28

CHAPTER 3

RESULTS AND DISCUSSION

3.1 METAL FATTY ACID SALTS AS CARBON SOURCE

FOR RHAMNOLIPID PRODUCTION

3.1.1 Growth curve analysis

The growth curve for Pseudomonas aeruginosa was obtained in

different media containing metal fatty acid salts as sole carbon source,

PPGAs, and nutrient broth. The turbidity of the culture was measured using

Hitachi model 3010 UV-spectrophotometer. As shown in the Figure 3.1 the

culture growth was confirmed through the increase in optical density in every

1 hour of analysis. The optical density was measured at 600 nm. The growth

pattern had a usual lag, log and stationary phases. The growth curve was also

compared with culture grown in nutrient broth.

Figure 3.1 Growth curve of P.aeruginosa in different media

29

Figure 3.2 Rhamnose standard graph

3.1.2 Rhamnolipid production in metal fatty acid containing media.

Rhamnolipid production was quantified for 7 days using standard

orcinol method (Chandrasekaran and Bemiller 1980). Rhamnolipid was

extracted and quantified from the culture supernatants obtained from the

different media. Rhamnose standard as shown in Figure 3.2 was used to

quantitate the rhamnolipid production. It was observed that calcium stearate,

calcium palmitate containing mineral media as shown in Figure 3.3 gave the

maximum yield of an average of 1100 µg/ml when compared to the reported

production media PPGA of 1070 µg/ml. Though the difference in production

is not significant, fatty acids salts are inexpensive and could be an alternative

to the existing production media. The reason for the better production of

rhamnolipids could be that fatty acid containing substrates stimulates, las/rhl

pathway for more rhamnolipid production so as to assimilate hydrophobic

substrates in the media.

30

Figure 3.3 Rhamnolipid production in metal fatty acid salts containing

media

3.1.3 Thin layer chromatography analysis of rhamnolipid

Rhamnolipids produced in PPGAs media were analyzed by thin

layer chromatography using chloroform: methanol: acetic acid in the ratio of

65:15:2. Mono and di-rhamnolipids were observed as separated spots as

shown in Figure 3.4 as they differ in polarity due to the difference in number

of rhamnose group in rhamnolpid molecules.

31

Figure 3.4 TLC of mono and di rhamnolipid

3.1.4 High Performance Liquid Chromatography analysis of

rhamnolipids

The type of carbon source, nitrogen sources, influences the types of

rhamnolipids produced by the organism. In this study, rhamnolipid congeners

produced in different media composition were analyzed through HPLC and

mass spectrometry. 4-Bromophenacyl bromide derivatized rhamnolipids from

both reported PPGAs media and metal fatty acid salts containing media were

analysed through C18 column separately. Derivatized rhamnolipids from

PPGAs media gave peaks as shown in Figure 3.5, which are different from

their media containing metal fatty acid, salts.

32

Derivatized rhamnolipid from PPGAs media showed four different

prominent peaks at retention time of 15 min, 23 min, 25 min, and 27 min. The

peak at 15.12 min corresponded for 4-Bromophenacyl bromide of molecular

weight of 198. Rest of the peaks corresponded to derivatized rhamnolipid.

The two peaks 15.12 min and 27.93 were analyzed by mass spectrometry.

Rhamnolipids from media containing metal fatty acid salts as carbon sources

showed a single peak other than p-Bromopehnacyl bromide peak. The peaks

for rhamnolipid derivatives appeared at 15.182, 15.186, and 12.816 mins for

calcium stearate, calcium laurate, and calcium palmitate respectively as

shown in Figures 3.6, 3.7, 3.8. All fractions corresponding to the peaks were

collected and analyzed using mass spectrometry.

