cellulose Nacke et al., 2012

Cellulase Soil Fosmid Hydoxyethyl cellulose Alvarez et al., 2013

Endocellulase Soil Plasmid Carboxymethyl-cellulose Bhat et al., 2013

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Lipase Marine

sediment

Plasmid p-Nitrophenyl (pNP)

esters

Peng et al., 2014

Lipase Soil Fosmid Tributyrin agar Fu et al., 2013

Lipase Sediment Fosmid Triolein Glogauer et al., 2011

Pectinase Lagoon Plasmid Polygalacturonic acid Singh et al., 2012

Protease Soil Plasmid Azocaesin Biver et al., 2013

Serine proteases Soil Plasmid Skimmed milk Neveu et al., 2011

Xylanase Compost soil

rumen

BAC Oat spelt xylan Gong et al., 2013

2. 6. 3 Screening strategies

Metagenomic libraries are screened for novel microbial gene coding ORFs. The libraries

containing small-sized insert DNA are suitable for identifying novel enzymes whereas those with

large inserts are also frequently used and sometimes preferred (Lee et al., 2004).

Table 2. 4 shows pros-and-cons of using small-insert and large-insert cloning strategies:

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Table 2. 4: Advantages and Disadvantages of small insert and large insert libraries (Table

courtesy: Monika and Marek, 2008)

Meticulous screening of metagenomic libraries has been quite successful in identifying

numerous enzyme coding genes and bioactive principles (Iqbal et al., 2012). Two approaches for

screening metagenomic libraries are frequently used viz. sequence based (genotypic) and

functional (phenotypic) screening.

(a) Functional (phenotypic) screening

A metagenomic library is screened for enzyme functionality to detect gene expression in the

heterologous host. Although codon-bias operates in expression of a majority of eukaryotic gene

expression, function-based screening is still the most promising approach to explore for novel

ORFs and enzymes (Simon and Daniel, 2011). Lately there have been reports where the

conventional functional screening strategies have been modified wherein, researchers have opted

for direct detection of metabolic function of the enzyme(s) in question (Rondon et al., 2000;

Simon and Daniel, 2011). Also, genetically engineered host strains are being used for identifying

gene function trans-complementation in the library clones (Wang et al., 2006) and another

development in functional screening has been coupled to high-throughput screening to detect

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inducible/constitutive gene expression with substrates, auto-inducers and exogenously supplied

metabolic products (Uchiyama et al., 2005; Williamson et al., 2005).

The next approach for function-based screening of metagenomic libraries is known as substrate

induced gene expression screening (SIGEX). It is most often used for screening for catabolic

enzyme coding genes whose activity can be screened in an end-point plate assay in response to

an environmental/chemical/biochemical stimulus such as a chemical compound (Uchiyama et

al., 2005). This screening strategy is based on the knowledge of inducible gene expression

systems/operons which can be coupled to the cloning site of the gene so as to yield a fused gene

product. In high-throughput screening, an operon trap-GFP vector has been used, which allows

high-throughput DNA sequencing as well as fluorescence activated cell sorting (FACS) for

sorting positive cells in a liquid culture (Valdivia and Falkow, 1997; Dunn et al., 2003).

Overview of different methods used in screening metagenomic libraries is represented in Figure

2. 4.

Figure 2. 4: Overview of metagenomic screening methods

(Image courtesy: (Streit et al., 2004)

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(b) Sequence-based screening

Sanger sequencing or chain-termination sequencing is the traditional metagenomic sequencing

method which yields longer reads than 700 base pairs (bp) with relatively lower error rate

(Thomas et al., 2012). The sequence based screening provides us insight of not only the gene

fragment present in the vector but it can also be used for assessing the microbial diversity in an

ecosystem. Researchers have cloned the 16S r-RNA gene in cloning vectors and sequenced the

genes to build phylogenetic trees of various ecological niches (Knietsch et al., 2003). A new

sequence-based screening protocol is based on the developments made in the next-generation

sequencing (NGS). Direct DNA sequencing by NGS is now being extensively used to assess the

metagenomes of different microbial communities (Dinsdale et al., 2008). NGS of metagenomic

DNA generates a vast amount of sequencing data which needs to be assembled and annotated

using various tools of bioinformatics (Teeling et al., 2012). Function based and sequence based

screening can be coupled to high throughput sequencing techniques to increase the frequency of

detection of novel ORFs.

Relative merits and demerits of screening techniques used in metagenomics is presented in Table

2. 5

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Table 2. 5: Merits and Demerits of screening methods used in metagenomics (Table

courtesy: Jiae and Sangryeol, 2005)

Function-based

screening

Sequence- based screening SIGEX

Screening

principle

Detecting changes by

enzymatic reactions (e. g.

halo formation around the

colonies)

PCR or Southern

hybridization based on

DNA sequence consensus

Trapping the operon

induced by a substrate

and sorting using FACS

Advantages Secures a complete form

of gene or gene cluster

required for desired traits

Potentially obtains

completely novel genes

Overcomes limitations of

the heterologous expression

Fast and economical

Any substrates that can

be introduced into

cytoplasm can be used

in its native forms

Disadvantages Must satisfy the

expression

conditions(transcription,

translation, folding and

secretion) in heterologous

host

Requires a database and

analyses of the DNA

sequence consensus

Does not guarantee the

acquisition of complete

forms of genes or gene

cluster

Sensitive to the

orientation of the genes

with desired traits

Cannot use substrates

that do not migrate to

cytoplasm

Sensitive to the initial

FACS setting

Examples Antibiotics (Macneil et

al., 2001; Gillespie et al.,

2002), agarases (Voget et

al., 2003), amylase (Yun

et al., 2004), lipases

(Henne et al., 2000)

Amylases(Richardson et al.,

2002), polyketide synthases

(Piel, 2002; Piel et al.,

2004)

Benzoate- degradative

or catechol degradative

operon, P450

enzyme(Uchiyama et

al., 2005)

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Recently, metagenomic libraries have been replaced by meta-transcriptomic libraries. This step

was taken in order to gain access to a significantly existing eukaryotic population in

environmental samples which includes various fungi, yeasts and protistans. The idea behind this

development is to isolate m-RNA from environmental samples, their reverse-transcription to c-

DNA followed by construction of c-DNA libraries, resulting in meta-transcriptomic libraries

(Starkey et al., 1998; Lehembre et al., 2013).

2.7 Enrichment strategies to improve the number of active clones in

Metagenomic Libraries

The most significant drawback in metagenomic analysis is the low success rate in terms of

obtaining the desired gene function/ORF out of the metagenome of a particular ecosystem. In

order to increase the number of positive clones, in a library, an alternative technique that is being

used these days is enrichment culture.

Enrichment can be achieved in several ways as represented in Table 2. 6.

Table 2.6: Overview of enrichment technique used in Metagenomics (Table courtesy:

Schwarz et al., 2006; Sul et al., 2009)

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2.7.1 Enrichment for GC content

Enrichment for GC content of the genomes is a simple and convenient method. Those organisms

having high GC content in their genomes, such as actinomycetes, acidophiles etc. can be

enriched by extracting the metagenomic DNA followed by ultracentrifugation to separate out

high GC DNA. This provides the researchers with a crudely enriched metagenomic DNA for GC

rich regions.

2.7.2 Sample Enrichment

Screening of metagenomic libraries is usually supported with the number of positive clones

being very low (usually ≥ 0. 01%) (Cowan et al., 2005). This suggested that without enrichment

of the sample the discovery of particular genes in a complex metagenome may be challenging

and difficult. A small proportion of the entire DNA fragment represents the target gene(s). Target

gene discovery can be increased by using any one of the several enrichment procedures available

ranging from enrichment of target genes and genomes to whole-cell enrichment (Cowan et al.,

2005). Pre- enrichment of the sample by varying physical, chemical and nutritional criteria thus

provides an attractive means of enhancing the screening hit rate but substrate utilization is most

commonly employed. Enrichment of the culture on a particular medium enables the growth of

target microorganisms. Although this approach undoubtedly causes a huge loss of a great part

of the microbiota by allowing the selection of only the fast-growing species which can be

cultured. However, it can be reduced to some extent by decreasing the pressure for a short period

of time (Knietsch et al., 2003).

