STUDY OF EFFECT OF PHYTASE ON PLANT GROWTH

A PROJECT REPORT
Submitted in partial fulfilment of the requirement for the award of degree of

BACHELOR OF TECHNOLOGY
IN
BIOTECHNOLOGY ENGINEERING














ACKNOWLEDGEMENT


TABLE OF CONTENTS
Abstract

CHAPTER

1.Introduction
2.Objectives
3.Literature Review
4.Materials and Methods
5.Results and Discussion
6.Process Flow Chart
7.Conclusion
8.Future Prospects
List of References
Appendix 1












ABSTRACT

The phytin molecule in its uncomplexed state is referred to as phytic acid. Phytic acid
(phytate; myo-inositol 1,2,3,4,5,6 hexakisphosphate is the primary source of inositol and
storage phosphorus in plant seeds contributing ~70% of total phosphorus .The abundance of
phytic acid in cereal grains is a concern in the foods and animal feeds industries because the
phosphorus in this form is unavailable to moogastric animals due to a lack of endogenous
phytases; enzymes specific for the dephosphorylations of phytic acid. In addition, the strong
chelating characteristic of phytic acid reduces the bioavailability of other essential dietary
nutrients such as minerals ,proteins and amino acids.Phytase is an extracellular enzyme that
can catalyse the hydrolysis of phytic acid to inorganic monophosphate and lower myosin. The
ability of phytase to make phytate phopsphorus available for plant nutrition during the low
phosphate concentration , contributes to plant growth promoting activity. In order to study the
effect of phytase enzyme in plants growth, the legume seeds (chickpea seeds) were grown
with and without the addition of enzyme in two sets of paper cups and were incubated for 15
days.Then the two sets were compared for chlorophyll content,root weight,shoot weight,root
length and shoot length.

Key words: phytin,phytic acid,phytase












CHAPTER 1
INTRODUCTION



A phytase (myo-inositol hexakisphosphate phosphohydrolase) is any type of phosphatase
enzyme that catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate) --an
indigestible, organic form of phosphorus that is found in grains and oil seeds-- and releases a
usable form of inorganic phosphorus.While phytases have been found to occur in animals,
plants, fungi and bacteria, phytases have been most commonly detected and characterized
from fungi.








1.1CLASSES OF PHYTASE

Four distinct classes of phytase have been characterized in the literature: histidine acid
phosphatases (HAPS), B-propeller phytases, purple acid phosphatases, and most recently,
protein tyrosine phosphatase-like phytases (PTP-like phytases).

Histidine acid phosphatases (HAPs)

Most of the known phytases belong to a class of enzyme called histidine acid phosphatases
(HAPs). HAPs have been isolated from filamentous fungi, bacteria, yeast, and plants. All
members of this class of phytase share a common active site sequence motif (Arg-His-Gly-X-
Arg-X-Pro) and have a two-step mechanism that hydrolyzes phytic acid (as well as some
other phosphoesters). The phytase from the fungus Aspergillus niger is a HAP and is well
known for its high specific activity and its commercially marketed role as an animal feed
additive to increase the bioavailability of phosphate from phytic acid in the grain-based diets
of poultry and swine. HAPs have also been overexpressed in several transgenic plants as a
potential alternative method of phytase production for the animal feed industry and very
recently, the HAP phytase gene from E. coli has been successfully expressed in a transgenic
pig.

-propeller phytases

-propeller phytases make up a recently discovered class of phytase. This first examples of
this class of enzyme were originally cloned from Bacillus species,[2] but numerous
microorganisms have since been identified as producing -propeller phytases.[7] The three-
dimensional structure of -propeller phytase is similar to a propeller with six blades. Current
research suggests that -propeller phytases are the major phytate-degrading enzymes in water
and soil, and may play a major role in phytate-phosphorus cycling.