Figure 3.5 HPLC of rhamnolipid derivatives from P. aeruginosa in

PPGAs media

33

Figure 3.6 HPLC of rhamnolipid derivatives from P.aeruginosa in

media with magnesium palmitate

Figure 3.7 HPLC of rhamnolipid derivatives from P.aeruginosa in

media with calcium stearate

34

Figure 3.8 HPLC of rhamnolipid derivatives from P.aeruginosa in

media with calcium laurate

3.1.5 Mass spectrometry analysis of rhamnolipids

Mass spectrometric analysis of rhamnolipid showed several

homologues ranging from 874 mz 1 and 475 mz 1 respectively. The proton

abstraction for rhamnolipid molecules yielded [M-H] 1 anions for the

fragment Rha-C10-C8 at 437 mz 1. The fragments obtained for rhamnolipids

were similar to earlier published reports (Haba et al 2003). The fragments can

broadly be classified as mono-rhamno mono-lipidic, mono rhamno di-lipidic,

di-rhamno mono-lipidic and di-rhamno di-lipidic (Arino et al 1996). The

fragments obtained in our study revealed only as mono-rhamno-mono-lipidic,

mono-rhamno-di-lipidic, and di-rhamno-di-lipidic congeners. HPLC analysis

of derivatized rhamnolipids from PPGAs revealed the presence of mono and

di-rhamnolipids in equal proportion. While derivatized rhamnolipids from

metal fatty acid salt containing media showed only one peak containing

mostly of di rhamnolipids. The reason could be the presence of hydrophobic

metal fatty acid salts in the media, made them to produce di rhamnolipids.

Apart from Pseudomonas aeruginosa, there are several other bacteria known

to produce different congeners of rhamnolipids (Abalos et al 2001 Haba et al

35

2003, Gunther et al 2005; Sharma et al 2007, Dubeau et al 2009). For

instance, Mono- and di-rhamnolipid with C10 -C10 from Acinetobacter

calcoaceticus (Rooney et al 2009), rhamno-amino-lipids from Myxococcus sp.

(Ohlendorf et al 2009), L-ornithine lipids, composed of L-ornithine coupled to

iso-3-hydroxyfatty acid (C15 C17) and iso-fatty acid (C15 or C16) from

Myroides sp. have been reported (Maneerat et al 2006)

In our study, rhamnolipid derivative fractions collected from HPLC

were analyzed through mass spectrometry. Rhamnolipids obtained from

different media showed different fragmentation pattern. Though rhamnolipid

derivatives from metal fatty acid salt containing media had shown single peak

at same retention time, mass spec analysis revealed a difference in

fragmentation pattern. Interestingly rhamnolipid derivative from PPGAs

media had shown three prominent peaks in HPLC, out of which two peaks,

were selected for mass spectrometry analysis. Both the fractions contained

different proportions of mono and di rhamnolipid. Molecular weight of 4-

Bromophenacyl bromide (after derivatization mol.wt-198) was taken in to

account while calculating m/z values for fragments. The 23rd min fraction

from PPGAs as shown in Figure 3.10 had shown mostly fragments containing

di-rhamnolipids like (Rha2-C10-C12+D)-874 m/z. As shown in the Figure 3.9

the 27th fraction of HPLC from PPGAs media had shown mostly mono

rhamnolipids. The Table 3.1 lists the possible fragments obtained for 23rd min

and 27th fractions from HPLC of derivatized rhamnolipids from PPGAs

media. This could be due to less polarity of mono rhamnolipid than di

rhamnolipid causing retention in the column. The rhamnolipid derivatives

from other media containing metal fatty acid salts as carbon source as shown

in Figures 3.6, 3.7, 3.8, were showed peaks with different retention time in

HPLC and revealed mostly di rhamnolipid fragments like Rha2-C10-C12-D

with m/z 872 during mass spectrometry as shown in figures 3.11, 3.12, 3.13

and list of fragments were summarized in tables 3.2, 3.3, 3.4 respectively.