2.7.3 BrdU enrichment

Bromodeoxyuridine (BrdU) is a base analogue which provides a non-radioactive label for

labeling the DNA. The basic principle followed in BrdU enrichment is that metabolically active

microbes take up BrdU which gets incorporated into the genomic DNA. These microbes can then

be explored by isolating the metagenomic DNA and separating the labeled DNA by anti-BrdU

antibodies a process called as immunocapture (Urbach et al., 1999). This strategy can be used to

enrich the microbes that can metabolize substrates such as starch, cellulose and polypeptides to

find amylase, cellulase and proteases respectively of interest in constructed metagenomic

libraries.

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2. 7. 4 Stable isotope probing (SIP)

An alternative can be radioactive labeling of DNA a method called stable isotope probing (SIP)

(Radajewski et al., 2000). It provides a 13

C labeled substrate to microbes. The actively dividing

bacteria incorporate the label into their DNA, which makes the labeled DNA denser than the

normal DNA containing 12

C labeled substrate.

2. 7. 5 Gene-specific PCR method

This method can be specifically used for screening the microbial communities for metabolic or

biodegradative capabilities. The major drawback of this method is that the designing of primers

is based only on the information available about the sequences. The specific set of primers

cannot identify genes that are similar in function (Liu et al., 1995; Futamata et al., 2001).

2. 7. 6 Differential display (DD) technique

The genes obtained from metagenomes of different environment are studied directly or by

analysis of microbial transcripts without the need of targeting a particular organism (Liang et al.,

1992; Brzostowicz et al., 2000; Taxman et al., 2001) with the help of RT–PCR.

2.8 Software and Tools for Metagenomic Analysis

16s rRNA gene isolation followed by sequencing is the approach used when there is a need for

metagenomic sequences for phylogenetic analysis. Although numerous online and offline

softwares and analytical tools are available for sequence and structural analysis (Table 2. 7).

Warren et al., (2010) devised a method based on sequence analysis to obtain the left out

sequences from prokaryotic gene annotations. White et al., (2010) designed algorithms based on

phylogenetic markers for sequence alignment for studying microbial diversity in different

environmental samples.

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Table 2. 7: In- silico tools used for Metagenome analysis

2. 9 Metagenomics: Commercial success in biotechnology

The coalition of two commercial metagenomic projects in the 90s took place between

Recombinant Biocatalysis Ltd and TerraGen Discovery Inc. The Diversa Corporation (www.

diversa. com) is the leading name in the area of metagenomics with significant collection of

libraries sourced from ‘global biomolecules’. Although there are only a few reports of

successfully commercialized therapeutics derived from metagenomic screening initiatives, the

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usual timelines for the identification, development, testing and approval of important products

for the pharmaceutical market is bound to take much more time than what would be expected out

of most scientific researches aimed at drug discovery and so on.

Table 2. 8 gives an overview of commercialization status of metagenomic technologies.

Table 2. 8. Commericialized Metagenomic technologies (Table courtesy: Monika and

Marek, 2008)

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2. 10 Major breakthrough in Metagenomics

2. 10. 1 Sargasso sea microbiome

The vast and significant metagenomic microbial study was the shotgun sequencing (SGS) in the

ocean of microorganisms in the Atlantic Ocean near Bermuda of Sargasso Sea (Venter et al.,

2004; Tringe et al., 2005; Foerstner et al., 2006) ranging from 0. 1 to 3. 0 μm in size. Marine

organisms are known to be the nearest living descendents of the ancestoral life forms and are

regarded as a main element of the geochemical cycles of the universe. Sargasso Sea a completely

studied marine environment and is known to possess “considerably” less microbial community

as an outcome of deprived-nutrient circumstances. The main highlight included was the

extension of bacterial occupation of proteo rhodopsin genes. This study also provided a

framework of unique genes possessing great potential in the discovery and development of

several novel biocatalysts (Venter et al., 2004).

2. 10. 2 Methane oxidizing archaea from deep sea sediments

In marine deep sediments anaerobic methane oxidation by archaea plays an important part in

decreasing methane released into the atmosphere from oceans. On the uncultured communities

thriving in a methane seep in Eel River Basin (California coastline) a metagenomic study was

carried out. An enrichment step in this study was introduced in order to decrease the complexity

of the sample taken. This step included selection based on size for sulfate-reducing bacteria and

for archaeal cells by size-fractionation with the help of density centrifugation before construction

of the library. Targeted and random sequencing produced ~ 120 Map of DNA from foamed

clones which were studied for pathways such as methanogenesis (Hallam et al., 2003; 2004).

2. 10. 3 Metagenomics of the deep mediterranean ocean

Metagenomic analyses of the marine samples have been specifically devoted to photic waters.

From 3, 000 m-deep Mediterranean plankton, a fosmid metagenomic library was constructed.

The deep Mediterranean ocean shows a lot much warmer (~14oC) temperature than waters of

similar oceans and depth(~2oC). The library was identified both by 16S rRNA amplification of

the clones and by screening (phylogenetic) based on sequencing (Martin-Cuadrado et al., 2007).

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2. 10. 4 Human distal gut communities

There are trillions of bacteria, in the human intestine representing hundreds of species and

thousands of sub species. (Xu et al., 2007). There is present a huge community of microbes in

the human gut. The metagenomic libraries constructed from two healthy humans fecal flora

generated a data of ~78 Mbp on random sequencing. The human genome and sequenced

prokaryotic genomes were compared with this metagenomic data and revealed a number of

pathways for synthesis of vitamins, polysaccharide degradation and methanogenesis. These

results showed that they might have applications for curing obesity and cancer (Gill et al., 2006).

2. 10. 5 Global ocean sampling (GOS) expedition

The mixture of micro-organisms which is highly complex and are present in the world’s oceans

are largely uncharacterized both biochemically and genetically. Rusch et al., (2007) reported a

marine planktonic microflora metagenomic study in which mostly surface water samples were

studied. A total of 41 samples were analyzed. This study showed a large dataset consisting of 7.

7 million sequencing reads (6. 3 billion bp). Although only some microbial clades envelope the

marine planktonic niche, the dataset consists of 85% microbial sequence which is assembled and

57% of the data which is unassembled leading to novelty at a 98% of sequence identity cut off.

2. 11 Examples of metagenomic studies

Isolation of new Antibiotics

According to the study (Gillespie et al., 2002) DNA was extracted directly from soil and an

average size of 44. 5 kb of the insert were ligated into a BAC vector. A library of 24, 546

recombinants in Escherichia coli was screened for haemolytic activities. Earlier, only one clone

that displayed a clearance zone on agar ( blood) was identified. Subsequently, three more clones

gave colonies of melanin like-dark brown color. The structural study of these clones suggested

that they were triarylcations, designated turbomycin A and turbomycin B, having a vast-

spectrum of antibiotic activity. Chang and Brady reported a gene cluster bor from soil that

encodes indolotryptoline based compounds, a small and relatively rare family of natural products

that exhibit potent activity against certain tumor cell lines (Chang et al., 2013). These discoveries

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have triggered extensive search operations for the quest of novel and biomedical important drugs

as evidenced by various studies (Allen et al., 2009; Feng et al., 2012).

