Heterologous Expression and Characterization of Cinnamoyl CoA Reductase from Leucaena leucocephala for Paper Industry

Heterologous Expression, Purification and
Characterization of Cinnamoyl Coenzyme A
Reductase (CCR) from Leucaena leucocephala
Project thesis
Submitted in partial fulfillment for
Master’s Degree in Science in
Biotechnology
Submitted by
Pratik B. More
Under the guidance of
Dr. Bashir Mohammad Khan
to
The University Of Pune
Work done at
National Chemical Laboratory,
Dr.Homi Bhaba Road, Pune - 411 008

Declaration
I, the undersigned hereby declare that the Project / Dissertation work presented
in the “Heterologous expression, Purification and Characterization of
Cinnamoyl Coenzyme-A Reductase (CCR) from Leucena
leucocephala” has been carried out under the guidance and supervision of
Dr. B. M. Khan (Scientist ‘G’) Head, Plant Tissue Culture Division,
National Chemical Laboratory, Pune. I have myself collected the literature
required for the project and have presented the same following standard
publication guidelines. I have not submitted the review presented here to any
other university for award of any degree.
Pratik B. More

ACKNOWLEDGEMENT
I sincerely express my deep sense of gratitude and immense respect to my
guide Dr. Bashir Mohammad Khan, Scientist and Head Plant Tissue Culture
Division (PTC), National Chemical Laboratory (NCL), Pune for scholarly
guidance, generous encouragement and suggestions throughout the course of my
dissertation work. I am heartily thankful to him for giving me chance to work in
his laboratory.
I express my thanks to Dr.Girish R. Pathade, Head of Biotechnology,
Fergusson College, Pune, for supporting me to utilize this opportunity to gain such
a wonderful experience and knowledge. I am thankful to all professors and
lecturers of my Department for their encouragement and guidance.
I would like to extend my sincere thanks to my co-guide Mr. Prashant D.
Sonawane who has always been my inspiration and also for constantly bringing the
best out of me. I am also thankful for his support and encouragement throughout
the year.
I am very glad to thank all the members of our laboratory including Mr.
Parth Patel, Mr. Santosh Jadhav, Mr. Rishi V., Miss Trupti, Mr. Krunal Patel, Mr.
Somesh Singh, Miss Ruby, Miss Neha, Mr. Shakeel, Mr. Kannan, Mr. Santosh
Kumar and Mrs. Sumita Gupta for giving me their valuable advice and helping me
in times of difficulty.
I express my special thanks to all my laboratory friends Neha, Amar,
Disha, Anuja, Neerja, Zoheb, Sudarshan, Shrikant, Sneha, Gayatri, Sudhir, Asmiti
and others for their constant cooperation, which helped me a lot in successful
completion of my work.
I wish to express my gratitude to my loving parents for their affection,
inspiration, and encouragement to put all the best efforts for achieving betterment
in my life.
Last but not the least; I thank GOD who is above all the errors.
Pratik More

Contents
List of Abbreviations
List of Table
Abstract
Particulars Page No.
Chapter 1 Introduction 1
1.1 Review of Literature 3
1.2 Aims and Objectives 16
Chapter 2 Materials & Method 18
2.1 Materials 18
2.1.1 Glassware 18
2.1.2 Preparation of Glassware 18
2.1.3 Plastic ware 18
2.1.4 Chemicals 19
2.1.5 Equipments 19
2.1.6 Different Media Used for Studies 19
2.1.7 Host cells 20
2.1.8 Buffers and Solutions 20
2.1.9 Solutions for Protein Purification 22
2.2 Methods
2.2.1 Expression and Purification CCR Proteins 24
CCRH1 & CCRH2
2.2.2 Affinity Purification of CCR Protein 25
2.2.3 Sample Conditioning 1 (Concentration, Desalting) 28
2.2.4 Anion Exchange Chromatography 28
(Intermediate Purification)
2.2.5 Sample Conditioning 2(polishing, concentration) 30

Contents
Particulars Page No.
2.2.6 Qualitative and Quantitative analysis of CCR proteins 30
2.2.7 Western Blot Analysis 32
2.2.8 ESI-MS Analysis 33
2.2.9 Crystallization 35
2.2.10 Enzyme Kinetics 36
Chapter 3 Results and Discussions 37
3.1 Expression studies of CCRH1 and CCRH2 in pET 30b (+) 37
3.1.1 Screening for over Expression of Recombinant Proteins 37
3.1.2 Standardization of protein expression in E. coli BL21 38
3.2 Expression studies of CCRH1 and CCRH2 in pET 41a (+) 40
3.2.1 Screening for over-expressing recombinant CCR proteins 40
3.2.2 Standardization of protein expression in E. coli BL21 40
3.3 Purification of recombinant CCRH1 pET30b (+) clone 42
3.3.1 Affinity chromatography 42
3.3.2 Sample Conditioning 1 43
3.3.3 Ion Exchange Chromatography 43
3.3.4 Sample conditioning 2 45
3.4 Western Blot Analysis 47
3.5 ESI-MS Analysis 48
3.6 Crystallization 49
3.7 Enzyme Kinetics 49
Chapter 4 Summary 56
4.1 Summary 56
4.2 Future Prospects 57
Chapter 5 References 58

List of Abbreviations
CCR Cinnamoyl coenzyme A reductase
Co-A Coenzyme-A
°C Degree centigrade
µg Microgram
µL Micro liter
DW Distilled water
AA Amino acid
SMQ Sterile MilliQ water
E.coli Escherichia coli
µg/ml micrograms per milliliter
gm/L Gram per liter
Hrs Hour(s)
Mg Milligrams
Min Minute(s)
mL Milli liter
mM Milli molar
RPM Rotations per minute
SDS Sodium dodecyl sulphate
PAGE Polyacrylamide Gel Electrophoresis
β Beta
v/v Volume per volume
w/v Weight per volume
IPTG Isopropyl β-D-1- thiogalactopyranoside
BSA Bovine serum albumin
DEAE Diethylaminoethyl
cm Centimeter
Ni Nickel

His Histidine
DW Distilled water
O/N Overnight
PMSF Phenylmethanesulfonylfluoride
KDa Kilo Daltons
N Normality
ng Nanogram
nm Nano meter
O/N Overnight
RT Room temperature
pmol Picomole
SSC Saline sodium citrate
s Second
TE Tris EDTA buffer
TEMED Tetramethylethylenediamine
UV Ultra violet
ELISA Enzyme Linked Immuno Sorbent Assay

List of Figures
Figure Number Title Page number
Fig.1.1 Seed, branch and tree of Leucaena leucocephala 3
Fig.1.2 Monolignols and Lignin units 6
Fig.1.3 Dimerization of coniferyl alcohol 7
Fig.1.4 Cross coupling of coniferyl alcohol 7
Fig.1.5 Lignin biosynthetic pathway 9
Fig.1.6 Schematic view of some branches of
phenylpropanoid metabolism
11
Fig.2.1 Strategy used for purification of CCR from cell
lysate
26
Fig.2.2 The hanging-drop vapor-diffusion method for
protein crystallization
35
Fig.3.1 Over expression of CCRH1 and CCRH2 Protein 37
Fig.3.2 Over-expression of CCRH1 protein in soluble form
harvesting at different Temperature after 0.5 mM
induction
38
harvesting at different times after 0.5 mM induction
at 20 °C
39
Fig.3.4 Inclusion bodies of over-expressed CCRH1 protein
at different IPTG concentration
39
Fig.3.5 Over expression of CCRH1 and CCRH2 Proteins
cloned in pET 41a (+)
40
harvesting at different Temperature after 0.5Mm
induction
41
harvesting at different times after 0.5Mm induction
at 18 °C
41
Fig.3.8 Chromatogram for Ni Sepharose 6 column affinity
chromatography (ÄKTAexplorer™)
42

Fig.3.9 Affinity Chromatography SDS PAGE (Gel Picture) 42
Fig.3.10 Chromatogram for HiPrep Sephadex G-25
Desalting (ÄKTAexplorer™)
43
Fig.3.11 Chromatogram for DEAE-Sepharose Anion
Exchange chromatography
44
Fig.3.12 Anion Exchange SDS PAGE (Gel Picture) 44
Fig.3.13: Chromatogram for HiPrep Sephadex G-25
Desalting (ÄKTAexplorer™)
45
Fig.3.14 Western blot analysis for CCR 47
Fig.3.15 ESI-MS analysis showing sequence of CCR
Proteins and contaminants
48
Fig.3.16 Graph of A340 vs. Time for Cinnamaldehyde 51
Fig.3.17 Graph of A340 vs. Time for Coniferaldehyde 51
Fig.3.18 Graph of A340 vs. Time for sinapaldehyde 52
Fig.3.19 Graph of residual activity (%) vs. pH 53
Fig.3.20 Graph of residual Enzyme activity (%) Vs.
Temperature
53
Fig.3.21 Graph of residual activity (%) Vs. Time (in Hours)
at 4ºC (pH stability)
54
at 25ºC (pH stability)
54
(Temperature stability)
55

List of Tables
Table number Title Page number
Table2.1 Lab Equipments and Instruments 19
Table2.2 Different Media Used for Studies 19
Table2.3 Host Cells 20
Table2.4 Solutions for induction of E. coli 20
Table2.5 Buffers and Solutions for Gel Electrophoresis
(PAGE)
21
Table2.6 Buffers and Solutions for Protein Extraction 21
Table 2.7 Solutions used in Affinity Chromatography 22
Table2.8 Solutions used in Ion Exchange Chromatography 22
Table 2.9 Solutions used in Western Blot 22
Table 2.10 Solutions used in Enzyme Kinetics 23
Table2.11 Solutions used in ESI-MS 23
Table3.1 Overview of the optimized purification protocol for
CCR
46
Table3.2 Antibodies for Western blot analysis with their
respective dilutions used
47
Table3.3 Percent homology of CCR protein with available
sequence in database
49
Table 3.4 Specific activities of purified CCR enzyme for
different aldehydes
52

ABSTRACT
Paper industry in India mainly uses bamboos, Eucalyptus sp., Casuarina sp. and
Leucaena sp. as a source for pulp. Although all these plant species are of importance to
the paper industry, Leucaena sp. is extensively used in India and about 25% raw
material for pulp industry comes from this plant.
In the production of pulp (which is mainly cellulose), the hemicellulose and much of
the lignin is removed using alkali. To produce bright paper, pulp may require a further
bleaching treatment to remove lignin residues. This delignification process consumes
large quantities of energy and hazardous chemicals and is neither economical nor
environment friendly. Reduced lignin content or altered quality of lignin in pulp wood
species without compromising the mechanical strength of the plant is desirable for
paper industry. The alteration of the lignin components has to be achieved in a manner
where in the sinapyl alcohol (S unit, less compact lignin) to guaiacyl alcohol (G unit,
more compact lignin) ratios are in favour of sinapyl alcohol (higher S/G ratio desirable
for paper industry).
It has been hypothesized that CCR plays a key regulatory role in lignin biosynthesis as
the first committed step in the production of monolignols from phenylpropanoid
metabolites. Cinnamoyl CoA Reductase (CCR) catalyzes the reduction of
Hydroxycinnamoyl-CoA thioesters to the corresponding aldehydes. This reaction is
considered to be a potential control point that regulates the overall carbon flux towards
lignin. Studies have shown that altering the expression of CCR gene (down-regulation
and up-regulation) has a direct effect on quality and quantity of lignin produced.
In this context, the present study was aimed at a better understanding of the enzymes
involved in phenylpropanoid pathway; the one responsible for monolignols
biosynthesis in plants, and study its structure-functional relationships. The enzyme
Cinnamyl Co-A Reductase (CCR) involved in reversible reduction of cinnamaldehydes
to hydroxycinnamyl alcohols was targeted in the study due to its key role in the
monolignols biosynthesis in plants.

The CCR gene was directionally cloned in expression vector pET 30-b (+) using the
restriction sites Nde1 and Xho1, and into pET 41 a (+) using BamH1 and Xho1, and
expressed in E.coli BL21 DE3. A protein with a molecular weight of 40 KDa was
expressed in these cells.
A simple and efficient purification protocol employing two chromatography steps- Ni-
Sepharose affinity and DEAE-Sepharose anion-exchange chromatography were
optimized for purifying His Tagged CCRH1 which was confirmed by Western Blot
analysis.
Kinetic studies on the purified CCR showed that Coniferaldehyde is the preferred
substrate for CCR. The results can be further validated by enzyme kinetic studies.
Purified CCR showed a temperature and pH optima at 30 °C and 9 pH, respectively.
Attempts were made to crystallize purified CCR.