36

Figure 3.9 Mass spectrometry of rhamnolipid derivatives of 27th min

fraction (PPGAs) collected from HPLC

Figure 3.10 Mass spectrometry of rhamnolipid derivatives of 23rd

fraction (PPGAs) collected from HPLC

37

Table 3.1 List of possible Mass spectrometry fragments for

rhamnolipids from PPGAs media

Possiblefragments(4th

fraction)

m/z Possible fragment(2nd

fraction)m/z

C8+D 340 Rha-C8-C10 +D 476

Rha-C10-C8+D 537 Rha2-C12-C10 +D 628

Rha-10-C10+D 702 C6-C8 254

Rha-C10-C12:1 +D 727 C8+D 339

Rha-C10-C12 +D 729 Rha-C10-C10 +D 702

C10-C12 +D 564 Rha-C12-C12 +D 760

Rha2-C10-C12:1 874 C8+C8+D 475

Rha-C8-C10 476 C6-C8+D 453

Rha2-C10-C10 650 Rha-C6 +D 475

Rha2-C10-C12 678 Rha-C6-C10 +D 642

Rha2-C12-C10+D 678 Rha-C6-C12 +D 679

C6-C8+D 453 Rha-C8-C12 +D 702

Rha-C10-C12 +D 726

Rha-C12-C12 +D 756

Rha-C14-C12 +D 784

D: 4-Bromophenacyl bromide Mol. Wt 198

38

Figure 3.11 Mass spectrometry analysis of rhamnolipid derivative from

magnesium palmitate containing media

Table 3.2 List of possible mass spectrometry fragments for

rhamnolipids from magnesium palmitate media

Mg PalmitatePossible fragments M/z valueC8+D 340Rha-C10-C8-D 537C8-C6-D 453Rha2-C10-C10 650Rha2-C12:1-C10 674Rha2-C10:1-C12 674Rha-C10 335Rha-C12-C10 534Rha2-C10-C10 649Rha2-C10-D 534Rha-C10-C8 474Rha2-C8-C10+D 672

39

Figure 3.12 Mass spectrometry analysis of rhamnolipid derivative from

calcium stearate media

Table 3.3 List of possible mass spectrometry fragments for

rhamnolipids from calcium stearate media

Possible fragments(fraction)

m/z

Rha-C10-D 537

Rha-C10-C10+D 701

Rha-C10-C12:1+D 727

Rha-C8+C10 476

Rha2-C10-C12 +D 678

Rha2-C12-C10 +D 678

C6-C8-D 453

Rha-C10 333

Rha2-C10-C12:1 530

40

Figure 3.13 Mass spectrometry analysis of rhamnolipid derivative from

calcium laurate media

Table 3.4 List of possible Mass spectrometry fragments for

rhamnolipids from calcium laurate media

Possiblefragment(fraction) m/z

C8-D 476

Rha-C10-C10-D 702

Rha2-C10-C12-D 872

Rha-C8-C10 476

Rha2-C10-C12 678

Rha-C12-C10 678

Rha2-C10+C8 618

C8+D 339+2H

C8 +C8-D 480

C6-C8 +D 453

41

3.2 SYNTHESIS OF RHAMNOLIPID CAPPED

NANOPARTICLES

3.2.1 Atomic Force Microscopy analysis of rhamnolipid capped CdS

nanoparticle

Rhamnolipid capped CdS nanoparticles were synthesized using

cadmium nitrate and sodium sulphide solution. The particles formed were

analyzed through atomic force microscopy (AFM). It was found that the

sample contained spheres of 50 nm size as shown in Figure 3.14.

Figure 3.14 AFM Image of rhamnolipid capped CdS nanoparticles

3.2.2 SYNTHESIS OF RHAMNOLIPID CAPPED ZNS

NANOPARTICLES

3.2.2.1 Fourier Transform Infra Red Spectroscopy analysis

Rhamnolipid and rhamnolipid capped zinc sulphide nanoparticles

were subjected to FTIR analysis in transmittance mode. As shown in Figure

3.15 the capping agent rhamnolipid was found to have all the necessary

functional groups attached. The spectra appeared as -OH (Hydroxyl) 3307

42

cm-1, -CH2-CH3 (2922 cm-1),C=O(Carbonyl) 1731cm-1,-COO(Carboxyl) 1646

cm-1, C-O-C(epoxy)(1052cm-1). The appearance of the peak of 702 cm-1

confirms the capping of rhamnolipid with zinc sulphide nanoparticles.