Table 2. 9 shows the antibiotics discovered by functional screening of metagenomic clones

Table 2. 9: Antibiotics discovered from metagenomic libraries (Table courtesy: Yasir et al.,

2014)

Antibiotics Habitat Library type Reference

Beta-lacatamases Soil Plasmid Allen et al., 2009

Fasamycin A and B Soil Cosmid Feng et al., 2012

Indirubin Soil Fosmid Lim et al., 2005

Terragine Soil Cosmid Wang et al., 2000

Turbomycins A and B Soil BAC Gillespie et al., 2002

Violacein Soil Cosmid Feng et al., 2012

Isolation of new Polyketide Synthase genes

The natural compounds which contain alternate methylene and carbonyl groups are known as

Polyketides (Baerson and Rimando, 2007). Examples of Polyketide antibiotics are tetracyclin,

erythromycin A, clarithromycin, and azithromycin. Courtois et al., (2003) in the study

constructed and screened a “shotgun” environmental DNA library of 5, 000-clone. The

metagenomic library was highly diverse on the basis of the phylogenetic content mostly

representing microbiota that has remained unexploited. On screening the library new genes in

approximately eight clones were discovered. This study suggested that exploring the earlier

unculturable microorganisms for the discovery of novel natural products has marked potential

value for establishing a technology as a drug discovery tool.

Identification of novel biocatalysts

The search for bioactive molecules using a metagenomic approach has generally been conducted

using either homology-based methods or functional screening. Novel sequences found in

homology-based screens can be examined for the ability to encode the biosynthesis of novel

small molecules in heterologous expression experiments (Banik and Brady, 2010). To fully

exploit the potential within metagenomic libraries development of expression systems with new

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selective, specific, and sensitive testers plus high-throughput platforms is needed. An interesting

system, designated METREX, has been designed containing an expression host which carries a

GFP reporter sensitive to compounds capable of inducing quorum sensing; clones expression

from metagenomic DNA library encoding genes required for synthesis of novel bioactive

molecules that induced the reporter which could then be detected by fluorescence ( Williamson

et al., 2005). Therefore, using culture-independent methods, the-yet-uncultured bacteria which

are likely to be a rich source of novel bioactive molecules can be explored.

Metagenome from an unplanted field soil was used to construct cosmid libraries (Voget et al.,

2003). 20 to 40 kb DNA fragments were cloned and the library constructed was analyzed by

sequencing as well as functional approaches. For identification of agarases the cosmid clones

were first grown on LB medium. The grown cells were then pelleted by centrifugation and lysed.

The cell extracts were added to solutions- 0. 2% of LMA and incubated at 37°C overnight. The

extracts showing activity (agarolytic) enzymes were recognized in microtitre plates, consisting of

agarose which was liquefied by heating. This experiment gave four clones that encoded for 12

putative agarase genes (Beja et al., 2000). From the same library constructed the clones

displaying lipolytic activity were identified by clear halos formation around the clones after

growth on LB agar plates supplemented with tributyrin (substrate). Two lipase genes as a

conserved cluster encoding a type I secretion system were identified (Kim et al., 2009).

Furthermore, numerous other biomolecules-encoding genes, including two cellulases, xylose

isomerase, an α-amylase, genes for a putative stereoselective amidase and two pectate lyases

(Knietsch et al., 2003).

Table 2.10 summarizes the success of metagenomics in the discovery of novel enzymatic genes.

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Table 2. 10: Metagenomic discovery of novel enzymatic genes in recent years

2. 12 State-of-the-art in Metagenomics

Our view of the microbial world and its impact on our lives is rapidly changing. Until recently

we have considered ourselves as largely independent from the microbial ecosystem we live in

(Blaser, 2006; Lozupone et al., 2008; Davies, 2009). As such microorganisms offer great hopes

for scientific research and potential biotechnological applications. The increasing interest in

understanding the function of the microbial life forms in planet ecology, pharmacology as well

as other biotechnological applications promises very important economic and societal benefits

for those countries that are involved in such research.

As for every new field, many tools and approaches need to be invented from scratch or adapted

in order to answer questions and gain insight on the studied topic, and metagenomics is no

exception. It is overwhelming to see the pace at which the whole experimental and analytical

framework is being built as a consequence of international teamwork and different collaborations

at national level. This very recent technology is already challenging the very basis of scientific

practice in molecular biology. Some authors have even stated that “the hypothesis-driven science

may find it hard to keep up” (Gilbert and Dupont, 2011). In spite of the scientific and industrial

excitement about the new possibilities of unraveling microbial diversity, a remaining challenge is

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to develop technologies at competitive prices and to turn metagenomics into commercial

successes. As Lorenz and Eck put it, “Metagenomics, together with in vitro evolution and high-

throughput screening technologies, provides industry with an unprecedented chance to bring

biomolecules into industrial application” (Lorenz and Eck, 2005). The afore mentioned

technology and experiments have given to wealthy nations a vast library of data and tools to

analyze them with a great potential for applications and discoveries.

The very young field of metagenomics is encountering a great success and is offering the

possibility to explore in high-resolution places whose landscape we could only have imagined

before. Seeing the outstanding diversity of the microbial world at the gene level is not enough to

understand the functions and dynamics of the constituting parts. Ultimately, integrated analysis

of metagenomes, metatranscriptomes, metaproteomes and meta-metabolomes will be needed to

understand the microbial systems biology (Sleator et al., 2008). Achieving such integration

necessitates interdisciplinary efforts and continuous development of appropriate bioinformatics

tools to decipher the complex biological networks underlying molecular, functional and

community structure. The in silico investigation of biological networks could be quite effective

in identifying central connected components that could bring, at a later time, more insight on

their functionality and dynamics within the system.

International projects such as MetaHIT and HMP have already released a wealth of data on

ecosystems along with the corresponding reference catalogues and their available functional and

phylogenetic annotations. Being able to improve human health as a result of metagenomics

studies would not only help taking an important medical burden away from the society but also

preserve active individuals that would participate to the economy. Therefore, in the years to

come, invaluable contributions to evolutionary biology, enzymology, medicine and agro-science

are in order from research in the field of ‘life beyond the unseen’ -Metagenomics.

2. 13 Search for hydrolases through metagenomics: Emphasis on Proteases

There is a rat race of procuring better, faster and high yielding enzymes, so the industrial

processes could be rendered more economical. The microbes have indeed, proven to be the

single most comprehensive source of industrially important enzymes like amylases, proteases,

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cellulases, lipases, esterases, nitrilases and the list is endless which belong to the class hydrolases

involved in reactions such as hydrolysis, condensations and alchoholysis. But, we cannot now or

not unlikely in the coming future, will be able to culture all the microbes out there for obtaining

novel enzymes. Scientific estimates conclude that less than 10% native microbes of most habitats

are culturable (Amann et al., 1995; Handelsman et al., 1998; Couto et al., 2010), which clearly

means that the great genetic diversity in the environment still lies undiscovered. The 16S rRNA

study from diverse sources of environment has provided strong evidence for the existence of new

lineages of microbes (Pace, 1997; Lagesen et al., 2007). Assigning function to uncultured

microorganisms in various environments (in absence of pure culture) presents immense

challenge for microbial ecologists. (Fuhrman et al., 1993; Kaeberlein et al., 2002). And it is no

wonder people have now shown increasing interest in metagenomic science. The word

‘metagenomics’ was put forth to signify the potential applications of uncultured microorganisms

(Handelsman et al., 1998). It is a culmination of molecular techniques that allow evaluation of

the structural, dynamic and metabolic potential of environment samples. Metagenomic research

over the past 15 years or so has yielded a plethora of enzymes which have made industrial

processes much more viable from an economic point of view. The metagenomic approach

provides the researchers with an effective tool for exploring the various ecosystems for unknown

ORFs and enzymes, which are immediately needed for industrial products and processes (Olalla

et al., 2013). This holds true because the uncultivable mirobiota in extreme niches provide with

suitable enzymes with high specificity capable of working at all ranges of temperature and pH.

