“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

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.

CONTINUED...• Present study (focus):

• Fungal diversity of mangrove ecosystem prevailing in Kutch

region of Gujarat, India.

• Diversity study of mangrove fungi:

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.

CONTINUED…• Well known endophytic fungi:

Mycorrhizal fungi

E.g., Conversion of insoluble inorganic phosphate salts of Ca,

Al, or Fe into soluble/available form.

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.

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.

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

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).

MECHANISM OF ACTION

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.

MATERIALS,

METHODS

WITH

RESULTS & DISCUSSION

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.

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.

Of the 13 fungal isolates screened, 7 isolates were found to be good protease producers.

Plate showing positive protease activity by strain AF7

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.

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:

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.

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:

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.

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.

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.

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.

THE RESULTS OF SCREENING:

SCREENING OF SELECTED STRAINS FOR

L-ASPARAGINASE PRODUCTION

L-asparaginase production by the selected 5 strains show peak activity upon 96 h.

incubation

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.

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.

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.

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.

SALT TOLERANCE OF STRAIN T2:

Plot showing growth of fungal strain T2 at different NaCl concentrations

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.

FUNGAL IDENTIFICATION

Genomic DNA of AF9 & AF10

repectively

Amplified ITS region of their respective genomic DNA.

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.

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.