Figure 3.15 FTIR analysis of rhamnolipid capped ZnS nanoparticles

The characteristic vibrational modes of rhamnolipid molecules

appeared to be suppressed when they capped zinc sulphide nanoparticles.

While the intensity of C-O-C and -COO vibrations were the same, the -CH2-

CH3 vibrational modes were reduced. This may be due to the formation of

complex between zinc and rhamnolipid. The oscillator vibrations associated

with different functional groups such as -CH2-CH3 are also weakened.

43

3.2.2.2. High resolution Transmission Electron Microscopy analysis of

rhamnolipid capped zinc sulphide nanoparticles

Rhamnolipid capped ZnS nanoparticles were subjected to high

resolution TEM analysis. As shown in figure, the sample contained spherical

particles. It was found that the radius was in the range of 2.5 nm to 5 nm. The

images analyzed were in different resolution like 50 nm, 20 nm, and 5 nm as

shown in Figure 3.16, 3.17, and 3.18.

Figure 3.16 HRTEM analysis of rhamnolipid capped ZnS nanoparticles

in 50nm resolution.

44

Figure 3.17 HRTEM Image of capped ZnS nanoparticles in 20 nm

resolution.

Figure 3.18 HRTEM Image of capped ZnS nanoparticles in 5 nm

resolution.

45

3.2.2.3 Small angle X-ray scattering analysis

Small angle X-ray scattering analysis was performed for both

aqueous and powder sample of rhamnolipid capped ZnS nanoparticles. Size

and distribution of rhamnolipid capped ZnS nanoparticles were obtained

using an Anton Paar slit collimation compact Kratky type SAXS camera fitted

on to a rotating anode X-ray generator running at 100mA.CopperS k

radiation was used in this study. As shown in Figure 3.19 the particle radius

was found to be 4.55 nm from Guinier plot in the small q (=4 .sin ) range.

The particles were spherically shaped. Primary particles were combined to

form aggregates which in turn form agglomeration giving rise to mass fractal

dimension of -2.55 as shown in Figure 3.21. The spatial distribution and

correlation of the particles gives rise to the structure factor and manifests

itself in the intermediate q range. The log -log plot of aqueous solution of

rhamnolipid capped ZnS nanoparticles prepared at 80ºC as shown in Figure

3.20, reveals the q dependence on the intensity (Sinha et al 1988, Teixeira

1988). The power law behavior was also seen in the linear variation of log

intensity, implying self-affine agglomeration in the dried powder sample

where as self-affine agglomeration was not observed in dilute aqueous

solution. This property was due to concentration change in dilute solution.

46

Figure 3.19 Guinier plot of rhamnolipid capped ZnS nanoparticles

Figure 3.20 Log-log plot of rhamnolipid capped ZnS nanoparticles in

aqueous medium

47

Figure 3.21 Log-log plot of dried rhamnolipid capped ZnS nanoparticles

and aqueous solution (circles-dried powder, squares -

aqueous solution)

3.2.2.4 Optical property analysis by fluorescence spectroscopy

The max of the excitation spectrum of the rhamnolipid-capped

ZnS nanoparticle was found to be 340 nm, using a Hitachi UV-visible

spectrophotometer. The nanoparticles were found to give a narrow absorption

and broad emission in Hitachi make F- 2310 fluorescence spectrophotometer.

It gave a broad emission peak, as shown in Figure 3.22, at about 450 nm when

excited by 340 nm wavelength radiations. Such a broad spectrum can be a

combined result of the quantum confinement and the modification of the

surface states due to the capping process. At this stage, it is difficult to

delineate the different possibilities and ascribe specific mechanism of

fluorescence to the various regions of the broad spectrum. However, while the

reason for the broadening of emission spectra was unknown, the emission

shift parameters were used to calculate the average particle radius in the

suspension. A particle radius of 5.5 nm was obtained based on the formulation

48

of Brus (1986). The particle size corroborates with the HR-TEM and SAXS

results.