(Christel et al., 2007).

Here is a brief account of metagenomic research put into fruitful yield of various industrially

important hydrolases, viz., amylases, cellulases, lipases and proteases, as these enzymes are

undoubtedly the most dominant players in the industrial application regime.

2. 14 Hydrolases

Hydrolases are the enzymes that possess the characteristic activity of water driven cleavage of

anhydride bonds. Systematically, Hydrolases are named as “substrate hydrolases”. However,

they are also called sometimes as “substratease”. Typically, hydrolases find their immense

application in the food, pharmaceutical, cosmetic, textile, leather processing, paper, beverages,

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confectioneries and detergent industries. Best known examples of industrially important

hydrolases are proteases, amylases, lipases, cellulases, esterases, nitrilases and many more

(Enzyme Nomenclature, 1992). While proteases are key components of various detergents

(Niehaus et al., 2011, Showell 1999; Gupta et al., 2002), cellulases find extensive applications in

bioethanol research and industry (Collins et al., 2005; Ando et al., 2002; Lynd et al., 2002;

Xiong et al., 2012). Amylases are key components of food industry where sugar syrup and starch

liquefaction are required (Kiran and Chandra 2008; Syed et al., 2009). Lipases, especially

enantioselective ones, are key components of pharmaceutical industry where drug selectivity is

the key parameter for drug synthesis (Teena and Prerna, 2013).

Metagenomics is the newest tool in the market for searching novel enzyme coding genes. And

for a fact, this tool is being called into service by scientists world around. Moreover, the

ecological habitats supplying the gene pool for these genes are scattered all over the planet. Right

from tropical forest soils to the Antarctic coastal sediments, all possible permutations and

combinations of ecological niches have been and are currently being explored(Gilbert and

Dupont, 2011) There is an enormous variety of hydrolase enzymes for example, amylases,

cellulases, lipases and proteases that can be explored and studied through metagenomic

approaches from such a vast ecological habitats.

2. 15 Amylases

Amylases are starch hydrolyzing enzymes that include α-endo-amylases that hydrolyze α-1, 4-

glycosidic linkages, exo-amylases that include β-amylases which break α-1, 4 linkages from non-

reducing ends, glucoamylases which perform hydrolysis of α-1, 4 as well as α-1, 6 linkages and

the de-branching enzyme which primarily cleaves the α-1, 6 linkages in starch. There are three

different types of amylases:

1. α-Amylase

2. β-Amylase

3. γ – Amylase

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2. 16 Sources

The ability to utilize starch as a carbon and energy source is widely prevalent among animals,

plants and microorganisms. Some of the microbial sources of α-amylases are from bacteria (B.

acidocaldarius, B. amyloliquejaciens , B. cereus), Fungi (A. niger, F. oxysporum, H. lanuginose) and

yeast (E. jibuligera, L. starkeyi).

2. 17 Assay Methods

Amylase activity is estimated by measuring the amount of reducing monosaccharides released by

action of amylases on starch, as per the protocol of Somogyi and Nelson. (Somogyi, 1952 and

Nelson, 1944). Various other assays are also used to calculate amylase activity:

1. Measuring the reducing sugars released colorimetrically using soluble starch as substrate

and by the addition of 3, 5- dinitrosalicylic acid reagent (Sasmita and Niranjan, 2008).

2. The amylase activity in yeast strains can be estimated by introducing an equal number of

cells respective yeast isolates into agar diffusion holes. The activity is detected by

staining with Lugol’s solution and measuring the diameter of halo zone (Bertrand et al.,

2005).

2. 18 Industrial Applications

The utility of an enzyme becomes multiple folds if it is a thermostable/thermophilic enzyme.

Such enzymes have definitive potential applications, one of the notable ones is liquefaction and

saccharification, which requires high temperature of 100-110 oC. the thermostable amylases that

are being used on industrial scale for starch processing (Gomes et al., 2003). Some of the

applications are discussed in detail as follows:

2. 18. 1 Starch processing industry

α-amylases are the major stakeholders of the starch industry owing to their application in

liquefaction of starch for production of fructose and glucose syrups. The three main steps of this

process are: Gelatinization liquefaction saccharification, each stage utilizing a wide variety

of enzymes. This process is rendered even more efficient if coupled to high temperature steps.

This calls for better enzymes capable of functioning at high temperatures (Regulapatti et al.,

2007).

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2. 18. 2 Detergent industry

The use of enzymes in industries is well established because of the fast process and higher

efficiency of enzymes, coupled to eco-friendliness of the process (Hmidet et al., 2008).

Amylolytic enzymes are present in as much as 90% of all detergent formulations.

2. 18. 3 Biofuel industry

Among all the types of biofuels (biodiesel, bioethanol, biohydrogen) bioethanol is the most

widely used. For bioethanol production, fermentable sugars need to be produced from complex

polysaccharides, which can be directly fermented to ethanol. Starch is the most economically

available polysaccharide of all, which can be hydrolysed using amylolytic enzymes to yield

fermentable sugars (Moraes et al., 1999; Sanchez et al., 2008).

2. 18. 4 Paper industry

For preparing paper of writing quality, natural starch is unfit because of very high viscosity.

Thus, α-amylases are used to partially hydrolyze the starch to obtain a slurry of optimal

viscosity. This improves the writing quality as well as the coating quality of the paper. (Fryer et

al., 2009; Kuddus, 2010).

2. 19 Amylases from Metagenomes

With the advent of new frontiers in biotechnology, the metagenomics Dina et al., (2013) reported

the identification of unique enzymes from uncultured thermophilic microorganisms. An α-

amylase from an uncultured organism was identified encoding an ORF of 1, 461 bp. The gene

designated as amy13A was cloned in Escherichia coli, for over expression of the recombinant

amylase. Amy13A was highly thermoactive in nature. The optimum temperature of the enzyme

was 80 °C, and was active in 25 % (w/v) NaCl exhibiting high salt-tolerant property. The

novelty of this amylase was that it could be a suitable candidate for starch processing under harsh

conditions. Jalaja et al., (2011) reported a novel amylase from a metagenomic library constructed

from the soil samples collected from the Western Ghats of Kerala, India. The amylase was

studied for its thermostability, pH and salt tolerance, metal ion and inhibitor effects. The enzyme

was stable at high temperature with 60°C being the optimal temperature. The enzyme retained

more than 30% activity after 60 min incubation at 80°C. The pH optimum of the enzyme was at

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pH 5. 0. The enzyme activity enhanced in the presence of chelating and strong reducing agents at

5 mM β-mercaptoethanol, dithiothreitol and N-bromosuccinimide. However, the enzyme activity

was almost complete inhibited with 5 mM EDTA, while activity enhancement was observed

upon incubation with Ca2+

suggesting the enzyme be a Ca2+-

dependent α-amylase.

Wang et al., (2011) identified and characterized a novel thermostable gh-57 gene (α amylase)

from metagenomic fosmid library of the Juan De Fuca Ridge hydrothemal vent. The gene was

overexpressed in E. coli using pQE expression system. The recombinant protein showed an

optimal pH of 7. 5, an optimal temperature of 90°C, and its thermostability at 90°C retained over

50% enzyme activity for 4 h. The enzyme activity increased by DTT and Mg2+

while an

inhibitory effect was observed with EDTA and Ca2+

. These results showed that the gh-57 gene

was a novel thermostable amylase from oceanic microorganisms. An α-amylase was isolated by

screening libraries constructed from Himalayan soil as reported by Sharma et al., (2010) with

activity at 10°C to 30°C against amylose, soluble starch, glycogen and maltose,

2. 20 Cellulases

Cellulases are the enzymes that perform hydrolysis of cellulose. Cellulases act on the β-1, 4

glycosidic linkages of cellulose and hemi-cellulose and other cellulosic macro-complexes.