1
Chapter 1
Introduction
Plants and paper industry:-
Nature serves the mankind, in one way or the other, in every aspect of life. Trees are
reservoirs of many economically and biotechnologically significant products. Wood is
one such gift of nature, which has diverse applications for mankind. One of the most well
known applications of wood is paper and paper products.
In the past forty years, the world wide annual production of paper has increased more
than three folds. Wood, agricultural residues and many other plant materials, which can
be used for pulp and paper production, consist largely of ‘lignocellulose’, which is a
composite of mainly cellulose, hemicellulose and lignin.
Lignin, one of the most abundant natural (terrestrial) organic polymers and a major
constituent of wood, forms an integral cell wall component of all vascular plants. It
represents, on an average, 25% of the terrestrial plant biomass.
In the production of pulp (which is mainly cellulose), the hemicellulose and much of the
lignin is removed using alkali. To produce bright paper, pulp may require a further
bleaching treatment to remove lignin residues. This delignification process consumes
large quantities of energy and hazardous chemicals and is neither economical nor
environment friendly. Reduced lignin content or altered quality of lignin in pulp wood
species without compromising the mechanical strength of the plant is desirable for paper
industry. The alteration of the lignin components has to be achieved in a manner where in
the sinapyl alcohol (S unit, less compact lignin) to guaiacyl alcohol (G unit, more
compact lignin) ratios are in favour of sinapyl alcohol (higher S/G ratio desirable for
paper industry).
Although lignin has been studied for over a century, many aspects of its biosynthesis still
remain unresolved. The monolignol biosynthetic pathway has been redrawn many times
and yet remains a matter of debate. The enzymes involved in lignin biosynthesis include:
phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), coumarate 3-

2
hydroxylase (C3H), 4-coumarate CoA ligase (4CL), caffeoyl CoA 3-O-
methyltransferase (CCoAOMT), caffeate O-methyltransferase (COMT), ferulate 5-
hydroxylase/ coniferaldehyde 5-hydroxylase (F5H/CAld5H), cinnamoyl CoA reductase
(CCR), cinnamyl alcohol dehydrogenase (CAD), sinapyl alcohol dehydrogenase (SAD),
UDP-glycosyltransferase (UDP-GT), coniferin β glucosidase (CBG), peroxidases and
laccases.
It has been hypothesized that CCR plays a key regulatory role in lignin biosynthesis as
the first committed step in the production of monolignols from phenylpropanoid
metabolites. Cinnamoyl CoA Reductase (CCR) catalyzes the reduction of
hydroxycinnamoyl-CoA thioesters to the corresponding aldehydes. This reaction is
considered to be a potential control point that regulates the overall carbon flux towards
lignin. Studies have shown that altering the expression of CCR gene (down-regulation
and up-regulation) has a direct effect on quality and quantity of lignin produced.
Leucaena sp. as a source for pulp. Although all these plant species are of importance to
the paper industry, Leucaena sp. is extensively used in India and about 25% raw material
for pulp industry comes from this plant. Leucaena leucocephala is native of Central
America and belongs to the family Fabaceae. It is one of the most versatile and fast
growing tree species.
Lignin biosynthesis genes have not been studied so far in Leucaena sp. Study of these
genes will help in understanding the lignin biosynthetic pathway in Leucaena sp. and its
manipulation so as to meet the needs of pulp and paper industry.
Few research groups in India are engaged in raising transgenic Leucaena sp. with low or
altered lignin content. One of the prominent groups at National Chemical Laboratory
(Council of Scientific and Industrial Research) is actively engaged in pursuing research
on genes involved in lignin biosynthetic pathway in L. leucocephala. I had an opportunity
to work under the guidance of Dr. B. M. Khan at this institute and the work carried out is
presented here.

3
1.1 Review of literature
Leucaena leucocephala
Leucaena is represented by 22 species. Of these, 6 are intraspecific taxa and 2 are
widespread spontaneous hybrids. Most of the species are diploid 2n=52 or 56. However,
4 species are tetraploid 2n = 4X=104 or 112 (Hughes, 1998). L. leucocephala is a
member of the genus related to the other species within the Mimosoideae sub-family, its
subspecies and other related genera. Leucaena leucocephala variety K - 636 have been
used in this study.
Fig.1.1: seed, branch and tree of Leucaena leucocephala
Classification of Leucaena leucocephala
Division: Magnoliophyta
Class: Magnoliopsida
Sub- class: Rosidae
Order: Fabales
Family: Fabaceae
Sub-family: Mimosoideae
Tribe: Mimoseae
Genus: Leucaena
Species: leucocephala
Leucaena is a native of Central America and has been naturalized pan-tropically.
Members of the genera are vigorous, drought tolerant, highly palatable, high yielding,
and rich in protein and grow in a wide range of soils (Jones, 1979; Hughes, 1998).

4
However, these attributes are limited by the occurrence of anti-nutritive factors in the
fodder, such as tannins and mimosine (Jones, 1979; Hegarty et al., 1964b; Hammond et
al., 1989 a, b). Leucaena occupies 2 to 5 million hectares of land worldwide (Brewbaker
and Sorenson, 1990). They are recognized as some of the fastest growing and most useful
trees in the tropics (NRC, 1984).A major pest, the psyllid, has infested leucaena stands
around the world with particular susceptibility expressed by varieties of Leucaena
leucocephala. Leucaena vary widely in leaf and tree shape, ranging from shrubs to stately
trees. Leaves are alternate and bipinately compound. Flowers range from bright yellow
and pink to white.
Why Leucaena leucocephala?
Paper industry in India mainly uses Bamboo, Eucalyptus sp., Casuarina sp. and
Leucaena sp. as a source for paper pulp. Selection of the species depends upon
availability, price and acceptability by any one given industrial unit. In bamboo growing
country like India, the proportionate use of bamboos and hardwood species is in the ratio
of 15:85. Although all these plant species are of importance to the paper industry,
Leucaena sp. is exclusively used in India and about 25% of raw material for pulp and
paper industry comes from this plant. Leucaena sp. is a fast growing multipurpose tree
adapted to a variety of soils and climatic conditions. Leucaena wood is among the best
hardwoods for paper and rayon making. It produces pulp that is high in holocellulose,
low in silica, ash, lignin, alcohol-benzene soluble and hot water soluble. Pulp yield is 50
to 52%. Its short fiber is suitable for rayan production (Pottinger and Hughes, 1995). To
meet the increasing demand of high quality wood for paper industry, it is essential to
provide designer plant species. However, as a safeguard for the future no plant should be
harvested from areas that may challenge sustainability. It will thus be crucial to raise
plantations of the plant species with elite materials and or genetically modified plants that
meet the demands of the pulp and the paper industry in economical and sustainable
manner. However no study has been done on Lignin Biosynthesis gene(s) so far in
Leucaena sp. and study of these gene(s) will help in understanding the Lignin
Biosynthetic Pathway in Leucaena sp. and its manipulation so as to meet the needs of
pulp and paper industry.
Leucaena leucocephala species growth forms
L. leucocephala has three growth forms that are commonly found in tropics (Hughes,
1998). The first and common type is a shrubby free shading form known as the Hawaiian

5
Leucaena, which is weedy and low yielding. The second growth form is giant type,
which is a tall tree of about 20 meters, with large leaves, pod and seeds, and almost
without branches. It is native of Central America and Mexico, and produces twice the
biomass of the common type. It is suitable for timber, wood product and industrial fuel.
The last is Peru type, which are a multi-branched, semi-erect, medium height (about 10
meters) and the most productive form.
Wood
Leucaena wood has a thin bark, which is about 8% dry matter at the age of 5 years. The
sapwood is yellow-white, while the heartwood is yellow to reddish brown. Bole wood
has a specific gravity of 0.54 at the age of 6 to 8 years. This is similar to the density,
tensile, compression, bending and shear strength of oak, ash, birch and sugar maple. It is
fine textured and workable. It absorbs preservatives, and can be treated against termites
(Pottinger and Huges, 1995). Leucaena wood is among the best hardwoods for the paper
and rayon making. It produces pulp that is high in holocellulose, low in silica, ash, lignin,
alcohol-benzene soluble and hot water soluble. Pulp yield is 50 to 52%. Its short fiber is
suitable for rayan production (Pottinger and Hughes, 1995). Wood from giant Leucaena
has a heating value of 4640 Kcal.kg-1 at the age of 2 to 4 years, and 7000 Kcal.kg-1 at
the age of 8 years, which is equivalent to 70% of the heating value of fossil fuel.
Uses
Under suitable conditions, leucaena have produced wood yields similar to the best of
tropical trees. Mean annual wood increments fall in the range of 20-60 m3
/yr in short
rotation (3-5 yr) trials. Wood of a 4-yr-old tree has about 46% moisture and a specific
gravity of 0.52. This medium hard wood serves well for posts, house-building, utensils
and parquet flooring. It is an excellent pulpwood (Hu 1987), and a preferred fuel wood
that burns with little smoke or ash. Charcoal is of high yield and quality. Leucaena is
perhaps best known as a fodder plant for ruminant animals, with high acceptability and
dry matter digestibility (55-70%). The Leucaena has been the model tree for alley
farming, a system in which trees are planted in crop fields and managed as bedgrows.
Lignin
Lignin (from Latin lignum meaning wood) is the most abundant natural (terrestrial)
organic polymer, next only to cellulose, and a major constituent of wood. It is a vital cell
wall component of all vascular plants and represents on an average of 25% of the

6
terrestrial plant biomass. Lignins provide mechanical support and are functional in water
transport, and disease resistance in higher terrestrial plants. The ability to synthesize
lignin has been essential in the evolutionary adaptation of plants from an aquatic
environment to land and provides crucial structural integrity to the cell wall stiffness and
strength of the stem (Chabannes et al., 2001; Jones et al., 2001).
Occurrence
Lignin is primarily synthesized and deposited in the secondary cell wall of specialized
cells such as xylem vessels, tracheids and fibers. It is also deposited in minor amounts in
the periderm where in association with suberin it provides a protective role against
pathogens (Sarkanen and Ludwig, 1971). In addition, lignin waterproofs the cell wall;
enabling transport of water and solutes through the vascular system. Lignin does not
occur in algae, lichens or mosses (Nimz and Tutschek, 1977), whereas the “lignins” of
bark differ in their structure from typical wood lignins (Zimmermann et al., 1985).
Lignin content is higher in softwoods (27-33%) than in hardwood (18-25%) and grasses
(17-24%). The highest amounts of lignin (35–40 %) occur in compression wood on the
lower part of branches and leaning stems (Sarkanen and Ludwig, 1971).
Chemistry of lignin
Lignins are complex aromatic heteropolymers synthesized from the dehydrogenative
polymerization of monolignols, namely p-coumaryl, coniferyl and sinapyl alcohol
monomers differing in their degree of methoxylation (Fig.1.2 a-c) (Freudenberg and
Neish, 1968).
Fig.1.2:Monolignols and Lignin units: Monolignols (a) p- coumaryl (b) coniferyl, and

7
(c) sinapyl alcohol and Lignin units (d) p-Hydroxyphenyl or H unit, (e) Guaiacyl or G
unit, and (f) Syringyl or S unit.
These monolignols as such are toxic to the plant cell and hence are converted to
respective glycosides, which are transported to the cell wall via cell membrane (Whetten
and Sederoff, 1995; Boerjan et al., 2003). In the cell wall the glycosides are again
hydrolyzed to form Hydroxyphenyl, Guaiacyl and Syringyl units respectively (Fig.1.2 d-
f). These monomers are linked together via end-wise and radical coupling reactions
(Sarkanen and Ludwig, 1971; Freudenberg and Neish, 1968) to produce H, G and S
lignin respectively. This process is known as lignification. Monolignol dimerization and
lignification are substantially different processes (Adler, 1977), explaining why
lignification produces frequencies of the various units that are different from those
produced by dimerization or bulk polymerization in vitro.
Fig.1.3: (a) Dimerization of coniferyl alcohol produces only three dimers, in each of
which at least one of the coniferyl alcohols is coupled at its β position. The 5–5 and 5–O–
4 dimers (crossed out) do not arise in any significant way from monomer dimerization
reactions.
Fig.1.4: (b) Cross coupling of coniferyl alcohol with a G unit gives only two main
products, explaining why there are more β-ethers formed during lignification than in
monolignol dimerization. Coupling of preformed oligomers is the source of most of the
5–5- and 5–O–4 units. Sites of further coupling reactions during lignification are
indicated by arrows (Baucher et al., 1998; Boerjan et al., 2003).