Figure 3.22 Excitation and emission spectrum of rhamnolipid capped

ZnS nanoparticles

3.2.2.5 High resolution scanning electron microscopy (HRSEM)

analysis of rhamnolipid capped ZnS nanoparticles.

Rhamnnolipid capped ZnS nanoparticles were analysed for

rhamnolipid's capping with zinc metal ion using FEI Quanta FEG 200 - High

resolution scanning electron microscope coupled with energy dispersive X -

ray analysis (EDX). The sample was spread on thin glass plate and resolution

was kept at 1µ. The particles were hierarchically aggregated. As shown in

Figure 3.23 the EDX analysis revealed that metal ions were completely

capped by rhamnolipid and the presence of the peaks corresponding to carbon

49

and oxygen denotes rhamnolipid composition. The appearance of peaks for

Zn metal ion and S revealed the presence of ZnS with the capping agent

rhamnolipid.

Figure 3.23 HRSEM and EDX analysis of rhamnolipid capped ZnS

nanoparticles

50

3.3 RHAMNOLIPID CAPPED ZNS – BIOMOLECULE

INTERACTIONS

3.3.1 Interaction of rhamnolipid capped ZnS nanoparticles with

bovine serum albumin (BSA)

3.3.1.1 Fluorescence spectroscopy analysis

Fluorescence spectroscopy analysis of rhamnolipid capped ZnS

nanoparticles and bovine serum albumin (BSA) revealed maximum

absorption and emission wavelength as shown in Figure 3.24. It was observed

that there was an overlap region of 10 nm between the emission peak of

protein and absorption peak of nanoparticles. This minimum distance

influenced förster’s or resonance energy transfer from protein molecules to

rhamnolipid capped ZnS nanoparticles. Bioconjugate was made between

protein molecules and the rhamnolipid capped ZnS nanoparticles. The

interaction between them was found to be an electrostatic interaction.

Aromatic amino acid residues tyrosines, tryptophans of protein molecule

transfer energy from protein molecules to ZnS nano particles. A constant

amount 100 g of protein bovine serum albumin (BSA) along with varying

concentration of 5mM rhamnolipid capped ZnS nanoparticle was taken for the

conjugation. As the concentration of nanoparticle increased in the conjugation

reaction, protein emission at 340 nm reduced and nanoparticle emission at

450 nm increased due to energy transfer when excited at 280 nm as shown in

Figure 3.25. The saturation point for the concentration of rhamnolipid capped

ZnS nanoparticles in the bioconjugation was found to be 240 l. As shown in

Figure 3.26 saturation point of capped ZnS nanoparticle yielded maximum

fluorescence under ultra violet illumination. The FRET efficiency was found

to be 0.45 using standard calculations. The forsters distance Ro for BSA found

to be 1.14 nm, J (8.49X10-17) and donor to acceptor distance r found to be

1.19 nm.

51

Figure 3.24 Excitation and emission spectrum of BSA and ZnS

nanoparticles

Figure 3.25 FRET between BSA and capped ZnS nanoparticles

52

Figure 3.26 Fluorescence emissions of 1.BSA-ZnS conjugates and 2.

BSA alone

3.3.1.2 Agarose gel electrophoresis

Bioconjugate of nanoparticles with BSA protein and CRL on 1%

native agarose gel electrophoresis showed FRET. The binding of capped

nanoparticle with protein was confirmed by the appearance of bands on the

gel under ultra violet illumination, which was corroborated using Coomassie

brilliant blue staining as shown in Figure 3.27. Due to the aggregation of CRL

protein failed to enter the gel and appeared as band at the well. Heat

denaturation did not affect protein’s binding to rhamnolipid capped ZnS

nanoparticles and exhibited energy transfer on the agarose gel electrophoresis

as exhibited in fluorescence spectroscopy analysis.