Structurally, cellulases contain 3 functional domains viz. catalytic core domain, cellulose binding

domain and linker domain.

2. 21 Classification

There are three different types of cellulases each having specific hydrolytic properties.

1. endo-1, 4-β-D-glucanase

2. exo-1, 4-β-glucanase

3. β-D-glucosidase

2. 22 Sources

Cellulases are known to be produced chiefly by fungi, bacteria (Aerobic thermophilic, Aerobic

mesophilic, Anaerobic thermophilic and Anaerobic mesophilic), protozoans, and termites, which

catalyzes the hydrolysis of cellulose (Watanabe et al., 1998 and Lee et al., 2000).

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2. 23 Assay methods

Different assays are designed to detect overall hydrolytic activity on selected substrates .

Principle methods used for detecting enzyme activity include:

1. Estimation of change in viscosity of substrate solution

2. Estimation of reducing sugars after substrate hydrolysis

3. Quantification of release of dye molecules from substrate-dye

The most widely accepted and used assay for overall cellulose activity is filter paper activity

(FPA) assay. It measures the capacity of an enzyme preparation to hydrolyze Whatman#1 filter

paper stripes into reducing sugars (Nieves et al., 1998).

Carboxymethyl-cellulase (CMCase) activity: CMCase activity is assayed using 1% CMC. The

reaction is stopped by adding 10% dinitrosalicylic acid (DNS) reagent and optical density is

recorded at 575 nm against the blank buffer (Wood and Bhat, 1998)

2. 24 Industrial Applications

2. 24. 1 Textile and laundry industry

Cellulases are used in textile industry for the process of stonewashing of denims (Biostoning),

which was earlier done by rubbing with pumice stones. Cellulases are also applicable for

removal of lint from clothes. Lint is small fibers of cellulosic material projecting outwards of the

clothes, giving them a rough appearance. Cellulases break down those non-cross linked fibers to

make the cloth appear shiny and glossy, a process called as Bio-polishing.

2. 24. 2 Paper Processing

Cellulases and hemicellulases are used in paper pulping industry for manufacturing paper. In the

process referred to as “bio-pulping”, the cellulases increase the saccharification, reduce the

viscosity so that the drainage property of the waste is increased and the mill runs faster (Sharyo

et al., 2002). The cellulases are also used for de-inking of laser and xerographic inks from used

paper (Hsu et al., 2002).

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2. 24. 3 Beer and wine industry

Breweries use barley for beer production, which contains high amount of cellulosic material. The

process of brewing includes saccharification of cellulosic material, followed by fermentation of

sugars by an alcohol tolerant strain of yeast or bacteria. When barley is malted, the germinating

seeds produce enzymes amylases, carboxypeptidases and endoglucanases, which hydrolyze the

reserve stored material in barley seeds.

2. 24. 4 Extraction of fruit/vegetable juices and olive oil

Cellulases, hemicellulases and pectinases form the class of macerozymes, which are used for

clearing of fruit and vegetable juices (Galante et al., 1998). These enzymes work by aiding in the

liquefaction of fruit pulp, to increase the yield of juice extraction. This enzyme mix is now also

being used in olive oil extraction and also enrichment of vitamin E and anti-oxidants in olive oil

(Cinar, 2005).

2. 25 Cellulases from Metagenomes

Metagenomics has been used to unlock novel cellulases from various natural environments,

namely, compost soils, soil from cold regions, rumen samples, and so forth by constructing the

metagenomic libraries followed by screening of the biologically active clones (Nacke et al.,

2012; Alvarez et al., 2013). These biocatalysts have gained considerable importance owing to

their potential candidature for the bioconversion of biomass into renewable liquid fuels. In fact

there is a major funding from Department of Energy, USA, for three bioenergy research centers:

one led by the Berkeley Argonne National Laboratory, one led by Oak Ridge National

Laboratory, and one led by the University of Wisconsin that will study all aspects of liquid

biofuel production from biomass sugars (http://genomicscience. energy. gov/centers/).

A breakthrough study in this regard was carried out by Hess et al. and identified 27, 755

candidate genes with a significant match to at least one relevant catalytic domain or

carbohydrate-binding module (Hess et al., 2011). They generated tremendous data in order to

demonstrate the potential of deep sequencing of a complex community to accurately reveal

cellulolytic genes at a massive scale and to generate draft genomes of uncultured novel

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organisms involved in biomass deconstruction. Other studies focusing on unlocking novel

cellulases of industrial importance from nature using metagenomics technique have met a lot of

success (Yeh et al., 2013). In addition, Alvarez et al. isolated and characterized a novel cellulase

from a sugarcane soil metagenome (Alvarez et al., 2013) while Duan et al. isolated and

characterized metagenomic gene encoding acidic cellulases from buffalo rumen metagenome

(Duan et al., 2009) and Voget et al. also characterized a metagenome-derived halotolerant

cellulase which is highly stable revealing the importance of metagenomics cellulases. The

metagenome-derived cellulase is ideal for industrial applications (Voget et al., 2006). Its

unconventional characteristics render it as a potential candidature to be employed in

biotechnological processes that require the more tolerant and alkaline cellulases.

Archana et al., (2013) discovered a gene (cel8M) encoding cold-active endocellulase (CEL8M)

from the cold desert soil of Ladakh region which represents a place for the search of new ‘cold-

adapted’ and functionally relevant enzymes to biotechnological and industrial applications.

cel8M was expressed in pET expression system . CEL8M displayed maximal activity at pH 4. 5

and remained significantly active when the temperature decreased to 10°C. resulting in the

application of cold-active endocellulase in textile industry at low temperature which can result in

energy savings. Recently, Ueda et al., (2010) reported a cellulase complex that could convert

cellulose directly into glucose from an earthworm living in a cold environment. Relatively at

elevated temperatures of 50–60°C, cellulose is converted to ethanol with an increase energy

consumption and high production costs. In this report, cellulosic material directly produced

ethanol at reduced temperature . This may prove to lead the production of biofuels at low

temperatures from cellulosic waste.

Given the amount of research that is underway in order to unlock novel cellulases from nature, it

is expected that the vast gene mining for cellulase enzymes will become literally possible in the

near future.

2. 26 Lipases

Lipases (triacylglycerol acylhydrolases), perform the breakdown of lipids to form glycerol and

long-chain fatty acids. Their activity is much similar to the esterase family of enzyme. The key

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50

difference is that esterases are capable of functioning in purely aqueous solution containing any

ester whereas lipases need an emulsion or oil- water interface for their activity. (Martinelle et al.,

1995). Several criteria have been used to distinguish true lipases also known as triacylglycerol

lipases, from carboxylesterases also called esterases. However, substrate specificity is the only

criterion completely valid now a day for this distinction due to the presence of several exceptions

with respect to other criteria previously used (Jaeger and Eggert, 2002; Fojan et al., 2000 and

Bornscheuer, 2002). Lipases are serine hydrolases.

2. 27 Sources

Lipases are known to be produced by almost every life form but the majority of industrially

applicable lipases are sourced yeast, fungi and bacteria. An example of mammalian lipase is the

pancreatic lipase, an exocrine enzyme of digestive pancreatic juices. Pancreatic lipase is obtained

from human and pig pancreas. Among all lipolytic enzymes, pancreatic lipase is the best known

and the most often investigated (Ferrer et al., 2001). This enzyme can be purchased from

different suppliers. Among bacterial lipases, attention has usually been focused on lipases from

the genus Pseudomonas which are especially interesting for biotechnology because they exhibit

the most versatility, reactivity and stability in catalyzing reactions in a non-aqueous environment

(Gao et al., 2000). Lipases from Geotrichum candidum and Rhizopus are attractive catalysts for

lipid modification. (Burkert et al., 2004).