8
Dimerization and Lignification: Dimerization (a) differs substantially from simple
Lignification (b) of monolignols.
As the monolignols differ in their degree of methoxylation, the respective lignins
produced are also different in their compactness. Syringyl unit has less number of
potential radical forming sites than guaiacyl unit and hence the lignin formed from S unit
monomers is less compact than that formed from G unit monomers.
The three monolignols (S, G and H) are incorporated at different stages of cell wall
formation. In angiosperms, H units are deposited first, followed by G and S units
(Donaldson et al., 2001; Terashima et al., 1993). Lignin in vessels is generally enriched
in G units, whereas lignin in fibers is typically enriched in S units (Saka and Goring,
1985). A large proportion of S units are also found in secondary walls of ray parenchyma.
In gymnosperms, the lignin deposited in compression wood is enriched in H units
(Timell, 1986). The difference in timing of monolignol deposition is associated with
variations in lignin condensation in the individual cell wall layers, as shown by
immunocytochemistry with antibodies raised against pure H, pure G, or mixed G-S
synthetic lignins (Chabannes et al., 2001). The amount and composition of lignins vary
among taxa, cell types and individual cell wall layers, and are influenced by
developmental and environmental cues (Campbell and Sederoff, 1996).
Lignin Biosynthesis:
Lignin biosynthesis is a significant part of Phenylpropanoid biosynthetic pathway which
itself can be broadly divided into common phenylpropanoid pathway and monolignol
specific biosynthetic pathway. Monolignol specific branch of phenylpropanoid pathway
leads to the formation of monolignols and subsequently lignin. Phenylpropanoid
metabolism comprises a complex series of branching biochemical reactions that provide
the plant with a host of important phenolic compounds. Phenylpropanoids have a range of
important functions in plants, including as structural components (such as lignin),
protectants against biotic and abiotic stresses (antipathogenic phytoalexins, antioxidants
and UV-absorbing compounds), pigments (particularly the anthocyanins) and signaling
molecules (e.g. flavonoid nodulation factors).
The synthesis of lignin represents one of the most energy demanding biosynthetic
pathways in plants, requiring large quantities of carbon skeletons. The monolignols that
make up the lignin polymer are derived from the sequential action of three key
biosynthetic pathways (Lewis & Yamamoto, 1990; Whetten & Sederoff, 1995; Boerjan et

9
al, 2003). Shikimic acid metabolism gives rise to the amino acid phenylalanine, which in
turn is used by general phenylpropanoid metabolism to synthesize the
hydroxycinnamoyl-CoA-thioesters that are used in the two-step reduction, and phenyl
substitution reactions of monolignol biosynthesis.
The monolignol biosynthesis pathway, functions of its products, and regulation of
expression of biosynthesis genes, have been discussed in several recent reviews.
Manipulating the phenylpropanoid metabolism holds significant commercial potential.
Increasing or reducing the expression of particular genes in transgenic plants raises many
possibilities to alter phenylpropanoid biosynthesis both quantitatively and qualitatively.
However, making a predictable change may not be always possible as the pathways are
complex and alternative routes to the synthesis of particular compounds may be present.
Fig.1.5: Lignin biosynthetic pathway.

10
The lignin biosynthetic pathway can be subdivided into four steps starting with
(a) The biosynthesis of monolignols
(b) Transport of monolignols from the site of synthesis to the site of polymerization
(c) Dehydrogenation and
(d) Polymerization of monolignols to give the final product.
Biosynthetic pathway of Monolignols:
During the last two decades, there has been a great deal of interest in cloning and
characterization of the genes controlling monolignol biosynthesis pathways in trees and
other plants. A number of reviews have been done about the advancements of monolignol
biosynthesis pathways (Whetten and Sederoff, 1995; Whetten et al., 1998; Humphreys
and Chapple, 2002; Boerjan et al., 2003).
There is an enormous variation in lignin content and composition among plant species,
tissues, and cell types. Developmental stages of plant and environmental conditions play
a significant role in deciding the content and composition of lignin and its deposition. It is
debatable whether lignin biosynthesis, in all plants, follows the exact same pathway or
not. To date, most of the genes for monolignol biosynthesis have been identified and
characterized in various plant species.

11
Fig.1.6: Schematic view of some branches of phenylpropanoid metabolism (Weisshaar
and Jenkins, 1998).
Cinnamoyl Coenzyme A Reductase (CCR):
As discussed above, the biosynthesis of lignin begins with the common phenylpropanoid
pathway starting with the deamination of phenylalanine and leading to the cinnamoyl-
CoA esters which are the common precursors of a wide array of end-products such as
flavonoids, coumarins and many small phenolic molecules, (Hahlbrock and Scheel, 1989;
Boerjan et al., 2003) that play key roles during plant development and defense.
Cinnamoyl-CoA esters are then channeled into the lignin branch pathway to produce
monolignols via two reductive steps using Cinnamoyl CoA Reductase (CCR; EC:
1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD). The first reductive step in lignin

12
biosynthetic pathway is acted upon by CCR and it controls the over-all carbon flux
towards lignin (Lacombe et al., 1997). The reduction of cinnamoyl CoA esters to
cinnamaldehydes is the first metabolic step committed to monolignol formation.
Transport of monolignols:
After the synthesis, the lignin precursors or monolignols are transported to the cell wall
where they are oxidized and polymerized. The monolignols formed are as such insoluble
and toxic to the plant cell and hence are converted to their respective glucosides by the
action of enzyme UDP-glycosyltransferase. This conversion renders the monolignols,
soluble and less toxic to the plant cells. These glucosides can be stored in plant vacuoles,
which are transported to the cell wall as the need arises. In gymnosperms and some
angiosperms, monolignol 4–O–β-D-glucosides accumulate to high levels in the cambial
tissues (Steeves et al., 2001). It has been hypothesized that these monolignol glucosides
are storage or transport forms of the monolignols. A uridine diphosphate glucose (UDP-
G) coniferyl alcohol glucosyl transferase (Steeves et al., 2001), together with coniferin-
beta-glucosidase (CBG), may regulate storage and mobilization of monolignols for lignan
and/or lignin biosynthesis (Dharmawardhana et al., 1995).
Dehydrogenation:
After the transport of the monolignols to the cell wall, lignin is formed through
dehydrogenative polymerization of the monolignols. The dehydrogenation to monolignol
radicals has been attributed to different classes of proteins, such as peroxidases, laccases,
polyphenol oxidases, and coniferyl alcohol oxidase, which of these enzymes or a
combination thereof are responsible for the dehydrogenation of the monolignols in plants
and whether monolignol oxidation occurs through redox shuttle-mediated oxidation are
still unclear (Onnerud et al., 2002).
Polymerization:
It is believed that lignin is polymerized at the outside of the plasma membrane in
secondary cell walls. Thus, monolignols that are synthesized inside plasma membrane
need to be transported across plasma membranes for polymerization. Based on
biochemical and cellular evidence, it has been suggested that laccases and peroxidases
may be two types of possible enzymes involved in lignin polymerization (Christensen et
al., 1998 ;). However, convincing genetic evidence to support this suggestion is lacking.

13
Whether lignin is polymerized from monomer units randomly or in a guided way remains
an enigma (Ralph et al., 2004; Davin and Lewis, 2005). A gene encoding a dirigent
protein was cloned and the biochemical results suggested that the dirigent protein might
play a role in guiding a stereo-specific lignin polymerization (Davin et al., 1997). This
hypothesis still remains to be confirmed.
The actual process of lignin polymer formation occurs without the rigid biochemical
controls seen in the biosynthesis of the precursor monolignols, giving rise to a unique
class of polymers. Lignins are racemic mixtures (Ralph et al., 1999), derived from radical
coupling reactions under chemical (but no apparent biochemical) control between
phenolic radicals (monolignol radicals) in an essentially combinatorial fashion. The
accepted model for lignin polymerization is based on simple chemically controlled
combinatorial coupling reactions, which was recently challenged by Davin and Lewis
(2000). According to Davin and Lewis group, the macromolecular assembly of lignin is
not based on “random coupling” of monolignols. Instead, this group proposed a strong
biological control over the outcome of phenoxy radical coupling in vivo. The new theory
arose from the discovery of a fascinating class of dirigent proteins implicated in lignin
biosynthesis (Boerjan et al., 2003); lignans are de-hydrodimers of monolignols and are
typically optically active. The first such dirigent protein discovered; guided the
dimerization of coniferyl alcohol radicals to produce an optically active lignan,
pinoresinol. The corresponding gene was cloned and shown to encode a cell wall–
localized protein. The finding was extrapolated to lignification, suggesting that such
proteins would logically be responsible for specifying the exact structure of the lignin
polymer, bringing lignins in line with proteins and polysaccharides that are more
carefully biosynthesized (Davin and Lewis, 2000).
Radical generation and radical coupling:
After their dehydrogenation, the radicals, which are relatively stable owing to electron
delocalization that provides single-electron density to the side-chain β position, are
coupled. The most important reaction is cross-coupling to the growing polymer to extend
the complex three-dimensional lignin network (Fig.1.4). But, such coupling reactions are
radical quenching. Each extension of the polymer requires new radicals on each of the
two coupling partners. Radicals on the growing lignin polymer are thought to be
generated by radical transfer from monolignols or other intermediaries (Takahama and
Oniki, 1994).

14
Similar radical transfer mechanisms can be envisioned between the monolignols and the
growing polymer, i.e. the monolignols may act as the radical shuttles. When a
monolignol radical encounters a polymer radical, it may cross-couple with it, but when
the polymer is not electron-deficient, radical transfer may occur and the monolignol will
diffuse back to the peroxidase/laccase to be reoxidized. Alternatively, redox shuttles,
such as a Mn2+/Mn3+ system (Onnerud et al., 2002), may be involved.
Lignin deposition:
Lignin deposition begins at the cell corners in the region of the middle lamella and the
primary wall when S1 (outermost layer of secondary wall) layer formation has initiated.
It is one of the final stages of xylem cell differentiation and mainly takes place during
secondary thickening of the cell wall (Donaldson et al., 2001). The deposition of lignin
proceeds in different phases; each proceeded by the deposition of the polysaccharide
matrix in the S2 (middle layer of secondary wall) layer. The bulk of lignin is deposited
after cellulose and hemicellulose have been deposited in the S3 (innermost layer of
secondary wall) layer. Generally, lignin concentration is higher in the middle lamella and
cell corners than in the S2 layer of secondary wall (Baucher et al., 1998; Donaldson et
al., 2001; Saka and Goring, 1985).
Given that the middle lamella and the cell corners are rich in Ca2+ pectate and are the
first sites to be lignified, Ca2+ pectate-bound peroxidases may conceivably play a role in
the spatial control of lignin deposition, and changes in Ca2+ and H+ concentrations may
modulate the location of these peroxidases (Carpin et al., 2001). The negatively charged
pectins are also good binding sites for polyamines (Carpin et al., 2001) and, hence, may
be suitable sites for H2O2 generation by polyamine oxidases. Also, evidence is available
to show that ferulates and diferulates may act as attachment sites for monolignols (Ralph
et al., 1995)
Lignin Modification through Genetic Engineering:
The lignin biosynthetic pathway has been studied for more than a century but has
undergone major revisions over the past decade. Significant progress has been made in
cloning new genes by genetic and combined bioinformatics and biochemistry approaches.
Molecular breeding of forest trees will soon be possible due to advances in gene transfer
methods. A possible application would be to influence the quality of lignin. If lignin

15
could be changed into a more easily removable form, paper pulp would require
significantly less cooking and bleaching. Within the lignin biosynthesis theme, the aim of
research is to isolate the lignin-polymerizing enzyme and its gene, and to transfer the
gene to forest trees.
Lignin genetic engineering is promoted as a promising strategy to improve fiber
production but the drawbacks of anti-sense manipulation and transgene stability are not
seriously dealt with. Trees genetically modified to produce low lignin are called "super"
trees with little consideration of pest resistance and genetic stability. Field and pulping
performance of transgenic poplars with altered lignin was evaluated to be superior by the
developers of the poplar and abnormal pest damage was not found. However, the pest
damage studies were cursory and not compared with experimental controls, but with
norms reported by government agencies.
Lignin Biosynthesis and proteomics:
National Chemical Laboratory (Council of Scientific and Industrial Research) is actively
engaged in pursuing research on genes involved in lignin biosynthetic pathway in L.
leucocephala.
The eight genes involved in lignin biosynthesis pathway were isolated, cloned, expressed
and partially characterized. Those genes are cinnamate 4-hydroxylase (C4H), 4-
coumarate CoA ligase (4CL), caffeoyl CoA 3-O- methyltransferase (CCoAOMT),
caffeate O-methyltransferase (COMT), ferulate 5-hydroxylase/ coniferaldehyde 5-
hydroxylase (F5H/CAld5H), cinnamoyl CoA reductase (CCR), cinnamyl alcohol
dehydrogenase (CAD), peroxidases. As we know that, CCR plays a key regulatory role
in lignin biosynthesis as the first committed step in the production of monolignols
from phenylpropanoid metabolites. This is the first step of phenylpropanoid pathway
specifically dedicated to the monolignol biosynthesis branch.
Thus we planned to carry out detailed studies of this enzyme with respect to structure and
function. Basically these studies involves structure determination through
crystallography, Enzyme Kinetics studies, substrate binding studies, Fluorescence
studies, Protein Refolding studies etc. The protein structure of this enzyme is still
unknown; so attempts have been made to determine three dimensional structure of
protein through X-RAY crystallography. The basic need of crystallography is to get
pure protein (>97%).This is the most tedious task with respect to time, energy and
money.

