LIPASE MEDIATED DEGRADATION OF

VARIOUS POLYESTERS

By

Misbah Amin
(M. Phil UAF)

2004-ag-51

A Thesis Submitted in Partial Fulfillment of the Requirements for the

Degree of

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

DEPARTMENT OF CHEMISTRY
FACULTY OF SCIENCES
UNIVERSITY OF AGRICULTURE, FAISALABAD.

2019
To,

The Controller of Examinations,

University of Agriculture,

Faisalabad.

“We, the Supervisory Committee, certify that the contents and form of thesis submitted
by Miss Misbah Amin, 2004-ag-51, have been found satisfactory and recommend that it be
processed for evaluation, by the External Examiner(s) for the award of degree”.

SUPERVISORY COMMITTEE:

1. Chairman __________________________

(Prof. Dr. Haq Nawaz Bhatti)

2. Co-Supervisor __________________________

(Prof. Dr. Muhammad Zuber)

3. Member __________________________

(Prof. Dr. Ijaz Ahmed Bhatti)

4. Member __________________________

(Prof. Dr. Muhammad Asgher)

 DECLARATION

I hereby declare that the contents of the thesis “Lipase mediated degradation of various
polyesters” are product of my own research and no part has been copied from any published
source (except the references, standard mathematical or genetic
models/equations/formulate/protocols etc). I further declare that this work has not been
submitted for award of any other diploma/degree. The University may take action if the
information provided is found inaccurate at any stage. (In case of any default the scholar will be
proceeded against as per HEC plagiarism policy).

MISBAH AMIN
2004-ag-51
 DEDICATED TO

AMMI & ABBU

“Who have provided me a secure upbringing with esteemed love which

enabled me to get the success”
 ACKNOWLEDGEMENT

All praises to Almighty ALLAH, the most benevolent and merciful, the creator of the universe,
who enabled me to complete this research work successfully. I offer Salaam to the Holy Prophet
Muhammad (Peace Be Upon Him) who is a blessing for all the worlds.
I pay my special thanks and recognition to the efforts of my knowledgeable and worthy
supervisor Prof. Dr. Haq Nawaz Bhatti, Department of Chemistry, University of Agriculture,
Faisalabad, whose valuable guidance, constant motivation and kind attention made it possible for
me to accomplish my research work and thesis write up.
My thanks are extended to Prof. Dr. Muhammad Zuber, Prof. Dr. Ijaz Ahmad Bhatti and
Prof. Dr. Muhammad Asgher for their support and assistance.
I offer my cordial thanks to Dr. Alison Paul, Department of Chemistry, Cardiff University,
Cardiff, UK for her nice behavior and co-operation during my research work in Cardiff
university.
I highly appreciate the Higher Education commission, Pakistan for the financial support in all
respects during my study period, without which it was impossible for me to achieve the
objectives of my research work. My appreciation and thanks are extended to organizers of
International Research Support Initiative Program (IRSIP) for giving me opportunity to do
research work abroad.
I am lucky enough to have the support of many good friends. Special thanks are due to my
friends and all lab fellows for their co-operation during my research work. I want to express my
special gratitude to my whole family, without their prayers and moral support; it would not be
possible for me to complete my degree. Words always seem shallow whenever it comes to my
dearest and loving husband Waqas Safdar, whose continuous help, motivation and criticism gave
me strength and encouragement to go on and complete this thesis. When all seemed quite lost, he
just kept pushing me till the very end to ensure the submission of this dissertation.
I would like to thank all those people who helped me and made this study to complete
successfully and apologize that I couldn’t mention personally one by one.

Misbah Amin
 LIST OF CONTENTS

Sr. No TITLE Page No.

1 Introduction 1

2 Review of Literature 6

3 Materials and Methods 25

4 Results and Discussion 38

5 Summary 118

Literature Cited
 LIST OF TABLES

Sr. No TITLE PAGE NO.