2. 28 Assay Methods

Numerous methods for detection or quantification of lipolytic activity have been developed and

improved . Agar media methods are mainly applied in the detection of microbial lipases and are

classified into two categories:

i) Methods where lipolysis results in a change of substrate appearance: clear zones around colony

are observed after hydrolysis of tributyrin while opaque halos of calcium oleate appear in the

case of Tween 80 hydrolysis (Sierra, 1957).

ii) Methods where lipase activity is detected with indicator dyes: liberation of fatty acids during

lipid hydrolysis decreases the pH of the medium around the lipolytic colony. This pH decrease

can be detected by a colour change of an indicator dye present in the medium viz. Victoria blue

B, night blue, Nile blue sulphate and Spirit blue (Shelley et al., 1987). Fluorogenic reagents can

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51

also be used to detect lipolytic activity on solid media. For instance, substrate hydrolysis can be

visualised with rhodamine B dye which formed an orange fluorescent halo around lipolytic

colony visible under UV light (Kouker and Jaeger, 1987).

Usually these methods are applied for qualitative detection of lipolytic activity. Various

quantitative methods have been developed to measure lipase activity and most of them are based

on the time profile of product synthesis such as photometry, titrimetry, chromatography,

conductimetry, and IR spectroscopy methods. Turbidimetric method is based on the clearing of

substrate emulsion during hydrolysis.

2. 29 Industrial Applications

2. 29. 1 Detergent industry

Lipases are immediately applicable in laundry industry as well as household as detergent

additives because they hydrolyze fats and greases. The most important parameters that must be

kept in mind for an effective lipase preparation are: low substrate specificity so that the enzyme

can degrade various compositions of fats and greases, stability at high pH and temperatures as

most detergents provide a highly alkaline pH and are used at warm to hot temperature to remove

oils and fats from clothes and utensils (Yeoh et al., 1986, Wang et al., 1995 and Cardenas et al.,

2001) and protein engineering (Kazlauskas and Bornscheuer, 1998).

2. 29. 2 Food industry

Lipases have the capability to carry out trans-esterification reaction, which are highly applicable

for replacing the constituent fatty acid chains. For example, lipase from Rhizomucor miehei is

used in immobilized state for transesterification of palm oil, which replaces palmitic acid with

stearic acid, a saturated fatty acid. By using this method, a non-desirable oil/lipid can be

converted into a better nutritional value oil/fat (Colman and Macrae, 1980; Pabai et al., 1995;

Undurraga et al., 2001).

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2. 29. 3 Organic synthesis

Lipases can be used to perform numerous chemoselective, regioselective, and stereoselective

transformations of organic compounds (Rubin and Dennis, 1997, Kazlauskas and Bornscheuer,

1998 and Berglund and Hutt, 2000). A substantial proportion of such lipases is sourced from

microbes and is used extensively in organic synthesis. Another important application of lipases is

synthesis of enantiopure compounds, because of the enantioselectivity of lipases (Berglund and

Hutt, 2000).

2. 30 Lipases from Metagenomes

The hunt for novel lipases continues unabated as evidenced by the discovery of new families of

microbial lipases mostly by metagenomic approaches (Nagarajan, 2012). Among the hundreds of

sequences encoding lipases that have been identified Peng et al. isolated a novel alkaline-stable

lipase from a metagenomic library constructed from marine sediments and concluded that this

novel lipase may be used to impart a distinctive and desirable flavor and odor in milk fat flavor

production (Peng et al., 2014). Lee et al. isolated and characterized a novel metagenomic lipase

from tidal flat sediments which provided an evidence of a bacterial lipase belonging to a new

family (Lee et al., 2006). Hårdeman and Sjöling also isolated from uncultured bacteria of marine

sediment a novel low-temperature active lipase. The conserved regions, including the putative

active site and catalytic triad, were found to be similar to the culturable lipases (Hardeman and

Sjoling, 2007). A novel halotolerant lipase was isolated following a functional screening of a

marine sponge fosmid metagenomic library (Selvin et al., 2012). The stability and activity over a

wide range of salinity, pH, and temperature and in the presence of organic solvent and metal ions

suggest a utility for this enzyme in a variety of industrial applications. In the recent past, lipases

isolated and characterized by (Ngo et al., 2013; Fu et al., 2013; Chow et al., 2012; Glogauer et

al., 2011) from various metagenomic libraries showed novel characteristics, namely, thermal

stability, alkaline stability, organic solvent tolerance, cold active nature.

Tirawongsaroj et al., (2008) discovered patayin-like phospholipase (PLPl accession no.

EF413636) from a hot spring metagenomic library, which was expressed in E. coli. The enzyme

was a thermostable lipase, with an optimum activity at 70oC and pH 9. 0. The enzyme showed

remarkable stability at 70oC and retained its activity even after an incubation of 2 hours at 70

oC.

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The estimated molecular weight of PLP was found to be 29 kDa with an affinity for PNP-

butyrate. Elend et al., (2007) discovered a lipase from forest soil that played an important role in

biosynthetic reaction, displaying its promising application in hydrolyzing stereo-selectively

ibuprofen-pNP ester, with a high preference for the (R) enantiomer of >91% . In another study,

Mark et al., (2008) reported the screening of a metagenomic library which resulted in ~350 novel

lipases and esterases from environmental DNA samples, which showed high affinity for the

synthesis of 1, 2-Oleoyl-3-palmitoyl-sn-glycerol (OOP) and 1, 3-Oleoyl-2-palmitoyl-sn-glycerol.

The vast mining of genetically untapped sources for lipases of certain unique and desired

features like substrate specificity, enantioselectivity, extreme temperature, pH, tolerance, and so

forth using culture-independent metagenomic approach has proved it to be a promising approach

for biotechnological advancement.

2. 31 Proteases

Proteases and their potential applications have been known to the scientific world for a long time

now. Peptide Hydrolases have caught a lot of scientific attention not only because of their

indispensability in metabolic processes but also their industrial applicability. Proteases are

ubiquitous biocatalysts that are found in large amounts and diversity in the microbial world

(Gupta et al., 2002). Proteases or peptidases can be classified as exopeptidases and

endopeptidases depending on the location of the peptide bond under question (Bendtsen et al.,

2004; Pfennig et al., 1966).

2. 32 Sources

Proteolytic enzymes are available in plant, animal and microbial sources (Rao et al., 1998).

However, microbe are considered preferred source of protease because of ease of genetic

manipulation and protein expression. Microbial proteases account for approximately 40% of the

total worldwide enzymes (Horikoshi, 2008) and hold roun about a share (two- third) of the entire

commercial production of protease in the world (Horikoshi, 2011). Several protease types

(Toyokawa et al., 2010) and the protease research by applying different molecular biology tools

(Ningthoujam et al., 2009., Zhang et al., 2008) has been undertaken and studied.

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2. 33 Classification of Proteases

Based on the functional groups in their active sites proteases are classified into the following

types as shown in Table 2. 11.

Table 2. 11: Classification and EC number of proteases (Table courtesy: Hartley, 1960)

2. 33. 1 Endopeptidases

Metalloproteases

Aspartate proteases

Cysteine proteases

Serine proteases

2. 33. 1. 1 Metallo proteases

They are named so because of the presence of a metal ion in their active site, which is in most

cases Zinc divalent ion (Jeff et al., 2009). Their optimal range of pH is from 7. 0 to 9. 0.

Important representative members of this group are matrix metalloproteases (MMPs) the key

players in the process of apoptosis and ECM composition (Oliver et al., 1999).

2. 33. 1. 2 Aspartic proteases

Aspartic proteases are named so because of the presence of an aspartate residue in their active

site. The target sites for these enzymes generally contain amino acids phenylalanine, methionine,

leucine or tryptophan. They optimally work in acidic pH range and most of them have a pH

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55

optima near 2. 0. The most common example of this group is the digestive enzyme pepsin.

