Production of tannase from microbes

1. COMPARITIVE STUDY BETWEEN FUNGAL
AND BACTERIAL sp., FOR THE
PRODUCTION OF TANNASE ENZYME
Project work

2. ENZYMES
 Catalytic proteins are called as enzymes. Enzymes stimulate the rate
of several reactions in the body without themselves getting affected in
the process
 Enzymes play a diversified role in many aspects of everyday life
including aiding in digestion, the production of food and several
industrial applications.
.

4. TANNASE
A tannase (EC 3.1.1.20) is an enzyme that catalyzes the
chemical reaction
digallate + H2O = gallate
 Thus, the two substrates of this enzyme are digallate and
H2O, whereas its product is gallate
 This enzyme belongs to the family of hydrolases,
specifically those acting on carboxylic ester bonds. The
systematic name of this enzyme class is tannin
acylhydrolase. Other names in common use include
tannase S, and tannin acetylhydrolase.

5. TANNIN
 The term tannin refers to the use of wood
tannins from oak in tanning animal hides into
leather; hence the words "tan" and "tanning"
for the treatment of leather.
 Tannin is an astringent, bitter plant
polyphenolic compound that binds to and
precipitates proteins and various other
organic compounds including amino acids
and alkaloids.

6. OBJECTIVES
 Isolation and Identification of microorganisms from
natural sources.
 Screening of microbes for the production of tannase
enzyme.
 Extraction and purification of tannase enzymes
 To compare, the production and molecular
determination of tannase enzyme between fungal
and bacterial species

7. COLLECTION OF SAMPLES
 The effluents were collected freshly from
vegetable tanning industry by using sterile
conical flasks. The flasks were covered with
paper and plastic cover and transported as
quickly as possible to the laboratory by
keeping them in a ice –box. The samples
were processed and stored in refrigerator

8. ISOLATION OF MICROBES
BACTERIA
The collected samples were serially diluted upto 10-5
dilution.
↓
0.1 ml of each dilution was transferred to sterileplates
containing nutrient agar medium adnd spreaded with a sterile
L – rod.
↓
Then the plates were incubated at 337ºc for 24 hrs.
↓
The bacterial growth was observed and the colony morphology
was noted down periodically

9. FUNGI
The effluent sample collected was serially diluted up to 10 –7
dilutions
↓
1ml of each of the dilutions was transferred to a sterile plate
and pour plate technique was carried out using Potato dextrose
agar.
↓
Plates were incubated at 21- 25 °C for 48 hrs.
↓
Fungal growth was observed and the colony morphology
was noted down periodically.

10. SCREENING OF MICROBES
 A selective media called as tannic acid agar medium is used to
isolate the tannase producing fungal specimen.
 PROCEDURE
 The medium was sterilized with all the ingredients (except
tannic acid)
 10ml of filter-sterilized tannic acid was added after the
sterilization and the medium was poured after mixing well.
 The medium was allowed to solidify and inoculated with fungal
isolates.
 The plates were incubated at 37 °C for 24hrs.
 The halo zone around the colonies was then observed.

11. In tannic acid agar medium, all organisms were
inoculated to detect their ability to produce
tannase enzyme. After incubation all organisms
showed clear zones around the colonies. In this
stage, we can conclude that isolated organisms
were tannase producers

12. IDENTIFICATION OF MICROBES
 Bacterial and fungal organisms were isolated from soil.
According to morphological and biochemical properties the
organisms were identified as Bacillus, Lactobacillus, Mucor and
Penicillium
 As a result of spread plate technique, the Mucor produced
blackish to brown mycelia and Penicillium produced greenish to
yellow mycelia on PDA medium
 And the Bacillus produced pale white color, irregular, lobate and
flat colonies on nutrient agar medium and Lactobacillus
produced pale white color, powdery colonies on specific MRS
medium

13. BIOCHEMICAL RESULTS
BIOCHEMICAL
TEST
Bacillus Lactobacillus
Indole test Negative Negative
Methyl red test Positive Positive
Voges proskauer
test
Negative Negative
Citrate utilization
test
Negative Negative