16
Thus I had carried out my work on purification and characterization of enzyme
Cinnamoyl CoA reductase (CCR), which was already expressed as recombinant protein
in E.coli expression system. Two full length CCR genes (isoforms) were isolated, Cloned
and expressed in E.coli by Dr. Sameer Srivastava in National Chemical Laboratory.
Different chromatographic techniques like affinity chromatography, ion exchange and gel
filtration were used to purify CCR protein.

17
1.2 Aims and Objectives
The present work can be divided into 3 parts-
1- Expression of CCR protein in E.coli
2- Purification of the expressed protein from the cell lysate.
3- Structural and Functional studies of the purified enzyme.

Chapter 2
Materials and Methods

18
Chapter 2
Materials and Methods
2.1 Materials
This chapter deals with the general laboratory equipments and techniques
routinely followed during the course of work. Other important specific methodologies
followed are discussed separately.
2.1.1 Glassware
Glassware used in all the experiments was procured from “Borosil”, India.
Test tubes (25 mm x 150 mm), glass bottles (70 mm x 125 mm), petridishes (85 mm x
15 mm; 55 mm x 15 mm), conical flasks (100, 250 & 500 mL; 1, 2 & 5 L capacity)
Micropipettes (2,10, 20, 200,1000 μL capacity) and pipettes (1, 2, 5, 10 and 25 mL
capacity) were used during the course of study.
2.1.2. Preparation of Glassware
Glassware used for all the experiments were cleaned by boiling in a saturated
solution of Sodium bicarbonate for 1h followed by repeated washing in tap water.
Thereafter, it was immersed in 30% HNO3 solution for 30 min followed by repeated
washing in tap water and rinsed with distilled water. Washed glassware was thereafter
dried at room temperature. Test tubes and flasks were plugged with absorbent cotton
(Mamta Surgical Cotton Industries Ltd., Rajasthan, India). Autoclaving of the
glassware and above items was done at 121 0
C and 15 psi for 1 hrs.
2.1.3 Plastic ware
Sterile disposable petridishes (55 mm and 85 mm diameter) were procured
from “Laxbro”, India. Eppendorf tubes (1.5 mL and 2 mL capacity), microtips (10,
200 and 1000 μL capacity) were obtained from “Tarsons” and “Axygen”, India.

19
2.1.4 Chemicals
Tris, IPTG, SDS, EDTA, APS, Kan, Imidazole and Ethidium bromide were
purchased from Sigma-Aldrich (USA), Bio-world (USA). Agarose, lysozyme was
obtained from Bioworld (USA). Bradford Reagent was purchased from Biorad. All
other chemicals and solvents of analytical grade were purchased from HiMedia,
Qualigens Fine Chemicals and E-Merck Laboratories, India. The Sucrose, glucose
and agar-agar were obtained from “Hi- Media”.
2.1.5 Equipments
Table2.1: Lab Equipments and Instruments
Sr. No. Equipment Company
1 Balances Contech / Sartorious
2 Water bath Fisher Scientific
3 Dry Bath Eppendorf
4 Incubator New Brunswick
5 Centrifuge Sorvall / Eppendorf
6 Spectrophotometer Perkin Elmer
7 Power pack Bio-Rad
8 Protein Gel Electrophoresis Units GE HealthCare
9 Speed Vacuum concentrater Eppendorf
10 pH-Meter Digital corp.
11 Water purification system Millipore Unit (Milli RO/ Milli Q)
12 Microwave oven Electrolux
13 Fridge/ Deep freezer Vestfrost / Godrej
14 Magnetic rotator REMI
15 Laminar Air Flow Microfilt India
16 Nanovue GE Healthcare
17 FPLC (AKTA Explorer) GE Healthcare
18 ELISA plate reader Bio-Rad
19 Sonication system Misonix
20 Icematic Saksham Technologies
2.1.6 Different Media Used for Studies
Table2.2: Different Media Used for Studies
Name Ingredients Preparation and
storage
Luria
Bertani
Broth (LB)
1% Tryptone type I
0.5%Yeast extract
1% NaCl
pH adjusted to 7.0 with NaOH, Store
at room temperature or at 4 0
C

20
2.1.7 Host Cells
E.coli Genotype
XL-10 Gold endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte Δ(mcrA)183
Δ(mcrCB-hsdSMR-mrr)173 tetR
F'[proAB lacIq
ZΔM15
Tn10(TetR
Amy CmR
)]
BL 21 F-, ompT hsdSB (rB – mB -) gal dcm (DE3) pLysS (CamR)
Table2.3: Host Cells
2.1.8 Buffers and Solutions
Name Ingredients Preparation and Storage
IPTG
solution
200 mg/mL in SMQ
Sterile filtration and storage at
-20 0
C
Kanamycin 50 mg/mL in SMQ Sterile filtration and storage at
-20 0
C
Table 2.4: Solutions for induction of E. coli
Name Ingredients Preparation and
Storage
Monomer
solution
29.2% acrylamide
0.8% bis-acrylamide in water
Stored at 4 0
C in the
brown bottle
Stacking gel
(10%)
1 M Tris-HCl (pH 6.8)
Acrylamide/Bis 30%
10% (w/v) SDS
10% (w/v) APS
TEMED 5 μL
Store at room
temperature.
Separating
gel (10%)
Distilled Water 3.3 mL
1.5 M Tris-HCl (pH 8.8) 2.5 mL
Acrylamide/Bis 30% 4.0 mL
10% (w/v) SDS, 0.1 mL (SDS-PAGE)
10% (w/v) APS, 0.1 mL
TEMED, 4 μL
Store at room
temperature
5X Protein
loading
buffer
Distilled Water, 2.7 mL
0.5 M Tris-HCl (pH 6.8), 1.0 mL
Glycerin, 2.0 mL
10% (w/v) SDS, 3.3 mL (SDS-PAGE)
β-Mercaptoethanol, 0.5 mL
0.5% (w/v) Bromophenol blue, 0.5 mL
Store at 4 0
C

21
10X SDS-
electrode
buffer
Tris base 15 g
Glycine 94 g
SDS 5 g
Water up to 500 mL
Store at 4 0
C, dilute
1:10 before use
Staining
solution
Coomassie-blue R 250,
Methanol, 100 mL
Acetic acid, 20 mL
Water up to 200 mL
Store at room
temperature
Coomassie
Blue
Coomassie-blue R 250, 0.5 g
Water up to 50 mL
Dissolve the dye in
water and filtre
Destaining
solution
Methanol 45%
Acetic acid 10% Freshly prepared
Silver
staining
Fixing
solution
Methanol 45%
Acetic acid 10% Freshly prepared
Sensitizing
solution
30% ethanol
0.5 M sodium acetate
0.2% Na2S2O3
Store at room
temperature
Silver
solution
0.2% silver nitrate
0.03 % formaldehyde Freshly prepared
Developing
solution
6% Na2CO3
0.02% formaldehyde
0.001% sodium thiosulphate
Freshly prepared
Stop
solution
1.5% Na2EDTA Store at room
temperature
Table2.5: Buffers and Solutions for Gel Electrophoresis (PAGE)
Name Ingredient Preparation and Storage
Lysis
buffer
50mM Tris-HCl
1% Triton-X 100
500mM NaCl
10% Glycerol
Lysozyme100µg/mL (Added freshly)
Stored at 4 0
C
Sonication
Buffer
100 mM Tris-HCl, pH 8.0; 50 mM
Glycine Stored at 4 0
C
Dispersion
buffer
100 mM Tris-HCl, pH 8.0; 50 mM
Glycine; 8 M Urea Stored at 4 0
C
Table2.6: Buffers and Solutions for Protein Extraction

22
2.1.9 Solutions for Protein Purification
Binding
buffer
50 mM Tris
500 mM NaCl
20 mM imidazole
Adjust pH by adding
concentrated HCl and Store at 4 0
C
Wash buffer
50 mM Tris
500 mM NaCl
30 mM imidazole
Adjust pH by adding
C
Elution
buffer
50 mM Tris
500 mM NaCl
250 mM imidazole
Adjust pH by adding
C
Table 2.7: Solutions used in Affinity Chromatography
Binding/Equilibration
buffer
20 mM Tris-HCl,
pH. 7.5
Adjust pH by adding
concentrated HCl and
Store at 4 0
C
Elution Buffer 20 mM Tris-HCl,
0.5 M NaCl, pH. 7.5
Adjust pH by adding
concentrated HCl and
Store at 4 0
C
Table2.8: Solutions used in Ion Exchange Chromatography
Name Components Preparation and storage
Blocking solution
Ultra filtered water 14 mL
Diluent part A 4 mL
Diluent part B 2 mL
Room temperature.
Antibody wash solution
Ultra filtered water
Antibody wash solution
(16X) 10.00 mL
Room temperature.
Chemiluminiscent
substrate
Chemiluminiscent substrate
2.375 mL,
Chemiluminiscent substrate
enhancer 0.125 mL
Room temperature.

23
Table2.9: Solutions used in Western Blot
Enzyme purified CCR Store at 4 0
C
Substrate/s Cinnamaldehyde
Coniferaldehyde
Sinapaldehyde
Store at 4 0
C
Co substrate Coenzyme A Store at 4 0
C
Coenzyme NADP+ Store at 4 0
C
Buffer 200 mM Tris ( pH 8.8 ) Store at 4 0
C
Table2.10: Solutions used in enzyme kinetics
Destaining solution (50% acetonitrile / 50%
50 mM NH4HCO3)
Store at 4 0
C
Dehydration solution 100% acetonitrile Store at 4 0
C
Digestion Buffer in 10 mM DTT in 100
mM NH4HCO3
Store at 4 0
C
IAM
55 mM iodoacetate in
100mM NH4HCO3 Store at 4 0
C
Trypsin Stock 0.1μg/μL of Trypsin Store at -20 0
C
Table2.11: Solutions used in ESI-MS

24
2.2 Methods
2.2.1 Expression and Purification CCR Proteins CCRH1 & CCRH2.
2.2.1.1 Screening for over-expressing recombinant CCR proteins:
E.coli BL 21 (DE3) cells transformed with recombinant pET 30b+ plasmid were
screened for over-expression. The E.coli BL21 cells harboring recombinant plasmid
were grown O/N in 5 mL LB (Kanamycin 30 μg/mL) tubes. The above primary
culture was re-inoculated in fresh 5 mL tube containing LB (Kanamycin 30 μg/mL).
As the A600 reached 0.5, it was induced with 1 mM IPTG solution and kept at 37 °C
for 6 h. An uninduced control was taken out before inducing with IPTG. After 6 h of
induction, samples were centrifuged at 10000 g for 2 min and re-suspended in 200 μL
2X SDS-Gel loading buffer. The solution was boiled for 5 min, centrifuged at 10000 g
for 2 min and loaded onto a 10%.SDS- PAGE (as described earlier). The gel was run
at 100 V for 2 h and stained in Coomassie blue (R 250) staining solution.
2.2.1.2 Standardization of protein expression in E. coli BL21:
Eukaryotic proteins expressed intracellularly in E.coli are frequently sequestered into
insoluble inclusion bodies. Inclusion bodies are insoluble aggregates of denatured or
partly denatured protein that are less significant in further studies due to lack of
biological activity. Thus in order to obtain functionally active soluble protein (also
termed as Lysate), expression conditions are optimized by reduction in growth
temperature following induction, altering IPTG concentration and time of culture
incubation after induction.