1 2

53

Figure 3.27 Agarose gel electrophoresis of BSA and CRL Staining using

1.Coomassie brilliant blue R-250 2.Capped ZnS under UV

illumination

3.3.2 INTERACTION OF RHAMNOLIPID CAPPED ZNS NANOPARTICLES

WITH Candida rugosa LIPASE (CRL)

3.3.2.1 Fluorescence spectroscopy

Fluorescence spectroscopy analysis was performed to analyze the

interaction between rhamnolipid capped ZnS nanoparticles and Candida

rugosa lipase (CRL). The interaction revealed maximum energy transfer to

rhamnolipid capped ZnS nanoparticles. Emission spectrum of CRL and heat

denatured CRL with and without ZnS nanopartices showed no difference in

the intensity as shown in Figure 3.28. As the concentration of nanoparticles

increased in the reaction, the intensity of CRL protein emission at 340 nm

54

reduced and the intensity of nanoparticle emission at 450 nm increased as

shown in Figure 3.29. The FRET efficiency of CRL was lower than BSA

protein and was found to be 0.19 which was contributed by the number

aromatic amino acids present in the CRL protein. The forsters distance Ro

found to be 1.151 nm, J (8.38X10-17), the distance between donor and

acceptor r found to be 1.46 nm. The saturation point for rhamnolipid capped

ZnS nanoparticles with CRL conjugation was found to be 240 l. It exhibits

maximum FRET with 240 l of 5mM capped ZnS nanoparticles.

Denaturation of CRL protein did not affect its interaction with nanoparticles

and exhibited energy transfer.

Figure 3.28 Emission spectrum of native and denatured CRL Protein

55

Figure 3.29 FRET between CRL and capped ZnS nanoparticles

3.3.2.2 Agarose gel electrophoresis & zymography analysis

The effect of rhamnolipid capped ZnS nanoparticle on protein

functionality was analyzed by zymography or activity staining method.

Zymography was done for CRL-nanoparticle bioconjugate and run on 1.5%

native agarose gel electrophoresis. The activity of CRL protein was confirmed

by sandwiching agarose gel with tributrin substrate containing agar. The

appearance of a zone of clearance around CRL protein band at the well as

shown in Figure 3.30 corroborate the functionality of the enzyme conjugated

with rhamnolipid capped ZnS nanoparticles. In another zymography

experiment with 1.5% agarose gel electrophoresis of CRL-along with

lysozyme protein validated the binding and activity of CRL with capped ZnS

nanoparticles. The lysozyme protein did not show any activity where as it

appeared as blue band under UV illumination as shown in Figure 3.31. Part of

CRL protein found to enter gel in 1% agarose gel and showed activity in two

zones during zymography as shown in Figure 3.33. The activity of CRL with

56

capped ZnS nanoparticles was found to be unaffected. The zone of clearance

was similar in the zymography of CRL with and without capped ZnS

nanoparticles as shown in Figure 3.32.

Figure 3.30 Zymography analysis of Candida rugosa lipase (CRL) with

capped ZnS nanoparticles (Left panel), UV illumination

(right panel)

57

1 2 3

Figure 3.31 Zymography and UV illumination of CRL and lysozyme

protein

Figure 3.32 Effect of nanoparticles on zymography of CRL 1.water, 2.

CRL alone, 3. CRL with capped ZnS nanoparticles

CRL protein (Zone ofclearance)

Lysozyme protein

58

Figure 3.33 Zymography of CRL in 1% agarose gel showing two zones

of clearance. UV illumination (left) zymogram (right)

3.3.3 Interaction of rhamnolipid capped ZnS nanoparticles with

plasmid deoxyribonucleic acid (DNA)

3.3.3.1 Fluorescence spectroscopy

Rhamnolipid capped ZnS nanoparticles were conjugated with

plasmid DNA and fluorescence spectroscopy analysis was performed.

Plasmid DNA alone was excited at 260 nm and emission was hardly seen.

After conjugation with capped ZnS, emission appeared at 450 nm. As the

concentration of rhamnolipid capped ZnS nanoparticles were increased in the

reaction, emissions of nanoparticle were also increased at 450 nm when

excited at 260 nm. As shown in Figure 3.34, DNA also had revealed Förster’s

or resonance energy transfer to rhamnolipid capped ZnS nanoparticles. The

forsters distance R0 was found to be 3.8 nm.