3.1 Experimental ranges and levels of independent variables 29

4.1 ANOVA results for production of lipase by Penicillium fellutanum 53

through RSM
4.2 ANOVA results for production of lipase by Aspergillus melleus 54
through RSM
4.3 Analysis of variance (ANOVA) results for response parameters 56

4.4 Experimental design and result of CCD of response surface 57

methodology for production of lipase by Penicillium fellutanum and
Aspergillus melleus

4.5 Validation of model showing lipase production at optimum level of all 74

parameters
4.6 Summary of partial purification of Penicillium fellutanum lipase 76

4.7 Summary of partial purification of Aspergillus melleus lipase 76

4.8 Kinetic and thermodynamic parameters for irreversible thermal 86

denaturation of lipase from Penicillium fellutanum
4.9 Kinetic and thermodynamic parameters for irreversible thermal 87
denaturation of lipase from Aspergillus melleus
4.10 Values of glass transition and melting temperature of polyester 111
samples before and after Penicillium fellutanum lipase treatment by
DSC analysis

4.11 Values of glass transition and melting temperature of polyester 112

samples before and after Aspergillus melleus lipase treatment by DSC
analysis
 LIST OF FIGURES

Sr. No TITLE PAGE NO.

3.1 Standard curve for protein estimation 31

3.2 Polyvinyl acetate (PVAc) film sample 35

4.1 Screening of substrates for lipase production by Pencillium fellutanum 39

and Aspergillus melleus in SSF
4.2 Effect of moisture content on lipase production by Pencillium 41
fellutanum and Aspergillus melleus

4.3 Effect of incubation time on lipase production by Pencillium 42

fellutanum and Aspergillus melleus

4.4 Effect of pH on lipase production by Penicillium fellutanum and 43

Aspergillus melleus
4.5 Effect of amount of substrate on lipase production by Penicillium 45
fellutanum and Aspergillus melleus
4.6 Effect of inoculum size on lipase production by Penicillium fellutanum 46
and Aspergillus melleus
4.7 Effect of temperature on lipase production by Penicillium fellutanum 48
and Aspergillus melleus
4.8 Effect of olive oil concentration on lipase production by Penicillium 49
fellutanum and Aspergillus melleus

4.9 Effect of additional carbon sources on lipase production by Penicillium 51

fellutanum and Aspergillus melleus

4.10 Effect of nitrogen sources on lipase production by Penicillium 52

fellutanum and Aspergillus melleus

4.11 Normal probability plot of Residuals for lipase production by 59

Penicillium fellutanum
4.12 Normal probability plot of Residuals for lipase production by 59
Aspergillus melleus
4.13 3-Dimentional response surface plot for the interaction effect of pH and 61
incubation time on lipase production by Penicillium fellutanum while
other factors i.e., temperature and carbon source were fixed at one level
4.14 Contour plot showing the interaction of pH and incubation time on 62
lipase production by Penicillium fellutanum while other factors i.e.,
temperature and carbon source were fixed at one level
4.15 3-Dimentional response surface plot for the interaction effect of pH and 62
temperature on lipase production by Penicillium fellutanum while other
factors i.e., incubation time and carbon source were fixed at one level
4.16 Contour plot showing the interaction of pH and temperature on lipase 63
production by Penicillium fellutanum while other factors i.e.,
incubation time and carbon source were fixed at one level
4.17 3-Dimentional response surface plot for the interaction effect of 63
incubation time and temperature on lipase production by Penicillium
fellutanum while other factors i.e., pH and carbon source were fixed at
one level
4.18 Contour plot showing the interaction of incubation time and 64
temperature on lipase production by Penicillium fellutanum while other
factors i.e., pH and carbon source were fixed at one level
4.19 Interaction plot showing the interaction of pH and carbon source on 65
lipase production by Penicillium fellutanum while other factors i.e.,
incubation time and temperature were kept constant
4.20 Interaction plot showing the interaction of incubation time and carbon 66
source on lipase production by Penicillium fellutanum while other
factors i.e., pH and temperature were kept constant
4.21 Interaction plot showing the interaction of temperature and carbon 66
Source on lipase production by Penicillium fellutanum while other
factors i.e., incubation time and pH were kept constant
4.22 3-Dimentional response surface plot for the interaction effect of pH and 68
incubation time on lipase production by Aspergillus melleus while
other factors i.e., temperature and nitrogen source were fixed at one
level
4.23 Contour plot showing the interaction of pH and incubation time on 68
lipase production by Aspergillus melleus while other factors i.e.,
temperature and nitrogen source were fixed at one level
4.24 3-Dimentional response surface plot for the interaction effect of 69
incubation time and temperature on lipase production by Aspergillus
melleus while other factors i.e., pH and nitrogen source were fixed at
one level
4.25 Contour plot showing the interaction 69
of incubation time and temperature on lipase production by
Aspergillus melleus while other factors i.e., pH and nitrogen source
were fixed at one level
4.26 Interaction plot showing the interaction of incubation time and nitrogen 70
source on lipase production by Aspergillus melleus while other factors
i.e., pH and temperature were kept constant
4.27 Interaction plot showing the interaction of temperature and nitrogen 71
source on lipase production by Aspergillus melleus while other factors
i.e., pH and incubation time were kept constant