25
2.2.2 Affinity Purification of CCR Protein
ÄKTAexplorer™ FPLC (GE Healthcare) was used for purification of CCR due to its
advantage of reproducibility, quality of protein separations, large sample volume
handling, etc. over manual purification. A Capture-Intermediate-Polishing-Protocol
(CIPP) strategy was devised in order to obtain highly pure protein.
Here, the capture phase was devised to isolate and concentrate CCR from rest of the
cell proteins. During the intermediate purification phase the objective was to remove
most of the bulk contaminating proteins. The final polishing step was incorporated to
achieve higher purity by removing any remaining trace impurities or closely related
substances. Different chromatographic techniques like Affinity, Ion-Exchange,
Hydrophobic Interactions, and Gel Filtration Chromatography were tried in order to
purify CCR.
However, a simple two step protocol comprising of an Affinity Chromatography
followed by Anion-Exchange Chromatography was found to be best suited for CCR,
and hence this protocol was optimized for purification of the protein from the cell
Lysate. All the protocols were performed at 4 °C to rescue the purified protein in
active form.
In order to monitor the purification at each step, the protein was checked in terms of
quantity, purity and activity using Bradford Assays (for protein quantification), SDS-
PAGE and Western Blot Analyses to monitor the purity of protein fractions, and,
Enzyme Assays specific for CCR for assessment of active CCR protein fractions. A
more detailed description of these qualitative and quantitative methods is given at the
end of this chapter.

26
Fig.2.1: ÄKTAexplorer™ FPLC (GE Healthcare) and Strategy used for purification
of CCR from cell lysate. AC-Affinity Chromatography, IEX-Ion exchange
Chromatography, GF-Gel Filtration

27
Affinity Chromatography (Capture)
Polyhistidine-tags are often used for affinity purification of recombinant proteins that
are expressed in Escherichia coli or other prokaryotic expression systems (Hengen,
1995). Histidine-tagged proteins have a high selective affinity for Ni2+
and several
other metal ions that can be immobilized on affinity matrix column such as Ni
Sepharose High Performance (HP) and Ni Sepharose 6 Fast Flow (FF) (GE
Healthcare). Consequently, a protein containing a Histidine tag will be selectively
bound to metal-ion-charged media while other cellular proteins will not bind or bind
weakly. In general, the Histidine-tagged protein is the strongest binder among all the
proteins in a crude sample extract. Moreover, Histidine tags are small and generally
less disruptive than other tags to the properties of the proteins on which they are
attached. Because of this, tag removal may not always be a priority. Due to its
advantage in retaining maximum amount of CCR from other cell proteins, Ni-
Sepharose High Performance Affinity Column was used at the Capture step of this
purification protocol. A simple method, comprising of binding the His-tagged protein
to the column, followed by washing to remove all the unbound proteins, and
subsequent elution of the bound protein was devised by using the following buffers.
Before loading onto the column, the Lysate was adjusted to the composition and pH
of the binding buffer by: adding buffer, NaCl, Imidazole, and additives from
concentrated stock solutions; by diluting the sample with binding buffer; or by buffer
exchange. The sample was passed through a 0.22 μm filter immediately before
applying to the column.
The Column was pre-equilibrated with Binding Buffer containing 20 mM Imidazole at a
constant flow-rate of 1 ml/min prior to sample application until a baseline for A280, pH
and conductivity were obtained. The filtered lysate was applied onto the column at a
constant flow-rate of 0.5 ml/min. All the unbound proteins were washed out by
washing the column with Wash Buffer 1 at a flow-rate of 1 ml/min till A280 fell below
1.0 m A.U. The column was further washed with Wash Buffer 2 having 40 mM
Imidazole till A280 fell below 1.0 m A.U. The protein bound onto the Ni-Sepharose
column was eluted by passing Elution Buffer which had 150 mM Imidazole at a flow-
rate of 1.0 ml/min. Eluted peaks were collected as 1.0 ml fractions using a fraction

28
collector. All the fractions were quantified for protein content by using Bradford
reagent, loaded onto 10% SDS-PAGE and were visualized using silver staining
protocol.
2.2.3 Sample Conditioning 1 (Concentration, Desalting)
The above capture phase of purification concentrated the His-tagged protein
considerably, however for further purification; even more concentrated sample was
required. Thus, fractions collected from Ni-Sepharose column were pooled together
and concentrated ten folds using Macrosep Centrifugal Devices (Pall Life Sciences)
with a molecular weight cut-off at 10 kDa. The protein solution was added into the
reservoir of the device and centrifuged at 5000x g at 4 ºC (Eppendorf 5810 R).
However, this came with a loss of about 2-3% of the protein which resulted due to
some non-specific binding to the filtration device.
This concentrated protein was further purified by DEAE-Sepharose anion exchange
chromatography. However, before applying the sample onto DEAE-Sepharose
column, the protein needed to be completely free from salts (Imidazole, NaCl, etc.)
resulting due to elution from Ni-Sepharose column, as well as, the sample needed to
be adjusted to the composition and concentration of the equilibration buffer to be used
for DEAE-Sepharose column. Thus, prior to anion exchange, an additional desalting
step using Sephadex G-25 gel filtration column had to be incorporated into this
purification protocol. This provided the necessary conditioning of the sample required
for ion-exchange chromatography.
2.2.4 Anion Exchange Chromatography (Intermediate Purification)
Ion-Exchange (IEX) chromatography separates molecules on the basis of differences
in their net surface charge. Molecules vary considerably in their charge properties and
will exhibit different degrees of interaction with charged chromatography media
according to differences in their overall charge, charge density and surface charge
distribution. Since all molecules with ionizable groups can be titrated, their net
surface charge is highly pH dependent. Proteins, which are built up of many different
amino acids containing weak acidic and basic groups, their net surface charge will
change gradually as the pH of the environment changes. Each protein has its own

29
unique net charge versus pH relationship which can be visualized as a titration curve.
This curve reflects how the overall net charge of the protein changes according to the
pH of the surroundings.
IEX chromatography takes advantage of the fact that the relationship between net
surface charge and pH is unique for a specific protein. In an IEX separation,
reversible interactions between charged molecules and oppositely charged IEX media
are controlled in order to favor binding or elution of specific molecules and achieve
separation. A protein that has no net charge at a pH equivalent to its isoelectric point
(pI) will not interact with a charged medium. However, at a pH above its isoelectric
point, a protein will bind to a positively charged medium or anion exchanger and, at a
pH below its pI, a protein will behind to a negatively charged medium or cation
exchanger. Both Anion and Cation exchange chromatography were attempted to
further purify Ni-Sepharose captured CCR. However, Anion Chromatography gave
far better results than its counterpart, and hence discussed below. As calculated from
the deduced amino sequence of CCR, its theoretical pI was calculated to be 6.4, and
thus, Anion exchange separation was devised at pH 7.5 for proper binding of CCR
onto DEAE-Sepharose matrix.
A simple method, comprising of binding the negatively charged protein to the surface
of DEAE-Sepharose Anion Exchanger column, followed by washing to remove all the
unbound proteins, and subsequent elution of the bound protein by setting up a NaCl
gradient was devised by using the following buffers.
The column was pre-equilibrated with Equilibration Buffer at a flow rate of 1.0
ml/min until a stable baseline for A280, pH and Conductivity were obtained. Sample
was loaded onto the column with a flow-rate of 0.5 ml/min. The column was washed
with Equilibration Buffer at same flow-rate until A280 fell below 1.0 m A.U. The
bound protein was eluted by applying a gradient elution against Elution Buffer with
1.0 M NaCl. The gradient was set up for 50 % of Elution Buffer over a period of one
hour at a constant flow rate of 0.5 ml/min.

30
2.2.5 Sample Conditioning 2 (polishing, concentration)
Q-Sepharose provided with necessary purification of CCR needed for further studies.
However, the protein still needed to be completely salt-free with high concentrations
in order to proceed with crystallization experiments. So, the fractions obtained from
above step were pooled together and concentrated to one tenth using Macrosep
Centrifugal Devices (Pall Life Sciences) with a molecular weight cut-off at 10 kDa.
The protein solution was added into the reservoir of the device and centrifuged at
5000x g at 4 ºC (Eppendorf 5810 R). The concentrated sample was then desalted
using Sephadex G-25 gel filtration column. This provided the necessary conditioning
of the sample required for further experiments to be set up.
2.2.6 Qualitative and Quantitative analysis of CCR proteins.
2.2.6.1 Bradford Assay
All the fractions arising in different purification steps were quantified for protein
content using Bradford assays. Titer plates (96-well) were used for quantifying
multiple fractions at a time arising from different purification steps using Bradford
reagent (Sigma), 250 μl of which was added to each well containing 1-5 μl of the
protein sample and A595 measured using xMark microplate reader (Bio-Rad). A
standard graph was made for BSA (1- 10 microgram) and concentration of unknown
sample was determined by plotting on standard graph.
2.2.6.2 Polyacrylamide gel electrophoresis (PAGE)
PAGE system is the widely used electrophoresis system for protein separations
(Laemmeli, 1970). The resolution in a Laemmeli gel is excellent because the treated
peptides are stacked in a stacking gel before entering the separating gel.
2.2.6.3 Preparation of the Separating Gel
A vertical slab gel was assembled using 1.0 mm spacers. In a side armed vacuum
flask, 10% separating gel solution was made for the addition of ammonium persulfate
and TEMED. The solution was degassed by applying vacuum while stirring on a
magnetic stirrer. TEMED and ammonium persulfate were added, and mixed gently

31
without generating bubbles. The solution was pipetted into the gel cassette leaving 1.5
cm from the top unfilled. The gel solution was overlaid with water saturated n-butanol
to remove trapped air bubbles and to layer evenly across the entire surface. When a
sharp liquid-gel inter-surface was observed after the gel polymerization, the slab was
tilted to pour off the overlay.
2.2.6.4 Preparation of the Stacking Gel
Stacking gel solution was prepared excluding ammonium per sulfate and TEMED. As
in the separating gel, this solution was degassed. TEMED and ammonium per sulfate
were added, mixed and overlaid on the separating gel. A comb was inserted taking
care not to trap air bubbles beneath the comb teeth. The gel was left to polymerize.
2.2.6.5 Preparation of the sample
Equal parts of the protein sample and the loading buffer were mixed in a eppendorff
tube and kept in a boiling water bath for 10 mins for SDS-PAGE. Then the samples
were centrifuged at 10,000g for 10 min, supernatant collected and used for
electrophoresis. Gel was run at room temperature at 100 V. The protein samples for
native PAGE were mixed with native dye and gel was run at 80 V.
2.2.6.6 Loading and Running the Gel
The comb was removed from the gel, the wells were flushed with distilled water and
drained off completely. The wells were filled with tank buffer and the samples under
laid using a syringe. Lower and upper buffer chambers were filled with tank buffer.
Voltage was set between 70 and 80 volts. The run was stopped when the dye reached
the bottom of the gel.
2.2.6.7 Silver Staining
The introduction of silver staining of proteins in Polyacrylamide gels in 1979 (Switzer
et al. 1979) has been a breakthrough in the field of protein detection, raising the
sensitivity from the microgram range obtained with dyes such as Coomassie blue to
the nanogram range. This breakthrough as well as the numerous problems
encountered with this first protocol (cost, background, non specific surface staining)
led to extensive research and to the description of various silver staining methods

32
(reviewed in Rabilloud, 1990). To summarize these data, we can consider that every
staining protocol is divided into five main phases.
Protocol:
1. After complete the procedure of SDS PAGE gel electrophoresis, gel was
transferred in fixer solution for 1 hr and kept on the dancing shaker.
2. After 1 hr gel was transferred in fixer solution II 50% ethanol for 30 mins
on dancing shaker.
3. Sodium thiosulphate solution (0.02% w/v) was added and kept for two mins
on dancing shaker.
4. Gel was washed thrice with distilled water.
5. Silver nitrate solution (0.2% w/v) containing formaldehyde (0.75% v/v)
was added and kept for 20 mins in dark on dancing shaker.
6. Gel was washed thrice with distilled water.
7. Gel was transferred in developer solution (6% Na2CO3 w/v, 0.05%
formaldehyde v/v and 0.02% sodium thiosulphate w/v) and kept till the
protein bands appear and stored in fixer solution.
2.2.7 Western Blot Analysis:
Detection and Purification of both CCR proteins were also monitored by Western
blot analysis. Here, western analysis was conducted using primary antibodies against
CCR enzyme as well as the Histidine Tag. Different antibodies and substrate used are
shown in Table 2.10. Protein (20 µl) was loaded onto 10% SDS-PAGE and run. The
protein on the gel was then transferred onto PVDF membrane through iBlot Dry
Blotting System (Invitrogen) according to the manufacturer’s protocol. One
membrane was used for hybridization with anti-His tag antibody, while other was
used for hybridization with anti-CCR Ab. The membranes were incubated in
appropriately diluted antibodies for 2 hours each and then visualized by adding the
substrate for alkaline phosphatase.

