This proposal outlines a research project to produce and purify the enzyme tannase from fungal sources. Tannase is an extracellular enzyme produced by fungi and bacteria in the presence of tannic acid. The research aims to identify the best method for tannase production and purification by comparing liquid surface fermentation, solid state fermentation, and submerged fermentation using different fungal species. Several purification techniques will be tested and compared, including ion exchange chromatography, gel filtration, and ammonium sulfate precipitation to determine the highest purification level. The candidate has an MSc in biotechnology and relevant research experience producing and purifying tannase, giving them qualifications to complete the proposed work.

1. PROPOSAL
Introduction:
All enzymes are high molecular weight proteins. Enzymes may be defined as
biocatalysts synthesized by living cells. They are protein in nature, colloidal thermo labile in
character, and specific in their action.
In 1878, Kahne used the term enzyme to indicate the catalysis taking place in the
biological systems. The active site, of an enzyme represents as the small region at which the
substrate(s) binds and participates in the catalysis. Enzymes in general have the ability to
function under relatively mild conditions of temperature pH and pressure.
Tannase or tannin acyl hydrolyses (E.c.3.1.1.20) Catalyses the hydrolysis of ester
bonds present in the hydrolysable tannins and Gallic acid esters, releasing glucose and Gallic
acid as final products. Enzymes are sometimes considered fewer than two broad categories.
Intracellular enzyme, they are functional within cell, where they are synthesized.
Extracellular enzyme, these enzymes are active outside the cell, all the digestive enzymes
belong to this group.
Tannase is and Extracellular, inducible enzyme produced in the presence of tannic
acid as and inducer and sole carbon source. This enzyme is produced by certain filamentous
fungus such as Aspergillus, Pencilliuml, Fusarium and by certain Bacteria and yeast
.Production of tannase can be done by various methods like liquid surface, submerged and
solid-state fermentation. At Industries level, it is produced by microbial means using
submerged fermentation (SmF), where the activity is expressed mainly in intracellur form;
however, the commercial forms are partly pure and have low tannase activity. Tannase
produced by solid-state fermentation (SSF) recently. The enzymes produced in SSF is
extracellulaar.Lekha and Lonsane (1994) reported the production of an Extracellular
thermostable tannase,with higher titres in SSF as compared to SmF.Species of Aspergilli
were reported as the best tannase producers in submerged and solid state fermentation .
Tannase enzyme can purified by various methods, such as Ion exchange
chromatography, Gel filtration, SDS-PAGE, ultracentrifution and ATPE.This purified

2. tannase was able to cleave the β,1-4 linkage of a disaccharide cellobiose,resulting in a novel
function for the tannase from A.niger.
The enzyme is utilized in a number of industrial applications, including the
manufacture of instant tea, wing and Gallic acid. Tannase is also used in production of food,
juice, cosmetic, pharmaceutical and coffee-flavoured drinks. It has also become an important
aid in the efficient treatment of wastes and pollutants, especially effluents and solid wastes
rich in organic constituents. But it has and important application in pharmaceutical industry
for the production of Trimethoprim, an antibiotic derived from gallic acid.
The reason for the research work:
I have a lot of interesting in enzyme activity and microbiology, as well as I have done my
research on Tannase enzyme in my masters. So I would like to do my thesis work related to
that field because it will be more curious to me with interesting.
Aim of the thesis:
My research aims to provide a grate method for production and purification of Tannase
Enzyme from the fungal sources.
Objective of the study:
Enzymes can produce from different sources as well as it can be purified by different
methods, however, the purification level may be differing among the methods. So according
to the purification level, easily find out the proper production method whereas purification
techniques like Ion-exchange chromatography, Gel filtration method, Ammonium sulphate
precipitation and Aques two phase extraction will also use to know the higher purification level
containing best method.
Review of literature:
Methodology:
A) Tannase:
Tannase (tannin acyl hydrolase, Ec 3 .1. 20) is an enzyme produced in the presence of
tannic acid by various filamentous fungi, principally Aspergillus and penicillium. Other
tannase producers include bacteria and yeast.
Tannase is an Extracellular, inducible enzyme produced in the presence of tannic acid
as a carbon source. The Tannase enzyme produced by different methods such as Liquid
surface fermentation (LSF), Solid state fermentation (SSF) and Submerged fermentation
(SmF). Various fungal Species are also use to produce these enzymes.

3. Tannase used in the preparation of instant tea and manufacture of acorn liquor. Tannase is
also used in the production of Gallic acid, which is an important intermediary compound in
the synthesis of antibacterial drugs.
Tannase enzyme can be observed from different sources like plants, animal and microbial
resources, but microbes are the major source of Tannase.
Production of Tannase:
The foundation of my research work for production of Tannase found in the study which is
the ‘Tannase is an enzyme produced in the presence of tannic acid by various filamentous
fungi, principally Aspergillus and Penicillium and other Tannase producers include bacteria’
(Deschamps et al., 1980; Kumar et al., 1999; Mondal and Pati 2000) and Yeast (Aoki et al.,
1976). As my thesis work focus on Tannase enzyme production and different purification
process and also identify the techniques, which provide the higher level of purification.
Fungal sample will be used in sub-merged fermentation method and solid state fermentation
method for the production of Tannase enzyme.
Work plan:
My work will have six main chapters include the introduction and conclusion, production
process will have to do till get the best purified sample by two different species like
Aspergillus and Pencilium. Already, I have studied some research papers regarding my work,
and also have done much work related to this topic on my Master degree in a research
institute, Chennai, India. I’m planning to do my work on production of Tannase by other
different fungal species, definitely, contamination will affect the process. However, it will be
reduced through the proper handling.
The selected purification techniques are ion-exchange chromatography, gel
permeation chromatography, SDS-page, ATPE, affinity chromatography, Ammonium
sulphate precipitation method, HPLC and ultrafiltration etc. I’m able to do this all techniques
with good perfection to get a good level of purification, and regarding the suggestion of my
prof, I can add extra other processes also. Although, I have selected different methods, I have
a confident to complete all of the process, report and my dissertation before the end of the
period.
Qualifications of the Candidate:
In 2006, I graduated with second class (Lower division) from the Bachelor of Science in
Jaffna University, Srilanka. After that I have completed my Master degree in Bio-technology
in Madras University, India. My dissertation, entitled in central leather research institute,

4. Chennai: The production and purification of Tannase enzyme from fungal culture. As well as,
when I was doing masters, I have done so many presentations, and joined with the students,
whose had followed research work for their PhD, for covered my training period in a research
institute. I believe that these are important starting point to me to having an interest and
continue my research with encouragement. If I had completed my research work and
dissertation lonely, I have strength also to finish my work with successfully. Furthermore, I
got good comments and grade from our lecturers to my dissertation and presentation work in
both my University and Research institute. Since my graduation, I have been interesting to do
the MPhil or PhD in any of the recognised universities, this proposal may lead that position to
me.