Purple acid phosphatases
A phytase has recently been isolated from the cotyledons of germinating soybeans that has
the active site motif of a purple acid phosphatase (PAP). This class of metalloenzyme has
been well studied and searches of genomic databases reveal PAP-like sequences in plants,
mammals, fungi, and bacteria. However, only the PAP from soybeans has been found to have
any significant phytase activity. The three-dimensional structure, active-site sequence motif
and proposed mechanism of catalysis have been determined for PAPs.
Protein tyrosine phosphatase-like phytases
Only a few of the known phytases belong to a superfamily of enzymes called protein tyrosine
phosphatases (PTPs). PTP-like phytases, a relatively newly discovered class of phytase, have
been isolated from bacteria that normally inhabit the gut of ruminant animals.All
characterized PTP-like phytases share an active site sequence motif (His-Cys-(X)5-Arg), a
two-step, acid-base mechanism of dephosphorylation, and activity towards phosphrylated
tyrosine residues, characteristics that are common to all PTP superfamily enzymes. Like
many PTP superfamily enzymes, the exact biological substrates and roles of bacterial PTP-
like phytases have not yet been clearly identified.Interestingly, the characterized PTP-like
phytases from ruminal bacteria share sequence and structural homology with the mammalian
PTP-like phosphoinositide/-inositol phosphatase PTEN and significant sequence homology to
the PTP domain of a type III-secreted virulence protein from Pseudomonas syringae
(HopPtoD2).
1.2.BIOCHEMICAL CHARACTERISTICS
Substrate specificity
Most phytases show a broad substrate specificity, having the ability to hydrolyze many
phosphorylated compounds that are not structurally similar to phytic acid such as ADP, ATP,
phenyl phosphate, fructose 1,6-bisphosphate, glucose 6-phosphate, glycerophosphate and 3-
phosphoglycerate.Only a few phytases have been described as highly specific for phytic acid,
such as phytases from Bacillus sp., Aspergillus sp., E. coli and those phytases belonging to
the class of PTP-like phytases.
Pathways of phytic acid dephosphorylation
Phytic acid has six phosphate groups that may be released by phytases at different rates and
in different order. Phytases hydrolyze phosphates from phytic acid in a stepwise manner,
yielding products that again become substrates for further hydrolysis. Most phytases are able
to cleave five of the six phosphate groups from phytic acid. Phytases have been grouped
based on the first phosphate position of phytic acid that is hydrolyzed. The Enzyme
Nomenclature Committee of the International Union of Biochemistry recognizes three types
of phytases based on the position of the first phosphate hydrolyzed, those are 3-phytase (EC
3.1.3.8), 4-phytase (EC 3.1.3.26), and 5-phytase (EC 3.1.3.72). To date, most of the known
phytases are 3-phytases or 4-phytases, only a HAP purified from lily pollen and a PTP-like
phytase from Selenomonas ruminantium subsp. Lactilytica have been determined to be 5-
phytases.
1.3.BIOLOGICAL RELEVANCE
Phytic acid and its metabolites have several important roles in seeds and grains, most notably,
phytic acid functions as a phosphorus store, as an energy store, as a source of cations and as a
source of myo-inositol (a cell wall precursor).Phytic acid is the principal storage forms of
phosphorus in plant seeds and the major source of phosphorus in the grain-based diets used in
intensive livestock operations. The organic phosphate found in phytic acid is largely
unavailable to the animals that consume it, but the inorganic phosphate that phytases release
can be easily absorbed. Ruminant animals can use phytic acid as a source of phosphorus
because the bacteria that inhabit their gut are well characterized producers of many types of
phytases. However, monogastric animals do not carry bacteria that produce phytase, thus,
these animals cannot use phytic acid as a major source of phosphorus and it is excreted in the
feces.
Phytic acid and its metabolites have several other important roles in Eukaryotic physiological
processes. As such, phytases, which hydrolyze phytic acid and its metabolites, also have
important roles. Phytic acid and its metabolites have been implicated in DNA repair, clathrin-
coated vesicular recycling, control of neurotransmission and cell proliferation. The exact
roles of phytases in the regulation of phytic acid and its metabolites and the resulting role in
the physiological processes described above are still largely unknown and the subject of
much research.
Phytase has been reported to cause hypersensitivity pneumonitis in a human exposed while
adding the enzyme to cattle feed.
1.4.AGRICULTURAL AND INDUSTRIAL USES

Phytase is produced by bacteria found in the gut of ruminant animals (cattle, sheep) making it
possible for them to use the phytic acid found in grains as a source of phosphorus.Non-
ruminants (monogastric animals) like human beings, dogs, birds, etc. do not produce phytase.
Research in the field of animal nutrition has put forth the idea of supplementing feed with
phytase so as to make available to the animal phytate-bound nutrients like calcium,
phosphorus, other minerals, carbohydrates and proteins
Phytase is used as an animal feed supplement - often in poultry and swine - to enhance the
nutritive value of plant material by liberation of inorganic phosphate from phytic acid (myo-
inositol hexakisphosphate). Phytase can be purified from transgenic microbes and has been
produced recently in transgenic canola, alfalfa and rice plants. Phytase can also be produced
on a large scale through cellulosic biomass fermentation using genetically modified (GM)
yeast. Phytase can also be isolated from basidiomycetes fungi. A strain of transgenic pig can
produce phytase, thus reducing their environmental impact.
CHAPTER 2
OBJECTIVE


This project aims to achieve the following objects
1.To study the effect of phytase on legume plants(chickpea) by estimating the chlorophyll
content
2.To compare the length and weight of plants grown with and without the addition of
enzymes

