33
2.2.8 ESI-MS Analysis:
As described earlier, two step purification protocol was used for purification of CCR
proteins. First, Affinity chromatography which removed majority of contaminants
protein and yielded 90 % purified CCR protein along with some minor contaminants.
Two of them are interesting having molecular weight of approximately 32 KDa and
27 KDa because both were still present even after second purification i.e. anion
exchange (DEAE Sepharose). Optimization for purification was done to remove the
contaminants by altering binding, washing and elution conditions; but yielded same
results. Different chromatography technique combinations were used in order to
remove these contaminants : Anion exchange ( Q Sepharose, DEAE Sepharose)
followed by cation exchange ( SP Sepharose, CMSepharose) or vice versa; Ion
exchange followed by Hydrophobic interaction( phenyl Sepharose ) and vice versa
but resulted in co-purification of contaminants along with CCR protein. Also it is
difficult to separate these proteins from CCR by gel filtration due to small differences
in molecular sizes. Co-purification of these proteins with CCR clearly shows that they
have similar physio-chemical properties like CCR. These proteins can be degradation
products of same CCR protein due to premature termination of translation (Molecular
level) or degradation of CCR protein by proteases. Thus different combinations of
Protease inhibitors cocktail were used but ended up with same results as described
above. Thus identification of theses contaminants proteins is necessary in order to get
some understanding about nature, physio-chemical properties of these which will help
to design purification strategies to get purest form of CCR protein. Thus ESI-MS
technique was used to identify the contamination proteins along with CCR.
For ESI-MS analysis, 10% SDS-PAGE was run for affinity and ion exchange
purified CCR protein. Gel was then stained with coomassie Blue. Expected
stained protein bands were excised from the gel. The gel pieces were then
destained by Destaining solution (50% acetonitrile / 50% 50 mM NH4 HCO3 )
till color was gone. Gel was then dehydrated by treating with 100% acetonitrile.
After dehydration acetonitrile was completely removed by evaporating briefly in
speedvac till noticeably shrunken and white. Then gel pieces were dissolved in
10 mM DTT in 100 mM NH4HCO3 and proteins were reduced for 45-60 min. at

34
56 ºC. Cooled to room temperature, DTT solution removed and 55 mM
iodoacetate in 100mM NH4 HCO3 was added. This mixture was vortexed, spun
briefly and incubated for 45 min in dark place at room temperature. This was
followed by iodoacetamide removal and gel pieces were washed with 100 mM
NH4 HCO3 for 5 min. Again gel pieces were washed twice with 50% acetonitrile /
50% 50 mM NH4 HCO3) and dehydrate with 100% acetonitrile as mentioned
above. Then enough Trypsin solution was added to cover the gel pieces (usually
around 20 µL) and the gel pieces were rehydrated at 4 ºC for 30 min in buffer
containing 50 mM NH4 HCO3 and Trypsin. Spun briefly and more NH4 HCO3 was
added to cover gel pieces (typically another 25 µL). This was followed by
overnight digestion at 37 ºC. The digested solution (supernatant) was
transferred into clean 1.5 mL eppendorf tube. Add 50 % acetonitrile /
2% formic acid solution to the gel pieces which was incubated and vortexed for
20 min. This was spun and sonnicated for 5 mins in a water bath with no heat.
Supernatant was then removed and combined with initial digestion solution
(supernatant). Extracted digests was then vortexed, evaporated to reduce to 5-10 µL.
The remaining 5-10 µL was spun at 14k rpm for at least 10 min. to remove any micro
particulates. The supernatant was carefully transferred to a fresh 1.5 mL eppendorf
tube. The sample was then ready for loading onto ESI-MS. Analysis was done
using different softwares like Protein Lynx Global server, PLGS by searching the
databank sequences in UniProt.

35
2.2.9 Crystallization
The first step considered crucial in protein structure determination is the growth of
diffraction quality single crystals. In the absence of any single concrete theory behind
the mechanism of crystallization, we have treated the protein crystallization as a trial
and error procedure invoking experience and crystallization reports as guiding
principles. It is accepted that the presence of impurities, ionic strength, pH,
temperature, precipitating agent and several unspecified factors play role in
crystallization process.
Among various crystallization techniques known, hanging drop vapor diffusion
method is widely adopted and has produced more crystallized proteins than all other
methods combined (Chayen, 1998).
This method is simple, consumes less protein and it is easy to monitor the progress of
crystallization. In a typical experimental set up using multiwell trays, 2 μl of CCR
protein solution was placed on a siliconized cover slip, mixed with 2 μl of the
precipitant solution and allowed to slowly equilibrate against 0.5 ml reservoir solution
of the precipitant. Different Crystal screen solution Kits were used for screening
crystallization conditions, such as Index, PEG/Ion 1 & 2, Crystal Screen 1 & 2
(Hampton Research), CSS 1 & 2 (Molecular Dimensions), JCSG I & II etc. which
contained pre-prepared solutions.
Fig.2.2: The hanging-drop vapor-diffusion method for protein crystallization

36
2.2.10 Enzyme Kinetics
CCR carries out both; the oxidation of aldehydes and the reduction of the
corresponding Hydroxycinnamoyl CoA esters. However, hydroxycinnamoyl CoA
esters (Forward substrates) are not commercially available; thus these have to
chemically synthesize according to method described by Stockigt and Zenk (1975).
Here, Cinnamaldehyde, Coniferaldehyde and sinapaldehyde were used as substrates
for enzymatic reactions and reverse reaction was determined here by monitoring the
change in A420 spectrophotometrically (Lambda 650, Perkin Elmer) for
sinapaldehyde; A400 for coniferaldehyde and A290 for cinnamaldehyde due to the
disappearance of respective aldehydes. Molar extinction coefficients for each
aldehyde were determined in 0.2 M Tris (pH 8.8) buffer. For coniferaldehyde 12900
M cm; sinapaldehyde 14200 M cm; cinnamldehyde were used for calculation of
enzyme activity.
The assay was carried out in 1.0 ml of reaction mixture containing, 200 mM Tris (pH
8.8), 100 μM respective aldehyde, 200 μM NADP+, 200 μM CoA and 5-8 μg of
purified CCR. This reaction mixture was monitored for decrease in absorbance at 400
nm for coniferaldehyde, 420 nm for sinapaldehyde, 290 nm for cinnamaldehyde over
10 mins at an interval of 1 second. All the reactions were carried out in triplicates and
the experiments were performed twice. The readings were plotted against time, and
the slope was calculated.

Chapter 3
Results and Discussions

37
Chapter 3
Results and Discussions
3.1 Expression studies of CCRH1 and CCRH2 in pET 30b (+)
3.1.1 Screening for over Expression of Recombinant Proteins
The CCR genes was cloned in pET 30b (+), and transformed into the expression host
E.coli BL 21 (DE3) cells. The expression of the protein was checked by SDS PAGE.
A ~38 KDa CCRH1 and CCRH2 (Fig1) proteins were found over expressed in these
clones, and were used for further studies.
Fig.3.1: Over expression of CCRH1 and CCRH2 Protein; M: Protein Molecular
weight marker, Lane 1: Un-induced; Lane 2: Induced CCRH1 protein; Lane 3:
Induced CCRH2 protein.

38
3.1.2 Standardization of protein expression in E. coli BL21:
Eukaryotic proteins expressed intracellularly in E.coli are frequently sequestered into
insoluble inclusion bodies. Inclusion bodies are insoluble aggregates of denatured or
partly denatured protein that are less significant in further studies due to lack of
biological activity. Thus in order to obtain functionally active soluble protein (also
termed as Lysate), expression conditions are optimized by reduction in growth
temperature (Fig.3.2), following induction, altering IPTG concentration (Fig.3.4) and
time (Fig.3.3) of culture incubation after induction.
Studies have shown that, 0.5 mM IPTG, 200
C and 14 hour incubation gives optimum
CCRH1 protein and 0.5 mM IPTG, 200
C and 16 hour incubation gives optimum
CCRH2 protein in soluble form.
Fig.3.2 Over-expression of CCRH1 protein in soluble form harvesting at different
Temperature after 0.5 mM induction
Lane M: Molecular weight marker (Bangalore Genei, India), Lane 1: 30 ºC induced
lysate, Lane 2: 30 ºC induced inclusion bodies, Lane 3: 25 ºC induced lysate, Lane 4: 25
ºC Inclusion bodies, Lane 5: 20 ºC induced lysate, Lane 6: 20 ºC induced Inclusion
Bodies

39
Fig.3.3 Over-expression of CCRH1 protein in soluble form harvesting at different
times after 0.5 mM induction at 20 °C: Lane M: Molecular weight marker (Bangalore
Genei, India), Lane 1: Un-induced lysate, Lane 2: 6 h induced lysate,
Lane 3: 16 h induced lysate, Lane 4: 18 h lysate, Lane 5: 20 h lysate
Analysis of result showed that 1 mM IPTG concentration was found to be less
effective for protein expression and other two tested concentration gave more or less
the same result 0.5 mM IPTG concentration was utilized for other experiments.
(Fig.3.4)
Fig.3.4: Inclusion bodies of over-expressed CCRH1 protein at different IPTG
concentration: Equal quantity of total protein was loaded in each well.
Lane 1: 0.1 mM IPTG, Lane 2: 1 mM IPTG and Lane 3: 0.5 mM IPTG.
1 2 3

40
3.2 Expression studies of CCRH1 and CCRH2 in pET 41a (+):
3.2.1 Screening for over-expressing recombinant CCR proteins:
Apart from pET 30 b (+), CCR genes were also cloned in pET 41a (+), and
transformed into the expression host E.coli BL 21 (DE3) cells. The expression of the
protein was checked by SDS PAGE. ~66 KDa CCRH1 and CCRH2 proteins
(Fig.3.5) were found over expressed and selected clones were used for further studies.
Fig.3.5: Over expression of CCRH1 and CCRH2 Proteins cloned in pET 41a (+);
M: Protein Molecular weight marker, Lane 1, 3: induced CCRH2;
Lane 2, 4: Uninduced CCRH2 protein; Lane 5, 7: Induced CCRH1 protein;
Lane 6, 8: Uninduced CCRH1 protein.
3.2.2 Standardization of protein expression in E. coli BL21:
In order to get active soluble CCR proteins, optimization was done with respect to
IPTG concentration, Temperature (Fig.2 .6), and time of culture incubation (Fig.3.7)
after induction. Studies have shown that, 0.5 Mm IPTG, 200
C and 14 hour incubation
gives optimum CCRH1 protein and 0.5 Mm IPTG, 200
C and 16 hour incubation gives
optimum CCRH2 protein in soluble form. .

41
Fig.3.6. Over-expression of CCRH1 protein in soluble form harvesting at different
Temperature after 0.5Mm induction:
Lane M: Molecular weight marker (Bangalore Genei, India), Lane 1: 30 ºC induced
lysate, Lane 2: 30 ºC induced inclusion bodies, Lane 3: 25 ºC induced lysate,
Lane 4: 25 ºC Inclusion bodies, Lane 5: 20 ºC induced lysate,
Lane 6: 20 ºC induced Inclusion Bodies Lane 7: 15 ºC induced lysate,
Lane 8: 15 ºC induced Inclusion Bodies
Fig.3.7: Over-expression of CCRH2 protein in soluble form harvesting at different
times after 0.5Mm induction at 18 °C: Lane M: Molecular weight marker (Bangalore
Genei, India), Lane 1: 4 h induced lysate, Lane 2: 8 h induced inclusion bodies, Lane 3:
12 h induced lysate, Lane 4, 5, 6: induced inclusion bodies at 4,8,12 hours respectively.

