Literature Review:

The research is based on the analysis of the secondary structure of phytase

enzyme acknowledging its immense importance to the economy and the

environment. This portion shall be dedicated to study the literature and the

work that is associated with phytase enzyme with special emphasis to FTIR

Analysis. As per Lei et al., 2007, phytase enzymes have been one of the

most important and the most researched enzymes during the last two

decades due to their ecological, environmental, and economical importance.

Detailed work has been dedicated to enhance and improve the thermo

stability and design of formulations of chemical coating for protecting the

enzymes from heat-denaturation (Lehmann et al., 2000). Dozens of

exogenous compounds have been discovered to stabilise the structure of

Phytase during palleting (Phillippy, 2002). Phytases are known to break the

ortho-phosphate groups from the inositol core of phytate, the major

chemical form of phosphorus in plants (Lei et al., 2007).

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Phytate is a polyanionic particle, which chelates healthfully significant

cations like iron, calcium and zinc (Bhonet al., 2008). Phytate is likewise

accumulated in other plant organs like pollens, tubers and roots (Raboy,

2003). Grouping of phosphorous in soil is high however it is for the most

part present in inaccessible structure as phytate. Plants require inorganic

phosphorous, whose presence is reliant on soil pH. It is invested in

monovalent structure and most extreme assimilation happens between pH

5.0 and 6.0 (Schachtman, 1998).

The binding of nutritionally significant cations has although a positive

aspect as they are stored but however, this leads to anti nourishing properties

to phytate. This brings down the bioavailability of phytate phosphorous and

minerals in people and other non-ruminant creatures like poultry and pigs

(Hanafi et al., 2013). Unhydrolyzed phytate when discharged during feed

preparing or in the gut of creatures, phytate ties minerals, essentially iron

and zinc, making them inaccessible as dietary components. This coupling

relies upon elements like pH, size and valancy of mineral and amount of

phytic acid. Likewise, unutilized phosphorous of phytate is discharged from

plant feed into condition prompting phosphorus contamination (Yao et al.,

2012 ; Greiner and Konietzny, 2006).

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Owing to low bioavailability of phosphorus, exogenous phosphate is

included eating regimen of domesticated animals to maintain a strategic

boundary from phosphorus insufficiency. Hence, enzymatic breakdown is

another fundamental pathway so as to free phosphorous alongside mineral

cations for ingestion as portrayed in Figure 2.1. This will likewise lessen

fecal discharge of phosphorus. Phytases are as a rule widely used to

assemble phosphorous as an added substance in feeds of swine and poultry

(Bhon et al., 2008; Yao et al., 2012).

Figure 2.1: Action of phytase on phytate. ((Yao et al., 2012).

A multitude of living beings are utilized as a source to acquire phytase

enzyme. Among bacterial strains, Peniophoralycii, E.coli and

Schizosaccharomycespombe species produce phytase which is being

utilized economically (Bhon et al., 2008). Other than bacterial and

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contagious sources, wheat phytases (EC 3.1.3.26) were first detailed by

Posternak and Posternak in 1929 and portrayal of chemical was finished by

Collatz and Bailey (1921), and Kolobkowa (1936). Whereas, the forst

commercial phytase product Natuphos was launched in 1991. (Lei et al.,

2007). Owing to this, the market volume surged up to ca. 150 million euro

and is still expected to rise with the newer applications. However, the major

application so far is the augment the phosphorus bioavailability in feed

supplements (Lei et al., 2007)

Every single diverse examination proposes that wheat phytases are most

dynamic at feeble acidic to impartial pH. Their ideal action lies between pH

4.5 and 7.2, with certain special cases that shows most astounding

movement at pH 6 (Bhon et al., 2008). Major mechanical use of phytase is

its utilization an added substance in feed of poultry, pigs and somewhat for

fish feed. Swine, poultry and fish guts have stomach related tract at unbiased

pH and have lower body temperature. These life forms are monogastric with

constrained measure of gut microbial compound which can hydrolyze

phytate.

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Until now, four classes of phosphatase enzymes have been found out that

degrade phytic acid including Histidine acid phytase (HAP), Beta-Propeller

Phytase (BPP), Cystine Phosphatase (CP) & and Purple Acid Phosphate

(PAP). They all have individual structural convolutions that enable them to

utilise phytic acid as a substrate from multiple environments. (Lei et al.,

2007)

The basic features of phytase enzymes have been linked and associated to

previously known classes of phosphatases (Chu et al., 2004). While in some

cases, X-Ray crystallographic studies have linked the structure of phytase

to the novel catalytic mechanism. However, the current research has

enlightened our understanding of the multi dimensional structure of phytase

enzyme and it has also established the connected linkage between the

structure and the catalytic functions to broader extents (Lei et al., 2007).

Structural data on protein–protein interactions can't be effectively

determined and hence the major research on proteins have been limited to

the examination of single proteins. Techniques, for example, cryo-electron

microscopy, X-ray scattering, mass spectrometry, X-raycrystallography,

NMR spectroscopy, surface plasmon resonance, atomic force microscopy,

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isothermal calorimetry, fluorescence spectroscopy, circular dichroism

spectroscopy, Raman spectroscopy and Fourier transform infrared

spectroscopy can be involved to consider protein–protein interaction.

Fourier Transform Infrared Spectroscopy (FTIR) that is mainly indulged in

the analyses of the secondary structure of biological molecules. Other than

that, protein mis-foldings and aggregate formation can also be analysed and

studied through FTIR (Miller et al., 2013). FTIR is a powerful yet relatively

inexpensive technique for the analyses of the membrane lipids in their

plethoric polymorphic phases (Lewis & McElhaney, 2012). The vibrational

modes within protein can be harnessed to excavate the protein secondary

structure, amino acid side chain structure as well as protein dynamics and

stability (Haris 2013).

X-ray crystallography and NMR spectroscopy are without a doubt the most

dominant procedures as they can be utilized to determine the structure of a

protein–protein complex at nuclear resolution. Be that as it may, they are

not free from issues. Just 1.08% of the settled protein X-Ray structures.

(Haris 2013) This is an extremely modest number considering the way that

approximately 20–30% of qualities in many genomes encode for membrane

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proteins (William et al., 1998). On the other hand, FTIR has a few highlights

that make it especially profitable for auxiliary examination of film dynamic

proteins and peptides (Haris & Chapman, 1995)

The primary structures of proteins at a nuclear resolution were resolved in

the late 1950s (Kendrew et al., 1958). From that time to the mid 1990's,

around 300 protein structures were added to the protein data bank, basically

utilizing X-ray crystallography. At present more than 20,000 structures are

settled, a portion of these utilizing the more current strategy of

multidimensional nuclear magnetic resonance spectroscopy (NMR).

As it is known, the functional-structural of biological molecules relationship

vary in various environments and forms and hence the analyses and study

of these proteins also becomes complex to intimidating extents. However,

FTIR has the ability to study the structure (and functions consequently) in

various environments such as solid states, aqueous solutions and suspension

forms. FTIR also is known to not damage the protein-protein membrane

complexes at various temperature and pH and FTIR independent of the size

of protein to be studies (Haris 2013)

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One novel approach to retain all the features of FTIR alongwith giving the

ability to charaterize samples in fluids which is elemental in biophysical

research. This is done through combining hybrid infrared spectroscopy with

SPM platforms involving the mating of SPM scanner with ATR crystal

holder (Li et al., 2013).

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