1. “SCREENING OF MANGROVE FUNGAL ISOLATES
FOR THEIR BIOPROSPECTS: PARTIAL
OPTIMIZATION OF PHYSICO-CHEMICAL
CONDITIONS FOR
PRODUCTION OF L-ASPARAGINASE BY
UNIDENTIFIED MANGROVE FUNGAL ISOLATE, T2”
-DEBJYOTI PAUL
MSc (IBT), IVTH
SEM

2. INTRODUCTION
• Mangrove ecosystem:
• A dynamic ecotone (or transition zone) between terrestrial &
marine habitats (Gopal & Chauhan 2006).
• Harbors unique microbial diversity.
• Present in coastal areas of tropical countries & supports
abundant life through a food chain that starts with the trees
and the micro-biota (Smith et. al, 1991).
• Mangrove ecosystem largely stochastic & thus the need to
understand & predict ecosystem response to human induced
stresses that may directly affect coastal ecosystem.

3. CONTINUED...
• Present study (focus):
• Fungal diversity of mangrove ecosystem prevailing in Kutch
region of Gujarat, India.
• Diversity study of mangrove fungi:

4. CONTINUED…
• SAPROPHYTIC FUNGI:
• Fundamental to many aspects of decomposition & energy flow
in mangrove forests in addition to litter degradation.
• Significant role in nutrient recycling.
• ENDOPHYTIC FUNGI:
• Symbiotic association between fungi & photosynthetic
organisms: both ancient & ubiquitous.
• Protects their hosts against various aggressions.
• Good source of triterpenes: folk medicines for various
diseases.

5. CONTINUED…
• Well known endophytic fungi:
Mycorrhizal fungi
E.g., Conversion of insoluble inorganic phosphate salts of Ca,
Al, or Fe into soluble/available form.

6. CONTINUED…
• PATHOGENIC FUNGI:
• Foliar diseases : significant effects on plant survival, growth &
fitness in natural ecosystems.
• Usually infect top of mangrove plants.
• Reports suggest unique defense mechanisms such as salt
extraction & microbial metabolites protecting mangrove plants
from fungal attacks.

7. Why explore ‘Manglicolous’ fungi?
• They possess unique structures, metabolic pathways,
reproductive systems, sensory & defence mechanisms which
need to be identified.
• Urgent need to develop a fundamental understanding of the
genetic, nutritional, and environmental factors that control the
production of primary and secondary metabolites in mangrove
fungi, as a basis for developing new and improved products.

8. Fugal identification by ITS 1 & ITS 4 primers
• PCR targeting the 18S rDNA and internal transcribed spacer
(ITS) regions are increasingly used to study fungal communities
(Prosser, 2002; Korabecna, M., 2007).
• In these methods, DNA is extracted from the environmental
sample and purified. Target DNA (16S, 18S or ITS) is amplified
using universal or specific primers and the resulting products are
separated in different ways.
• ITS 1(TCCGTAGGTGAACCTGCGG): forward primer
• ITS 4(TCCTCCGCTTATTGATATGC): reverse primer

9. L-asparaginase
• L-asparaginase (EC 3.5.1.1) is an enzyme which catalyzes the
hydrolytic reaction of L-asparagine into L-aspartic acid and
ammonia.
• Occurs abundantly from prokaryotes to vertebrates.
• Fungal asparaginase were reported from molds like
Aspergillus niger or Aspergillus oryzae and patented for
industrial use (Laan et al., 2008; Matsui et al., 2008).

10. MECHANISM OF ACTION

11. OBJECTIVES
 To screen mangrove fungal isolates as source for various
industrially important enzymes
 To study and optimize the production of any one enzyme
based on screening results by a selected fungal isolate.
 To identify all the fungal isolates on the basis of ITS sequence.

12. MATERIALS,
METHODS
WITH
RESULTS & DISCUSSION

13. THE FUNGAL ISOLATES
• 13 fungal isolates in pure form were made available to me.
They are coded as: AF (Aerial pneumatophore) (1, 2, 3, 7 ,8,
9, 10, 11), UF (Unsterile Underground pneumatophore) (1, 2,
3) and T1, T2.
• These fungal cultures were isolated from sediments, twigs,
leaves, roots of mangrove from Jakhao, Kutch, Gujarat, India.
• They were maintained by repeated subculturing on Potato
Dextrose Agar plates amended with 3% (w/v) NaCl.

14. Screening of fungal isolates for various
enzyme activities
• PROTEASE:
• Fungal cultures spot inoculated on PDA amended with 3% (w/v)
NaCl and 1% (w/v) casein.
• Upon incubation the plates were flooded with Frazier’s reagent
(15g/100ml HgCl2(w/v) dissolved in 2N HCl up to final volume
of 100 ml).
• Transparent zone of casein hydrolysis around fungal growth
represented protease production.

15.  Of the 13 fungal isolates screened, 7 isolates were found to be
good protease producers.
Plate showing positive protease activity by strain
AF7

16. CONTINUED…
• CELLULASE:
• The fungal cultures were screened for cellulase activity by spot
inoculating them on basal salt agar medium + 1%
Carboxymethylcellulose (CMC) + 3% (w/v) NaCl.
• Upon incubation the plates were flooded with 0.1 % Congo red
followed by treatment with glacial acetic acid.
• Development of whitish- purple zones around the fungal
growth ascertained positive cellulase activity against
background of deep - violet colored complex.