59

Figure 3.34 FRET between plasmid DNA and capped ZnS nanoparticles

60

CHAPTER 4

CONCLUSION

Rhamnolipids are extracted and purified from media containing

metal fatty acid salts as carbon source through HPLC and Mass spectrometry.

Nanoparticles are synthesized using rhamnolipid as capping agent.

Rhamnolipid capped ZnS nanoparticles and its interaction with biomolecules

are investigated. Summary of the study are as follows.

4.1 RHAMNOLIPID PRODUCTION IN METAL FATTY ACID

SALT CONTAINING MEDIA

1. Mono and di-rhamnolipids are extracted, and purified through

HPLC and mass spectrometry

2. Metal fatty acid salts are prepared using standard method and

used as sole carbon source in minimal media for rhamnolipid

production.

3. Rhamnolipids produced by Pseudomoas aeruginosa in metal

fatty acid salts containing media are 4-Bromophenacyl

bromide derivatized.

4. The derivatives are isolated and purified through high

performance liquid chromatography (HPLC). Individual

peaks are collected as fractions and mass spectrometry

analyzed.

61

5. Mass spectrometry analysis reveals that Pseudomonas

aeruginosa produces three diferent rhamnolipid congeners of

mono-rhamno mono-lipid, mono-rhamno di-lipid, di-rhamno

di-lipids in metal fatty acid salts containing media.

4.2 SYNTHESIS OF NANOPARTICLES USING

RHAMNOLIPID AS CAPPING AGENT

1. Rhamnolipids are used as capping agent to synthesize ZnS and

CdS nanoparticles.

2. Capping of rhamnolipid with metal ions are optimized at

alkaline pH.

3. Rhamnolipid capped CdS nanoparticles are characterized by

using atomic force spectroscopy (AFM) and size of particles

are in the range of 50-100 nm.

4. Rhamnolipid capped ZnS nanoparticles are characterized by

using UV-Vis spectrophotometer, fluorescence spectroscopy,

fourier transform infra red spectroscopy (FTIR), small angle

X-ray scattering (SAXS), transmission electron microscopy

(TEM), scanning electron microscopy (SEM) and energy

dispersive X-ray analysis (EDX). The particles are stable,

uniform in the size of 4-5 nm. The shapes of the particles are

in uniform spheres and widely dispersed.

62

4.3 INTERACTION BETWEEN RHAMNOLIPID CAPPED ZNS

NANOPARTICLES AND BIOMOLECULES

1. Rhamnolipid capped ZnS nanoparticles interact with bovine

serum albumin (BSA) and exhibit Försters resonance or

fluorescence energy transfer.

2. Fluorescence spectroscopy analysis revealed that capped ZnS-

BSA conjugate emits fluorescenc at 450 nm when excited at

280 nm of protein molecules.

3. During interaction of rhamnolipid capped ZnS nanoparticles

with BSA on agarose gel electrophoresis, it exhibits FRET

phenomenon. The FRET efficiency is about 0.45 and R0 is

1.153 nm and the distance between donors to acceptor is 1.19

nm

4. Fluorescence spectroscopy analysis of Capped ZnS - Candida

rugosa lipase (CRL) bioconjugate also transfers fluorescence

resonanance energy (FRET) from lipase molecules to capped

ZnS nanoparticles in aqueous media. The FRET efficiency is

about 0.19 for CRL and R0 is 1.151 nm and the distance

between donor and acceptor is 1.46 nm.

5. Interaction between capped ZnS nanoparticles and CRL

protein is not affected by protein heat denaturation.

6. Capped ZnS nanoparticles – CRL bioconjugate exhibits

maximum FRET efficiency on agarose gel electrphoresis.

7. Zymography analysis of rhamnolipid capped ZnS - CRL

bioconjugate revealed that interaction is not affecting the

lipase function.