42
3.3 Purification of recombinant CCRH1 pET30b (+) clone
3.3.1 Affinity chromatography:
The CCRH1 protein was purified by affinity chromatography which was checked on
SDS PAGE by silver staining. It was observed that the elution started after passing 10
ml of elution buffer, and a single peak was eluted (Fig.3.8) which was collected and
quantified. SDS PAGE (Fig.3.9) showed a band of about 38kDa present in all
fractions, along with some contaminants. 30 mg of protein was recovered after this
step with 22 folds purification.
Fig.3.8: Chromatogram for Ni Sepharose 6 column affinity chromatography
(ÄKTAexplorer™) Blue-A280
.
Fig.3.9: M- Mol. Wt. Marker, Lanes 1 and 2 - Fractions collected from Ni Sepharose
column

43
3.3.2 Sample Conditioning 1
Ni-NTA purified CCRH1 was concentrated using Macrosep Centrifugal devices (Pall
Lifesciences). These concentrated protein samples were further desalted using
Sephadex G-25 column. As shown in the chromatogram, a peak at A280 of 275mA.U.
was observed which was completely free from salts, as well as some low molecular
weight proteins were also separated during the process.
Manual run 6:10_UV1_280nm Manual run 6:10_Cond
0
50
100
150
200
250
300
mAU
0.0 5.0 10.0 15.0 ml
Fig.3.10: Chromatogram for HiPrep Sephadex G-25 Desalting (ÄKTAexplorer™)
3.3.3 Ion Exchange Chromatography
But, Affinity purification did not give complete purification of desired CCRHI protein
due to non specific binding of host cell proteins (<95% purity). Thus charge property
was exploited that is ion exchange chromatography was used for purification of
protein. As we know that the isoelectric point (pI) of CCRH1 is 6.4; thus anion
exchanger i.e. DEAE Sepharose XL matrix was tried for purification of protein.
The bound protein was eluted by applying a gradient elution against Elution Buffer
with 1.0 M NaCl. The gradient was set up for 50 % of Elution Buffer over a period of
one hour at a constant flow rate of 0.5 ml/min. As seen in the chromatogram
(Fig.3.11), one peak was eluted using such a gradient elution. This peak was collected
as 1.0ml fractions, checked on 10% SDS PAGE and was visualized by silver staining.

44
The fractions collected showed a 38kDa band (Fig.3.12). CCR was purified 50 folds
using this step; however, two contaminating proteins still persisted.
Fig.3.11: Chromatogram for DEAE-Sepharose Anion Exchange chromatography
Fig.3.12: M- Mol. Wt. Marker, Lanes 1 to 3- Fractions collected from DEAE -
Sepharose column

45
3.3.4 Sample conditioning 2
DEAE purified CCR was concentrated and desalted using Sephadex G-25 gel
filtration column. As shown in the chromatogram (Fig.3.13), a peak at A280 was
observed which was completely free from salt. This peak was collected as 1.0ml
fractions and quantified. These fractions were pooled together and further
concentrated using smaller Microsep Centrifugal device (Pall Lifesciences) by
centrifuging protein at 5000x g at 4ºC to a final volume of 1.0ml. The concentrated
protein was quantified and checked on 10% SDS PAGE.
This provided the necessary conditioning of the sample required for further
experiments to be set up.
Manual run 3:10_UV1_280nm Manual run 3:10_Cond%
0
100
200
300
400
mAU
100.0 105.0 110.0 115.0 ml
Fig.3.13: Chromatogram for HiPrep Sephadex G-25 Desalting
(ÄKTAexplorer™)

45

Fraction Total
Vol. (ml)
Total
Protein
(mg
Protein
Conc.
(mg/ml
Enzyme
Activity*
(nKat/ml)
Total Activity
(nkat) †
Specific
Activity
(nkat/mg)
Fold
Purification
Yield
(%)
Cell Lysate 60 900 15 12 720 0.8 ‐ ‐
Ni‐sepharose 6
Fast Flow
10 30 3 52.8 528 17.6 22 73.3
DEAE ‐Sepharose
Fast Flow
7 13 1.85 74.28 520 40 50 72.2

† nkat is nanokatals. 1 katal= 0.059 Enzyme Units.
Table 3.4: Overview of the optimized purification protocol for CCR.

47
3.4 Western Blot Analysis
It was observed that anti-His Tag antibody resulted in more than one band in each
Samples, i.e. in Lysate, Ni-Sepharose purified, and DEAE-Sepharose purified samples
with increasing intensity of desired CCR and a faint band below it with low intensity.
This was an indication of increasing concentration of the His-tagged protein with
these purification phases. Purification of CCR can also be seen from the membrane
developed using anti-CCR, wherein, the Lysate showed multiple hybridizations with
the antibody, while Ni-Sepharose and Q-Sepharose fraction showing a few more.
Table3.2: Antibodies for Western blot analysis with their respective dilutions used
CCR His-Tag
Primary Ab Rabbit anti-CCR IgG (
(1:2000)
Mouse anti His IgG (Novagen)
(1:10,000)
Secondary Ab Goat anti-rabbit IgG
(1:20,000)
Goat anti-mouse IgG
(1:20,000)
Linked Enzyme Alkaline Phosphatase
Substrate BCIP/NBT, purple (Bioworld)
Fig.3.14: A) Western blot analysis for CCR by Antibody against CCR protein;
B) Western blot analysis for CCR by Antibody against His-Tag
Western blot analysis for CCR by
Antibody against CCR protein;
1: Ni-Purified
2: ion exchange Purified,
3: Lysate
Western blot analysis for CCR by
Antibody against His-Tag 1: Ni-
Purified
2: ion exchange Purified,
3: Lysate

48
3.5 ESI-MS Analysis
Purified CCR, as well as, both contaminating proteins were subjected to Peptide
Mass Fingerprinting using MALDI-TOF (MaldSynpt, Waters). Analysis of the
peptide fragments generated was done using different softwares like Protein Lynx
Global server, PLGS by searching the databank sequences in UniProt (Fig.3.15).
(A)
(B)
(C)
Fig.3.15: ESI-MS analysis showing sequence of A: ~ 38 KDa CCR Proteins;
B: Contamination protein of size ~32 KDa;
C: Contamination Protein of size ~27 KDa
CCR fragment showed 97% sequence similarity to CCR protein from Leucaena
glauca with almost 149 peptide hits (Table3.2). It was also observed that the two
contaminating proteins showed a striking similarity to bona fide CCR protein. This
may be due to either truncated expression in the host cells or, protease digestion
during purification procedure.

49
Table3.3: Percent homology of CCR protein with available sequence in database
CCR
Protein
Entry Description Peptides Coverage
(%)
~38
KDa
Q08GL0_LEUGL Cinnamoyl CoA Reductase OS
Leucaena glauca GN CCR PE 2 SV 1
149 97.0238
~32KDa Q08GL0_LEUGL Cinnamoyl CoA Reductase OS
107 69.161
~27
KDa
Q08GL0_LEUGL Cinnamoyl CoA Reductase OS
77 52.952
3.6 Crystallization
Initial crystallization trials of CCR did not yield crystals. Subsequently it was found
that the presence of formates or acetates was essential for growing crystals in
presence of polymers like polyethylene glycol (PEG) at slightly alkaline pH. PEG/Ion
Screens showed that the protein crystallized in a variety of conditions but the crystals
were of small size and possessed different morphologies. These tiny crystals were not
used for X-ray measurements owing to their small size and growth defects. However,
attempts are being made towards optimizing these conditions to obtain better quality
crystals with good diffraction properties.
3.7 Enzyme Kinetics
Cinnamoyl-CoA Reductase (EC 1.2.1.44) is an enzyme that catalyzes the chemical
reaction
Cinnamaldehyde + CoA + NADP+
Cinnamoyl-CoA + NADPH + H+
This enzyme belongs to the family of Oxidoreductases, specifically those acting on
the aldehydes or oxo group of donor with NADP+ as acceptor. The systematic name
of this enzyme class is Cinnamaldehyde: NADP+ Oxidoreductases (CoA-
Cinnamoylating). Other names in common use include Feruloyl-CoA Reductase,

50
Cinnamoyl-coenzyme A Reductase, Ferulyl-CoA Reductase, feruloyl coenzyme A
Reductase, p-Hydroxycinnamoyl coenzyme A Reductase, and cinnamoyl-CoA:
NADPH Reductase. This enzyme participates in phenylpropanoid biosynthesis.
Cinnamaldehyde, Coniferaldehyde and sinapaldehyde were used as substrates for
enzymatic reactions by monitoring the change in 420 nm for sinapaldehyde; 400 nm
for Coniferaldehyde and 290 nm for Cinnamaldehyde.
Molar extinction coefficients for each aldehyde were determined in 0.2 M Tris (pH
8.8) buffer. For Coniferaldehyde 14200 M-1
cm-1;
sinapaldehyde 12900 M-1
cm-1
and
Cinnamaldehyde 18868 M-1
cm-1
were used for calculation of enzyme activity
The enzyme activity was calculated as moles of substrate converted to product per
unit time, and was expressed as Enzyme Units.
Enzyme Activity (units/ml) = ΔA × V
VE × E
Where,
ΔA = Slope of A340 vs. Time (t)
V = Volume of reaction
VE = Volume of the enzyme
E = Molar Extinction coefficient
However, the enzyme activity can also be expressed as Nanokatal (nKat), and
similarly, the specific activity as nKat/mg of protein.
1 Nanokatal (nKat) = 0.059 Enzyme Units (EU).
Km and Vmax for different aldehydes were determined for purified CCR using
Michelis-Menten plots (Fig.3.18, 3.19, 3.20). CCR showed maximum Vmax as well
Km values for Coniferaldehyde, followed by Sinapaldehyde, and, lowest for
Cinnamaldehyde (Table3.3). The results showed that Coniferaldehyde is the preferred
substrate for CCR. The results can be further validated by enzyme kinetic studies.

51
Fig.3.16: Graph of A340 vs. Time for Cinnamaldehyde
Fig.3.17: Graph of A340 vs. Time for Coniferaldehyde
Michaelis-Menten data
[Substrate]
enzymeactivity
0 5 10 15 20 25
0
200
400
600
[Substrate]
enzymeactivity
0 5 10 15 20
0
20
40
60
80
100

52
Fig.3.18: Graph of A340 vs. Time for sinapaldehyde
Table3.4: Specific activities of purified CCR enzyme for different aldehydes
Km ( μM) Vmax (units/min)
Coniferaldehyde 38.74 621.9
Sinapaldehyde 52.53 453.6
Cinnamaldehyde 136.9 166.8
3.7.1 Determination of optimum pH for CCR Enzyme
For optimum pH, enzyme assay was carried out using different buffers having pH
range of 2-12 at every 0.5 pH intervals. Coniferaldehyde was used as substrate as
showed higher specificity. Buffers used in enzymatic assay are as follows: Tris-
Glycine (PH 2-4); Citrate buffer (pH 4-6); Phosphate Buffer (pH 6-7.8); Tris-HCl (pH
7-9); Glycine-NaOH (pH 9-10).
[Substrate]
enzymeactivity
0 5 10 15 20 25
0
100
200
300
400

53
A graph of residual enzyme activity (%) Vs. pH was plotted to determine optimum
pH. The optimum pH for the enzyme was found to be in range of 8.5-9.5, with optima
at pH 9.
Optimum PH
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14
PH
ResidualActivity(%)
Series1
Fig.3.19: Graph of residual activity (%) vs. pH
3.7.2 Determination for optimum temperature for CCR Enzyme
For optimum Temperature, enzyme assay was carried out at different temperature
ranges from 4 – 80 at every 5 °C interval. Coniferaldehyde was used as a substrate
and graph of residual Enzyme activity (%) Vs. Temperature was plotted for
temperature optima determination. Significant activity was observed to be in range of
25-55 °C, with optima at 30°C.
Temperature Optima
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80 90
Temperature (° C)
ResidualActivity(%)
Series1
Fig.3.20: Graph of residual Enzyme activity (%) Vs. Temperature