17. Plate showing positive cellulase activity by strain UF1
Two strains (AF3 & AF10) of the total 13 isolates showed
very good cellulase activity while 8 strains showed good
activity .
RESULTS:

18. CONTINUED…
• L-ASPARAGINASE:
• The fungal isolates were incubated in agar (2%) (w/v) plates
supplemented with L-asparagine; dextrose (carbon source)
(0.2g/100mL); MgSO4 (10mg/100mL); K2HPO4 (50mg/100mL)
phenol red (0.009%) (w/v) (as pH indicator) and 3% (w/v)
NaCl for 48 hrs. at 30˚C.
• The production of L-asparaginase was detected by pink zone
around colonies of L-asparaginase producing cultures, formed
due to release of NH3 from L-asparagine, shifting pH towards
alkaline side and indicated by phenol red.

19. Plate exhibiting positive L-asparaginase activity (pink zone)
10 of the total 13 isolates showed good positive l-asparaginase
activity during plate assays as confirmed by the pink zones around
the cultures
RESULTS:

20. CONTINUED…
• LIPASE:
• The fungal cultures were screened for lipase activity by spot
inoculating them on BSM amended with 3% (w/v) NaCl and
1% tributyrin oil (v/v) (TBO) as the sole lipid (carbon) source.
• The production of lipase by fungal cultures was detected by
transparent zone of tributyrin hydrolysis around colonies.
• RESULTS:
 8 cultures showed positive lipase activity of the 13 isolates.

21. CONTINUED…
• AMYLASE :
• The fungal cultures were screened for amylase activity by spot
inoculating them on BSM agar amended with 3% (w/v) NaCl
and 1% (w/v) starch.
• Upon incubation the plates were flooded with iodine reagent and
transparent zone of starch hydrolysis around fungal growth
represented amylase production.
• RESULTS:
 Only 2 strains showed very good amylase activity while of the
rest of the total 13 isolates, 5 strains showed positive activity.

22. Plant Growth Promoting Activities:
1. INDOLE ACETIC ACID PRODUCTION:
• To test indole acetic acid production each isolate (actively
growing fungi) was inoculated in 5 ml of BSM medium and
incubated for 96 h under shaking condition (120rpm) at 30˚C.
• 1 ml culture supernatant obtained after centrifugation at 8000
rpm for 25 min. was mixed with 2 drops of o-phosphoric acid
(v/v) (35%), 2 ml 0.5M FeCl3(Salkowski’s reagent).
• Assay system was allowed to stand in dark at room temp. for 1
h and development of pink color indicated IAA production.

23. CONTINUED…
2. PHOSPHATE SOLUBILIZATION:
• The fungal cultures for their phosphate solubilizing ability
were screening by cultivation on Pikovaskaya’s agar (2%)
plate at 30˚C for 72 h.
• The appearance of transparent zone indicated phosphate
solubilization activity of fungal cultures.
• RESULTS:
 Other than strain T1, none of the fungal isolates exhibited
plant growth promoting activities.

24. THE RESULTS OF SCREENING:

25. SCREENING OF SELECTED STRAINS FOR
L-ASPARAGINASE PRODUCTION
L-asparaginase production by the selected 5 strains show peak activity upon 96 h.
incubation

26. DISCUSSION
• In this experiment, agar plate assays and spectrophotometric
methods are compared & it was found that some isolates had no
enzyme activity despite producing a large positive pink zone in
the agar plate assay.
• As many as seven cultures produced positive zone on agar plates
but two of the strains had no enzyme activity or insignificant
activity in the Nesslerization assay .
• Findings similar to the work reported by Holker et al., (2004):
enzyme production of fungi different in solid and submerged
fermentation.
• 5 strains selected for Nesslerization assay.

27. MONITORING L-ASPARAGINASE PRODUCTION OF HIGHER
PRODUCING ISOLATES FOR 6 D.
Plot exhibiting l-asparaginase production by AF7 & T2 for a period of 144 h.

28. OPTIMIZING CULTURE CONDITIONS FOR HIGHEST
L-ASPARAGINASE PRODUCER STRAIN
1. EFFECT OF CARBON SOURCE :
Effect of different C-source on production of L-asparaginase by strain T2 & biomass produced
in each case.

29. 2.EFFECT OF pH ON L-ASPARAGINASE PRODUCTION :
Effect of different pH on strain T2 for L-asparaginase production monitored upon
48h of incubation corresponding to peak activity.

30. SALT TOLERANCE OF STRAIN T2:
Plot showing growth of fungal strain T2 at different NaCl concentrations

31. CONCLUSION
 Of the total 13 mangrove fungal isolates, the strain coded
‘T2’, produced maximum extracellular asparaginase with
better half life and activity as compared to all the L-
asparaginase positive strains.
 Strain T2 exhibited better extracellular L-asparaginase activity
& production in presence of Lactose as carbon source &
showed optimum activity at pH 10.

32. FUNGAL IDENTIFICATION

33. Genomic DNA
of AF9 & AF10
repectively
Amplified ITS region of their respective
genomic DNA.

34. RESULTS :
• The DNA of all the fungal strains were isolated and subjected to
polymerase chain reaction for the amplification of ITS region
by using ITS1 (forward primer) and ITS4 (reverse primer) of
the respective genomic DNA of the fungal strains.
• However, only the genomic DNA of 2 of the isolates (AF9 &
AF10) could be amplified for ITS region. Further, the sequence
of ITS region obtained could not match with the pre-existing
known fungal ITS database.

35. DISCUSSION
• The unidentified ITS sequence of the fungal strains could have
paved way for identification of novel strains.
• The rest of the isolated DNA could not lead to ITS region
amplification which could be due to the following reasons:
 PCR inhibitors.
 Improper standardized conditions for PCR amplification of the
its region of the genomic DNA of the various fungal strains
having distinct morphology and characteristics.
 Shearing of the genomic DNA of the fungal isolates leading to
loss of ITS region.