14. TANNASE PRODUCING
MICROORGANISMS
UNGI
 Aspergillus
 Mucor
 Pencillium
 Trichoderma
 Rhizopus
BACTERIA
 Bacillus
 Lactobacillus
 Cryptococcus
 Citrobac Fer
 Corynebacterium

15. PRODUCTION OF ENZYME BY
SUBMERGED FERMENTATION
 The enzyme was produced by means of surface culture on a liquid medium
(Zapek Dox minimal medium) containing tannic acid as the sole source of
carbon
 PROCEDURE :
 All the ingredients were added and sterilized at 121°C for 15 lbs for 15 minutes.
 After sterilization 10 ml of 1% tannic acid solution was added to the production media.
 2-3 loopful of culture was inoculated into the bacterial and fungal production media.
 Both the media were incubated at 21-25°C for 3-4 days till a fine mat growth was seen
on the surface.
 Then these cultures were used as starter inoculums for the production of enzyme in the
conical flasks and then all the above procedures were followed.
 .

16.  In the tannic acid production medium both
fungal and bacterial colonies showed good
growth under the respective incubation
period.
 The produced enzyme was extracted by
filtration and centrifugation, without disturbing
the stability of the enzyme

17. EXTRACTION OF ENZYMES
 The extraction of the enzyme is done by means of
homogenization of the fungal mycelium and bacterial cells to
extract the intracellular enzyme.
 PROCEDURE
 The flask with fully-grown bacterial culture was taken and the
crude was filtered using Whatmann filter paper.
 It was then transferred into a sterile mortar and pestle and
homogenized.
 Equal volume of 0.01M acetate buffers was added to the crude
and homogenate was centrifuged at 10,000 rpm for 15 minutes
at 4°C using a cooling centrifuge.
 The supernatant obtained was transferred to a fresh tube and
this was used as a crude enzyme extract.

18. PURIFICATION OF ENZYME
The enzyme was purified by using the following method
• Ammonium sulphate precipitation
• Dialysis
• Ion Exchange chromatography

19. ESTIMATION OF PROTEIN: (Lowry et
al., 1951):
 Though proteins are estimated by various methods.
Hydrolyzing the proteins and estimating the amino acids alone
will give the exact quantification. The method developed by
Lowry O.H et al., (1951) is a standard method, which gives
moderately constant value, and hence this method is selected
for our studies. The estimation was made based on the blue
color developed by the reduction of the phosphomolybdate and
phosphotungstic components in the Folin-Ciocalteau reagent
by the amino acids, tyrosine and tryptophane present in the
protein and the color developed by the reaction of the protein
with the alkaline cupric tartarate were measured in the Lowry’s
method.

20. APPLICATIONS
 Tannase is a key enzyme in the degradation of
gallotannins, a type of hydrolysable tannins. It is present
in a diverse group of microorganisms, including rumen
bacteria.
 It hydrolyses tannic acid to gallic acid, a key intermediate
required fot the synthesis of an antibiotic drug,
trimethoprim (TMP)
 It is used in the manufacture of instant tea, beer, wine.and
ti is a inducible enzyme
 Partially purified tannase in acetone can be used as
clarifying aid of fruit juices.

21. Tannin rich fresh water draining into
Cox Bight from Freney Lagoon,
Southwest Conservation Area,
Tasmania, Australia

22. Food items with tannins

23. RESULT
 In the tannic acid liquid medium, the isolated microbes
were grown well. The enzyme production was started after 3
hours of incubation, and within two days enzyme production
reaches highest level. The Bacillus and Lactobacillus showed
optimum activity at 37oC and the Mucor and Penicillium
showed at 40 oC.
 The crude and purified enzyme activity of bacterial
tannase showed less activity than fungal tannase. In fungi,
Mucor showed more activity than Penicillium and in bacteria,
Bacillus showed more activity than Lactobacillus. Therefore we
can conclude that fungi are the best tannase producer than
bacteria. In this research work Mucor is the best tannase
producer, when compared to others.