Others include Cathepsin D, chymosin, penicillopepsin and rennin (from vertebrate kidneys)

(Powers et al., 1993).

2. 33. 1. 3 Cysteine proteases

Cysteine proteases contain the thiol group of cysteine in their active site, which work on the

principle of nucleophilic attack on the substrate molecule. The notable members of this group of

proteases are papain (from papaya seeds), cathepsins B and H, streptococaal proteinases, and

calpains (cytosolic calcium activated proteases) (Barret, 1986).

2. 33. 1. 4 Serine proteases

Serine proteases are well-studied and characterized types of hydrolytic enzymes, which can be

identified by a nucleophilic serine residue present in the enzyme active site. The optimum pH for

most serine proteases known till date is in the neutral range and they are studied in two

categories viz. mammalian serine proteases and bacterial serine proteases (Garcia et al., 1993;

Polgar, 1987). The best known example of this class of proteases is the enzyme Subtilisin, a

bacterial serine protease, whereas trypsin, chymotrypsin, kallikrein and elastase are mammalian

serine proteases. It is believed that both these categories have evolved from the same ancestors

and achieved different specificities over the course of evolution (Polgar, 1987).

2. 33. 1. 5 Subtilisin

The microbial proteases are usually extracellularly and are specific for hydrophobic or aromatic

residues. Towards PMSF they are extremely sensitive. There are two major classes of subtilisin

as described below.

2. 33. 1. 6 Subtilisin Carlsberg

An enzyme capable of converting ovalbumin to plakalbumin was discovered by Gutenlberg and

Ottesen (1952). This enzyme was known as subtilisin carlsberg. The source of this enzyme was

B. pumilis and B. licheniformnis. Subtilisin Carlsberg is widely used in detergents. Enzyme has a

wide pH range of 5. 0-11. 0 for stability. At pH 10. 0 they are most active with a 15-39 kDa

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molecular weight (Gupta et al., 2005). The enzyme has wide substrate specificity and does not

depend on Ca2+

for its stability.

2. 33. 1. 7 Subtilisin Novo or bacterial protease Nagase (BPN’)

This alkaline serine protease was first purified and crystallized by Hagihara, (1958). It is mostly

present as a side activity in commercial preparation of Bacillus α-amylases.

2. 33. 2 Exopeptidases

2. 33. 2. 1 Aminopeptidases

Aminopeptidases are the proteases which cleave their thatget polypeptide at the free N-terminal

and cleave out an amino acid, a di-peptide or tri-peptide. They have been reported to be isolated

from many species of bacteria and fungi (Watson, 1976). They are mostly intracellular, an

exception being Aspergillus oryzae aminopeptidase, which is extracellular (Rao et al., 1998).

2. 33. 2. 2 Carboxypeptidases

Unlike aminopeptidases, carboxypeptidases act at a free carboxy terminus of a polypeptide to

cleave out an amino acid or a di-peptide. They have been further classified as serine

carboxypeptidases, cysteine carboxypeptidases and metallocarboxypeptidases.

As per the optimum pH, the proteases are categorized as acidic proteases, neutral proteases and

alkaline proteases (Paul et al., 2011).

2. 33. 2. 3 Alkaline Proteases

They are produced and secreted out by many bacteria and fungi. Bacillus genus is particularly

well known for producing alkaline proteases (e. g. B. licheniformis, B. megaterium, , B. firmus,

B. amyloliquifaciens, and B pumilus) along with the species of Streptomyces (e. g. S. fradiae, S.

griseus, and S. rectus) as well as some fungi (e. g. Aspergillus niger, A. sojae, A oryzae and A.

flavus). Until today, detergent alkaline proteases hold the biggest market proportion, which work

efficiently and stably at alkaline pH (Gupta et al., 2005).

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2. 33. 2. 4 Neutral Proteases

They are also produced and excreted out by both bacteria and fungi including Bacillus subtilis,

B. cereus, B. megaterium, Pseudomonas deruginosa, Streptomyces griseus, Aspergillus oryzae, A

sojae and Percularia oryzae. They are active at a pH of 7. 0. Neutral proteases are used in leather

industry and food industry. They are relatively unstable.

2. 33. 2. 5 Acidic Proteases

They are active at a pH of 5-2. The important fungal genera producing rennin are Aspergillus,

Candida, Coriolus, Endothia and Mucor. They include rennin, which is an important enzyme for

producing cheese. Many other acid proteases are used in medicines.

2. 34 Assay methods

There are various methods to assay proteolytic activity. The most, widely employed procedure

for determining protease activity is using sulphanilamide azocasein substrate according to the

method of (Leighton et al., 1973). Azocasein is a chemically modified protein containing

sulphanilamide groups (orange in colour), covalently linked to peptide bonds of milk protein

casein. During incubation for 1h, proteases hydrolyse peptide bonds, liberating shorter peptides

and amino acids from the chain. Trichloroacetic acid (TCA) is then added to precipitate the

enzyme and native azocasein, which can be removed by centrifugation. The low molecular

weight oligopeptides and liberated amino acids are not precipitated by TCA and thus, remain in

solution, which is orange in colour. The intensity of colour is measured spectrophotometrically

to determine protease activity.

Other assay methods for determination of protease activity include:

1. The protease enzyme activity can be determined by checking for zone of clearance in

gelatin agar diffusion technique of Elwan et al., (1986) which was later standardized by

(Ammar et al., 1998; Safey and Abdul-Raouf, 2004).

2. Proteinase activity assayed against casein and activity measured with Follins method at

600nm. ( Kim et al ; 2001)

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3. Proteinase activity was assayed against casein and tyrosine content was measured using a

Folin-phenol method at 578 nm (Huang et al., 2006).

4. Protease activity determined by incubating azocasein with enzyme solution and

absorbance was read at 440 nm. (Folasade and Joshua, 2005)

5. Green fluorescent protein (GFP) tagged protein A can also be used as a substrate, which

on cleavage by a protease will give a particular amount of fluorescence. It is a sensitive

assay and can measure picograms of tyrosine release and relative fluorescence (Hiroyoshi

et al., 2002)

6. A fluorescent zymogram in-gel assay using SDS polyacrylamide gel copolymerised with

a peptide-MCA (4-methyl-coumaryl-7-amide) substrate helps to assess the specificity of

a protease as well as the molecular weight of the enzyme (Yasothonsrikul and Hook,

2000)

7. Developing a fluorescence polarization technique, where a change in molecular volume

due to cleavage of intact fluorescein thiocarbamoyl (FTC)-casein molecules to smaller

FTC peptides is measured. This assay is more sensitive than other non radioactive

protease assays and requires no separations, precipitations or transfers of the reaction

mixture (Bolger and Checovich, 1994)

2. 35 Purification

A better understanding of the function of enzyme could be determined by purification of

enzyme. The primary objective in the purification programme is the removal of excessive

amount of water in the cell free extract. A direct method is concentration of extract using salts or

solvents with a high affinity which results in the precipitation of protein. In addition to this,

purification can be achieved by chromatographic and electrophoretic procedures (Manonmani

and Joseph, 1993; Su and Lee, 2001).

2. 35. 1 Enzyme concentration

After the culture is separated from the biomass by centrifugation or filtration, the supernatant by

ultra- filtration and by ammonium sulphate salting out method is concentrated (Thumar and

Singh, 2007; Reza et al., 2008; Purohit and Singh, 2011). Besides, the salt precipitation

extraction methods using ethanol or acetone as solvents (Thangam et al., 2002) and ethanol are

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also effective in order to purify the enzymes. Many chromatographic techniques can be used in

different combinations.

1. Affinity Chromatography: Protease purification by this chromatography technique uses t an

adsorbent –a hydroxyapatite to separate and purify the proteases (Gupta et al., 2005). However,

the enzyme cost and the labile property of some affinity legends limit the use of this technique.