CHAPTER 4
MATERIALS AND METHODS


MATERIALS
Seeds
Paper Cups
Soil
Sand
Rice Bran
Measuring Cylinder
Beakers
Filter Paper

CHEMICALS USED
0.2M monobasic sodium phosphate(27.8g/L)
0.2M dibasic sodium phosphate(53.65 g/L)
10g of Phytase
Acetone
Water

INSTRUMENTS USED
Weighing machine
Spectrophotometer







PREPARATION OF ENZYME

27.8 g of monobasic sodium phosphate was weighed and dissolved in 1L water to give stock
solution A (0.2M monobasic sodium phosphate 27.8g/L).Similarly stock solution B was
prepared by adding 53.65g of dibasic sodium phosphate in 1L of water (0.2M Dibasic sodium
phosphate 53.65g/L).390 ml of stock solution A and 610 ml of stock solution B were mixed
together to prepare phosphate buffer of pH 7.To this buffer ,10g of phytase enzyme was
added and stirred well.
GROWING OF SEEDS
Six paper cups were taken and filled with soil,sand and 1g of rice bran in each cup.These
cups were divided into 2 sets,each with 3 cups.In the first set, chickpea seeds were sown .0.5
ml of phytase enzyme and 4.5 ml of water were added to make a total of 5ml.To the second
set,5ml of water was added .This was continued for 15days.
DETERMINATION OF LENGTH AND FRESH WEIGHT
The 15 days old plants were taken out from the cups carefully without the detachment of
root. Shoot and root length were measured using a 30cm scale. Then separate the root and
shoot using a blade and weigh them separately.Compare the results between the plants with
and without the enzyme.
CHLOROPHYLL ESTIMATION
Extraction of chlorophyll
For the estimation of chlorophyll ,colourimetric method of acetone preparation was
adopted.About 0.5g of both phytase positive and negative leaves were taken separately.6.6ml
of acetone was taken in a measuring cylinder .The leaves were crushed using a mortar and
pestle .The acetone solution was slowly added during crushing.The leaves were completely
crushed until a green solution was obtained.This is due to the fact that the chlorophyll has
been extracted from the leaves.the green solution is filtered and is collected in test tubes
covered with aluminium foils and they were subjected to colourimetric analysis.
Colourimetric Analysis
The absorbence of the extracts was found out at 4 different wavelengths.From standard
calculations ,chlorophyll content was estimated.







RESULTS AND DISCUSSIONS

5.1WEIGHT MEASUREMENTS
WITH ENZYME WITHOUT ENZYME

ROOT WT(g) 0.53 ROOT WT 0.24(g)
SHOOT WT(g) 0.74 SHOOT WT 0.45(g)

5.2LENGTH MEASUREMENTS
WITH ENZYME WITHOUT ENZYME

ROOT
LENGTH(cm)
ROOT
LENGTH(cm)

SHOOT
LENGTH(cm)
SHOOT
LENGTH(cm)


5.3.CHLOROPHYLL ESTIMATION

WAVELENGTH(nm)


665
645
ABSORBANCE
PHYTASE
POSITIVE
PHYTASE
NEGATIVE
2.084 1.586
1.367 0.944








PHYTASE POSITIVE

Amount of Chlorophyll a in the leaves:
C=12.65A
665
-2.79A
645

=21.715g/ml

PHYTASE NEGATIVE

Amount of Chlorophyll a in the leaves:
C=12.65A
665
-2.79A
645

=17.429g/ml
The amount of chlorophyll a ,the chief component of chlorophyll in pea plants,was found to
be higher in those plants which were grown in phytase enzyme.Thus we can conclude that
plants aided by phytase was healthier as they had an advantage of increased amount of
phosphorus ,which is an important nutrient for plants.












CHAPTER 6
PROCESS FLOW DIAGRAM




















CHAPTER 7
CONCLUSION


The degradation of phytate by phytase is a very effective means for the utilisation of phytate
as a phosphorus source .The total content of pigment is found to be the same in both phytase
positive and negative plant leaves, as they were from the same kind of seeds. But the amount
of chlorophyll a, the chief component of chlorophyll was found to be higher in those plants
grown in the presence of phytase enzyme. Thus we can conclude that the plants which were
aided by phytase were healthier as they had an advantage of increased amount of phosphorus,
an important nutrient for plants. As a result of phosphorus released by phytase, the plant
grown in positive medium had more phosphorus content. Hence the positive medium showed
more healthy growth.














CHAPTER 8
FUTURE PROSPECTS



Compare the plant growth factors like fresh weight, length etc

Development of fertilizers of high phytase content


Application of phytase in botanical garden as well as in agriculture

Finding out new methods to produce phytase on high scale