54
3.7.3 Determination of pH stability for CCR Enzyme
Phosphate buffer (6-7 PH) and Tris-Cl (7-9 PH) were used for pH stability
experiments. Enzyme (5-8 microgram) was incubated with phosphate and tris buffers
at different pH at two different temperatures i.e. 4 and 25 C. Readings were taken
after every 24 hours and enzyme activity was calculated. A graph of residual activity
(%) Vs. Time (in Hours) was plotted. The pH stability experiments were done and the
enzyme was found to be stable at pH 8, while the enzyme gradually lost activity with
higher as well as lower pH values at both temperatures.
pH stability at 4° C
0
20
40
60
80
100
120
0 50 100 150 200
Time(Hrs)
Residualactivity(%)
pH 6.0 phosphate buffer
pH 7.0 phosphate Buffer
pH 8.0 Tris-cl
pH 9.0 Tris-cl
pH 7.0 Tris-cl
Fig.3.21: Graph of residual activity (%) Vs. Time (in Hours) at 4ºC
pH stability at 25° C
0
20
40
60
80
100
120
0 50 100 150 200
Time(Hrs)
ResidualActivity(%)
pH 7.0Tris-cl
pH 8.0 Tris-cl
pH 9.0 Tris-cl
Fig.3.22: Graph of residual activity (%) Vs. Time (in Hours) at 25ºC

55
3.7.4 Determination of temperature stability for CCR Enzyme
Three different temperatures i.e. 4 °C, 25°C and 37 °C were used for determining
temperature stability. Enzyme (5-8 microgram) was incubated at respective
temperatures and activity assay was done after every 24 hours. A graph of residual
enzyme activity vs. time (in hours) was plotted. The temperature stability experiments
were done and the enzyme was found to be stable at 4 0
C with a loss of only 20%
after 1 week. However, a rapid loss in activity was observed at 37°C, CCR retained
only 20% activity after 1 week at 25°C.
Temperature Stability
0
20
40
60
80
100
120
0 50 100 150 200
Time(Hrs)
ResidualActivity(%)
Temerature 4°C
Temperature 25°C
Temperature 37°C
Fig.3.23: Graph of residual activity (%) Vs. Time (in Hours)

56
Chapter 4
Summary
4.1 Summary
Leucaena sp. as a source for paper pulp. Although all these plant species are of
importance to the paper industry, Leucaena sp. is extensively used in India and nearly
a quarter of raw materials for paper and pulp industry come from this plant.
Tremendous effort has been devoted to develop genetically engineered plants, with
the emphasis on reducing lignin content to improve wood pulp-production efficiency,
however, lignin chemical reactivity also is a critical barrier to wood pulp production.
Thus, the current tree-biotechnology emphasis on low lignin quantity must be
expanded to include greater lignin reactivity and, ultimately, a combination of low
and reactive lignin traits.
In this context, the present study was aimed at a better understanding of the enzymes
involved in phenylpropanoid pathway; the one responsible for monolignols
biosynthesis in plants, and study its structure-functional relationships. The enzyme
Cinnamyl Co-A Reductase (CCR) involved in reversible reduction of
cinnamaldehydes to hydroxycinnamyl alcohols was targeted in the study due to its
key role in the monolignols biosynthesis in plants.
In the present studies, CCR gene was directionally cloned in expression vector pET
30-b (+) using the restriction sites Nde1 and Xho1, and into pET 41 a (+) using
BamH1 and Xho1, and expressed in E.coli BL21 DE3. A protein with a molecular
weight of 38 KDa was expressed in these cells.
Subsequently, a simple and efficient purification protocol employing two
chromatography steps- Ni-Sepharose affinity and DEAE-Sepharose anion-exchange

57
chromatography were optimized for purifying His Tagged CCRH1. Following this
purification procedure, CCR showed a 50 fold increase in its specific activity than
from the bacterial cell lysate, and resulted in pure protein, which was confirmed by
Western Blot analysis.
Initial crystallization screens did not yield any crystals; however, different conditions
are being optimized in order to obtain diffraction quality crystals. Purified CCR
showed a temperature and pH optima at 30 °C and 9 pH, respectively. Kinetic studies
on the purified CCR showed that Coniferaldehyde is the preferred substrate for CCR.
The results can be further validated by enzyme kinetic studies. Even so this data gives
preliminary idea about substrate preference of Leucaena CCR enzyme.
4.2 Future Prospects
On the basis of this work, further studies on structural and functional aspects of CCR
have been planned which are enlisted below:
1. Crystallization protocol will be optimized in order to obtain good quality crystals
with detectable diffraction properties. This will include optimizing different
parameters like pH, compositions and concentrations of buffers, precipitants,
polymers, etc. An attempt will be made to elucidate the structure of CCR using X-ray
diffraction studies of CCR crystals.
2. Further biochemical Characterization of CCR, in terms of effects of different metal
ions, temperature, and pH will be carried out. Enzyme kinetic parameters for both
forward and reverse reactions will be determined, along with inhibition studies with
different inhibitor compounds, Active Site modification studies (using different
chemicals), Enzyme kinetics using different Hydroxycinnamoyl CoA esters
(Cinnamoyl CoA, Feruloyl CoA and Sinnapoyl CoA) will be carried out. Also the
studies on site directed mutagenesis will be carried out.

58
Chapter 5
Bibliography
1. Adler E. 1977. Lignin chemistry: past present and future. Wood Science and Technology 11,
169–218.
2. Baucher M, Halpin C, Petit-Conil M, Boerjan W. 2003. Lignin: genetic engineering and
impact on pulping. Critical Reviews in Biochemistry and Molecular Biology 38, 305–350.
3. Baucher M, Monties B, Van Montagu M, Boerjan W. 1998. Biosynthesis and genetic
engineering of lignin. Critical Reviews in Plant Science 17, 125–197.
4. Boerjan W, Ralph J, Baucher M. 2003. Lignin Biosynthesis. Annual Reviews in Plant
Biology 54, 519- 546.
5. Boerjan W 2003. Lignin Biosynthesis. Annual Reviews in Plant Biology 54, 519- 546.
6. Boudet AM. 2000. Lignins and lignification: selected issues. Plant Physiology and
Biochemistry 38, 81-96.
7. Boudet AM, Lapierre C, Grima-Pettenati J. 1995. Biochemistry and molecular biology of
lignification. New Phytologist 129, 203–236.
8. Boudet AM (2000) Lignins and lignification: selected issues. Plant Physiol. Biochem. 38: 81-
96.
9. Boerjan W, Ralph J, Baucher M. 2003. Lignin Biosynthesis. Annual Reviews in Plant
Biology 54, 519- 546.
10. Brewbaker JL, Sorenson CT (1990) Leucaena: New tree crops from interspecific hybrids.
In: Janick. J and Simon J. (Eds). Advances in new crops Timber Press, Oregon. pp 238.
11. Buxton DR, Redfearn DD (1997) Plant limitation to fiber digestion and utilization. J. Nutr.
127: S814-S818.
12. Campbell MM, Sederoff RR. 1996. Variation in lignin content and
composition.Mechanisms of control and implications for the genetic improvement of plants.
Plant Physiology 110, 3–13.

59
13. Chabannes M, Ruel K, Yoshinaga A, Chabbert B, Jauneau A, Joseleau J-P, Boudet A-
M. 2001. In situ analysis of lignins in transgenic tobacco reveals a differential impact of
individual transformations on the spatial patterns of lignin deposition at the cellular and
subcellular levels. Plant Journal 28, 271–82.
14. Davin LB, Lewis NG. 2005. Lignin primary structures and dirigent sites. Current Opinion in
Biotechnology 16, 407–415.
15. Davin LB, Wang H-B, Crowell AL, Bedgar DL, Martin DM, Sarkanen S, Lewis NG.
1997. Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein
without an active center. Science 275, 362–366
16. Dharmawardhana DP, Ellis BE, Carlson JE. 1995. A _-glucosidase from lodgepole pine
xylem specific for the lignin precursor coniferin. Plant Physiology 107, 331–339.
17. Dixon RA, Chen F, Guo D, Parvathi K (2001) The biosynthesis of monolignols: a
“metabolic grid,” or independent pathways to guaiacyl and syringyl units Phytochemistry 57:
1069–1084.
18. Donaldson LA. 2001. Lignification and lignin topochemistry—an ultrastructural view.
Phytochemistry 57, 859–873.
19. Freudenberg K, Neish AC, ed. (1968) Constitution and Biosynthesis of Lignin. Berlin:
Springer-Verlag. pp 129
20. Fengel D, Wegener, G (1984) Lignin. In Wood: Chemistry, Ultrastructure, Reactions : 132–
181.
21. Geiger, C.A., Napompeth B., Van Den Beldt R., 1995. An update on the status of leucaena
psyllid in Southeast Asia. In: Shelton H.M., Piggin C.M. and Brewbaker J.L. _eds_, Leucaena
– Opportunities and Limitations. Proceedings of an International Workshop held in Bogor,
Indonesia, 24-29 January 1994.
22. Goujon T, Ferret V, Mila I, Pollet B, Ruel K, Burlat V, Joseleau J-P, Barri`ere Y,
Lapierre C, Jouanin L. 2003. Down-regulation of the AtCCR1 gene in Arabidopsis
thaliana: effects on phenotype, lignins and cell wall degradability. Planta 217, 218–228.
23. Hammond AC, Allison MJ, Williams MJ. 1989b. Persistence of DHP-degrading bacteria
between growing seasons in subtropical Florida. Leucaena Research Report 10, 66.
24. Hegarty MP, Court RD, Thorne PM. 1964. The determination of mimosine and 3, 4- DHP
in biological material. Australian Journal of Agricultral Research 15, 168.
25. Hengen P. 1995. Purification of His-Tag fusion proteins from Escherichia coli. Trends in
Biochemical Science 20(7), 285-286.
26. Hosokawa M, Suzuki S, Umezawa T, Sato Y (2001) Progress of lignification mediated by
intercellular transportation of monolignols during tracheary element differentiation of isolated
Zinnia mesophyll cells. Plant Cell Physiology 42: 959– 68.

60
27. Hughes CE. 1998. Leucaena: A genetic resources handbook. Tropical Forestry Papers No.
37. Oxford Forestry Institute, Department of Plant Science. Oxford University, UK.
28. Hughes, M.A., 1993. Molecular genetics of plant cyanogenic b-glucosidases. In A Esen, ed,
b-Glucosidases: Biochemistry and Molecular Biology. American Chemical Society,
Washington, DC, pp 153–169
29. Humphreys JM, Chapple C. 2002. Rewriting the lignin roadmap. Current Opinion in Plant
Biology 5, 224–229.
30. Humphreys JM, Hemm MR, Chapple, C (1999) New routes for lignin biosynthesis defined
by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional
cytochrome P450-dependent monooxygenase. Proc. Natl. Acad. Sci. 96:10045–10050.
31. Jones RJ. 1979. The value of Leucaena leucocephala as feed for ruminants in the tropics
World Animal review 32, 10.
32. Kao, Harding SA, Tsai CJ (2002) Differential expression of two distinct phenylalanine
ammonia-lyase genes in condensed tannin-accumulating and lignifying cells of quaking
aspen. Plant Physiology 130: 796–807.
33. Kuc J, Nelson OE, Flanagan P (1968) Degradation of abnormal lignins in the brown-midrib
mutants and double mutants of maize. Phytochemistry 7: 1435–1436.
34. Lacombe E, Hawkins S, Doorsselaere JV, Piquemal J, Goffner D, Poeydomenge O,
Boudet, AM. 1997. Cinnamoyl CoA reductase, the first committed enzyme of the lignin
branch biosynthetic pathway: Cloning, expression and phylogenetic relationships. Plant
Journal 11, 429–441.
35. Larsen K. 2004. Cloning and characterization of a ryegrass (Lolium perenne) gene encoding
cinnamoyl-CoA reductase (CCR). Plant Science 166, 569–581.
36. Lauvergeat V, Lacomme C, Lacombe E, Lasserre E , Roby D, Grima-Pettenati J. 2001.
Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially
expressed during development and in response to infection with pathogenic bacteria.
Phytochemistry 57, 1187–1195.
37. Lee D, Meyer K, Chapple C, Douglas CJ. 1997. Antisense suppression of 4- coumarate:
coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition. Plant
Cell 9, 985– 998
38. Lee D, Meyer K, Chapple C, Douglas CJ. 1997. Antisense suppression of 4- coumarate:
coenzyme A ligase activity in Arabidopsis leads to altered lignin subunit composition. Plant
Cell 9, 985– 998
39. Lewis NG, Yamamoto E. 1990. Lignin: occurrence, biogenesis and biodegradation. Annual
Review of Plant Physiology and Plant Molecular Biology 41, 455–496.

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