4.28 Overlay Perurbation plot of three independent variables for lipase 72

production by Penicillium fellutanum using carbon source (a) glucose
(b) lactose (c) average of both
4.29 Overlay Perurbation plot of three independent variables for lipase 73
production by Aspergillus melleus using nitrogen source (a) sodium
nitrate (b) diammonium tartarate (c) average of both

4.30 Effect of pH on Penicillium fellutanum and Aspergillus melleus lipase 77

activity
4.31 Effect of temperature on Penicillium fellutanum and Aspergillus 78
melleus lipase activity

4.32 Arrhenius plot for the activation energy of substrate hydrolysis by 79

Penicillium fellutanum
4.33 Arrhenius plot for the activation energy of substrate hydrolysis by 79
Aspergillus melleus

4.34 Lineweaver-Burk plot for the determination of kinetic constants (Vmax, 81

Km) for pNPP hydrolysis at 45 °C, pH 8.5 by Penicillium fellutanum
lipase
4.35 Lineweaver-Burk plot for the determination of kinetic constants (Vmax, 81
Km) for pNPP hydrolysis at 40 °C, pH 7.5 by Aspergllus melleus lipase
4.36 Effect of pH on the stability of Penicillium fellutanum and Aspergillus 82
melleus lipase

4.37 Pseudo first order plots for irreversible thermal inactivation of 84

Penicillium fellutanum lipase
4.38 Pseudo first order plots for irreversible thermal inactivation of 85
Aspergillus melleus lipase

4.39 Arhennius Plot for the determination of Ea for irreversible thermal 85

denaturation of Penicillium fellutanum lipase
4.40 Arhennius Plot for the determination of Ea for irreversible thermal 86
denaturation of Aspergillus melleus lipase
4.41 Effect of metal ions on the activity of Penicillium fellutanum and 88
Aspergillus melleus lipase
4.42 Effect of urea on the stability of Penicillium fellutanum lipase 89

4.43 Effect of urea on the stability of Aspergillus melleus lipase 89

4.44 Effect of guanidine hydrochloride on the stability of Penicillium 90

fellutanum lipase
4.45 Effect of guanidine hydrochloride on the stability of Aspergillus 91
melleus lipase

4.46 Effect of organic solvents on the stability of Penicillium fellutanum and 92

Aspergillus melleus lipase
4.47 Enzymatic degradation of different polyester films in the presence of 94
lipases produced by Penicillium fellutanum and Aspergillus melleus

4.48 Effect of incubation time on the weight loss of different polyesters by 95

lipase from Penicillium fellutanum
4.49 Effect of incubation time on the weight loss of different polyesters by 95
lipase from Aspergillus melleus

4.50 Effect of pH on the weight loss of different polyesters by lipase from 96

Penicillium fellutanum

4.51 Effect of pH on the weight loss of different polyesters by lipase from 97

Aspergillus melleus

4.52 Effect of enzyme concentration on the weight loss of different 98

polyesters by lipase from Penicillium fellutanum
4.53 Effect of enzyme concentration on the weight loss of different 98
polyesters by lipase from Aspergillus melleus
4.54 Effect of temperature on the weight loss of different polyesters by 99
lipase from Penicillium fellutanum

4.55 Effect of temperature on the weight loss of different polyesters by 100

lipase from Aspergillus melleus

4.56 FT-IR spectra of PMMA before and after degradation by treating with 101
Penicillium fellutanum lipase

4.57 FT-IR spectra of PMMA before and after degradation by treating with 102
Aspergillus melleus lipase
4.58 FT-IR spectra of PEMA before and after degradation by treating with 103
Penicillium fellutanum lipase