2. Ion Exchange Chromatography: Generally, proteases alkaline in nature (positively

charged), cannot bind to anionic exchangers (Kumar, 1999). Therefore, the implication of

cationic exchangers can be the appropriate choice for the elution of the molecules that are bound

to the column by increasing the salt concentration or using pH gradient (Joshi et al., 2008). The

proteins that are positively charged (cationic proteins) require a negatively charged

carboxymethyl-cellulose (CM-cellulose) columns in order to get separated. The protein molecule

(adsorbed) is eluted by eluting buffer ionic strength or by a change in the pH gradient. 3.

Hydrophobic interaction chromatography: This approach uses the probability of external

residues of amino acid that are hydrophobic in nature on different proteins. These interactions

which are hydrophobic in nature are strengthened by increasing the salt concentrations and by an

increase in temperatures, and by the presence of detergents are weakened. Hydrophobic

interactions than ion exchangers are highly flexible. 4. Affinity precipitation: This type of

chromatography has a macromolecule regarded as soluble (macroligand and ligand polymer) that

possess two functions: (1) it has a polyvalent macromolecule, and (2) By changing the

temperature, ionic strength or pH it can be precipitated in many different ways. After elution of

proteins the polymer can be recycled. 6. Gel filtration: Keeping in view all the above

techniques, it is used for fast macromolecule separation on the basis of size. Currently, several

agarose based and cross-linked gels and highly rigid gels like Toyopearl, Superose, Sephacryl

and Superdex for purification purposes are also being used. An extreme halophilic bacterium

Chromohalobacter sp. Strain TVSP101 protease was purified using this chromatography to 180

fold with 22% yield (Vidyasagar et al., 2009). 7. HPLC: Through high-pressure liquid

chromatography (HPLC) the resolving power of all of the column techniques can be improved

substantially giving high resolution as well as rapid separation. Halophilic archaebacterium

strain 172 P1 was purified by HPLC technique (Seno, 2009). 8. Two-phase aqueous systems: It

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can be used for alkaline proteases purification by using mixtures of dextran and PEG or salts

such as H3PO4, MgSO4 and PEG (Sharma et al., 2006).

2. 36 Industrial Applications

Among all the enzymes used at industrial scale, the proteases are the largest stakeholders of the

enzyme market and are extensively used in a wide variety of applications (Gupta et al., 2002).

2. 36. 1 Food Industry

Milk contains specific proteins called caseins. Cheese production from milk requires curdling of

milk, which uses protease enzymes. Mainly, Rennet and Rennin are used in milk industry, from

which the chymosin isolates convert milk into cheese. Rennet and rennin are used to coagulate

milk in more than 70% of the milk products. Another well known application of proteases is of

the enzyme papain obtained from seeds of papaya (Carica papaya). Papain is used intensively in

meat tenderization. Bakeries also use crude and purified thermolabile proteases for gluten

hydrolysis in wheat flour (Abhijit, 2012).

2. 36. 2 Leather industry

Leather processing by chemical method involved pungent and harmful chemicals like hydrogen

sulphide and others, which create drastic environmental hazards. Thus it makes incredible sense

to use a more eco-friendly method for leather processing. Proteases offer such a solution for

tanning of leather as they are fast, specific, and easy to manipulate and produce lesser amounts of

waste. All these attributes make them eco-friendly. (Andersen, 1998). The proteases having

specificity towards keratin and elastin are the most suitable candidates for the leather industry.

These enzymes can efficiently perform the functions like leather tanning and de-hairing (Varela

et al., 1997).

2. 36. 3 Photographic industry

Extraction of used silver from used X-ray and photographic films is now being done by

bioprocessing using alkaline proteases, as compared to the previously used incineration method,

which produced a lot of air pollution and non-biodegradable tar (Abhijit, 2012).

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2. 36. 4 Detergent industry

The first application of proteases in detergent industry is by Rohm and Haas, who used proteases

isolated from pancreas in conjunction with washing soda (Sodium carbonate) as detergent

additives. But the first industrial application of a protease in detergents was reported in 1963

when alcalase, was added to detergent ‘BIOTEX’ as an alkaline protease by novo industry in

Denmark. Among proteases, a significant portion of the market share is held by the serine

protease called Subtilisin, isolated form Bacillus species. In order to be used as a detergent

additive, the protease enzyme should be compatible to detergents, surfactants as well as

bleaching reagents, which formulate the majority of washing powder. (Kumar et al., 1998), they

should possess high stability and efficiency at working temperatures and pH (Beg et al., 2002).

Conventionally, detergents have been used with luke warm to hot water, hence, currently there is

great interest in discovering alkaline proteases capable of working at high temperatures (Oberoi

et al., 2001).

2. 36. 5 Silk degumming

Silk industry is a recently recognized potential thrust area for protease applications. Degumming

of silk is aimed at processing of the protein sericin, which makes upto 25% of the raw weight of

silk (Kanehisa, 2002 and Annavarapu, 2011). Conventionally, sericin was removed from silk

for shrink proofing by use of starch (Kanehisa, 2002). It is a time consuming and expensive

process and can be replaced by use of enzymes such as proteases.

2. 36. 6 Medical usage

The application of proteases in pharmaceutical products is still under much R&D. an important

example is the enzymatic preparation called elastoterase from B. subtilis 316M protease, which

may be used for treating carbuncles, burn wounds and deep abscesses (Kudraya and Siminenko,

1994). Kim et al., (1996) from Bacillus strain CK 11-4 isolated an alkaline protease, which could

be used for dissolving blood clots confirming it to be a thrombolytic agent with fibrinolytic

activity. Proteases are also used in pharmaceutical products such as lens-cleaners and de-briding

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solutions, which help in wound healing by removal of dead cells at the wound site (Anwar and

Saleemuddin, 2000; Sjodahl et al., 2002).

2. 37 Proteases from Metagenomes

Several proteases have been discovered using metagenomic approach in the recent times Biver et

al, (2013) isolated an oxidant stable serine protease gene from functional metagenomic libraries

of forest soils suggesting its application in detergent and bleaching industries. The protease

SBCas3.3 had a molecular weight of 80kDa in its inactive form. Two serine proteases from

surface sand of deserts were found to be relatively resistant to detergent, making them interesting

for possible industrial applications. A novel protease belonging to chymotrypsin-like S1 serine

proteases was isolated by (Niehaus et al., 2011). Neveu et al. also isolated two serine proteases

from metagenomic libraries of the Gobi and Death Valley deserts (Neveu et al., 2011), while

Paul et al. identified and characterized metagenomic alkaline serine protease from the

metagenome of goat skin surface (Paul et al., 2011). This class of enzymes had not been

described earlier for use in laundry and cleaning applications.

Zhang and co-workers (2011) reported novel mesophilic protease from a cosmid library of

Antarctic coastal sediment. The library was constructed in the cosmid pWEB-TNC and over

expression was achieved in pET-His plasmid. This protease called ACPRO001 (accession

number CAQ57698) is a mesophilic protease belonging to the subtilase family, with an optimum

activity at 60oC and pH 9. The enzyme retained almost 3/4

th of its activity after an incubation of

2 hours at 50oC. ACPRO001 is reported to be a calcium-dependent serine protease inhibited by

PMSF.

Lee et al., (2007) reported a new fibrinolytic metalloprotease from a fosmid library of deep-sea

clam bed community sediment (accession no. EF100137). Their protease worked best at 50oC

and neutral pH and the activity was optimal in presence of Co2+

as well as Ca2+

. The peculiarity

of this protease was its differential affinity towards fibrin and hence the authors claim it to be a

candidate for use as a therapeutic agent against thrombosis.

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Proteases also find use in diagnostic industries and thus a lot of effort is needed to uncover novel

proteases with better characteristics with special emphasis on metalloproteases and serine

proteases from different habitats including extreme environments.

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