4.59 FT-IR spectra of PEMA before and after degradation by treating with 104
Aspergillus melleus lipase

4.60 FT-IR spectra of poly(ɛ-caprolactone) before and after degradation by 105

treating with Penicillium fellutanum lipase
4.61 FT-IR spectra of poly(ɛ-caprolactone) before and after degradation by 106
treating with Aspergillus melleus lipase
4.62 FT-IR spectra of polyvinyl acetate film before and after degradation by 107
treating with Penicillium fellutanum lipase

4.63 FT-IR spectra of polyvinyl acetate before and after degradation by 108
treating with Aspergillus melleus lipase

4.64 FT-IR spectra of polyester vylon-200 before and after degradation by 109
treating with Penicillium fellutanum lipase

4.65 FT-IR spectra of polyester vylon-200 before and after degradation by 110
treating with Aspergillus melleus lipase

4.66 SEM micrograph (X500) of untreated PCL film 113

4.67 SEM micrographs (X500) of PCL after degradation by treating with (a) 113
Penicillium fellutanum lipase (b) Aspergillus melleus lipase

4.68 SEM micrograph (X500) of untreated PV-200 film 114

4.69 SEM micrographs (X500) of PV-200 after degradation by treating with 114
(a) Penicillium fellutanum lipase (b) Aspergillus melleus lipase

4.70 SEM micrograph (X500) of untreated PVAc film 115

4.71 SEM micrographs (X500) of PVAc after degradation by treating with 115
 (a) Penicillium fellutanum lipase (b) Aspergillus melleus lipase

4.72 SEM micrograph (X500) of untreated PMMA film 116

4.73 SEM micrographs (X500) of PMMA after degradation by treating with 116
(a) Penucillium fellutanum lipase (b) Aspergillus melleus lipase

4.74 SEM micrograph (X500) of untreated PEMA film 117

4.75 SEM micrographs (X500) of PEMA after degradation by treating with 117
(a) Penicillium fellutanum lipase (b) Aspergillus melleus lipase
 Abstract

The present research project was aimed to produce microbial lipases, conduct their partial
purification and characterization to observe their prospective use to degrade the polyesters.
Penicillium fellutanum and Aspergillus melleus were used as lipase producing fungal strains and
canola seed oil cake as best fermentative substrate for the production of lipases using solid state
fermentation. Important parameters were optimized classically for lipase production. Results
revealed that the optimum conditions for lipase production by P. fellutanum were incubation
periods 48 h, moisture 50 %, pH 4, temperature 30 ºC and olive oil 2 % while by A. melleus,
these were incubation period 96 h, moisture 60 %, pH 4, temperature 30 ºC and olive oil 3 %.
Addition of carbon and nitrogen sources significantly affected lipase production. Response
surface methodology was also employed for lipase production and an overall 2.05 and 1.92-fold
increase in lipase production by P. fellutanum and A. melleus respectively, was being achieved.
Crude lipase extract from both fungal strains was subjected to partial purification and, 2.06 and
3.84 folds of purification were obtained after dialysis of P. fellutanum and A. melleus lipase
extract respectively. The partially purified lipase by both fungal strains was characterized by
means of optimum pH, temperature, pH and thermal stability etc. Results showed that P.
fellutanum lipase was alkaline and A. melleus lipase was neutral in nature. Both fungal lipases
showed stability up to 40 ºC and 45 ºC. Km and Vmax were estimated to be 0.75 mM & 83.33
µmol/min for P. fellutanum lipase while 0.29 mM & 142.86 µmol/min for A. melleus lipase
respectively. The activity of lipases produced in this study was evaluated for the degradation of
five different polyesters. Both the enzymes showed good degradation abilities. Optimization of
important parameters for biodegradation like incubation time, enzyme concentration, pH and
temperature was also carried out. Different techniques like FT-IR, DSC and SEM were applied
for the characterization of biodegradation process. FTIR spectra of PVAc, PV-200 and PCL
depicted significant decrease in ester functional group and many other transformations at
different regions after degradation which was the evidence of their significant weight loss during
biodegradation. DSC thermogram data revealed the noticeable reduction in Tg and Tm of PVAc,
PV-200 and PCL which confirmed the results. SEM exposed the appearance of widespread
cracks on the surface after 4 weeks of incubation with both microbial enzymes. The whole study
proved that lipases produced here can be utilized for the degradation of polyesters for solid waste
management.